Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a.To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations.S112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared.rBet v 1a and its misfolded variant rBet v 1a1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%.Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected. Escherichia coli are well established and rBet v 1 is also available as allergen reference standard from the European Directorate for the Quality of Medicines and Health Care, EDQM [The major allergen Bet v 1 is the main cause of birch pollen allergy affecting millions of patients in Central and Northern Europe –6. Heterre, EDQM .Physico- and immunochemical integrity and native-like protein conformation are prerequisites to evaluate suitability of rBet v 1 as an allergen biomarker for diagnosis and therapy of birch pollen-related allergy. In general, allergens of the Bet v 1 protein family mostly bind IgE only in their native protein conformation. However, heterologous expression and purification of rBet v 1 might produce a quantitatively unknown fraction of misfolded and thus non-functional (non-IgE binding) rBet v 1. In this regard it has been recently shown that protein conformation of rBet v 1 and thus IgE binding capacity is greatly affected by the conditions used for protein purification ,38. IgnoS112P/R145P, a variant harboring two proline residues which impair proper folding into the Bet v 1-type protein conformation. We used compositional mixtures of rBet v 1a, the major allergen isoform of birch pollen, and its derivative rBet v 1aS112P/R145P in defined molar ratios and tested them in immuno- and physicochemical assays routinely used for allergen characterization. Finally we correlated the experimental results with the theoretically expected values for the combinations of the two rBet v 1 proteins in order to i) correlate experimental and theoretically expected data and analytical resolution and to ii) determine the extent of variation depending on biological and technical replicates.These observations prompted us to systematically analyze the impact of misfolded rBet v 1 on quantitative IgE binding of rBet v 1 preparations. For this purpose we generated and purified rBet v 1aS112P/R145P, were made to generate 3 identical compositional mixtures with defined ratios of the two proteins to test both i) the influence of biological replicates and ii) the influence of method variation  on immuno-and physicochemical assays used in allergen characterization. Furthermore we wanted to define the range of quantitative variation within one method to evaluate the correlation of experimental and theoretical data. Finally we compared the resolution of the individual methods to evaluate the performance of the assays with respect to distinguish structural and IgE binding differences in the presence of various ratios of natively folded and unfolded allergen.Three independent preparations of rBet v 1a and rBet v 1aA/L (patients 1–4) or to birch pollen of 83.7->100 kUA/L (patients 5–10) as measured by ImmunoCAPTM . Patients were recruited at the University Medical Center Mainz, Department of Dermatology, Mainz, Germany, and at the Klinik für Dermatologie, Venerologie und Allergologie, University of Leipzig, Germany. Serum donations were approved by the local ethics committees of the clinical centers, namely the “Ethik-Kommission an der Medizinischen Fakultät der Universität Leipzig” and the “Ethikkomitee der Universitätsmedizin Mainz”. Study participants provided written informed consent and ethics approval included consent form and consent procedure. Serum from a non-allergic subject was used as negative control for the specific IgE measurements.10 allergic patients suffering from rhinoconjunctivitis or asthma to early flowering tree pollen were included as serum donors. Specific sensitization was documented by positive skin prick test responses and by detection of allergen-specific IgE to either rBet v 1a of 13.7–67.3 kUEscherichia coli was purchased from Geneart  and cloned into bacterial expression vector pET15b  using the Clontech InFusion Kit . Bet v 1a variant rBet v 1aS112P/R145P was generated with the QuickChange Multi Lighting mutagenesis kit  using the Bet v 1a ORF in pET15b_Bet v 1a as DNA template and two mutagenic primers Bet v 1S112P (acaccggatggtggtcccattctgaaaattagc) and Bet v 1R145P (ggtgaaaccctgctgcctgcagttgaaagctat) according to manufacturer’s instructions. The genes were expressed in E. coli strain BL21(DE3) cells, growing at 37°C in 1 liter of LB medium, containing 50 μg/ml carbenicillin to an OD600 value of 0.7. A final concentration of 1 mM IPTG was added to induce protein expression. The cells were harvested 4 h after induction by centrifugation , and the cell pellets were stored at −20°C. Upon expression rBet v 1a was found in both cytosolic soluble and in insoluble fractions (inclusion bodies) of bacterial lysates whereas rBet v 1aS112P/R145P was exclusively found in inclusion bodies. To ensure comparability of protein preparation rBet v 1a and rBet v 1aS112P/R145P were purified from inclusion bodies. The proteins were regained from protein pellets after cell lysis with 10 mM potassium phosphate (KPi), pH 7.4, 500 mM NaCl, 8 M urea and 10 mM imidazole, bound to Ni-NTA Superflow  and refolded by subsequently lowering the urea concentration in 10 mM KPi; pH 7.4, 500 mM NaCl, 10 mM imidazole during liquid chromatography. Proteins were eluted by gradual increase of imidazole to 500 mM. Fractions containing the respective protein were pooled and 6xHis-tag was cleaved off with Thrombin . Proteins were further purified by size-exclusion chromatography , concentrated, frozen in liquid nitrogen and stored at -80°C. Protein concentrations were determined with bicinchoninic acid using bovine serum albumin as standard .The open reading frame of Bet v 1a optimized for codon usage of S112P/R145P were generated to yield 3 individual rBet v 1a/rBet v 1aS112P/R145P combinations which were analyzed with 1 , 2 (MR assay), and 3 (ELISA) technical replicates each.Three preparations each of rBet v 1a and rBet v 1ai, pH 7.4. Each measurement comprised the average of 10 repeated scans between 255 and 185 nm.Far UV circular dichroism (CD) spectra of the rBet v 1a variants were acquired at 293 K using a Jasco J-810 spectropolarimeter  at a band width of 1 nm and a sensitivity of 100 mdeg in a 0.2 cm cell. All proteins were analyzed at a concentration of 10 μM in 10 mM KPexperimental) of rBet v 1a and rBet v 1aS112P/R145P at wave lengths characteristic for secondary structure  is the residual mean ellipticity of the non-folded rBet v 1a variant. Theoretical mean estimates of residual ellipticities  were obtained by multiplication of ΔΘexperimental determined with 100% rBet v 1a with 1.25, 1.67, 2.5, 5, 10, 100, 1000, or 10000 representing 80, 60, 40, 20, 10, 1, 0.1, 0.01, and 0.001% of rBet v 1a in the protein mixtures, respectively. The following formula was used:experimental (100% rBet v 1a) is the experimentally determined mean residual ellipticity of 100% rBet v 1a and x is the respective multiplication factor as described above. The experimental/theoretical comparisons for each rBet v 1a/rBet v 1S112P/R145P mix were calculated by dividing ΔΘexperimental by the corresponding ΔΘtheoretical.The mean estimates of residual ellipticities  in nm using a Zetasizer Nano ZS . 10 μM of recombinant Bet v 1a and Bet v 1aS112P/R145P in 10 mM KPi, pH 7.4 were analyzed at 25°C. Three individual measurements (3 x 10 runs per measurement) per protein were carried out. Data were analyzed by the proprietary software of the DLS instrument where the hydrodynamic radius (RH) is calculated from the diffusion coefficient (D) which is obtained by measurement of fluctuations of intensities of scattered light over time and fitting of the respective correlation curve to an exponential function from which the diffusion coefficient can be calculated. RH is then calculated by the Stokes-Einstein H is the hydrodynamic radius, k is the Boltzmann constant, T is the absolute temperature, η is the solvent viscosity, and D is the diffusion coefficient.Dynamic light scattering analysis was used to determine hydrodynamic radii  of IgE signals after 100 ms exposure were calculated by setting the IgE signal of rBet v 1a to 100% and the IgE signals of the rBet v 1a/rBet v 1aS112P/R145P combinations to 80, 60, 40, 20, 10, 1, 0.1, 0.01, 0.001, and 0% according to the respective molar content of rBet v 1a in the protein mixtures. The following equation was used:exp(100% Bet v 1a) is the densitometric IgE signal of 100% rBet v 1a, x is mol%  of rBet v 1a in the respective rBet v 1a/rBet v 1aS112P/R145P mixture, and metheoretical is the resulting calculated mean estimate of the IgE signal in dependence of rBet v 1a content. The experimental/theoretical comparisons for each rBet v 1a/rBet v 1aS112P/R145P mix was calculated by dividing the experimentally derived mean estimate  by the respective metheoretical and listed in IgE bound to rBet v 1 in Western blot analyses was analyzed by densitometry  and evaluated using ImageJ  Theoretical mean estimates  diluted 1:1000. As substrate 3,3′,5,5′-tetramethylbenzidine  was used for the horseradish peroxidase and the absorbances at 450 nm and 630 nm were measured after stopping the reaction with 25% H2SO4. For all data points absorbances at 630 nm were substracted from absorbances at 450 nm. Inhibition of IgE binding was calculated with the following equation:sample is the absorbance of the respective sample and absmax is the absorbance without inhibitor. Experimental half-maximal inhibition  of IgE binding to rBet v 1a (ELISA) was calculated using a four parameter sigmoid curve fit with the restrictions that slope, minimum and maximum asymptote had to be the same for all curves to ensure parallelism among curves. The following equation was used:50, and d is the maximum asymptote. Theoretical half-maximal inhibition  was obtained by multiplication of EC50 experimental obtained by 100% rBet v 1a with factors 1.25, 1.67, 2.5, 5, 10, 100, 1000, 10000 representing 80, 60, 40, 20, 10, 1, 0.1, 0.01, and 0.001% of rBet v 1a in the protein mixtures, respectively. The following equation was used:50 experimental (100% rBet v 1a) is the experimentally derived half maximal effective concentration with 100% rBet v 1a and x is the respective multiplication factor as described above. The experimental/theoretical comparisons for each rBet v 1a/rBet v 1aS112P/R145P mix was calculated by dividing EC50 experimental by the respective EC50 theoretical. For statistical analysis the EC50 values with 95% Confidence Intervals were estimated for all curves showing a significant regression and/or having values measured at least from minimum asymptote up to the inflection point of the curve. P values for significant horizontal shifts of the curves, i.e. significant different EC50 values were calculated .For IgE-ELISA inhibition experiments, Nunc Maxisorp plates  were coated overnight at room temperature with 50 ng Bet v 1/100 μl of 10 mM potassium phosphate-buffered saline (PBS). After blocking with PBS containing 2% BSA, plates were incubated with the human serum pool (dilution 1:80) and increasing molar ratios of rBet v 1a/rBet v 1aS112P/R145P compositional mixtures of defined ratios. Degranulation was quantified by photometric measurement of β-hexosaminidase activity in the culture supernatants. The percentage of β-hexosaminidase activity relative to cells lysed with Triton X-100  was calculated and corrected for spontaneous release . Experimental and theoretical half maximal β-hexosaminidase releases  were calculated in a similar fashion as described for the ELISA above using the similar formula:S112P/R145P mixture. Fit of dose-response curves and statistical analysis was done in a similar fashion as described for ‘Inhibition IgE ELISA’ above.The mediator release assay was performed as described . BrieflyS112P/R145P to homogeneity from Escherichia coli. The amino acid sequence of rBet v 1a and rBet v 1aS112P/R145P was confirmed by LC_MS with sequence coverage of 72% and 78% and a score of 11137 and 3957, respectively. The peptides harboring either S112 and R145 or S112P and R145P were individually double checked and detected with a mass error of 1.2 ppm and 2.5 ppm, or 2.4 ppm or 1.9ppm, respectively. The identity of rBet v 1aS112P/R145P was confirmed by liquid chromatography mass spectrometry (LC-MS) after SDS PAGE separation as described elsewhere [4HCO3; 10% aceto nitrile (ACN) and the digestion was stopped by adding 5% formic acid. The peptides were analysed by using a nano-ultra performance LC system coupled to a nano-ESI-MS  with a 5 μm symmetry 180 μm x 20 mm c18 pre-column and a 1.7 μm BEH 130 100 μm x 100 mm c18 separation column. A 30 minutes gradient (3% to 40% ACN at 500 nl/minute) after 3 minutes of trapping (99% water at 5 μl/minute) was applied to separate peptides. MS was operated in V mode, acquiring MSE data and applying standard parameters. Data analysis was performed with ProteinLynx Global Server version 2.4 (Waters), searching an in house database consisting of the Uniprot database  and the amino acid sequences of recombinant Bet v 1a and Bet v 1aS112P/R145P. Protein hits were accepted at a false positive rate of less than 4%.We expressed and purified rBet v 1a and rBet v 1alsewhere  with sliS112P/R145P  in dynamic light scattering (DLS) and the 15N spin relaxation rates of rBet v 1a in NMR. The RH of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) as well as the relaxation rates , thus over-estimating the quantity of IgE bound to rBet v 1a ≤ 1% in the protein mixtures blotted  yielding non-evaluable inhibition curves  from EC50 obtained for 100% rBet v 1a.Next we tested inhibition of IgE antibody binding to immobilized rBet v 1a with rBet v 1a/rBet v 1ain ELISA . Since on curves . Only haS112P/R145P protein mixtures induced mediator release up to a rBet v 1a content of 1% with experimental-theoretical values of half maximal mediator releases of 0.86 to 1.32 (20–99% rBet v 1aS112P/R145P). However lower contents of IgE binding rBet v 1a (<0.1%) did not allow valid determination of half-maximal mediator release. No significant differences could be observed between the EC50 values for 100% rBet v 1a and all rBet v 1a/rBet v 1aS112P/R145P compositional protein preparations. However, according to a confidence interval of 95%, only the rBet v 1a/rBet v 1aS112P/R145P protein combination containing 1% rBet v 1a resolved analytically from the mediator release obtained with 100% rBet v 1a.So far the immunoassays above addressed only serological IgE binding to rBet v 1a combinations. To analyze the biological activity of the recombinant allergens in a cellular system we carried out mediator release in humanized rat basophil leukemia cells sensitized with pooled sera IgE . We evalS112P/R145P protein combinations with up to 90% of unfolded rBet v 1aS112P/R145P . In particular for diagnostic purposes, recombinant Bet v 1 needs to bind IgE from sera of subjects allergic to birch pollen in a similar fashion as the native allergen since clinically relevant IgE interaction of Bet v 1 requires a native protein conformation. On the other hand recombinant unfolded Bet v 1 has been used in allergen immunotherapy trials and in this scenario refolding needs to be avoided to ensure the safety of study participants.S112P/R145P. We applied physico- and immunochemical assays that are routinely used to judge quality and IgE binding of rBet v 1a preparations according to two quantitative parameters, namely i) the accuracy with which the actual IgE-binding protein moiety is determined and ii) the precision according to the confidence intervals of 95% chosen with which the actual IgE-binding protein moiety is determined resolved quantitatively by the respective analytical method.The major goal of this study was to correlate changes of secondary/tertiary structure of recombinant variants of Bet v 1a with quantitative IgE binding of protein mixtures comprising defined molar fractions of IgE-reactive natively folded rBet v 1a and non IgE-binding artificially stably unfolded rBet v 1aS112P/R145P protein mixtures. However with molar ratios of rBet v 1a < 10% we found that all assays performed with large deviations from expected values. As opposed to ELISA and cellular mediator release, immunoblot analysis overestimated the actual amount of rBet v 1a-specific bound serum IgE.All assays showed experimental-theoretical comparisons (0.26–1.74) up to a molar fraction of 10% rBet v 1a in the rBet v 1a/rBet v 1aS112P/R145P mixture) of the total IgE binding content of rBet v 1a (i.e. 100% rBet v 1a) with an accuracy of 1.03 (CD222nm) and 0.93 (ELISA), respectively (S112P/R145P mixtures was observed with molar ratios of rBet v 1a of 80% (IB), 20% (ELISA), and 60% (CD222nm). No statistical significance was obtained for the cellular in vitro mediator release assay, because of large variation of the results in this biological assay. Our analyses revealed that experimental/theoretical comparisons of physicochemical and immunoassays with physically integer and IgE-reactive rBet v 1a in the range of 100–10% molar content lies within 0.26–1.74, with CD and ELISA correlating best with the theoretical values.Considering the accuracy, precision and resolution according to 95% confidence intervals of the individual assays we found that circular dichroism and ELISA could differentiate a 1.67-fold reduction (i.e. 60% of rBet v 1a in a rBet v 1a/rBet v 1aectively . In thisIn the European Pharmacopeia Monograph on Allergen Products (01/2010:1063) the total allergenic activity of an allergen product as assayed by inhibition of binding capacity of specific IgE may range from 50–150%. Considering that total IgE binding of a birch pollen extract is mainly caused by Bet v 1, we cover at least the 50–100% range which is represented by accuracies of > 0.48 (MR) to < 1.21 (IB) at 40% rBet v 1a to 1.0 at 100% rBet v 1a cf. . Thus, din vitro assays is one parameter that qualifies a rBet v 1a variant as potential hypoallergenic candidate molecule for AIT. In this regard the immunoassays used in this study are well suited to screen for potential therapeutics as they easily detected IgE responses of ≤ 1% of total IgE binding.Recombinant Bet v 1 has been generated as low IgE-binding  variants to test therapeutic potential . Reduced IgE binding capacity in S112P/R145P used in this study could be used as quality reference standards. However other attempts to assess Bet v 1-type protein conformation and thus potential allergenicity could also be considered to evaluate the quality of recombinant Bet v 1 preparations. The recently identified natural ligand of Bet v 1a and the determined structures of Bet v 1 isoforms with bound ligands could be employed for the development of quantitative ligand binding assays with clear-cut correlation to allergen conformation und thus IgE binding activity [Considering the potential use of recombinant Bet v 1a variants for AIT or as diagnostic reagents, the variants rBet v 1a and rBet v 1activity . Such apThe list of methods to analyze Bet v 1 conformation and correlated IgE interaction is not complete in this study. Surface plasmon resonance, infrared spectroscopy, isothermal titration calorimetry and other methods are also valuable additions to be included in a set of assays to assess the quality of recombinant Bet v 1-type allergens. However, lower IgE binding might not always be necessarily related to loss of Bet v 1-type conformation. Additional preparation/storage-dependent phenomena like modifications of single amino acid side chains  while maintaining native protein fold may also contribute to decreased IgE binding due to decreased antibody affinity on the epitope level.in vivo diagnostics and allergen-specific immunotherapies.Taken together the set of physico- and immunochemical assays used here to correlate rBet v 1a protein conformation and IgE binding revealed that ELISA and Circular Dichroism could accurately and precisely determine lower rBet v 1a contents (≤ 60%) in the context of absolute allergen concentration whereas immunoblot and cellular mediator release needed larger deviations of rBet v 1a ratios, ≥ 20% and ≥ 1%, respectively, to resolve the content of IgE-binding rBet v 1a in recombinant protein preparations. The principal findings in this study should be considered when employing methods for quality assurance of allergen preparations used in S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither  This enables the time- and space-specific translation of the most important signaling molecules for the meiotic cell cycle, e.g. cyclins and c-mos trans-acting factors such as Maskin and Pumilio Xenopus oocytes, analogous to Thr172 in porcine oocytes) activates the polyadenylation of a CPE-containing mRNA by excluding the poly(A)-specific ribonuclease (PARN) from its 3′UTR The CPE binds the CPE-binding protein 1 (CPEB1), which functions as a translational activator or repressor according to its phosphorylation state. The non-phosphorylated CPEB1 represses the translation initiation of CPE-containing mRNAs by recruiting other XenopusXenopus oocyte extracts the phosphorylation of CPEB1 at Ser174 occurred despite the depletion of AURKA or the inhibition of its activity. More often, CDK1 activated by speedy/RINGO (independently or in cooperation with AURKA) is proposed to be responsible for the CPEB1 activating phosphorylation in Xenopus oocytes 2+/calmodulin-dependent protein kinase II (CaMKII) has been found to be responsible for CPEB1 activation in neurons Aurora kinase A (AURKA) has been previously proposed to be the kinase responsible for the polyadenylation-activating CPEB1 phosphorylation in cyclin B1 mRNA precedes CPEB1 degradation. We therefore explored the effects of AURKA inhibition on meiotic resumption of porcine oocytes using MLN8237 and we have found that the MLN8237-treated oocytes remain arrested in the late diakinesis-like stage and that they cannot reach the metaphase I stage. However, neither cyclin B1 mRNA polyadenylation nor its translation is impaired in these oocytes. Using dual-luciferase assay, we further show that the inhibition of AURKA kinase does not prevent the translation of other CPE-containing mRNAs. Finally, using in vitro kinase assay, we demonstrate that CPEB1 is phosphorylated at Thr172 and/or Ser178 during oocyte meiotic maturation despite the inhibition of AURKA.In this study, we show that the cytoplasmic polyadenylation of both short and long forms of porcine 2 for 12 to 44 h. Oocytes were denuded, washed and stored at −80 °C until use. For evaluation of maturation, denuded oocytes were fixed in ethanol:acetic acid solution (3:1 v/v) for 48 h. Staining was performed with 1% orcein in 50% aqueous acetic acid and 1% sodium citrate followed by washing with 40% acetic acid. Oocytes were observed and photographed under a phase-contrast microscope .Porcine ovaries from non-cycling gilts were collected at a commercial slaughterhouse  and transported in physiological saline at 37 °C to the laboratory. Cumulus-oocyte complexes were aspirated from follicles and matured in M199 medium  supplemented with 10% fetal bovine serum  and 0.8 IU/mL P.G. 600 (Intervet). IVM was performed at 38.5 °C in a humidified atmosphere of 5% COFor inhibition of AURKA activity, MLN8237  was added to the IVM medium at concentrations of 1, 5 and 10 µM. Cumulus-oocyte complexes were cultured for 44 h to evaluate the effects of MLN8237 on meiotic maturation. For the western blot analysis, immunocytochemistry and poly(A)-test, oocytes were cultured in the presence of the inhibitor for 28 h, for the dual-luciferase assay, oocytes were collected after 3, 24 and 28 h of IVM.5′-GCG AGC TCC GCG GCC GCG TTT TTT TTT TTT-3′, cyclin B1 primer 5′-GCA TTT TCT TCG GAG AGC ATC CAA GAT T-3′ (amplification of both short and long cyclin B1 3′UTRs) and cyclin B1 long 3′UTR primer 5′-CTC ATT TGA ATG TGG CTA TTT CCC ACT TGA GG-3′ (specific for the long cyclin B1 3′UTR). cDNA was subjected to electrophoresis in 5% polyacrylamide gel and stained with GelRed . Gels were observed and photographed by Kodak Gel Logic 100/200 Camera , K. G. L. integrated illuminator cabinet  and KODAK MI SE software .The poly(A)-test was performed as described by Sallés and Strickland 2. Membranes were blocked, depending on the used antibody, in 10% gelatin, 2.5% or 5% skimmed milk dissolved in 0,05% Tween-Tris-buffer saline (TTBS), pH 7.4 for 1 h. After a brief washing in TTBS, membranes were incubated at 4 °C overnight with the following primary antibodies: CPEB (H-300) , cyclin B1 Ab-1 (Clone V152) , phospho-Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) , phospho-TACC3 (Ser558) , Anti-GAPDH , monoclonal anti-β-Tubulin antibody . The membranes were then washed 3 × 10 min in TTBS and incubated with a horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG antibody  for 1 h at room temperature. Proteins were visualized using an ECL-plus detection system  according to the manufacturer's instructions. Films were scanned using a GS-800 Calibrated Densitometer  and the results were quantified using Quantity One 1-D Analysis Software (Bio-Rad).Oocytes were lysed in 10 µL of 1 × Reducing SDS Loading Buffer  and heated at 100 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to Immobilon P membrane  using a semidry blotting system  for 25 min at 5 mA/cmOocytes were fixed in 4% paraformaldehyde (PFA) in PBS for 30 min, permeabilized for 15 min in 0.1% Triton X-100, and incubated overnight at 4 °C with primary antibody against phospho-Aurora A (Thr288) . After washing, oocytes were incubated for 1 h with an Alexa Fluor conjugated anti-rabbit antibody (Molecular Probes). Samples were visualized using an inverted confocal microscope in 16 bit depth . Images were assembled in Photoshop CS3.cyclin B1 3′UTR was a kind gift from Prof. Shin Murai, Department of Biochemistry, Toho University School of Medicine, Tokyo, Japan The pIVT plasmid containing firefly luciferase with mouse Porcine oocytes were microinjected as was described previously To exclude dead oocytes being collected for further analyses, we used trypan blue staining. Microinjected oocytes after the IVM in the absence or presence of the inhibitor were stained with 0.1% trypan blue (Beckman-Coulter) for 5 min. Dead oocytes (blue) were discarded and only live oocytes were used for the dual-luciferase assay.cyclin B1 3′UTR (0.5 pg/oocyte) and Renilla luciferase (0.25 pg/oocyte) were co-injected into GV-stage oocytes as described above. The oocytes were cultured in vitro for 3, 24 and 28 hours in the absence or presence of 1 µM MLN8237. Four live oocytes from each group were collected. The oocytes were lysed in 5 µL of Passive Lysis Buffer and stored at −80 °C until luciferase activity was measured by the Dual-Luciferase Assay System  according to manufacturer's instructions. Signal intensities were measured using a Glomax 20/20 Luminometer (Promega). Luciferase activity of firefly luciferase was normalized to that of Renilla luciferase.The cRNA of firefly luciferase with in vitro kinase assay was a modified version of the method published earlier 32P] ATP (Amersham Pharmacia Biotech). The reaction was stopped after 30 min by the addition of SDS-PAGE sample buffer and boiling for 3 min. After SDS-PAGE on 15% gel, the gels were stained with Coomassie Blue R250, destained overnight and dried. The phosphorylated substrates were visualized by autoradiography and analyzed using a BAS 2500 PhosphoImager .Phosphorylation of CPEB1 and LATS2 was measured in oocyte extracts via their capacity to phosphorylate external substrate peptides derived from CPEB1 and LATS2 sequence: RRSRLDTRPILDSRSS (underlined T corresponding to CPEB1 Thr172 activatory site) and YQKALREIRYSLLPFANESGT -test (PAT). We observed that both short and long form of cyclin B1 mRNA were gradually polyadenylated  (In enylated . These fon (IVM) . At thisXenopus oocytes is required for CPEB1 degradation and it depends on both CDK1 and PLK1 activities We observed the CPEB1 phosphorylation by a shift in its electrophoretic mobility starting at 20 h of IVM . Analogocyclin B1 mRNA and its translation are likely to precede CPEB1 phosphorylation by CDK1 and subsequent CPEB1 destruction. Therefore, our data suggest that the mechanism of cyclin B1 mRNA polyadenylation regulation in porcine oocytes probably differs from that in Xenopus oocytes and is dependent on CPEB1 activating phosphorylation rather than on its degradation.To follow the cyclin B1 expression more precisely during the critical time interval, we collected the oocytes before and around GVBD in the interval of 2 hours. Low expression of cyclin B1 was evident already after 14 h of IVM and the signal gradually increased in following time intervals . On the in vitro kinase assay using a peptide derived from another known substrate of AURKA, the large tumor suppressor kinase 2 (LATS2) in vivo, could be attributed to the mode of action of the inhibitor. MLN8237 inhibited AURKA activity in situ, but it did not prevent its activation and as such it could not prevent the peptide phosphorylation in kinase reaction with no inhibitor added. Altogether, the above-mentioned results document clearly that MLN8237 was able to inhibit AURKA activity and to prevent phosphorylation of its downstream targets in maturing porcine oocytes.MLN8237 is a highly selective AURKA inhibitor cyclin B1 mRNA polyadenylation and the role of AURKA in this process, we inhibited AURKA kinase activity in oocytes by adding MLN8237 at different concentrations ranging from 1 to 10 µM to the cultivation medium. At low concentrations of the inhibitor, the oocytes underwent GVBD but they were unable to reach metaphase I, and they became arrested in late diakinesis-like configuration of chromosomes even after 44 h of IVM , when 1 µM MLN8237 was used, the polyadenylation was similar to the polyadenylation observed in the control group . This result is in agreement with previous findings, although Zhang et al. cyclin B1 mRNA in porcine oocytes starts at 24 h, This difference in the timing of polyadenylation may be caused by the use of a more sensitive electrophoretic system in our case (polyacrylamide gel vs. agarose gel) together with the different speed of IVM in different conditions (MI – 30 h vs. 26–28 h in our conditions).It has been shown in many species and different cell types as well that cyclin B1 synthesis is regulated by the cytoplasmic polyadenylation of its mRNA cis-elements within the 3′untranslated region (3′UTR) of mRNAs, mRNA with short 3′UTR contains one CPE overlapping with polyadenylation signal, the hexanucleotide AAUAAA and one Pumilio binding element (PBE) in the distance of 32 nts from the hexanucleotide. According to Piqué et al. cyclin B1 mRNA should be theoretically polyadenylated soon after the beginning of oocyte maturation. Therefore, this polyadenylation should be dependent on CPEB1 activating phosphorylation. This is in a good agreement with our results showing that the long form of cyclin B1 mRNA is polyadenylated before GVBD and that there is no difference in the timing of polyadenylation between the long and short form.Regarding the regulation of cytoplasmic polyadenylation by cyclin B1 mRNA precedes CPEB1 phosphorylation and degradation. This suggests that the polyadenylation of porcine cyclin B1 mRNA is rather independent of CPEB1 degradation, although we cannot rule out the possibility that some partial degradation of CPEB1, which is below our detection limit, already occurs at this time.Nishimura et al. Xenopus and mouse oocytes, AURKA has been previously proposed to be the kinase responsible for the CPEB1 activating phosphorylation In Phosphorylation of AURKA at Thr288 is often used as a marker of its activity cyclin B1 mRNA is not affected. The slight inhibitory effect of higher concentrations on the polyadenylation of cyclin B1 mRNA is likely to be due to the delayed or blocked meiotic progression. Nevertheless, the expression of cyclin B1 protein is not inhibited even in the presence of 10 µM MLN8237. These results are confirmed by the dual-luciferase assay with the luciferase construct containing murine cyclin B1 3′UTR with a strong activating CPE in a distance of 21 nts from the hexanucleotide AAUAAA. In mouse oocytes microinjected with this construct, an intensive increase of firefly luciferase activity was observed during maturation In oocytes treated with MLN8237, polyadenylation of cyclin B1 mRNA is polyadenylated shortly before GVBD and not earlier. Moreover as we also show, CPEB1 peptide is not phosphorylated by recombinant AURKA at all suggesting that another kinase than AURKA may be involved in the CPEB1 activatory phosphorylation.Since AURKA phosphorylation at Thr288 and therefore its activity is present already in GV oocytes, we have explored the possibility that AURKA phosphorylates CPEB1 early in the GV stage and in such a case CPEB1 activation and subsequently cytoplasmic polyadenylation would not be inhibited by the inhibitor. However, our results show that this is not the case, because the peptide derived from CPEB1 is not phosphorylated by the GV-stage oocyte lysates even though it is substantially phosphorylated by lysates of oocytes cultivated for 28 h. CPEB1 activation in the early GV stage would also not explain why Altogether, our results show that although AURKA plays a crucial role in meiotic progression of oocytes to reach metaphase I, the CPEB1-dependent cytoplasmic polyadenylation is unlikely to be dependent on AURKA activity during meiotic maturation of porcine oocytes. Our further studies with different kinase inhibitors (unpublished) suggest that CDK kinases or MAP kinases could be involved in the regulation of cytoplasmic polyadenylation and CPEB1 activation, which was also proposed by other groups Figure S1Verification of MLN8237 functionality in porcine oocyte model system. (A) Oocytes were cultivated in the presence or absence of MLN8237  and collected after 28 h. The activity of AURKA in the samples was determined using western blot with phospho-TACC3 (Ser558) antibody. (B) In vitro kinase assay – LATS2 phosphorylation. Oocytes were cultivated in control medium for 0, 20 or 28 h, or in the presence of 2 µM MLN8237. Thereafter, in vitro kinase assay with LATS2 peptide as external substrate was performed in extracts prepared from 20 oocytes. MLN8237 was added to 20 h cultured oocyte extracts in final concentration of 2 or 10 µM in reaction buffer. Alternatively, recombinant AURKA (1 I.U. per assay) was used instead of oocyte extracts with or without MLN8237 in the concentration of 5 and 2 µM. The extracts were separated by SDS-PAGE and the phosphorylated substrates were visualized by autoradiography and analyzed using BAS 2500 PhosphoImager.(TIF)Click here for additional data file.Figure S2GVBD rate in oocytes treated with MLN8237. Oocytes were cultivated in the presence or absence of MLN8237  and collected after 20, 26 and 28 h.(TIF)Click here for additional data file.


Arabidopsis thaliana accessions is due to noncoding cis polymorphisms that define distinct haplotypes of FLOWERING LOCUS C (FLC). Li et al. find that a single natural intronic polymorphism in one haplotype affects FLC expression and thus flowering by specifically changing splicing of the FLC antisense transcript COOLAIR. Altered antisense splicing increases FLC expression via a cotranscriptional mechanism involving capping of the FLC nascent transcript.A large proportion of the natural variation in flowering in  Arabidopsis thaliana accessions is due to noncoding cis polymorphisms that define distinct haplotypes of FLOWERING LOCUS C (FLC). Here, we show that a single natural intronic polymorphism in one haplotype affects FLC expression and thus flowering by specifically changing splicing of the FLC antisense transcript COOLAIR. Altered antisense splicing increases FLC expression via a cotranscriptional mechanism involving capping of the FLC nascent transcript. Single noncoding polymorphisms can therefore be a major contributor to phenotypic evolution through modulation of noncoding transcripts.The extent to which natural polymorphisms in noncoding sequences have functional consequences is still unknown. A large proportion of the natural variation in flowering in  Arabidopsis thaliana accessions focusing on flowering time, a trait with considerable fitness consequences (FLOWERING LOCUS C (FLC)  . Functiothe gene .FLC regulation involves pathways that link different aspects of chromatin regulation with a set of antisense transcripts collectively called COOLAIR  than the Var-2-6 (5.2-fold) allele  allele C,D. ThisFLC alleles revealed 43 polymorphisms. One was in the FLC coding region and introduces a synonymous nucleotide change in exon 7, so the Var2-6 FLC allele encodes a protein identical to Col-0 . The other polymorphisms were in noncoding regions with one, SNP259 Col-0/Var2-6: G/T, located next to the acceptor splice site of the intron of class II-i COOLAIR  and five other polymorphisms within a 22-base-pair (bp) region (Ws-0/Ka-0: C-AAA/TTTTT). The T nucleotide at SNP259 associated with later flowering  in which the COOLAIR promoter had been replaced with an RBCS 3′ terminator region. Production of class I and class II COOLAIR transcripts was disrupted in this transgene, although some unspliced nascent antisense transcript was still produced . They also flowered considerably later after 4-wk vernalization  is sufficient to change the proportion of FLC transcripts carrying a 5′ cap. Changes in COOLAIR processing therefore appear to influence FLC expression via a mechanism involving the level of capping of the FLC transcript.Our earlier genetic analysis suggested that function C,D. FRI a 5′ cap , a struct of FRI A. Mutatihenotype B; thus, FLC RNA showed that the SNP259 effect on capping occurs on the FLC nascent transcript in the nucleus  experiments showed that RNA polymerase II (Pol II) levels were approximately twofold higher at the Var2-6 FLC allele introgressed into Col as compared with a Col FLC allele, similar to the effect of FRI on Col FLC . These data are all consistent with the primary effect of SNP259 resulting in a COOLAIR variant that up-regulates transcription of FLC (or represses less well than other COOLAIR class II variants). How a COOLAIR variant (an antisense transcript) influences transcription of FLC remains to be established, but COOLAIR class II variants are associated with chromatin at the 5′ end of FLC . Higher FLC expression and an increased vernalization requirement are likely to be adaptively important in these regions, enabling plants to avoid precocious flowering before winter is over. This significant change in reproductive timing behavior is the result of a single polymorphism in a noncoding region of a locus. This should provide an important paradigm in the many studies associating polymorphism with phenotypic variation. It will also influence the debate over the different types of molecular variation that underpin adaptive evolution  and FRI alleles . The association between bolting time and Ws-0 and Ka-0 FLC alleles was analyzed in the two subpopulations containing Ws-0 FRI and Ka-0 fri separately. The box plot and statistical analysis in this study were performed in Gene Stat edition 9 with default parameters , and bolting time was assayed. The individuals of the F2 population were genotyped with specific PCR markers for Ws-0 and Ka-0 rameters .FLC sequences were aligned and exported with BioEdit package version 7.0.9.0 with default parameters (rameters .COOLAIR class I was amplified with primers F1 (5′-TGGTTGTTATTTGGTGGTGTG-3′) and R1 (5′-GCCGTAGGCTTCTTCACTGT-3′). Class II was amplified with primers F3 (5′-GTATCTCCGGCGACTTGAAC-3′) and R1. PCR fragments were assayed in agarose gel, cloned into pGEM-T Easy Vector (Promega), and sequenced. For the COOLAIR class II-i intron splicing assay, cDNA was synthesized with the RT primer UBC-RP, F2, and unspliced COOLAIR in distal region LP and quantified with the quantitative PCR (qPCR) primers F2 (5′-CTCCTCCGGCGATAAGTA-3′) and R2 (5′-CTCACACGAATAAGAAAAGTAAAA-3′). Unspliced COOLAIR in the distal region was amplified with primers LP (5′-CGCAATTTTCATAGCCCTTG-3′) and RP (5′-CTTTGTAATCAAAGGTGGAGAGC-3′). SNP259/T-mediated class II splicing was quantified with primer F2 and R3 (5′-GAAAAAAACCAAACGGTAAGGAAA-3′). The intron splicing was calculated by normalizing spliced to unspliced COOLAIR. The PCR position is shown in UBC with primers 5′-CTGCGACTCAGGGAATCTTCTAA-3′ and 5′-TTGTGCCATTGAATTGAACCC-3′. The sequences of COOLAIR splicing variants in Var2-6 were submitted to GenBank (accession no. KP792767-792771).FLC introns 1 and 6. For intron 1, 2.5 μg of total RNA was treated with Turbo DNase-free kit (Ambion) and reverse-transcribed with primer 5′-CAAGGCTTTAAGATCATCAGCA-3′. Spliced FLC expression was assayed with primers 5′-GAGAGAAGCCATGGGAAGAA-3′ and 5′-AGGTTATCGCCGGAGGAG-3′, and unspliced FLC expression was assayed with primers 5′-AAAAGTGGAAATTVAGATGTGCT-3′ and 5′-TTGAAAAGGCCACTGGAAAC-3′. For intron 6, 2.5 μg of total RNA was transcribed with primer 5′-GGAGAGTCACCGGAAGATTG-3′. Spliced FLC expression was assayed with primers 5′-AAATGCTGAAAGAAGAGAACCAG-3′ and 5′-GGAGAGTCACCGGAAGATTG-3′, and unspliced FLC expression was assayed with primers 5′-TGGTTGTTATTTGGTGGTGTG-3′ and 5′-TCTCCATCTCAGCTTCTGCTC-3′. The intron splicing efficiency was calculated by normalizing the spliced FLC expression level to the unspliced FLC expression level for intron 1 and intron 6, respectively. The internal control for qRT–PCR was UBC.We analyzed the intron splicing in FLC RNA was performed using the FirstChoice RLM-RACE kit (Life Technologies) following the procedure described previously  and washed with urea/NP40 buffer . Next, the chromatin pellet was extracted with the hot phenol method for RNA preparation  and sequenced with M13 forward primer to confirm the sequence identity.The capping analysis of spliced eviously . After tparation . 5′ cappThe histone modification and Pol II occupancy assays were performed as described previously . The qPC


Phosphorylation offers a dynamic way to regulate protein activity and subcellular localization, which is achieved through its reversibility and fast kinetics. Adding or removing a dianionic phosphate group somewhere on a protein often changes the protein’s structural properties, its stability and dynamics. Moreover, the majority of signaling pathways involve an extensive set of protein–protein interactions, and phosphorylation can be used to regulate and modulate protein–protein binding. Losses of phosphorylation sites, as a result of disease mutations, might disrupt protein binding and deregulate signal transduction. In this paper we focus on the effects of phosphorylation on protein stability, dynamics, and binding. We describe several physico-chemical mechanisms of protein regulation through phosphorylation and pay particular attention to phosphorylation in protein complexes and phosphorylation in the context of disorder–order and order–disorder transitions. Finally we assess the role of multiple phosphorylation sites in a protein molecule, their possible cooperativity and function. Cellular regulatory mechanisms provide a sensitive, specific, and robust response to external stimuli and posttranslational modifications (PTMs) play an important role in these mechanisms and control protein activity, subcellular localization, and stability . Such dyIn this paper we summarize biological effects of phosphorylation which are explained through the lens of structural and dynamical changes. Below we will review a number of representative studies of computer simulations of the effects of phosphorylation on protein dynamics and stability together with experimental techniques to reveal the details and underlying mechanisms of phosphorylation events at atomistic scale.Phosphoryl group is dianionic at physiological pH and can form extensive hydrogen bond networks and salt bridges with neighboring residues of the same or different chains. One of the most dominant modes of interactions between phosphoryl and other residues is the interaction with the α-helical dipole at the C-terminal main chain nitrogen to neutralize the combined effect of carbonyl dipoles . AnotherAnalyses of phosphorylation in different proteins revealed the diversity and heterogeneity of its effects on protein structure , phosphoin silico phosphorylating several proteins and evaluating their conformations by OPLS-AA  force field and a Generalized Born implicit solvent model, it was shown that structures of phosphorylated regions and conformational changes induced by phosphorylation could be predicted in some cases with near-atomic accuracy compared to the actual phosphorylated conformations . In the unphosphorylated state myosin is auto-inhibited by interactions between the two catalytic domains, while phosphorylation of RLC at Ser19, which is rather distant from catalytic domain, disrupts these interactions and relieves the inhibition . The comAn interesting mechanism where phosphorylation inhibits disorder–order transition was reported for myelin basic protein (MBP), which includes a proline rich peptide containing two Thr-Pro motifs (-TPRTPPPS-) and an adjacent amphipathic a-helix which can bind to membrane . Both ThMany cellular control mechanisms operate at the level of protein–protein interactions, and main signaling pathways involve dense networks of protein–protein interactions and phosphorylation events. Phosphorylation may not only trigger the transitions between different conformation states of one protein but in some cases may modulate transitions between different conformations or oligomeric states in homooligomeric and heterooligomeric complexes and might represent an important mechanism for regulation of protein activity . RecentlTable 1.Calculation of binding energy differences upon phosphorylation showed that the majority of phosphorylation events did not affect protein–protein binding . It was Many proteins and protein regions are intrinsically disordered under native conditions, namely, they contain no or very little well-defined structure. Folding of disordered proteins into ordered structures may occur upon binding to their specific partners which in turn might provide high specificity even if binding affinity is low . On the Before describing specific cases of proteins involving the coupling between disorder, phosphorylation and binding, we would like to describe several studies which tried to generalize fundamental principles of such coupling in many different proteins. Mohan et al. analyzed relatively short (10–70 residues) segments called MoRFs (molecular recognition features) contained within longer disordered sequences that were structurally characterized in a complex with a larger protein . It was N-terminal disordered region and C-terminal histone-fold domain. The long disordered region is employed to bind to outer kinetochore complexes, namely, to Spc24/Spc25 subunits of Ndc80 complex . In addition, this phosphorylation site and salt bridge are conserved in many eukaryotic species which suggests that this mechanism is widespread for the CENP-T regulation. This example shows that phosphorylated residue can be critical for the complex formation through disorder even if it is located far away from the binding interface.While in many disorder-involving transitions phosphorylated residues may directly regulate binding orthosterically, this is not necessarily the case. Centromere protein T (CENP-T) is an essential component of the inner kinetochore and consists of  complex . NishinoSingle protein may contain multiple phosphorylation sites. Multisite phosphorylation can expand the patterns of regulation, give more accurate modulation of conformational change  and coopTD) to the pocket domain . Meanwhile, phosphorylation on Ser608 and Ser612 directly and orthosterically inhibits binding of E2FTD to the pocket. This mechanism involves a competitive binding between E2FTD and Ser608/Ser612 containing loop, namely, pSer608 stabilizes the association with the binding cleft, thereby mimicking and competing with E2FTD . A recent study showed that phosphorylation of Ser788 and Ser795 may also cause the inhibition of E2FTD binding to the pocket by inducing the association between RbC and the pocket domain  is one of the classical examples of a protein which has multiple phosphorylation sites and concerted phosphorylation patterns with very specific functional roles. Rb contains RbN, pocket, and RbC domains together with 13 different Ser/Thr phosphorylation sites that are phosphorylated by Cdk kinases. Phosphorylation sites are roughly grouped into eight clusters which mostly reside in flexible loop regions between structured regions or domains, and mediate domain–domain, domain–loop, and protein–protein interactions . For exat domain . This phProcesses of (de)phosphorylation on multiple sites can be classified by the order of (de)phosphorylation events, which can be sequential or random. In sequential phosphorylation sites are phosphorylated in a strict order of events where phosphorylation of one site depends on the phosphorylation state of another. Sequential phosphorylation has been observed for several kinases, especially Ser/Thr kinases . In contFigure 2B). Sic1 contains seven Ser/Thr sites in its N-terminal region, which are phosphorylated by Cdk1. Thr5 and Thr33 were previously identified as priming phosphorylation sites compared to downstream secondary phosphorylation sites . First, Sic1 binds to cyclin, which induces priming phosphorylation on Thr5 and Thr33 by Cdk1. This process is inhibited if priming Thr residues are placed closer to the cyclin docking sites, indicating the importance of maintaining the proper distance between cyclin-binding motif and the phosphorylation target residues of Sic1  is rate limiting for degradation and therefore crucial for circadian time keeping . This prReceptor tyrosine kinases (RTKs) represent another example. They transduce signals from the extracellular matrix to the cytoplasm of a cell and contain extracellular, transmembrane, and catalytic kinase domains and may include regulatory domains. In many cases binding of a ligand to the extracellular part induces dimerization or higher order oligomer formation and leads to the activation of intracellular kinase domain and its subsequent cross-phosphorylation. The interconversion between active and inactive states in kinases is highly regulated and kinases differ in their mechanisms of activation and inactivation . A key tPost-translational modification crosstalk occurs in those cases where the presence of one modification influences the modification of another site. Phosphorylation can change the activity of proteins that regulate other types of PTMs and, as a consequence, can promote or inhibit the modification of other sites. Several studies attempted to identify crosstalk between concerted phosphosites and other PTMs by looking at PTM sites within the sequence proximity from each other and by analyzing their evolutionary conservation and functional importance . Some ofPhosphorylation in some cases can promote subsequent ubiquitylation and the crosstalk between phosphorylation and ubiquitylation is reciprocal, namely, phosphorylation can be regulated by ubiquitylation and vice versa . InterplSeveral comprehensive statistical studies were recently performed trying to decipher the co-evolutionary links between different types of post-translationally modified sites. It was shown that phosphorylation associates with eleven other PTM types, followed by glycosylation and acetylation . In addiCrosstalk between phosphorylation and other PTMs can be illustrated by an example of histone tail modifications that are sometimes called the “histone code.” One important modification includes histone H3 Lys9 methylation that creates a binding site for chromodomain of heterochromatin protein 1 (HP1) which plays a key role in heterochromatin formation. Adjacent residue Ser10 is a known phosphorylation site. While the ultimate mechanism of Lys9 methylation and Ser10 phosphorylation crosstalk in H3 histone is still unknown, MD simulations showed that upon phosphorylation Ser10 forms a stable salt bridge with Arg8 rather than with the positively charged Lys9. It leads to a rearrangement of tail conformation and affects the binding of HP1 to methylated H3 Lys9 .In this paper we reviewed the present state of the structural and biophysical studies of protein phosphorylation. Physicochemical consequences of phosphorylation are very diverse which makes it difficult to summarize and deduce general mechanisms of phosphorylation events. However, recent experimental and computational studies point to several major mechanisms for how phosphorylation may ultimately affect and modulate protein function. They include orthosterical and allosterical effects of phosphorylation on protein structure and protein–protein binding, disorder–order and order–disorder coupled transitions upon phosphorylation and, finally, cooperativity and crosstalk between multiple phosphorylation sites or other PTMs. The structural and biophysical characterization of phosphorylation crosstalk is still in its infancy but in the future it will provide important clues about mechanisms of signal propagation, integration, and separation.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.


Nanosilver particles are used in various clinical settings because of their antibacterial properties. However, their safety evaluation when used for gynaecological disorders has not been established. Nanosilver particles were administrated in the vagina of New Zealand rabbits, and the pathological appearance of the surrounding tissue was examined by hematoxylin–eosin staining and transmission electron microscopy (TEM) after 1 and 3 days of treatment. The nanosilver content was assessed by plasma mass spectrometry, and the presence of particles in the hepatic portal vein blood was assessed by TEM. The results of our study show that the vaginal administration of nanosilver particles caused ultrastructural changes to the vaginal mucosa, urethra and rectum, with accumulation of particles in all tissues. These results demonstrate a new migration route of nanosilver particles following vaginal administration. They also demonstrate, for the first time, that the vaginal administration of nanosilver particles can enter the blood circulation system by examining the hepatic portal vein blood under the TEM which is the most direct visualized evidence. Among the numerous medical nanomaterials, nanosilver has been used extensively because of its unique antibacterial properties. Despite the fact that the safety evaluation of nanosilver particles has not been clearly established, nanosilver products are currently used in various clinical applications in China.in vivo studies conducted in animal models have demonstrated that nanosilver particles accumulated in various organs when administered through the oral, intraperitoneal, intravenous and subcutaneous routes [et al. [et al. [Over the past decade, a large number of s routes . In mices routes . In a su [et al.  showed t [et al.  showed tDifferent nanosilver products have been used for the treatment of gynaecological cervicitis or cervical erosion because of their antibacterial properties. However, the safety of nanosilver products following their vaginal administration has not been evaluated. In the present study, we assessed the ultrastructural morphology of the surrounding vaginal tissues. Our results highlight a new migration route of nanosilver particles, and provide a scientific basis for the risk assessment of nanosilver products in women.The nanosilver particles were purchased from Alfa Aesar China (Tianjin) Co., Ltd. (Batch No. B18Z014). As assessed by transmission electron microscopy (TEM), the nanosilver particles were round in shape, and in ∼90%, the diameter ranged between 20 and 40 nm.The absorption and accumulation of nanosilver particles in surrounding tissue following vaginal administration was assessed in healthy female New Zealand rabbits weighting 2.5–3.0 kg. Based on the published literature, the typical clinical dose and practical circumstances, a dose of 0.1 g/kg of nanosilver particle was administered topically in the vagina of the rabbits.The animals were divided into a control group and two treatment groups. The sampling time points were: (i) pre-administration (control group), (ii) 1 day (24 h) following treatment and (iii) 3 days (72 hours) following treatment. At each time point, the animals were killed, and tissue samples were taken from the vaginal mucosa near the end of the urethra, the vaginal mucosa near the end of the rectum, the urethra and the rectum. All samples were washed with normal saline, and 1/3 of each tissue samples were immersed in a 10% formalin fixation solution for hematoxylin–eosin (HE) staining, 1/3 were immersed in a 2.5% paraformaldehyde and 2.5% glutaraldehyde fixation solution for TEM sample preparation, and 1/3 were used for the detection of nanosilver particles following digestion. At each sampling time point, blood samples from the hepatic portal vein were drawn for TEM analysis.All tissue samples were prepared according to standard HE staining techniques, after which their appearance was evaluated for pathological changes using an optical microscope.All samples were placed in a digestion tube filled with 3 ml of nitric acid, and left to be digested for 24 h. The samples were then placed in a fume cupboard and heated at low temperature until the solution liquid turned clear. The samples were heated until almost dry and left to cool. The residue in the digestion tube was dissolved in ultra-pure deionized water, and diluted to 25 ml. The amount of silver in each solution (i.e. the silver content in each sample) was analysed using plasma mass spectroscopy. The specific particle mass per unit (mg) within each different type of tissue was calculated .Each sample was fixed in a 2.5% paraformaldehyde and 2.5% glutaraldehyde solution at 4°C for 2 h. The samples were then washed twice in PBS for 10 min before being placed in a 1% osmic acid fixation solution for 2 h at 4°C. Afterwards, the samples were washed three times in ultra-pure deionized water for 10 min, and dehydrated with graded ethanol. The samples were then subsequently placed in an epoxy propane–resin solution (1:1) for 1 h at room temperature, an epoxy propane–resin (1:4) for 1 h at room temperature, and a pure resin infusion for 2 h at room temperature. The samples were embedded in an Epon812 epoxy resin and cut into 1 µm semithin sections. Each section was stained with methylene blue-azure and analysed under an optical microscope. Ultra-thin sections were stained with uranyl acetate-lead citrate and analysed against a copper grid , 8.About 3.0 ml of blood were drawn from the hepatic portal vein, centrifuged for 5 min at 3000 rpm, and prepared using the same method as for the TEM tissue samples.Under a standard optical microscope, the epithelial cells in the innermost part of the vaginal columnar epithelium of the mucosa were orderly and complete. Below the mucosa, cells were loosely connected to tissue, with the presence of small blood vessels surrounded by minor oedema. Surrounding the mucosa was overlapping ring-shaped smooth muscles, which contained blood vessels. The outer membrane did not feature any conspicuous inflammation.The cross-sections of the urethra of the control animals revealed the following characteristics: some of the epithelial cells in the stratified transitional mucosa epithelium were loose and detached. Below the mucosa, cells were loosely connected to tissue, which contained blood vessels and fibrocytes. The surrounding tissue presented slight oedema, but was not conspicuously inflamed. Outer to the mucosa were intertwined smooth muscles, which also contained blood vessels. The outer membrane of the mucosa did not present any conspicuous changes.The cross-sections of the rectum of the control animals revealed the following characteristics: between the epithelium and the lamina propria were multiple intestinal glands. The lamina propria contained blood vessels, as well as a few lymph nodes that were scattered around the blood vessels and which invaded cells. Outside the lamina propria were smooth muscles arranged in circular rings on the inner layer and longitudinally in the outer layer. Outside these muscles was the placenta percreta and fatty connective tissue, with no conspicuous changes .Figure At 1 and 3 days following treatment, the nanosilver content in the vaginal mucosa, urethra and rectum had all increased as compared with the control group. Because the nanosilver content remained stable 3 days after treatment, it suggests that it caused a localized accumulation in surrounding tissue .Table 1An assessment of the ultrastructural changes of the tissue surrounding the vagina was performed at 1 and 3 days following treatment with nanosilver particles. At 1 day post-treatment, several high density round nanosilver particles were observed in the tissue of the vagina mucosa, the urethra and the cells of the rectum . At 3 daThese results demonstrate that ultrastructural changes occur in the vaginal mucosa, the urethra and the rectum during the first 3 days following the administration of nanosilver particles. In addition, high density round nanosilver particles  were observed in the tissue of the vaginal mucosa, the urethra and the rectum.As with tissues, analysis at 1 and 3 days after treatment revealed several high density round nanosilver particles in the hepatic portal vein blood. Some nanosilver particles formed conjugates with red blood cell, while others formed conjugates with blood platelet .Figure Most studies have evaluated the biological safety of nanosilver particles through the detection of blood silver content , 10 follThe present study revealed that, following the vaginal administration of nanosilver particles, silver accumulates in the tissue surrounding the vagina which caused ultrastructural pathological changes not only to the vaginal mucosa but also to the urethra and rectum. We also observed the migration of individual or multiple high density nanosilver particles between these tissues, thereby creating a cycle of cytotoxic reactions. Collectively, our results demonstrate new migration routes of nanosilver particles when administered intravaginally.Transmission electron microscopy observations performed at 1 and 3 days following treatment revealed several high density round nanosilver particles in the hepatic portal vein blood, with some nanosilver particles forming bonds with red blood cell and blood platelet. Because the blood from the esophagus, stomach, intestinum tenue, intestinum crassum, pancreas, cholecyst and spleen flows through the hepatic portal vein, it is possible nanosilver particles might accumulate in more distant tissues. We are not aware of other studies demonstrating the formation of nanosilver particle conjugates with circulating blood cells. This is clearly of scientific relevance as our observations were made using TEM, the most direct visualization technique to date. By entering the blood circulation, our results suggest new migration routes of nanosilver particles following vaginal administration, and should prompt further research into their systemic distribution.In summary, our results show, for the first time, that the vaginal administration of nanosilver particles leads to (i) their accumulation in the tissue surrounding the vagina, causing ultrastructural pathological changes, (ii) the potential promotion of cytotoxic reactions and (iii) by entering the blood circulation, it suggest additional migration routes to be elucidated.


Lonicera spp.) in the United States have been characterized as “ecological traps” by ecologists. Here we tested this hypothesis by investigating the effects of shrub honeysuckles on the nesting ecology of native birds in seven study sites in central Pennsylvania, USA. We examined how the abundance of shrub honeysuckles influenced the selection of nesting substrates and habitat for a community of common songbirds, and the parental-care behavior and nestling development of gray catbirds (Dumetella carolinensis). We found that birds had a strong bias towards nesting in honeysuckle shrubs, but not necessarily for nesting in honeysuckle-dominated habitats. Nest predation rates were affected by the density of nests in a habitat, but not by the overall abundance of honeysuckles in such habitats. Honeysuckle abundance in the habitat did show significant effects on some parental-care behavioral parameters: catbirds had higher nest visitation rates and shorter visit lengths in areas of high honeysuckle density. On average, Gray catbirds fed fruit 12%±0.31 s.e. of their nestling-feeding bouts, mostly fruits of shrub honeysuckles. Nestlings in sites with high honeysuckle density also showed higher mass:tarsus ratios, suggesting a good (possibly better) physiological condition of catbird nestlings at the time of fledging. Our study shows that honeysuckle-dominated habitats could have equivocal effects on nesting parameters of common species of native birds. We advise more caution in the widespread denomination of novel plant communities with high densities of honeysuckle as “ecological traps” as effects can be null or positive on native birds in certain localities.Invasive species have come to the forefront of conservation biology as a major threat to native biodiversity. Habitats dominated by shrub honeysuckles ( Lonicera spp.), now serve as foraging and nesting substrate for many species of native songbirds The ecological interaction between invasive plant species and bird communities has received a fair amount of attention over the past fifteen years Cardinalis cardinalis) and American robins (Turdus migratorious) have higher nest predation rates nesting in honeysuckle than nesting in native shrub species The effect of shrub honeysuckles on nest predation rates has been a major focus in the literature. For example, studies have shown that northern cardinals . For this we predicted that catbirds would show different parental-care behavioral patterns in honeysuckle dominated habitats than native habitats (prediction 3). We also hypothesized that nesting in honeysuckle dominated habitats would negatively affect the development of nestlings (prediction 4). Last, we report on nest predation rates as affected by nest placement in honeysuckle or native shrub substrates, and as a function of the amount of shrub honeysuckles in the habitats.Here we investigated the use of nesting habitat and nest substrates of birds at seven sites in central Pennsylvania, U.S.A to test several aspects of the ecological trap hypothesis. Each site contained areas of low, medium, and high abundance of shrub honeysuckles. Based on the ecological trap hypothesis, first we predicted that nest densities would be greater at higher abundances of honeysuckle in the habitat (prediction 1), and that birds will disproportionately build nests in honeysuckle shrubs as compared to other available shrubs (prediction 2). Next, we examined the effects of honeysuckle-dominated habitats on aspects of the parental care behavior and nestling development of gray catbirds  of the Pennsylvania State University. The protocol (including the portion that involved handling birds) was approved by the IACUC of the Pennsylvania State University (Permit Number: 37069), Pennsylvania State Game Commission (Permit Number: 77-2011), and by the U.S. Fish and Wildlife Service (Permit Number: MB44836A-0). No endangered species were used in this study and permission was obtained to conduct research on migratory birds, which are protected, from the U.S. Fish and Wildlife Service (Permit Number: MB44836A-0). In addition, permission was obtain from Pennsylvania State University Forestland Management and the Centre County Parks and Recreation to conduct this research on their lands.L. morrowii and/or L. maackii); Site 1–12.5 ha searched, Site 2–5.1 ha, Site 3–6.9 ha, and Site 4–1.7 ha). The other three sites  were all classified as dominated by native shrubs  of each classification in each site was then calculated. The total area of the three habitat classifications combined across all study sites was fairly even and amounted to: 12 ha for LHS, 10 ha for MHS, and 9 ha for HHS.Vegetation cover was measured using systematic surveys. Survey points were established by creating a thirty-meter grid at each site. At each point, we estimated the proportion of the area covered by all woody plants above 0.5 m tall within a two-meter radius. The total number of vegetation points per site was Site 1 (191), Site 2 (74), Site 3 (134), Site 4 (42), Site 5 (27), Site 6 (24), and Site 7 (58). The cover data was then used to create a honeysuckle density map with the inverse distance weighting (IDW) interpolation tool in ArcMap 10 (ESRI). ArcMap classified cover in three categories: high honeysuckle cover , medium honeysuckle cover , and low honeysuckle cover  and the other when most bird species lay a second clutch (June 19th–July 2nd). On every search day, two observers visited two sites in the morning and searched for approximately four hours, and then the observers switched between the sites in the afternoon, where they searched for an additional four hours . The whole area of each study site was searched for new nests once every week controlling for nest search effort , resulting in a total of 224 nest-search hours for each of the two nest search rounds . Multiple nest searching techniques were employed  to reduce the nest detectability biases across sites and habitats. Nests were mapped using a Magellan Professional sub-meter GPS with ArcPad 7 software (ESRI). We recorded the height of nests; the species of plant nests were built in; and the clutch size. The nests were then monitored through the whole breeding season by revisiting them every other day to assess their status. For predation analyses, nests were classified as either successful or predated. Successful nests fledged nestlings, while predated nests showed evidence of predation . Because we made detailed maps of the plant communities in each study site, our study design allowed us to examine the effects of background levels of honeysuckle both at the scale of the nest vicinity, and at the broader habitat-level scale.We conducted nest searches in the spring and summer of 2011: one at the start of the breeding season  and tarsus length (mm) were recorded for each nestling. To control for allometric effects of variation in body size, we divided the mass of the nestlings by the tarsus length To test the prediction (1) that honeysuckle-dominated habitats have higher densities of songbird nests (irrespectively of the nest substrate) we used a One-way ANOVA. We calculated nest density, the response variable, by dividing the number of nests found in each habitat classification within a site  and the average length of visits (minutes per visit) between HSD and NAT habitats. We used generalized linear models (GLMs) with binomial error distributions to compare nest visitation rates, as well as the proportion of visits in which parents fed fruit to nestlings, in HAS and Nat habitats. We addition, we used a one-sided t-test to compare the average mass to tarsus ratio of nestlings found in NAT habitats to those in HSD habitats to test our prediction that nestlings in HSD had reduced development (4).To test predictions that catbirds would exhibit different parental behavior in areas of high honeysuckle abundance (3), we used one sided To examine whether nest predation rates are higher in honeysuckle-dominated habitats we used a General Linear Mixed Model with binomial error distribution. As response variable we used both the number of predated nests and the total number of nests per habitat classification  per site. We included nest density per habitat in each site as a covariate since density is expected to influence predation rates We found 227 nests of 10 bird species . Number The density of nests was not influenced by habitat classification  . Howevert = 3.450, DF = 21, P = 0.002). However, there was no difference between HSD and NAT habitats in the proportion of feeding bouts , or in the proportion of feeding bouts in which nestlings were fed fruit . There was no difference in the average total time that catbirds spent at nest in HSD or NAT habitats . However, the average duration of visits was significantly higher for nests in NAT compared to HSD habitats .For the 23 catbirds nests we collected a total of 197.5 hours of video . The ratio between the body mass of the nestlings and the length of their tarsus was significantly higher for birds in HSD habitats compared to NAT habitats , with an average magnitude of increase of 4%  as compared to nesting in habitats dominated by native shrub species (NAT). Catbirds visited nests more frequently in HSD habitats than in NAT habitats, a pattern that could increase nest predation rates in HSD habitats More nests were built in honeysuckle shrubs than expected by chance across study sites, and the trend was consistent irrespectively of the abundance of honeysuckle in the plant community background. Even in habitats with very low honeysuckle abundance, such as those found in the Site 7 and Site 6, more nests were found in honeysuckle substrates, demonstrating a strong bias in favor of the introduced shrubs. Previous studies that have examined nest substrate selection in the context of honeysuckle invasions have focused only on the local nest environment to calculate measures of substrate availability e.g., , and onlCuriously we did not find positive correlations between nest densities and honeysuckle abundance, which refuting the hypotheses that the early leaf phenology of honeysuckle increases nest densities Catbirds had a 45% higher visitation rate which means they made significantly more trips to feed their nestlings but spent 32% less time per visit, in habitats of high honeysuckle cover (HSD). There are a few ways in which these results could be interpreted. One such way is that birds spend more time per visit in habitats dominated by native vegetation because the available food resource were of higher quality, providing more energy per volume and thus reducing foraging effort. The lower volume to energy ratio of fruit compared to invertebrates Our records of honeysuckle fruit being fed to catbird nestlings in both HSD and NAT habitats is remarkable, and it suggests that honeysuckle, or fruit in general, is an important resource for both adult catbirds and their nestlings, even at low dietary percentages. Underscored is the fact that honeysuckle fruits were fed to nestlings even in sites with a very low abundance of honeysuckle cover . In temperate regions, fruit densities are typically low during the nesting period of landbirds Unlike previous studies, we did not find nest predation rates to vary significantly among the different habitat classifications that reflected the abundance of shrub honeysuckles . NeitherWe did not observe a preference of birds to breed in habitats dominated by shrub honeysuckles across seven study sites in central Pennsylvania. We did find a preference for birds to build their nests in shrub honeysuckles in all sites, which is in agreement with what previous studies have reported. We also found that two aspects of the parental care behavior of gray catbirds  were different in areas dominated by honeysuckles in comparison to areas dominated by native vegetation. But those differences did not seem to cause nestlings to be in lower body condition at the time of fledging in honeysuckle dominated habitats, or to suffer more predation. In fact, mixed diets have been shown to be beneficial for many birds Our results show that relationships between introduced fleshy-fruited plants and native birds are complex and not easily characterized as purely harmful or beneficial because they can include negative, neutral, or positive outcomes Appendix S1Characteristics of the seven study sites.(DOCX)Click here for additional data file.Appendix S2Average honeysuckle cover per habitat classification for each study site.(DOCX)Click here for additional data file.Appendix S3Number of observed nests built during the study year for the bird community.(DOCX)Click here for additional data file.Appendix S4Number of observed nests built during the study year in each habitat classification and substrate type per study site.(DOCX)Click here for additional data file.Appendix S5Percent cover of plant species around nests.(DOCX)Click here for additional data file.Appendix S6The parental care data obtained from video recordings taken when the nestlings were 4–5 days old.(DOCX)Click here for additional data file.Appendix S7The morphological measurements of nestlings at age 8–9 days.(DOCX)Click here for additional data file.Appendix S8The number of nests predated followed by the number of nests monitored in each habitat classification and substrate type per study site.(DOCX)Click here for additional data file.