Mycobacterium tuberculosis ParB is phosphorylated by several mycobacterial Ser/Thr protein kinases in vitro. ParB and ParA are the key components of bacterial chromosome segregation apparatus. ParB is a cytosolic conserved protein that binds specifically to centromere-like DNA parS sequences and interacts with ParA, a weak ATPase required for its proper localization. Mass spectrometry identified the presence of ten phosphate groups, thus indicating that ParB is phosphorylated on eight threonines, Thr32, Thr41, Thr53, Thr110, Thr195, and Thr254, Thr300, Thr303 as well as on two serines, Ser5 and Ser239. The phosphorylation sites were further substituted either by alanine to prevent phosphorylation or aspartate to mimic constitutive phosphorylation. Electrophoretic mobility shift assays revealed a drastic inhibition of DNA-binding by ParB phosphomimetic mutant compared to wild type. In addition, bacterial two-hybrid experiments showed a loss of ParA-ParB interaction with the phosphomimetic mutant, indicating that phosphorylation is regulating the recruitment of the partitioning complex. Moreover, fluorescence microscopy experiments performed in the surrogate Mycobacterium smegmatis ΔparB strain revealed that in contrast to wild type Mtb ParB, which formed subpolar foci similar to M. smegmatis ParB, phoshomimetic Mtb ParB was delocalized. Thus, our findings highlight a novel regulatory role of the different isoforms of ParB representing a molecular switch in localization and functioning of partitioning protein in Mycobacterium tuberculosis.Here, we present for the first time that  Mycobacterium tuberculosis (M. tb), the etiologic agent of Tuberculosis, infected 8.6 million people and was responsible for the death of 1.3 million people in 2012 thus remaining a major threat to global health ATP , and 2 to 4 μg of kinase in order to obtain the optimal autophosphorylation activity for each mycobacterial kinase for 30 min at 37°C. Each reaction mixture was stopped by addition of an equal volume of 5 × Laemmli buffer and the mixture was heated at 100°C for 5 min. After electrophoresis, gels were soaked in 16% TCA for 10 min at 90°C, and dried. Radioactive proteins were visualized by autoradiography using direct exposure to films.E. coli strain carrying pDuet_parB and co-expressing the PknB kinase domain, was subjected to mass spectrometry without further treatment. Subsequent mass spectrometric analyses were performed as previously reported [Purified His-tagged hyper-phosphorylated ParB (ParB-P) from the reported –42. SpecM. tb H37Rv chromosomal DNA as a template which encompassed the parS region with respective primers pair listed in 32P]-ATP and T4 polynucleotide kinase. A typical assay mixture contained in 20 μl: 10 mM Tris-HCl, pH 7.5; 50 mM NaCl; 1 mM EDTA; 1 mM dithiothreitol (DTT); 0.1 μg of nonspecific competitor (polydI-dC); 5% (v/v) glycerol; radioactive DNA probe (2000 cpm.ml-1) and various amounts  of the purified ParB proteins. After 20 min of incubation at room temperature, 20 μl of this mixture was loaded onto a native 5% (w/v) polyacrylamide TBE Ready Gel (Bio-Rad) and electrophoresed in 1% TBE (v/v) buffer for 1 h at 100 V.cm-1. Radioactive species were detected by autoradiography using direct exposure to films.The DNA probe for electrophoretic mobility shift assays (EMSA) was generated by PCR using M. tb parB and parB_Ala genes were amplified from the corresponding pETPhos vector contructs and cloned into the shuttle vector pVV16 ATP, the proteins were resolved by SDS-PAGE and the protein phosphorylation status was analyzed by autoradiography. The presence of an intense radioactive signal indicated that ParB was phosphorylated by PknA, PknB, PknD, PknE, PknF and PknH  and subjected to mass spectrometric analysis after tryptic digestion. Noteworthy, we decided to use the PknB kinase previously described as involved in cell division processes among the kinases able to phosphorylate ParB in our in vitro assay ATP. Following separation by SDS-PAGE and analysis by autoradiography, total abrogation of the phosphorylation signal was observed , we confirmed in vitro phosphorylation of M. tb ParB by PknB from M. smegmatis (data not shown). Next, we investigated the in vivo phosphorylation status by western blotting using either anti-phosphothreonine, or anti-phosphoserine antibodies . El. ElparS t manner  as report manner , supportt manner . Our resM. smegmatis [Bordetella pertussis adenylate cyclase. Positive signals were observed with ParB_WT and ParB_Ala versus ParA, independently of the adenylate cyclase fragment and its position in the fusion protein suggesting that ParB_WT and ParB_Ala interact with ParA in vivo. Under the same conditions, no signal was observed when the interaction between ParB_Asp and ParA was tested indicating that ParB_Asp no longer interacts with ParA. Therefore, phosphorylation seems to also regulate ParB interaction with the cytoskeletal partner protein ParA. Noteworthy, BTH experiments indicated, that all ParB derivatives tested retain the ability to dimerise. This suggests, that replacement of the phosphorylation sites by alanine or aspartate does not prevent ParB dimerization, thus these results were not due to a tertiary or quaternary structure change due to the introduction of the mutations.A recent study highlighted the essential role of ParA in the proper positioning and number of ParB complexes in megmatis . ParA loin vivo. Wild-type ParB, phosphoablative and phosphomimetic ParB mutants were fused to EGFP using pVV16_egfp vector under hsp60 promoter and the resulting constructs were introduced into M. smegmatis mc2155ΔparB strain [M. tuberculosis ParB was fully functional in M. smegmatis cells, as demonstrated by the complementation of the parB deletion phenotype   . Such a  length) . This obn mutant . While mh strain .We propose that both phosphorylated and unphosphorylated states of ParB are required for proper chromosome segregation and that a subtle balance between the phosphorylated and unphosphorylated states of ParB would tune the segregation event during the cell cycle.parS, and this binding is modulated via the interaction with the cytoskeletal partner protein ParA, which is thought to enhance the binding of ParB to its DNA targets if ParB is complexed with ParA. In addition, ParB interaction with ParA is critical for a proper positioning of ParB in proximity to the cell pole. The PknB-mediated phosphorylation of ParB inactivates the DNA binding activity of this partitioning protein, as well as the interaction with ParA, thereby preventing the localization of ParB, thus the formation of the mitotic-like apparatus. Our results provide a foundation for further investigation of an important functional linkage between STPKs and the ParABS chromosome segregation system, which may contribute to proper mycobacterial cell division. Interestingly, it was shown before that ParA interacts with another phosphorylated protein Wag31 (DivIVA homologue) possibly providing another link between segregation apparatus and STPKs regulation. Although challenging, future studies will help identify extracellular cues sensed by kinases leading to the phosphorylation of ParB. This will allow us to understand how M. tuberculosis senses its environment and mediates its response in a coordinated manner to regulate chromosome partition.We have used a combination of genetic and biochemical approaches to provide the first reported evidence that the partitioning protein ParB, is negatively regulated by phosphorylation. Our work suggests that a shift in the unphosphorylated/phosphorylated ParB balance in favor of the phosphorylated form rapidly leads to the delocalization of ParB into the cytosol. An hypothesis arising from this work is that a regulatory role of the different isoforms of ParB represents a molecular switch in partitioning protein localization and function. The unphosphorylated form of ParB binds to its target DNA sequences, S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


PIT (passive integrated transponder) tags. PIT tags were implanted into 393 juvenile and adult brown trout Salmo trutta L. and European sculpin Cottus gobio L. in a boreal stream subjected to considerable ice formation. With weekly trackings over 6 months, we quantified apparent survival and detection probability in relation to biological, environmental, and methodological factors. Individuals with a higher physical condition in autumn showed a higher apparent survival; this pattern was consistent across all species and age classes. Detection probability decreased with increasing thickness of the surface ice layer; this effect was most pronounced for juvenile trout and benthic‐living sculpin, both tagged with smaller‐sized tags. Detection probability was reduced in structurally complex habitats. Our study demonstrates that apparent survival and particularly detection probability may show pronounced spatiotemporal variation. In order to compare results from different sampling occasions and sites, a good knowledge of the study site and of the regulating factors is crucial.Environmental fluctuations exert strong control on behavior, survival, and fitness of stream biota. Technical improvements increasingly allow for tracking the response of large numbers of individuals to environmental fluctuations, for instance, by remote detection of animals equipped with  River ecosystems are highly dynamic, with environmental conditions undergoing more frequent and more rapid spatiotemporal fluctuations than in terrestrial, marine, or lentic habitats Power . EnvironOver the past years, PIT (passive integrated transponder) technology has been improved and increasingly used in high‐altitude and latitude streams to explore how environmental fluctuations and particularly ice formation affect winter habitat use, activity, and physical condition in fish and how these factors influence demographic processes such as reproduction, mortality, immigration, and emigration  and European sculpin (Cottus gobio L) in a boreal stream over winter, by accounting for the influence of biological, environmental, and methodological factors. Our first objective was to determine to what degree apparent survival is affected by individual characteristics of fish such as physical condition in autumn or habitat use. Our second objective was to quantify detection probability under temporally dynamic environmental conditions such as the formation of different types of river ice. Third, we applied two different tag sizes which allowed us to account for the effect of tag size on detection probability, also by considering interactions with biological and environmental factors.We quantified apparent survival and detection probability of 393 PIT‐tagged brown trout , and in deep, structurally complex habitats. We expected apparent survival over winter to be positively correlated with physical condition of fish in autumn.The study was performed in a 550‐m long reach of Smörbäcken, a third‐order tributary to the Ume River in Västerbotten County, Sweden . In this boreal environment, winter lasts from early November to early April , and rivers are subject to pronounced formation of ice. Their flow regime is snowmelt‐driven, with the highest water levels in May–June and the lowest in late winter. The Smörbäcken catchment is characterized by crystalline geology (Precambrian gneiss) and mixed boreal forest. The study reach has a near‐natural morphology.In autumn 2010, we generated a detailed 3D map of the study reach by means of a total station . On average, one cross‐sectional profile consisting five evenly distributed points was set every 2 m along the river course. In addition to the topographical measurements, water depth was determined at each point. Furthermore, we collected the positions of 221 reference trees for fish tracking (see below), distinctly marked with a code of plastic tape. Field data were processed in ArcMap 9.3.1 .hi [m] across the study reach was approximated for weekly intervals following the Stefan equation approach .Average ice thickness h Ashton  by accouh Ashton  hi=2ks/iρ was set to 1000 g/m3  was accounted for by resetting thickness of the snow layer to 3 cm in −1; SD = 29 individuals 100 m−1) and brown trout  with various age classes present. There were occasional occurrences of European perch Perca fluviatilis L., European brook lamprey Lampetra planeri Bloch, river lamprey Lampetra fluviatilis L., and northern pike Esox lucius L. .The study reach harbors high densities of European sculpin , based on natural breaks in meso‐habitats  and juvenile trout of 110–150 mm length (n = 50). Adult trout >150 mm (n = 161) were tagged with 23‐mm HDX tags . Tags were surgically inserted into the body cavity through a small incision of 3 mm width for the 12‐mm tags and 6 mm width for the 23‐mm tags. For the sculpin, the incision was made about 2 mm off the mid‐ventral line, between the pelvic girdle and the anus  were PIT‐tagged in the October electrofishing campaign. We used 12‐mm HDX – half‐duplex – tags  for sculpin  using a mobile radio frequency identification system with a 90‐cm ring antenna mounted on a 2.5‐m pole . Reader and 12‐V DC rechargeable battery were enclosed in a backpack; the palmtop computer was mounted on the field map. Precedent laboratory tests in air following Linnansaari et al.  revealedFish locations were transferred into the electronic maps in ArcMap for data processing. For each fish, we built up a detection history covering the 26 tracking events, with detections within the entire study reach coded as 1 and missing detections coded as 0. Following Pradel et al. , we defiφ, and detection probability, p, in mark–recapture data  to estimate apparent survival, φ(g × t)p(g × t)} and 27 sampling occasions . In the starting model, t represents time and g stands for the grouping variable, that is, the three groups of fish. Transients were excluded from the analysis as classical CJS models assume that all individuals within a group have the same probability of subsequent detection (Cooch and White c), that is, the model which explained most of the variation in the data, while using the fewest model parameters. This is the model with the lowest QAICc value. From this top‐ranked model, estimates of apparent survival and detection probability were obtained. All models were run using the logit link function, and simulated annealing as an alternative optimization algorithm. A potential effect of the degree of maturity (spawning vs. spent) on the detection history of adult trout (transient vs. residents) was analyzed by means of the Mantel‐Haenszel chi‐squared test . Average measured ice thickness closely compared to the approximated values following the formula by Ashton  and LundP < 0.01). The probability of spawning trout to become transients was 2.1 times higher than the one for the spent trout.Over the entire study period, 297 individuals or 76% of all tagged fish showed discernible upstream or lateral movement, that is, they were treated as residents Fig. : 145 scuFor the 297 residents, we collected 3615 positions over the 26 weekly trackings, whereof 1183 or 33% belonged to sculpin, 341 or 9% to juvenile trout, and 2091 positions or 58% to adult trout. Among the individuals detected, the total number of detections varied between groups, with the smaller individuals tagged with 12‐mm tags being less frequently detected . More than 80% of all resident sculpin could be found at the early winter trackings, before the return rate dropped below 5% in late February. From early April on, a sharp increase in the return rate was observed with a return rate of 71% at the last tracking, leading to a total variation in return rate by factor 21.0 (CV = 0.80). A similar, but less pronounced pattern was found for the juvenile brown trout for which the return rate varied by factor 11.3 (CV = 0.59). Conversely, the return rate for the adult brown remained at a constantly high level of ≥70% and showed little temporal variation .c units from the top‐ranked model – the one with the lowest QAICc value, {φ(t × cond)p(g × ice + complex × t)} (Table Our data were slightly overdispersed as illustrated by a ĉ of 1.21 (SE = 0.01) resulting from the GOF‐testing of the fully time‐dependent model. The default value of 1 was therefore adjusted in MARK. Two of the 39 candidate models were within 20 ΔQAICIn the top‐ranked model, detection probability was driven by ice thickness (“ice”) which showed group‐specific effects, and the habitat parameter “structural complexity”  whose effect was time‐dependent. Detection probability was independent from the physical condition of the fish or any other biological variable. Biweekly detection probabilities as obtained from the top‐ranked model ranged from 0.05 to 0.86 for sculpin, from 0.20 to 0.86 for the juvenile trout, and from 0.85 to 0.97 for the adult trout Fig. . DetectiIn the top‐ranked model, apparent survival varied over time and as a function of the individual physical condition (“cond”). Habitat parameters had no effect on the apparent survival, nor did the grouping variable or the other biometric characteristics of the fish. Biweekly apparent survival as obtained from the top‐ranked model was generally high and varied between 0.995 and 0.999 Table . The phyOur results show that detection probability decreased with growing ice volume and was smallest in structurally complex reaches. This relationship held across all three groups considered, with the strongest effects in the smaller‐sized fish that were tagged with 12‐mm tags. Apparent survival was generally high and dependent on the physical condition of the fish in autumn.One‐fourth of all adult trout, but less than 10% of the juvenile trout and sculpin were transients. These fish had been tagged and released all across the study reach, that is, individuals tagged at the study reach borders did not have a higher probability of permanently leaving the study area, for instance, during post‐tagging movements . We believe this to be a reasonable, but rather restrictive approach as (1) at the spring electrofishing, we recaptured fish from all three groups with similar spatial patterns as the excluded tags, and (2) tag loss and post‐tagging mortality had been inexistent in precedent laboratory tests with sculpin and juvenile trout.The detection probability varied between groups and over time as a function of increasing ice thickness. The surface ice layer defined the position of the antenna. Accordingly, the tracking from mid‐December on was difficult, particularly for the sculpin and the juvenile trout that were tagged with 12‐mm tags which have a maximum detection range of 0.5 m only. The detection probability for these two groups decreased considerably with increasing thickness of the ice layer, and detection was restricted to locations where the ice was relatively thin or the water depth relatively shallow. However, with the decay and breakup of the ice in spring, detection probability for the small tags rose again. The effect of ice thickness on detection probability was visible, but much less pronounced for the adult brown trout tagged with 23‐mm tags. The detection range for these larger tags is much bigger (>80 cm), enabling the effective scanning of most habitats in our rather shallow study reach.Group‐specific differences in habitat use may have further increased the effect of the tag size and ice formation. Sculpin are bottom‐dwellers, which are reported to conceal themselves in the substratum during low‐flow conditions  which was reflected by differences in the variance of the maximal depth Table . IndividTraditional approaches to estimate winter mortality have compared fish densities in autumn with those in spring  and that frequent tracking is possible ., a critical determination of factors affecting return rate and the underlying detection and survival probabilities is needed for remote PIT‐tracking (Zydlewski et al. FurthermoreNone declared.


We measure the Accuracy (ACC) and Matthews Correlation Coefficient (MCC) of Rice_Phospho 1.0 to be 82.0% and 0.64, significantly higher than those measures for other predictors such as Scansite, Musite, PlantPhos and PhosphoRice. Rice_Phospho 1.0 also successfully predicted the experimentally identified phosphorylation sites in LOC_Os03g51600.1, a protein sequence which did not appear in the training dataset. In summary, Rice_phospho 1.0 outputs reliable predictions of protein phosphorylation sites in rice, and will serve as a useful tool to the community.Experimentally-determined or computationally-predicted protein phosphorylation sites for distinctive species are becoming increasingly common. In this paper, we compare the predictive performance of a novel classification algorithm with different encoding schemes to develop a rice-specific protein phosphorylation site predictor. Our results imply that the combination of Amino acid occurrence Frequency with Composition of K-Spaced Amino Acid Pairs (AF-CKSAAP) provides the best description of relevant sequence features that surround a phosphorylation site. A support vector machine (SVM) using AF-CKSAAP achieves the best performance in classifying rice protein phophorylation sites when compared to the other algorithms. We have used SVM with AF-CKSAAP to construct a rice-specific protein phosphorylation sites predictor, Rice_Phospho 1.0 ( Phosphorylation is one of the most important protein post-translation modification (PTMs) in eukaryotes. It plays essential roles in the majority of biological pathways, regulating cellular processes like metabolism, proliferation, differentiation and apoptosisConventional experimental identifications and recent advances in high throughput Mass Spectrometry (MS) techniques have generated a large number of phosphorylated substrates with confirmed phosphorylation sites. In parallel, a series of algorithms have also been developed to predict phosphorylation sites from amino acid sequence. These range from simple motif or pattern searches to more complex machine learning methods like Artificial Neural Networks (ANN) and Support Vector Machines (SVM). Examples of such predictive algorithms include NetPhosArabidopsisArabidopsis is found to perform better with Arabidopsis sequences than other generic predictorsMost computational phosphorylation site predictors are not organism-specific predictors. However, with the increases in experimentally verified protein phosphorylation sites for different organisms, an increasing goal is to develop organism-specific phosphorylation predictors, such has occurred for yeastOryza sativa L.). Rice is considered a model plant species of the monocots group, it has a sequenced genomeet al.We have focused our efforts on Rice (The performance of the three sole encoding schemes was measured by using different sizes of datasets and with SVM used as the classifier. CKSAAP performed best among the three sole encoding schemes . HoweverThe performance of AF combined with CKAAP (AF-CKSAAP) was better than the sole encoding scheme, AF or CKSAAP . The samThe performance of the classifiers with the six different encoding schemes were firstly compared. The best results of a DT classifier were for CKSAAP, with an ACC of 71.14% and MCC of 0.314 . The besWe then compared classifying ability of the 4 different classifiers on phosphorylation sites of proteins in rice. As shown in We used different phospho-amino acids datasets with different ratio of (+) sites to (−) sites to detect the performance of the top three predicting models . For phospho-serine, phospho-threonine or phospho-tyrosine sites, SVM with AF-CKSAAP out-performed SVM with CKSAAP or CKSAAP-KNN, even though the ratio of (+) sites to (−) sites in the dataset was unbalanced .et al. (2004) suggested that a real non-phosphorylation site had to be solvent-inaccessibleBlom t al. 200 suggesteWe used SVM with AF-CKSAAP to develop a new rice-specific predictor, Rice_Phospho 10. We applied the independent test dataset to compare the predicting performance of Rice_phospho 1.0 with the newly existing predictors, including Scansite, Musite, PlantPhos and PhosphoRice. The MCC of the prediction performance of Rice_phosphos 1.0 in comparison to Scansite, Musite and PhosphoRice were shown in Oryza sativa L.). The potential phosphorylation sites are retrieved after the user uploads a protein sequence in FASTA format into the text area and selects one of the encoding schemes  was experimentally identified to be phosphorylated at Thr349 site by a comprehensive mutagenesis methodhttp://bioinformatics.fafu.edu.cn/rice_phospho1.0). Rice_Phospho 1.0 provides state of the art levels of reliability in predicting protein phosphorylation sites in rice and will be a useful tool to the community.In summary, we have benchmarked the combination of several encoding schemes and classification routines to establish their relative effectiveness. This led to the choice of a SVM with AF-CKSAAP encoding scheme which we have incorporated into the development of an effective rice specific protein phosphorylation site predictor, Rice_phospho 1.0 , and the 25-mer sequences surrounding them were extracted and compiled within a negative dataset (The 25-mer sequences −12 to +12) surrounding the phosphorylation sites were extracted from the protein sequences2 to +12  dataset . We extrBecause the residues buried in the core of a protein would not be accessible to any kinasesA standard 10-fold cross validation was used to train the classifiers. We calculated the Sensitivity (Sn), Specificity (Sp), Accuracy (ACC) and the Matthew’s Correlation Coefficient (MCC) of each predictoret al. (2009)The KNN method is used to classify the samples based on their distances. For each sequence, the distances between all the sequences of positive datasets or negative datasets were measured using the formula introduced by Gao w = 12, sim—amino acid similarity matrix—is derived from the normalized BLOSUM62.where two protein sequences s1 = {s1(−w), s1(−w + 1),…, s1(w)} and s2 = {s2(−w), s2(−w + 1),…, s2(w)}, The K nearest distances were selected, and the average distance among them was calculated. This process was repeated for different values of K . The ratios of the average distances between positive datasets and negative datasets were extracted as the feature valuesAA is the composition of the corresponding amino acid pairs in the sequence fragment, while Ntotal represent the total composition of amino acid pairs in the sequence fragment. For instance, if there are n AxA pairs in the sequence fragment, the value of corresponding component of NAA is n  denote the number of instances of amino acid i in the sequence fragment and the length of the sequence fragment, respectively. vi illustrates the frequency of the amino acids in the sequence.where We combined the three sole encoding schemes  to construct three bi-encoding schemes. Because of the high dimensionality of the CKSAAP, relief-F was used to decrease the total dimension for the combined methods. Relief-F is the extension to the original Relief algorithm, which is able to deal with noisy and multi-class problems rather than two-class problemi, yi) = exp were selected and 25mer was input to the predictor with default settings. In Scansite, the setting of medium stringency level was selected and resulted in the production of Scansite_medium predictor. In PlantPhos, phosphorylation sites will be predicted with the score over −3.0 (HMMER bit score).Sn, Sp, ACC and MCC were employed to evaluate the performance of the different predictors.TP, FP, FN, and TN denote true positives, false positives, false negatives, and true negatives. Sn and Sp illustrate the correct prediction ratios of positive and negative datasets, respectively. Because MCC is much less susceptible to the ratio of positive samples and negative samples in the dataset, it is the most widely used prediction measure for two-class prediction programswhere We used SPSS 16.0 to create receiver operating characteristic (ROC) curves to measure the performance of different predictors. For each possible threshold, the sensitivity and specificity were evaluated, and the ROC curves [sensitivity versus (1-specificity) curve] were used to compare the predictive performance of different classifiers with different encoding schemes.How to cite this article: Lin, S. et al. Rice_Phospho 1.0: a new rice-specific SVM predictor for protein phosphorylation sites. Sci. Rep.5, 11940; doi: 10.1038/srep11940 (2015).


Contraction is opposed when myosin light chain phosphatase (MLCP) dephosphorylates MLC20. It is thought that Rho kinase (ROK) inhibits MLCP by phosphorylating Thr855 of the regulatory subunit MYPT, favouring platelet contraction. This study tested the hypotheses that in septic shock, (i) platelet function is inversely correlated with illness severity and (ii) ROK-dependent MLCP inhibition and myosin light chain phosphorylation are reduced.Impaired coagulation contributes to the morbidity and mortality associated with septic shock. Whether abnormal platelet contraction adds to the bleeding tendency is unknown. Platelets contract when Ca20 and [Thr855]MYPT phosphorylation quantified activation and inhibition of platelet MLC20 and MLCP, respectively. Data were analysed using Spearman's rank correlation coefficient, Student's t-test and Mann-Whitney test; p < 0.05 was considered significant.Blood was sampled from non-septic shock patients and patients in the first 24 h of septic shock. Platelet function was assessed using whole blood impedance aggregation induced by 1) ADP (1.6 and 6.5 μM), 2) thrombin receptor-activating protein , 3) arachidonic acid (500 μM) and 4) collagen (3.2 μg/ml). Arachidonic acid-induced aggregation was measured in the presence of the ROK inhibitor Y27632. Illness severity was evaluated using sequential organ failure assessment (SOFA) and acute physiology and chronic health evaluation (APACHE) II scores. Western blot analysis of [Ser19]MLCn = 22 to 34; p < 0.05). Aggregation correlated inversely with SOFA and APACHE II scores . Thr855 phosphorylation of MYPT from unstimulated platelets was not decreased in patients with septic shock (n = 22 to 24). Both septic shock and ROK inhibition attenuated arachidonic acid-induced platelet aggregation independent of changes in [Ser19]MLC20 and [Thr855]MYPT phosphorylation (n = 14).Agonist-induced aggregation was attenuated in septic shock patients (Impairment of whole blood aggregation in patients within the first 24 h of septic shock was correlated with SOFA and APACHE II scores. Attenuated aggregation was independent of molecular evidence of diminished platelet contraction or reduced ROK inhibition of MLCP. Efforts to restore platelet function in septic shock should therefore focus on platelet adhesion and degranulation. Septic shock is fatal in over 20% of cases in the developed world ,2. CoaguAnalysis of platelet function in the context of infection and sepsis has yielded equivocal data depending on the experimental paradigm. When platelets isolated from healthy volunteers are exposed to bacterial lipopolysaccharide (LPS) -7 or lip20) in in ,31 and w subunit .Figure 1Previous studies have evaluated aggregation of platelets from patients with sepsis; however, our analysis of platelet function was restricted to patients with septic shock. Since septic shock is characterised by uncontrolled vasodilation and recognising that vascular smooth muscle and platelets may share common contractile signalling pathways ,34, we e20 and the ROK substrate P-[Thr855]-MYPT were performed to test the following hypotheses: 1) whole blood aggregation of platelets is attenuated in patients with septic shock, 2) platelet aggregation in septic shock patients correlates with illness severity indexed by the SOFA and APACHE II scores, and 3) platelet aggregation in septic shock patients is associated with reduced (i) [Ser19]MLC20 phosphorylation and (ii) RhoA/ROK-mediated inhibition (Thr855 phosphorylation) of myofilament-associated MLCP.Both whole blood impedance aggregometry and Western blot analysis of P-[Ser19]MLCThe integrity of platelet contraction and its potential as a therapeutic target is an important gap in our understanding of the pathogenesis of platelet dysfunction in human septic shock.2 <32 mmHg, (iv) leucocyte count >12,000 or <4,000/mm3 or >10% bands, plus suspected or proven source of infection, organ dysfunction attributable to sepsis and hypotension/requirement for vasopressor support >1 h despite adequate fluid resuscitation. Patients were managed according to current international guidelines -MYPT.A volume of 12 ml of blood was drawn from an existing arterial line into 3.2% sodium citrate tubes . Samples were partitioned to be analysed either by whole blood impedance aggregometry or to undergo protein extraction for Western blot analysis of the phosphorylation state of [Ser19]-MLCn = 34, p > 0.05; data not shown).Whole blood impedance aggregometry  was performed within 1 h of blood collection according to the manufacturer's instruction. Samples were not normalised by platelet concentration since Multiplate whole blood impedance aggregometry by the Multiplate system has been shown to be relatively independent of platelet concentration when the count is >150,000/μl . Citrate20 and [Thr855]MYPT phosphorylation-dependent contractility, the aggregation response to agonists was measured: thrombin receptor-activating protein  and arachidonic acid (500 μM), both associated with RhoA/ROK-mediated inhibition of MLCP, and ADP (1.6 and 6.5 μM) and collagen (3.2 μg/ml), which are RhoA/ROK independent [aggregation agonists purchased were sourced from Multiplate®] [In order to ascertain whether there was a generalised impairment of platelet aggregation that was consistent with a reduction in [Ser19]MLCiplate®] .In order to identify whether platelet aggregation was impaired by inhibition of the RhoA/Rho kinase pathway, the aggregation response to arachidonic acid was also measured in the presence or absence of 20 min of incubation with the Rho kinase inhibitor Y27632 (1 μM)  and Y27632 alone. Additional controls included aggregation responses of platelets incubated in buffer alone or Y27632 alone.20 phosphorylation (P-[Ser19]MLC20) in 1) unstimulated platelets, 2) arachidonic acid-stimulated platelets, 3) arachidonic acid-stimulated platelets pre-incubated with Y27632 for 20 min and 4) platelets incubated with Y27632 only. To address whether the P-[Ser19]-MLC20 was associated with altered RhoA/ROK-dependent Ca2+ sensitivity, the Thr-855 phosphorylation state of the MYPT P-[Thr855]MYPT targeting subunit of MLCP was directly measured in the same platelets. Results were compared for platelets from both non-septic and septic shock patients.To identify whether the reduction in aggregation of platelets from patients suffering from septic shock was associated with reduced activity of the contractile proteins, Western blot analyses were used to measure the state of MLC2 receptor agonist (U46619)-stimulated rat mesenteric artery as a positive control  or mouse anti-P-[Ser19]MLC20 . The nitrocellulose was washed three times in TBS-T and incubated in TBS-T with a 1 to 10,000 dilution of biotin-conjugated goat anti-mouse or goat anti-rabbit secondary antibody  for 60 min before another three washes with TBS-T. The nitrocellulose was then incubated for 60 min in TBS-T with a 1 to 10,000 dilution of streptavidin conjugated to the 800-nm DyLight fluorochrome [Pierce/Thermo Scientific Australia]. Fluorescence was detected and quantified using the LI-COR Odyssey™ system. All samples were transferred, incubated and scanned together to ensure identical analysis conditions between septic shock and non-septic shock patients and between different platelet treatments. All samples used in the analysis were within the linear range of detection.A washed platelet-rich pellet from 2 ml of whole blood was solubilised in 200 μl glycerol-free sample buffer containing 50 mM Tris-HCl, pH 6.8, 1%  noradrenaline dose and platelet aggregation response to arachidonic acid and ADP, (ii) noradrenaline dose required to achieve the desired mean arterial pressure and Thr[855] phosphorylation state of MYPT, (iii) SOFA and APACHE II scores and aggregation responses to arachidonic acid and ADP and (iv) SOFA and APACHE II scores and the Thr[855] phosphorylation state of MYPT. A correlation between TRAP/collagen-induced platelet aggregation and illness severity scores was not performed as the number of samples was too small. Fisher's test was used to analyse the mortality difference between the groups. A mixed-effects regression analysis was undertaken (Stata/MP 13.1) to identify significant interactions between variables and to estimate the overall effect of septic shock on platelet aggregation.Western blot data was expressed as a ratio of total P-[Thr855]/total MYPT and total P[Ser19]-MLCh muscle . Statistp < 0.01) in septic shock patients.Patients with septic shock (37) and non-septic shock controls (40) were recruited and were well matched for demographic characteristics; clinical status at the time of blood sampling was recorded and represented in Table n = 16 to 20 per patient group, p < 0.05) .Platelet aggregation in response to each agonist was significantly attenuated in patients with septic shock. Compared with platelets from non-septic shock patients, platelets from patients with septic shock demonstrated attenuated whole blood impedance aggregation in response to low-dose ADP (74.3 ± 8.7 vs. 51.2 ± 6.7), high-dose ADP (78.1 ± 7.5 vs. 55.5 ± 6.8), thrombin receptor-activating protein (92.4 ± 6.8 vs. 69.3 ± 9.4) and collagen (108.8 ± 9.4 vs. 83.3 ± 11.1) (Platelet aggregation was strongly promoted by arachidonic acid. Arachidonic acid-mediated platelet aggregation was attenuated in whole blood (i) taken from patients with septic shock and (ii) incubated with the Rho kinase inhibitor Y27632.n = 26 to 33, p < 0.05). To illustrate the proof of principle that ROK inhibition can attenuate platelet function, platelets were incubated for 20 min with the ROK inhibitor Y27632. Incubation with Y27632 significantly attenuated arachidonic acid-mediated aggregation in whole blood from both non-septic shock and septic shock patients  In the presence of Y27632, arachidonic acid-induced aggregation was not significantly different from aggregation of unstimulated platelets (13.5 ± 7.1 vs. 15.03 ± 5.0 (non-septic shock) and 11.7 ± 4.8 vs. 11.4 ± 3.6 (septic shock); n = 10 to 12) .Arachidonic acid (500 μM)-mediated platelet aggregation was attenuated in whole blood taken from patients with septic shock . Furthermore, ADP- and arachidonic acid-induced aggregation of platelets from septic shock patients was also inversely correlated with their APACHE II score  and arachidonic acid (500 μM) was inversely correlated with their SOFA score . Following stimulation with arachidonic acid, P-[Ser19]MLC20 was significantly higher in platelets from both non-septic shock and septic shock patients , but the increase was not significantly different between non-septic shock and septic shock patients. Pre-incubation with Y27632 did not reduce the arachidonic acid-induced increase in P-[Ser19]MLC20 in platelets from either patient group (non-septic shock 1.5 ± 0.69 and septic shock 1.5 ± 0.63). The proportion of P-[Ser19]MLC20 in unstimulated platelets was not significantly reduced following pre-incubation with Y27632 (non-septic shock 0.31 ± 0.08 and septic shock 0.52 ± 0.23)  and incubation with Y-27632 (1 μM) attenuated U46619-mediated P[Thr855]MYPT.The fraction of the MLCP regulatory subunit MYPT which was phosphorylated at the inhibitory Thr855 site (i.e. the proportion of MLCP in the putative RhoA/ROK-dependent inactive state) was not different in unstimulated platelets taken from either non-septic shock or septic shock patients . There was no significant difference in the Thr855 phosphorylation state of MYPT in either group following stimulation with arachidonic acid of platelets that had been incubated with Y27632 (non-septic shock 22.25 ± 5.75 vs. septic shock 18.42 ± 5.91)  reduced RhoA/ROK-mediated inhibition of myofilament-associated MLCP was also tested.The global impact of septic shock is profound; in 2012, the Surviving Sepsis Campaign Guidelines Committee stated that severe sepsis and septic shock affect ‘millions of people around the world, killing one in four (and often more), and is increasing in incidence’ . Up to 7Our data add to the weight of evidence that platelet function is impaired in sepsis ,45, iden2 (TxA2) de novo  phosphorylation of MYPT or Ser19] phosphorylation of MLC9 phosphoRhoA/ROK .20. There are several alternative mechanisms by which ROK inhibition might attenuate platelet aggregation independent of changes in MLC20 phosphorylation MYPT and phosphorylation of MLCrylation , and therylation ,55, and MYPT and rylation ; (ii) merylation -59; (iiiecretion  and (iv)ecretion -63 and fecretion . It is n2+-mediated activation of MLCK and RhoA/ROK-mediated inhibition of MLCP. Animal studies suggest that diminished contraction of vascular smooth muscle in sepsis is associated with attenuated ROK activity and disinhibition of MLCP MYPT. Future efforts to ameliorate coagulopathy in sepsis should consider mechanisms that include platelet adherence and secretion.Platelets in whole blood derived from patients suffering from septic shock exhibit reduced aggregation to a range of agonists, indicating an increased bleeding risk despite a normal platelet count. The reduction in aggregation correlated with illness severity scores. Therefore, reduced platelet aggregation in whole blood from patients with septic shock may be valuable as an early prognostic marker associated with poor patient outcome. Reduced platelet aggregation in patients with septic shock was independent of molecular evidence of reduced platelet contraction, i.e. [Ser19]MLC


III complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr), have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer) and phosphothreonine (pThr), pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates. Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear Tb In nature, enzymatic phosphorylation and dephosphorylation of proteins control many biological events. Cellular pathways regulated by these enzymatic modifications of proteins are so versatile. In the course of signal transduction in cells, for example, Ser, Thr, and Tyr, residues in proteins are reversibly phosphorylated and dephosphorylated, resulting in desired modulation of the activity of relevant enzymes , 2. In tIn contrast with these overall detections of phosphorylations of Ser, Thr, and Tyr in proteins, this review focuses on selective detection of phosphorylation of Tyr alone . This TyAmong all the probes investigated, lanthanide ions and their complexes have been widely and successfully employed due to their unique light-emitting properties –77. The III ion) and their complexes are used. By combining unique properties of the emission from these metal ions with so-called “antenna effect,” the background signals are minimized, and only the signal from pTyr is selectively monitored [III complexes [This paper reviews recent developments in optical methods to selectively detect pTyr in proteins. The primary concerns are high sensitivity of pTyr detection and its sufficient specificity . As emission probes, lanthanide ions  is used; note that nucleotides and nucleic acids are also eminent antenna (vide infra) [The emission from lanthanide ions is intrinsically weak, since the corresponding f-f transitions are Laporte-forbidden. However, this luminescence is enormously strengthened, when a chromophore (“antenna”) is placed near the lanthanide ions and transfers its photoexcitation energy to these emission centers , 81–91. e infra) , 56.III center. The irradiated light (Ex) is first absorbed by this benzene ring, and the excitation energy is then transferred to TbIII (ET). Finally, the metal center emits luminescence from its photoexcited state (Em). On the other hand, the phosphate is essential to bind to TbIII and places the benzene ring near the metal ion as the emission center. Among the coexisting solutes , only pTyr possesses both notable antenna effect (benzene ring) and sufficient binding activity towards the TbIII complex (phosphate group). Thus, the selectivities (i) and (ii) are fulfilled. Furthermore, the selectivity (iii) to other coexisting phosphate-containing molecules is accomplished by using a bulky ligand which suppresses the access of these molecules to TbIII. Furthermore, nonspecific background signals can be removed by using time-resolved spectroscopy and analyzing only long life-time components of the luminescence emitted from TbIII.One successful solution to these problems (improvement of the selectivity of pTyr detection with respect to (i) nonphosphorylated Tyr, (ii) pSer and pThr, and (iii) other coexisting phosphate-containing biomolecules) is presented in III complexes. In the first part of III complex was prepared primarily to show the validity of the working hypothesis. In III complexes. As a result, useful tools to monitor enzymatic phosphorylation of Tyr (and its dephosphorylation) have been obtained and used for practical applications in the following sections. Among lanthanide ions, TbIII has been most widely employed, together with Eu(III), for biological applications, emitting the most intensive line at around 545 nm.Based on the strategy depicted in III ion by multicoordination of both the phosphate and the guanine (N7 and O6). Thus, TbIII ion itself is not directly applicable to the sensing. In order to suppress the emission due to these coexisting molecules and accomplish efficient sensing of Tyr phosphorylation, an appropriate ligand is necessary to prevent the access of these molecules to TbIII ion. For this purpose, DOTAM tetraacetamide) was used  and TbIII. On the other hand, the effect of DOTAM on the binding of the phosphate in pTyr to the TbIII is little  because of its smaller size.The sample solutions used for the sensing of pTyr always contain many other biological molecules, and some of them show notable antenna effects to induce the emission from lanthanide ions. Among them, nucleobases and nucleic acids especially deserve attention. For example, guanosine 5′-monophosphate (GMP) enormously enhances the luminescence through the binding to Tbwas used . This weIII-DOTAM complex  as a model compound of pTyr  was determined by luminescence life-time measurements. Interestingly and importantly, q value was always around 1, whether or not PhOP was binding to the TbIII-DOTAM complex. In other words, one water molecule was originally coordinated to TbIII in the TbIII-DOTAM complex, and this water molecule was never removed from TbIII when the TbIII complex interacted with PhOP. Thus, it has been concluded that the interaction between TbIII-DOTAM and PhOP  is an ion-pairing rather than direct coordination of the phosphate to TbIII. Nevertheless, the benzene ring of pTyr is placed in a sufficient proximity of TbIII and satisfactorily works as antenna.Exactly as designed, pTyr notably increased the intensity of luminescence from Tb peptide ) was sucIII complex has eminent selectivity for pTyr detection. However, the sensitivity is still rather limited, primarily because of its poor binding of phosphate group. In order to further increase the detection sensitivity of TbIII-DOTAM on pTyr, its binuclear complexes (TbIII2-L1 and TbIII2-L2) were developed . Importantly, the luminescence from these binuclear TbIII complexes was greatly enhanced when pTyr was added to the solution  more efficiently. Apparently, the doubled positive charges of these binuclear complexes (+6) are responsible for the tighter interactions with the negatively charged phosphate of pTyr . Furthermore, the TbIII center and the benzene ring of pTyr are in sufficient proximity for energy-transfer to occur smoothly. In addition to these enhancements in fluorescence intensity, the selectivity of pTyr detection of the binuclear TbIII complexes, with respect to other cosolutes in solutions, is kept sufficiently high and comparable with that of the mononuclear TbIII-DOTAM. By using these binuclear TbIII complexes, Tyr-phosphorylated nonapeptide (P1-pY) was clearly distinguished at pH 7 from nonphosphorylated nonapeptide (P1).As shown in the previous section, mononuclear Tbeveloped . In the III2-L1, the time-course of Tyr phosphorylation of peptides by PTKs can be straightforwardly monitored in real-time. For example, phosphorylation of the tyrosine residue in the center of a nonapeptide P1 by Src kinase was analyzed in III2-L1 and P1, as well as ATP and MnCl2 , Src tyrosine kinase was added and then the luminescence at 545 nm (5D4 → 7F5 transition) was measured. The luminescence intensity increased time-dependently, reflecting the Tyr phosphorylation. The magnitude of increase in luminescence intensity is exactly consistent with the difference in the concentration of P1. Without the substrate peptide, the luminescence was never enhanced. When Tyr-phosphorylated P1 was used as the substrate, the luminescence was strong from the beginning and not enhanced even after the addition of Src kinase. In order to confirm the validity of the method furthermore, the rate of this enzymatic phosphorylation was independently determined using TAMRA-labeled P1. There, the reaction was stopped at several reaction times and the products were analyzed by polyacrylamide gel-electrophoresis. The results of these two methods fairly agreed with each other, as expected.By using TbIII2-L1 in real-time . The luminescence intensity increased again. Apparently, the second Src kinase reaction was successfully monitored, even when the mixture was so complicated and contained many components .Similarly, the reverse reactions of the phosphorylations, dephosphorylations of a tyrosine-phosphorylated peptide by PTPs, were also visualized by Tbeal-time . Here, t fashion . When noIII complexes are advantageous in that no labeling is required and kinetic information can be straightforwardly obtained. Simply by adding the TbIII complexes to the reaction mixture and monitoring the photoluminescence, the time-courses of reactions can be directly obtained in situ in real-time. Accordingly, detailed kinetic results are precisely obtained, even when the enzymatic systems are highly complicated and the reaction rates do not strictly obey simple Michaelis-Menten equation .Most of previous kinetic studies on these enzymatic reactions involved either radiolabeling of the peptide/ATP or chemical labeling of peptide with chromophores. The labeling procedures are time-consuming and still, more importantly, could cause nonnegligible perturbations on the enzymatic reactions. Compared with these methods, the present methods using the TbIII complexes to screening of inhibitors from a pool of candidates. As described above, the binuclear TbIII complexes can visualize the enzymatic phosphorylation and dephosphorylation in a real-time fashion. Accordingly, the inhibition activity can be evaluated in terms of both kinetic aspects and static aspects, providing new kinds of information to these fields. In this section, the specificity and activity of well-known inhibitors on PTKs and PTPs were determined by using TbIII complexes and compared with the literature data primarily to confirm the validity of method.There are many kinds of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) in our bodies. Each of them takes an important role in the corresponding reaction and is strongly related to various diseases. Thus, inhibitors on a predetermined enzyme among these PTKs/PTPs have been regarded as promising targets for drug discovery and the subject of growing interest. This section presents the application of binuclear TbIII2-L1 as a chemical sensor, the inhibitory effects of PTK inhibitors on three kinds of PTKs  were analyzed. Among the PTK inhibitors investigated, staurosporine is a general kinase inhibitor with minimum selectivity [2, TbIII2-L1, and a PTK, gefitinib was added, and the reaction was started with the addition of ATP. The inhibitory effects were visually analyzed in terms of the real-time kinetics. Gefitinib drastically suppressed the enzymatic reaction of EGFR (its target PTK) depending on its concentration (μM (1000-fold larger than EGFR case), they showed sufficient activity. The inhibition specificity was completely identical with the known specificity.Using Tbectivity , 132. Onectivity , and dasectivity . Imatiniectivity . To the ntration . When [g50) was also successful  to the reaction mixtures. The method is simple, straightforward, and useful. However, the detection sensitivity is in some case rather small, especially when the peptide substrates are positively charged. There, the association of peptides with the positively charged TbIII complexes is suppressed by electrostatic repulsion, and thus the energy-transfer from pTyr to TbIII does not satisfactorily proceed (note that bindings are primarily due to electrostatic interactions). As a general strategy to overcome these drawbacks, substrate peptide and the TbIII complex were covalently connected, and the intermolecular association between them was converted to more efficient intramolecular one. In order to facilitate the synthesis of these conjugates, a new binuclear TbIII complex   by Abl kIII complex and the pTyr on the monitoring activity of the conjugate was analyzed by changing the length of linker peptide between the N-terminus of Abltide and the cysteine residue (1 to 5 amino acids). The rate of phosphorylation was almost the same for all the five conjugates. The TbIII complex bound to the Abltide imposes minimal steric hindrance on the enzymatic reaction. Furthermore, the luminescence increased in almost the same magnitude (10-fold) upon the phosphorylation. Apparently, the structure of the complex formed between the Tb2-Lc1yne and the pTyr residue in the peptide is similar irrespective of the length of the linker peptide. This ensures the applicability of this strategy to various substrates without strict structural restriction.The effect of the distance between the TbIII showing very high selectivity to pTyr has been developed. The photoemission from TbIII-DOTAM complex is notable only when pTyr exists in the solutions. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Accordingly, the emission is selective to pTyr, since nonphosphorylated tyrosine cannot efficiently bind to the TbIII complex, and neither phosphoserine nor phosphothreonine can satisfactorily provide an antenna effect. Furthermore, the binding of bulky cosolutes  to TbIII is suppressed by the steric hindrance of DOTAM. By the use of time-resolved luminescence analysis, only the long life-time luminescence from TbIII is analyzed and high signal-to-noise ratios are accomplished.The importance of phosphorylation of tyrosine residues in proteins and their dephosphorylation has been well recognized, and chemical sensors to monitor this phosphorylation selectively have been attracting interests. Recently, significant progress has been made in this field. A DOTAM complex of TbIII complexes (TbIII2-L1 and TbIII2-L2), in which two TbIII-DOTAM complexes are connected through the linkers in the ligands, are far more effective in the detection of pTyr than the monomeric TbIII complex. The increase in the sensitivity is primarily ascribed to the stronger binding of pTyr to these complexes, due to enhanced electrostatic interactions between them. With the use of these complexes as sensors, phosphorylation of tyrosine by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases are visualized in situ in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated by the TbIII complexes. This method should be useful in screening highly eminent inhibitors from a number of candidates. These enzymes take crucially important biological roles so that the information obtained by these studies should lead to development of new drugs for the therapy of relevant diseases. By immobilizing these TbIII complexes to some solid supports, the applications of the present methods should be further facilitated and widened.Binuclear TbIII complexes, phosphorylation of tyrosine in peptides by protein tyrosine kinases (PTKs) and dephosphorylation by protein tyrosine phosphatases (PTPs) can be successfully visualized in a real-time fashion. The activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method to efficient screening of eminent inhibitors from a number of candidates.By using binuclear Tb


To this end, native pCKII from Arabidopsis thaliana and Sinapis alba chloroplasts was enriched by Heparin-Sepharose chromatography and its activity on the microarray was compared to the activity of a recombinant Arabidopsis pCKII. All three kinase preparations phosphorylated a similar set of peptides that were clearly distinct from those phosphorylated by bovine heart protein kinase A (PKA) in control experiments. The majority of the pCKII phosphorylation targets are involved in plastid gene expression, supporting the earlier denomination of pCKII as plastid transcription kinase (PTK). In addition we identified Alb3 as pCKII substrate that is essential for the integration of light-harvesting complex subunits (LHC) into the thylakoid membrane. Plastid CKII phosphorylation activity was characterized in greater detail in vitro with recombinant wildtype Alb3 and phosphorylation site mutants as substrates, establishing S424 as the pCKII phosphorylation site. Our data show that the peptide microarray ChloroPhos1.0 is a suitable tool for the identification of new kinase downstream targets in vitro that can be validated subsequently by in vivo experiments.We report the development of a peptide microarray based on previously determined phosphorylation sites in chloroplast proteins. Altogether, 905 peptides were spotted as 15mers in nine replicates onto glass slides. We used the microarray for  Important aspects of this fast responding system are controlled by posttranslational modifications of resident proteins, e.g. phosphorylation  in vivo kinase targets. We used the microarray to identify the substrates of pCKII. Our analysis shows that ChloroPhos1.0 is a suitable screening tool to establish currently unknown kinase/substrate relationships in the chloroplast phosphoproteome network. It allows extracting preferred motifs of chloroplast kinases thus facilitating the prediction of additional targets that may have escaped mass spectrometric detection.Here we report the creation of the first chloroplast phosphopeptide microarray ChloroPhos1.0. The peptide microarray was specifically designed for the analysis of chloroplast protein kinases, using plastid phosphoproteomics data for peptide selection and phosphorylation site positioning (http://phosphat.uni-hohenheim.de/) using a chloroplast protein reference table  expression vector was transformed into competent BL 21 DE3 E.coli cells. The advantage of this system is the N-terminal MBP -tag on the protein, which enables fast purification with commercially available kits. The cloning of CK2 without transit peptide was done with the following primers NotI_FW, 5′ GTA TAG CGG CCG CGC TTC TCT TTA CCG TCA AC 3′ and BamHI_Rv, 5′ GTA TAG GAT CCT CAC TGG CTG CGC GGC G 3′. At an optical density of 0.5–0.8 at 595 nm, the protein expression was induced by the addition of 1 mM IPTG. The cells were shaken for 3 h at 250 rpm at room temperature and collected. Cell lysis and purification was done according to the NEB pMALc5× purification protocol. After the elution of the purified protein, some contaminating proteins were visible, which we deleted in the second purification step with XK 16/40 Sephacryl S100. A ∼85 kDa band (recombinant pCKII with MBP-tag) was obtained. The fractions were collected and concentrated. Kinase activity was tested with [γ-33P] ATP as described below.For bacterial overexpression of Sinapis alba seedlings were grown on soil under steady light conditions in a controlled environment chamber . Arabidopsis thaliana Col 0 seedlings were grown on soil under long-day conditions in a controlled environment chamber . Plants were harvested after 4 days  or after 17 days . Chloroplasts were isolated according to established protocols 4)2SO4 and applied on a 2-ml Heparin-Sepharose CL-6B column  equilibrated in buffer E , 0.1% Triton ×100 and 200 mM (NH4)2SO4). After 2 washing steps with equilibration buffer, the bound proteins were eluted in a single step with 1 M (NH4)2SO4 at a flow rate of 0.4 ml/min. Dialysis was performed overnight at 4°C in buffer E with 20% glycerol and without (NH4)2SO42, 1× PhosStop (Roche) and 0.1% protease inhibitor cocktail (Sigma P9599), 5 µM ATP, 60 nM [γ-33P] ATP). In case of recombinant pCKII we used 0.4 µg enzyme for the activity assays. Several controls were performed, in which the indicated ingredient was added or omitted from the reactions. These included omission of Mg2+, addition of EDTA, addition of non-radioactive GTP and addition of the CKII inhibitor heparin at a concentration of 15 µg/ml. Following incubation, the reactions were stopped by the addition of SDS sample buffer and the proteins therein were separated by SDS-PAGE All fractions were tested for kinase activity as described in 4) HCO3 and 0.1% RapiGest (Waters) to a final protein concentration of 1.6 µg/µl. In the presence of 10 mM DTT the protein mixture was reduced for 10 min at 60°C and alkylated with 30 mM iodacetamide for 30 min at room temperature in the dark. The tryptic digest was performed overnight at 37°C with a trypsin/protein ratio of 1/100. The digest reaction was acidified by HCl (pH<2) in order to stop tryptic activity and centrifuged for 10 min at 13000 g. The supernatant was analyzed by LC-MS using an ACQUITY UPLC System coupled to a Synapt G2-S mass spectrometer .Eluted proteins were precipitated in 90% acetone at −80°C and sedimented at 22.000xg for 15 minutes. The protein pellet was dissolved in 25 mM . MS acquisition was set to 50–2000 Da. Data analysis was carried out by ProteinLynx Global Server  with automated determination of chromatographic peak width as well as MS TOF resolution. Lock mass value for charge state 2 was defined as 785.8426 Da/e and the lock mass window was set to 0.25 Da. Low/high energy threshold was set to 180/15 counts, respectively. Elution start time was 5 min, intensity threshold was set to 750 counts. Databank search query (PLGS workflow) was carried out as follows: Peptide and fragment tolerances was set to automatic, two fragment ion matches per peptide, five fragment ions for protein identification, and two peptides per protein. Maximum protein mass was set to 250 kDa. Primary digest reagent was trypsin with one missed cleavage allowed. According to the digestion protocol fixed (carbamidomethyl on Cys) as well as variable (oxidation on Met) modifications were set. The false discovery rate (FDR) was set to 4% at the protein level. MSE data were searched against the modified A. thaliana database  containing common contaminants such as keratin and rabbit glycogen phosphorylase B (P00489) was used as internal quantification standard. Quantification was performed based on the intensity of the three most abundant proteotypic peptides. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository LC separation (140 min gradient) and HD-MS2, 1× PhosStop (Roche) and 0.1% protease inhibitor cocktail (Sigma P9599), 2 µM ATP, 240 nM [γ-33P] ATP). These incubations included 25 µg of the recombinant MBP-tagged pCKII, 90 µg of the Heparin Sepharose eluates or 20 units of commercially available bovine heart protein kinase A . The microarrays were washed in TBS pH7.5 to remove remaining substances of the incubation mixture, subsequently by H3PO4 (pH 2), to get rid of ATP bound to peptides and finally in H2O, to neutralize the microarray surface. The dried microarrays were exposed on a Phosphorimager Screen for 1 week and scanned by a BAS-1800 reader (Fujifilm) with 50 µm pixel size. Analysis was performed with the GenePixPro 6.1 software (Molecular devices). The grayscale intensity median of all spot pixels were determined after background subtraction and averaged over all 9 spots/peptide. These signals were classified into 4 categories, which were defined as 3 – strong (>50% over background intensity [BI]); 2 – medium (<50%–>20% BI); 1 – weak (<20%–>10% BI); 0 – none (<10% BI). Due to the possible occurrence of signal artefacts such as dust particles, we included an optical control to exclude false positives.The active kinase preparations were incubated on the microarrays for 2 h at 25°C in kinase activity buffer B  (Novagen) and shaken at 37°C until an optical density (600 nm) of 0.6–0.7 was reached. Protein expression was induced by the addition of 1 mM IPTG and continued for 1 h at 30°C. Afterwards the cells were harvested by centrifugation and immediately frozen to −20°C until use. His-pCKII was purified under native conditions using Ni-NTA resin (Qiagen). Protein purification was carried out as the manufacturer suggested using the following buffers: lysis/washing buffer 20 mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl, 5 mM MgCl2 and 20 mM imidazole; elution buffer 20 mM Na2HPO4/NaH2PO4 pH 8.0, 300 mM NaCl, 5 mM MgCl2, 1 mM ATP, 5% (v/v) glycerol and 250 mM imidazole. Alb3 phosphorylation assays for the determination of the phosphorylation site contained 10 µg of His-tagged Alb3 WT or mutant protein and 10 µg of His-pCKII in 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM MgCl2 and 100 µM [γ-32P] ATP . After incubation for 30 minutes at 30°C the phosphorylation reactions were stopped by the addition of SDS sample buffer and heating. Samples equal to 3 µg of each protein were separated by SDS-PAGE and the dried gels were subjected to autoradiography.His-tagged Alb3 wildtype and mutant constructs were expressed and purified as described previously 2, 1× PhosStop (Roche) and 0.1% protease inhibitor cocktail (Sigma P9599), 5 µM ATP, 25 nM [γ-33P] ATP). All reaction mixtures were separated by SDS-PAGE and analyzed via an autoradiography screen. In parallel a [γ-33P] ATP dilution series was spotted onto a dried SDS-gel and exposed on an autoradiography screen in the same way as the phosphorylation reactions. The autoradiograms were analyzed and quantified by the BAS Reader V2.26 and the TINA 2.09 software. The grayscale intensities of the pixels were quantified after background subtraction. Pixel intensities of the ATP-dilution were compared with the Alb3 phosphorylation signal. The resulting PSL values were correlated to the concentration of incorporated [33P] thus resulting in quantitative values for phosphate incorporation into Alb3.For kinetic studies, we phosphorylated increasing amounts of wildtype Alb3 in two replicates for 2 and 4 minutes at 25°C using 0.3 µM recombinant pCKII. The Alb3 concentration was varied in a range between 50 nM and 5 µM. The reaction was performed in kinase activity buffer . Table S1 in We extracted information on Arabidopsis thaliana pCKII in E. coli as a control. Phosphorylation activity of these two kinase preparations on the microarray was determined to identify pCKII phosphorylation targets. MBP-tagged pCKII was overexpressed in E. coli and purified in two steps by utilizing the maltose-binding protein tag on an Amylose column in the first step and by size-exclusion chromatography on Sephacryl S100 in the second step . This is most likely due to low kinase concentrations and difficulties with the phosphorylation of peptide substrates because of competition from suitable protein substrates in close proximity to the active kinase. We therefore decided to enrich native chloroplast CKII by Heparin-Sepharose chromatography http://weblogo.berkeley.edu/) and found that PKA prefers a motif with basic residues at positions −3 to −1 relative to the phosphorylation site, while pCKII prefers motifs with acidic residues at positions +1 to +3 relative to the phosphorylation site (We performed a first microarray experiment and compared pCKII activity with that of bovine heart protein kinase A (PKA) to assess phosphorylation activity and specificity  A. PKA pion site  B. Theseion site .Sinapis alba and Arabidopsis thaliana chloroplasts by Heparin-Sepharose chromatography. Sinapis alba was introduced as a preparation control because the pCKII (PTK) purification protocol was originally established for this plant Sinapis alba with minor modifications. The enrichment of pCKII in the eluates was analyzed in vitro using dephosphorylated and heat inactivated stroma extracts as substrate mixture. The specificity of the kinase preparation was assessed in control experiments using heparin as inhibitor and an excess of non-radioactive GTP as phosphate donor. These data indicate that pCKII was successfully enriched in the eluate fractions  and peptide KYEGKKLSELSDDED of TAC10 (AT3G48500) . Both pein vivoE. coli with its wildtype sequence and as the phosphorylation site mutants S416A, S418A and S424A . By exclusively using peptides that were found phosphorylated in vivo, we increase the specificity of a positive in vitro phosphorylation result on the microarray with a chloroplast protein kinase. By combining prior knowledge on the few established chloroplast kinases with knowledge about their in vivo targets, we introduce a meaningful constraint in the design of a large-scale experiment. Such constraints reduce false assignments of kinase/substrate relationships, as previously shown for kinase motif prediction in vivo substrates of this kinase, provided that the constraints are fulfilled . The specificity of this assignment is supported by the fact that we do not find any overlap in the set of phosphorylated peptides between pCKII and PKA  is an endosomal contaminant in the chloroplast reference proteome. Nonetheless, these proteins are excellent targets for CKII and it is very likely that they are phosphorylated by one of the other CKII alpha subunits in the nucleo-cytoplasmic compartment in vitro transcription activity The set of phosphorylated peptides on the microarray is relatively small, i.e. much smaller than the set of predicted CKII targets based on phosphorylation motif recognition A function of pCKII in the regulation of posttranscriptional processes of plastid gene expression is supported by the phosphorylation of the DEAD box RNA helicase RH3. RH3 has a predicted transit peptide length of 60 amino acids and the phosphorylation site is located at Ser80. Thus, this phosphorylation site is N-terminal to the helicase domain and close to the N-terminus of the mature protein. Because of this location it seems unlikely that phosphorylation alters the catalytic properties of RH3. It is more likely that phosphorylation alters its interactions with other proteins in RNA degrading complexes. The RNA-binding protein RNP31 is already a known pCKII target. This protein belongs to a family of RNA-binding proteins that have an acidic domain and two tandem RNA recognition motifs (RRM). They were among the first identified pCKII targets in chloroplasts In addition to targets in the gene expression system, pCKII appears to be involved in the regulation of the plants central energy metabolism. This entails the carbohydrate metabolism via phosphorylation of the starch branching enzyme (SBE2.1), the small subunit of RubisCO, and RubisCO activase, and photosynthesis via phosphorylation of Alb3. A regulatory role of CKII in carbohydrate metabolism was reported earlier for mammalian systems. Here, CKII phosphorylates glycogen synthase, phosphoglucose isomerase and glycerol-3-phosphate acyltransferase (GAT) in vivo phosphorylation sites of proteins that co-localize with chloroplast kinases, we aim for stringent substrate recognition and a low false positive rate in the assignment of in vivo kinase substrates. This requires that the constraints are correct, i.e. that the substrates truly co-localize with the kinase, which is not the case for PKA. At the same time, we expect a relatively high false negative rate for structural reasons such as the lack of interaction domains and docking sites that facilitate substrate recognition by its cognate kinase in vivo. The fact that we were unable to measure phosphorylation activity with complex extracts supports this hypothesis. Because of its design, the microarray is restricted to assay targets for Arabidopsis chloroplast protein kinases and may be extended to the analysis of closely related species such as mustard. We will constantly update the microarray and the number of peptides will increase as more information about chloroplast protein phosphorylation becomes available.We show here that the peptide microarray ChloroPhos1.0 is a suitable screening tool to identify novel kinase substrates and to characterize the preferred phosphorylation motif of currently uncharacterized plastid kinases. By using File S1File S1 includes the following: Figure S1. Purification and activity assay of recombinant pCKII from E coli. Figure S2. Potential phosphorylation sites in the C-terminal region of Alb3. Figure S3.In vitro phosphorylation of Alb3. Figure S4. Excerpt of a multiple sequence alignment of Alb3 homologs in different plant species. Table S1. MS-based phosphoproteomics studies used for the peptide library. Table S2. List of peptides included on the microarray ChloroPhos1.0. Table S3. List of peptides phosphorylated by recombinant pCKII and protein kinase A. Table S4. Proteins identified and quantified from the Heparin-Sepharose eluate fraction with kinase activity. Table S5. List of peptides phosphorylated by recombinant pCKII and the isolated native plastid casein kinases from Arabidopsis thaliana (A.t.) and Sinapis alba (S.a.).(PDF)Click here for additional data file.


Most proteins are regulated by posttranslational modifications and changes in these modifications contribute to evolutionary changes as well as to human diseases. Phosphorylation of serines, threonines, and tyrosines are the most common modifications identified to date in eukaryotic proteomes. While the mode of action and the function of most phosphorylation sites remain unknown, functional studies have shown that phosphorylation affects protein stability, localization and ability to interact. Two broad modes of action have been described for protein phosphorylation. The first mode corresponds to the canonical and qualitative view whereby single phosphorylation sites act as molecular switches that either turn on or off specific protein functions through direct or allosteric effects. The second mode is more akin to a rheostat than a switch. In this case, a group of phosphorylation sites in a given protein region contributes collectively to the modification of the protein, irrespective of the precise position of individual sites, through an aggregate property. Here we discuss these two types of regulation and examine how they affect the rate and patterns of protein phosphorylation evolution. We describe how the evolution of clusters of phosphorylation sites can be studied under the framework of complex traits evolution and stabilizing selection. The rate of discovery of new protein forms is increasing with the growing sensitivity of biochemical, analytical and bioinformatics tools . We now Phosphorylation sites play roles in allosteric and orthosteric regulation of proteins . AllosteFigure 1). In the second case, groups of phosphorylation sites are organized into modules of multiple sites found in a short distance in a particular domain or disordered region of a protein and each cluster regulates a particular and independent function. Each of the mechanisms described above has its own complexity in terms of effects on the protein and the dynamics of activation, and may thus affect the evolution of these sites . It is important to note that these mechanisms are not mutually exclusive. They are typically combined with various types of logic to encode patterns of substrate protein activity. For example, phosphorylation of some sites might “prime” (or increase the probability of phosphorylation) of other sites within the same region through the binding of other proteins that enhance the efficiency of the kinase , depending on several parameters such as enzyme and substrate concentrations, binding parameters and the kinase/phosphatase processivity . Sites could be gained and lost within a given region with limited effect on the overall function, but contributing to an increased rate of evolution if one considers sites individually. Different clusters could also play similar roles and diverging species could lose and gain these clusters without any significant change in function . These proteins show conserved regulation in animals and fungi but diverge in the position and number of clusters of CDK phosphorylation sites, suggesting that functions may be preserved despite changes in phosphorylation patterns . AdditioFigure 1). As mutation-selection-drift equilibrium is reached after a long divergence time, there will be a large diversity of “genotypes” that encode the trait. Even if the optimal trait value is the same in all species, if the number of phosphorylation sites (loci) that contribute to the trait is large, a minimal level of conservation will be observed for the actual phosphorylation sites. For example, CDK-mediated inhibition of Ste5 membrane localization is mediated by 8 phosphorylation sites in S. cerevisiae , corresponding to different positions and numbers of phosphorylation sites in the PM domain. This stabilizing selection model has been applied to explain the rapid evolution of transcription factor binding sites in highly conserved developmental enhancers in drosophila (More work will be needed along these lines to test whether the stabilizing selection model can apply to clusters of phosphorylation sites. Under this model, a phosphorylation cluster would be considered as a quantitative trait where selection does not act on a particular site but rather on the number or density of sites. In this model, each site represents a single locus that contributes to a “complex trait.” Deviation from the trait optimum is associated with a fitness cost that would increase or decrease following a given function (see, e.g., osophila . In one osophila . WhetherGiven the current knowledge on the function of clusters of phosphorylation sites, there is a need for the development of new evolutionary models or adaptation of existing ones that take into account the fuzziness of phosphorylation site position and density. Although to our knowledge the stabilizing selection model has not been explicitly applied to the evolution of phosphorylation sites, the distribution of kinase site consensus matches in the absence of selection has been calculated . This reThe study of protein phosphorylation will also need more experimental studies on the function of cluster of phosphorylation sites in order to learn the general principles by which their aggregate properties emerge from their combination. This will allow for instance to estimate the relationship between the number of sites, their density and positions, and protein function and organismal fitness. Most evolutionary studies performed so far, with few exceptions , rely onChristian R. Landry drafted the manuscript with contributions from Alan M. Moses. Luca Freschi contributed concepts, discussion and to manuscript edition. Taraneh Zarin and Luca Freschi contributed the figure.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.


Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.The intergeneric hybrids between GISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.Both GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated. Vanda, Ascocenda, Phalaenopsis, Renanthera, Rhynchostylis and Aerides, which are characterized by a monopodial growth habit and are mainly found in tropical Asia [Ascocenda John De Biase ‘Blue’ is an intergeneric hybrid between Ascocentrum and Vanda species [Phalaenopsis is one of the most popular and ecologically important orchids worldwide [P. Chih Shang's Stripes is an interspecific hybrid, and its genetic background includes species of P. amboinensis, P. lueddemanniana, P. amabilis, P. aphrodite, P. equestris, P. schilleriana, P. sanderiana, and P. stuartiana [Phalaenopsis cultivars have been registered in the orchid database of Royal Horticultural Society (RHS) [A. John De Biase ‘Blue’ and P. Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study [Comprising approximately 35,000 species, Orchidaceae is considered the largest family of flowering plants in the world . Vandacecal Asia . Althougcal Asia , interge species . The culorldwide ,8 with horldwide . P. Chihuartiana . To datety (RHS) . Intergeor study . Hence, A. John De Biase ‘Blue’  and P. Chih Shang's Stripes  based on GISH, PCR-RFLP and RFLP analyses of nrDNA.Apomixis is a unique type of asexual reproduction in many plant species that results in the formation of seeds that are genetically identical to the female parent, reflecting seed formation without fertilization. Apomixis has been found in a few agriculturally important crop species, such as citrus, apple, mango, and orchid . Apomixiin situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analyses are powerful molecular analytical techniques. GISH was developed by Schwarzacher et al. [Phalaenopsis [A. John De Biase ‘Blue and P. Chih Shang's Stripes.Both genomic r et al.  and has r et al. –18. RFLPr et al.  and has r et al. . RFLP haaenopsis . In addiaenopsis . In highaenopsis . Both thaenopsis –27. Receaenopsis ,28–33. TA. John De Biase ‘Blue’ and P. Chih Shang's Stripes were triploid (2n = 3x = 57) and tetraploid (2n = 4x = 76), respectively , medium (2–2.5 μm) and small (<2.0 μm) chromosomes [P. Chih Shang's Stripes) was either 2–2.5 μm or <2.0 μm. The chromosome size of the female parent (A. John De Biase ‘Blue’) varied between>2.5 μm, 2–2.5 μm, or <2.0 μm. By contrast, the male parent (P. Chih Shang's Stripes) did not have chromosomes>2.5um but female parent (A. John De Biase ‘Blue’) had chromosomes>2.5um. The result of giemsa staining reveals that the chromosome size of hybrids were 2–2.5 μm in which four chromosomes, and the others belongs to size of <2.0 μm (Giemsa staining showed that ectively . The putome size –35. The omosomes . In this <2.0 μm .A. John De Biase ‘Blue’ (stained with PI) and 34 chromosomes derived from P. Chih Shang's Stripes (stained with FITC) (A. John De Biase ‘Blue’ (stained with PI) and 36 chromosomes derived from P. Chih Shang's Stripes (stained with FITC)  and P. Chih Shang's Stripes . In addition, GISH analysis showed no chromosome recombination in the hybrids  . Anotherth FITC) . These r hybrids .A. John De Biase ‘Blue’, P. Chih Shang's Stripes and the 27 putative hybrid seedlings were approximately 880 bp. The PCR products were separately digested with HaeIII and electrophoresed  contained DNA fragments of 4,000, 1,100, 900, and 540 bp  and may have been inherited from P. amboinensis and P. lueddemanniana. The remaining Phalaenopsis chromosomes are small (< 2.0 μm) and may have been inherited from P. amabilis, P. aphrodite, P. equestris, P. schilleriana, P. sanderiana, or P. stuartiana [A. John De Biase ‘Blue,’ is triploid and can be used as the female parent for hybridization with P. Chih Shang's Stripes to obtain F1 progeny via embryo rescue [Lilium, which generated progeny that were predominantly pentaploid or near-pentaploid [Generally, the number of chromosomes in diploid  2x = 38 –37, alth greatly . Based ouartiana . Kao et uartiana  examineduartiana ,36. The uartiana –35. Geneo rescue . The F1 ntaploid . In addintaploid . This phTriticeae species [Phalaenopsis species [A. John De Biase ‘Blue’, or from the male parent, P. Chih Shang's Stripes, can be clearly distinguished without the use of blocking DNA. These results indicate that the parents do not have a close genetic relationship, which is in agreement with their classification into two separate genera [Festuca pratensis × Lolium multiflorum [Lycopersicon esculentum × Solanum tuberosum [GISH has proven to be a useful technique to distinguish genetically recombined genomes and assess the genomic relationships between different hybrid plants ,18,41. I species  and Phal species . In thise genera –46. Onlytiflorum  and Lycouberosum . Compareuberosum ,49.Gossypium [Cardamine [Rheum [Acer [Paeonia [Musa [Nicotiana tabacum [Hordeum vulgare L. × Hordeum spontaneum L. [Both cytoplasmic and nuclear inheritance are complex phenomena that can be divided into three modes: maternal, paternal and biparental. Usually, nuclear inheritance is biparental, and the cytoplasm is generally inherited uniparentally . In prevossypium , Cardamiardamine , Rheum [e [Rheum  and Acerum [Acer . In this[Paeonia , Musa [2ia [Musa , Nicotia tabacum –62, and aneum L. . Howeveraneum L. . Becauseaneum L. –56, theyaneum L. . Generalaneum L. , the conaneum L. , or the aneum L. . The malFurthermore, two types of ETS regions inherited from the male parent were identified in the F1 intergeneric hybrids; samples 2, 8, 18, 20, 21, 22, and 23 inherited two DNA patterns that were derived from the male parent . This reAscocenda John De Biase ‘Blue’  and Phalaenopsis Chih Shang’s Stripes  and evaluating the genetic inheritance of these hybrids. The results showed that the GISH and RFLP method can effectively identify all F1 hybrid seedlings are intergeneric hybrids. Moreover, the use of PCR-RFLP analysis to assess the inheritance of the putative hybrids should be carefully evaluated.To sum up, GISH and RFLP analyses are useful for identifying intergeneric hybrids derived from Ascocenda John De Biase ‘Blue’ female parent and a P. Chih Shang's Stripes male parent. The intergeneric hybrids were obtained via embryo rescue [The plant materials used in this study consisted of F1 intergeneric hybrid seedlings derived from an o rescue . All plaYoung root tips were collected from the two parents and the putative hybrids. They were then treated with 2 mM 8-hydroxyquinoline on a shaker (100 cycles min-1) at 18°C for 5 hr, fixed in Carnoy’s solution  and stored at 4°C for 24 hr. The root tips were then hydrolyzed in 1 N HCl at 60°C for 5–10 min, treated with 1% pectinase for 1 hr, crushed in 45% acetic acid on microscope slides that had been pretreated with Vectabond , and covered with cover glasses. The slides were frozen in liquid nitrogen, and the cover glasses were removed with a razor blade. The slides were then air-dried, and stained with Giemsa stain for 5 min. The slides were destained with formamide and stored in a dessicator at -20°C until use. Non-overlapping chromosomes were selected for the GISH experiments.A. John De Biase ‘Blue’, or from the male parent, P. Chih Shang's Stripes, can be clearly distinguished without the use of blocking DNA.More than 15 samples were examined by GISH analysis. Genomic DNA was extracted from young leaves of the two parents and the putative hybrids using the CTAB method . DNA wasSetaria italica (AB197128), Linum (EU307117), and Sarcochilus falcatus (AF321598). The primers were ETS-1 (5’-TGTCTGGCATGCAAGCACTG-3’) and ETS-2 (5’-CATGCATGGCTTAATCTTTGAGAC-3’) -specific primers for PCR amplification were designed based on reference sequences from the GenBank database, including maize (GenBank accession no. X03990), wheat (X07841), rice (X54194), AGAC-3’) . Each PCXbaI and DraI, separated on a 1.0% agarose gel by electrophoresis, and transferred to a Hybond N+ nylon membrane  via the capillary method. Probe preparation, hybridization, and detection were conducted using a DIG High Prime DNA Labeling and Detection Starter Kit II  according to the manufacturer’s instructions. The probe was prepared via PCR amplification of the ETS region using primers ETS-1 and ETS-2  and sequenced with vector-specific primers (SP6 and T7) according to the manufacturer’s recommendations. To validate the sequences of the ETS primer binding sites in all of the studied samples, inverse PCR (iPCR) was conducted as described by Ochman et al. [The PCR products were recovered using a MinElute PCR Purification kit with the provided spin columns . The recovered PCR products were ligated into a T-vector , and the resulting recombinants were transformed into n et al. . Sequencn et al. .