Results indicated chamaejasmine showed more notable anticancer activity than taxol against PC-3 cells, with IC50 values of 2.28 and 3.98 µM, respectively. Furthermore, Western blot analysis showed that chamaejasmine was able to increase the expression of β-tubulin, but not α-tubulin. In silico simulations indicated that chamaejasmine specifically interacts with the active site which is located at the top of β-tubulin, thanks to the presence of strong hydrophobic effects between the core templates and the hydrophobic surface of the TB active site. The binding energy (Einter) was calculated to be −164.77 kcal·mol−1. Results presented here suggest that chamaejasmine possesses anti-cancer properties relating to β-tubulin depolymerization inhibition, and therefore is a potential source of anticancer leads for the pharmaceutical industry.In this work, the anticancer activity of chamaejasmine was studied by evaluating its  Cancer, which is a great threat to human life, is a serious disease with a complex pathogenesis. Many chemotherapeutic drugs have been developed to treat cancer, including DNA-alkylating agents and antimitotic agents ,2. A greIn the past three decades, microtubules continue to be one of the most successful cancer chemotherapeutic targets ,5,6,7,8.Stellera chamaejasme L., belongs to the Thymealaeaceae, widely distributed in northwest and southwest parts in China. The roots of Stellera chamaejasme L., can be used as pesticide on bugs, flies and maggots, and can also control pests on crops, and pastures . The lysates were collected by scraping from the plates and then centrifuged at 10,000 rpm at 4 °C for 5 min.PC-3 cells were treated with chamaejasmine  for 72 h or with 2 µM chamaejasmine for 0, 24, 48 and 72 h. For isolation of total protein fractions, cells were collected, washed twice with ice-cold PBS, and lysed using cell lysis buffer  mM Tris 2 for 2 h. Membranes were blocked at room temperature for 2 h with blocking solution (1% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) plus 0.05% Tween-20). Membranes were incubated overnight at 4 °C with primary antibodies  at a 1:1000 dilution  in blocking solution. After thrice washings in Tris Buffer Solution Tween (TBST) for each 5 min, membranes were incubated for 1 h at room temperature with an alkaline phosphatase peroxidase-conjugated anti-mouse secondary antibody at a dilution of 1:500 in blocking solution. Detection was performed by the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. Bands were recorded by a digital camera .Total protein samples (20 μg) were loaded on a 12% of SDS-polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes  at 0.8 mA/cmThe docking and molecular dynamics (MD) simulations were performed with the different modules of InsightII 2005 software package on Linux workstations, using the consistent-valence force-field (CVFF) . The CVF+ ions were added to neutralize the system [−1·Å−1. The atomic charge and structural parameters of chamaejasmine (The atomic coordinates of the αβ-tubulin dimer were obtained from the PDB database (PDB Accession number: 1JFF) . For cone system . The resejasmine  were der−1·Å−1. The CVFF force-field was used for the optimizations. For more details about the calculations readers are referred elsewhere [The active site of TB protein was identified with the aid of Binding-site module ,23. Withlsewhere ,31.A 4.0-ns MD simulation was performed on the energy-minimized chamaejasmine -TB complex by Discover 3.0 module, using the CVFF force-field. The canonical ensemble (NVT) was employed and the non-bonded interactions were calculated by the Cell-Multipole approach. The velocity scaling thermostat was used for the temperature controls, which allows the MD simulation temperatures to change within a small range around the defined value (300K) . Integrap < 0.05 was considered to be statistically significant.The Student’s t-test was used to compare the difference between two different groups. A value of The current study demonstrated the anticancer activity of chamaejasmine as well as the inhibition of binding process between α-tubulin, β-tubulin and chamaejasmine. Further pharmacological investigations are necessary to provide firm evidence about the anticancer mechanism of chamaejasmine.


The amount of macromolecular tracer P(St-co-AMMA) does not affect the residence time distribution of polystyrene during single-screw extrusion processing, meanwhile, the minimum residence time decreases and the residence time distribution becomes narrow as the screw speed increases, that is, the axial mixing capacity of the single-screw extruder decreases as the screw speed increases.In this study, 9-anthracenemethyl methacrylate (AMMA) and styrene (St) as monomers and benzoyl peroxide as an initiator were used to synthesize P(St-co-AMMA), a macromolecule tracer with a fluorescence effect, via free radical copolymerization. A fluorescent online detection device was built on the basis of the principle of fluorescence online detection by using the single-screw extrusion platform of a torque rheometer to explore the effect of the amount of macromolecular tracer and screw speed on the residence time distribution of polystyrene in single-screw extrusion. Fourier transform infrared spectroscopy,  As onMost of tracer-related studies have used small-molecule tracers ,25. HoweAt present, the preparation of macromolecular tracers via chemical synthesis has become a novel method. A benzene ring-containing compound generally has good fluorescence properties and excellent heat resistance if it is successfully grafted in a polymer segment; a macromolecule with fluorescent properties can also be prepared ,27. Hu aThis study mainly aims to develop a new route to obtain a macromolecular tracer by incorporating anthracene moieties to polymer backbones. Specifically, self-made 9-anthracenemethyl methacrylate (AMMA) and styrene (St) were used as monomers, and benzoyl peroxide (BPO) was utilized as an initiator to obtain a new macromolecular tracer P(St-co-AMMA) with a fluorescence effect via free radical copolymerization . A fluor2.2.1.St  was used after it was further purified, the polymerization inhibitor and water were removed, and reduced pressure distillation was conducted. 9-Anthracenemethanol (9-AM) (98%) was purchased from J&K Scientific Company. Methacryloyl chloride (95%), chloroform (1.471–1.478 g/mL), and 4-dimethylaminopyridine (DMAP) (95%) were purchased from Sigma-Aldrich. BPO (AR) was purified by dissolving in chloroform, precipitating in methanol, and vacuum drying at room temperature for use. Toluene (AR) was purchased from Hangzhou Gaojing Fine Chemical Company. BPO, pyridine (AR), sodium sulfate, sodium hydroxide, sodium chloride, and deionized water were purchased from Aladdin.2.2.−1; and maximum torque, 160 Nm. From the feeding port to the die, the single-screw extruder was divided into four heating zones, and different temperatures could be set in accordance with various experimental schemes for precise temperature control.All of the experiments were completed at Zhejiang Sci-Tech University. In addition to the conventional instruments used in the laboratory, the following special equipment was used in this test. Rotary evaporator was purchased from Tokyo Physicochemical Company, instrument model N-1100. High-pressure reactors were purchased from Parr Instruction Company, instrument number 4848. Single-screw extruder was purchased from Harbin Harper Electric Technology Company, instrument model RM-200C. Some basic parameters of the single-screw extruder were as follows: screw diameter, 20 mm; length-to-diameter ratio, 25; maximum temperature, 350 °C; maximum speed, 150 min2.3.2SO4, filtered, and vacuum distilled. The obtained yellow crystallization product was purified through recrystallization from hexamethylene. This monomer was prepared via the substitution reaction of methacryloyl chloride and 9-AM. First, 10.0 g of 9-AM and 58.7 mg of DMAP were dispersed in 150 mL toluene. Second, 7.8 mL of pyridine was added to the mixture, and the solution was cooled to 0 °C. Subsequently, 7.5 g of methacryloyl chloride was added dropwise with a syringe to ensure a total addition time of more than 10 min and stirred at 0 °C for 1 h and then at 25 °C for 3 h. 2.4.n-heptane to ensure the complete removal of the unreacted monomer and initiator. Afterward, the resulting product was dried at room temperature for 5 h, vacuum dried at 30 °C for 12 h, and vacuum dried at 60 °C for 24 h. In this experiment, P(St-co-AMMA) was prepared via solution radical polymerization. The composition of the reactants is shown in 2.5.2.5.1.−1, mixing temperature of 220 °C, and mixing time of 8 min. Second, the mixture was extruded into a strip shape by a 2 mm die of a capillary rheometer and granulated on a granulator to obtain a particle size comparable with polystyrene particles.First, the polystyrene and P(St-co-AMMA) were mixed at a mass ratio of 100:1 on the mixing platform of the torque rheometer under the following conditions: rotor speed of 45 min2.5.2.3.3.1.3.1.1.−1, the scan rate was 32 s−1, and the resolution was 4 cm−1. −1, which corresponds to the characteristic stretching vibration peak of the 9-AM hydroxyl group (–OH). The – OH group on 9-AM and the – Cl of methacryloylchloride undergo a substitution reaction; hence, the peak of AMMA at 3448 cm−1 disappears, and two new peaks that correspond to the ester group appear at 1716 and 1160 cm−1. At the same time, a weak peak, which corresponds to the carbon–carbon double bond in AMMA, is observed at 1639 cm−1. The characteristic peak of the two aforementioned functional groups indicates that 9-AM and methacryloylchloride undergo a substitution reaction to form AMMA. In comparison with the infrared spectrum of AMMA, P(St-co-AMMA) has a strong peak at 700 cm−1, which corresponds to the monosubstituted group peak of benzene. Moreover, two group peaks, which correspond to the characteristic peaks of saturated hydrocarbon and the stretching vibration peak of C–H in the aromatic ring, appear at 2800–3000 and 3000–3100 cm−1, respectively. The appearance of these characteristic group peaks strongly indicates that the intermediate product AMMA reacts with the St monomer to synthesize the target product P(St-co-AMMA). In addition, the ester peak of P(St-co-AMMA) still exists but seems to become less intense, suggesting that AMMA reacts with other monomers and occupies a portion of the product. The most obvious change compared with the infrared spectrum of polystyrene is that P(St-co-AMMA) has a carbonyl peak at 1716 cm−1, which also reflects the smooth progress of the reaction from another aspect. Therefore, the obtained final product is the target product P(St-co-AMMA) [FT-IR spectroscopy was conducted to determine whether the target product was successfully synthesized in this experiment. FT-IR analysis was conducted using a Nicolet 5700 Fourier infrared tester. The measurement range was 4000–400 cm3.1.2.1H-NMR spectrum. In this experiment, the equipment of 1H-NMR spectral analysis was Switzerland Bruker Avance DMX400 nuclear magnetic resonance instrument. Deuterated chloroform was used as a solvent for AMMA and P(St-co-AMMA). Tetramethylsilane was the internal standard, and the concentration of the test sample was approximately 2 wt%. 1H-NMR spectrum of AMMA. Some peaks attributed to the presence of the anthracene ring are observed at 7.45 (e), 7.21 (a), 7.01 (d), 6.51 (b), and 6.38 ppm (c). A peak is also detected at 5.22 ppm (i) due to the existence of H next to the ester group. Another peak appears at 5.19 ppm (f), which is attributed to the presence of H on the carbon–carbon double bond. Moreover, a peak forms at 1.46 ppm, which is attributed to the existence of H on the methyl group. The synthesized product is AMMA.The chemical structures of AMMA and P(St-co-AMMA) were further characterized by 1H-NMR spectrum of P(St-co-AMMA). Some peaks attributed to the H of anthracene ring appear at 8.45 (e), 8.27 (a), 7.96 (d), 7.52 (b), and 7.45 ppm (c). Some peaks corresponding to the H of benzene ring form at 7.20, 7.10, and 6.93 ppm respectively. A peak is also detected at 5.99(o) due to the existence of H next to the ester group. The peaks at 1.74 (l), 0.98 (k), and 0.78 ppm (i) are due to the presence of methylene. Two peaks are found at 5.45 ppm (p) and 5.24 ppm (n) because of the existence of H on the carbon–carbon double bond. A peak also appears at 1.85 ppm (m) due to the presence of methyne. The 1H-NMR spectrum of P(St-co-AMMA) shows that P(St-co-AMMA) is the synthesized product.3.2.−1 gradually increases, but other characteristic peaks do not change significantly. The amount of St used is consistent in each set of experiments, and the infrared peaks associated with polystyrene are clearly similar. 3.3.The samples were thermogravimetrically analyzed using a Mettler-Toledo thermogravimetric analyzer. The sample to be tested was placed in a drying oven at 100 °C for 12 h to remove moisture, and 5–8 mg of the sample was taken for each test. The test temperature range was 30 °C to 600 °C, the heating rate was 20 °C/min, and the nitrogen flow rate was 40 mL/min. The TGA and DTGA curves of P(St-co-AMMA) with different AMMA contents are shown in 3.4.Gel chromatography analysis was performed using a gel permeation chromatograph from the Waters Company, USA (model number Waters 1525-2414-2487-717). The GPC test results of the copolymerized product only have one macromolecule elution peak without the elution peak of the small molecules. Thus, the product is only one polymer without byproducts and small molecular residues. Other specific data can be obtained by integrating the test results. The molecular weight and molecular weight distribution of P(St-co-AMMA) with different AMMA contents are presented in 3.5.The samples in this experiment were tested in terms of their fluorescence properties by using an F-46,001 fluorescence spectrophotometer . During this test, the incident wavelength was 360 nm, the scanning speed was 240 nm/min, the excitation grating was 10.0 nm, and the radiation grating was 5.0 nm. Substances that contain fluorophores likely emit fluorescence, whereas substances that fluoresce generally contain a conjugation structure in which benzene ring and biphenyl structures are the most common. 3.6.−1 to 100 s−1. The rheological properties of P(St-co-AMMA) were tested on an Anton Paar MCR 301 rheometer. The sample was pressed into a circular sheet with a diameter of 25 mm and a thickness of 2 mm by using the press vulcanizer to enable the direct testing on a rheometer. The pressing temperature was 180 °C, the test temperature was 200 °C, and the shear rate was 0.1 sThe storage modulus and loss modulus of polystyrene and macromolecular tracers P(St-co-AMMA) were put together as shown in 3.7.The concept of the probability distribution can be used to quantitatively describe the residence time distribution of a material in a reactor because the process of extruding a material from a single-screw extruder is continuous and random. Two kinds of probability distribution functions, namely, the residence time distribution density function and the cumulative residence time distribution function, describe the residence time . The resThe cumulative residence time distribution function, also known as the F function, is usually represented by F(t):where c(t) is the voltage signal strength of the tracer at the single screw extruder, and t = 0 when the tracer is added from the feed port.3.7.1.3.7.2.4.In this experiment, a fluorescent macromolecular tracer P(St-co-AMMA) was successfully synthesized via free radical polymerization. The same mechanism as in most macromolecules was developed on the basis of the principle of polymerization by introducing specific groups into the macromolecular chain to obtain a functionalized macromolecular chain. However, this experiment had some differences. A double bond, which can be polymerized, was introduced to a small functional molecule and then copolymerized with a St monomer to prepare a random copolymer. The target product P(St-co-AMMA) was detected and analyzed in terms of three aspects:The structure of each step product was analyzed through FT-IR spectroscopy and nuclear magnetic resonance spectroscopy, and the accuracy of the synthesized product was determined.Fluorescence property detection indicated that the synthesized product P(St-co-AMMA) could emit fluorescence under specific conditions, indicating that P(St-co-AMMA) had the most basic conditions as a tracer.Thermal stability analysis indicated that the synthesized product P(St-co-AMMA) had certain thermal stability. Rheological property analysis showed that the synthesized product P(St-co-AMMA) had rheological properties similar to those of polystyrene. The results of these two experiments demonstrated that P(St-co-AMMA) could act as a tracer to indicate the residence time distribution of polystyrene in a single-screw extruder.Subsequently, the macromolecular tracer P(St-co-AMMA) was used to characterize the residence time distribution of polystyrene in a single-screw extruder. In this test, the effect of the amount of tracer added and the screw speed on the residence time distribution was mainly discussed. Through a series of experiments, the added amount of the tracer did not affect the residence time distribution of polystyrene in the single-screw extruder, the difference in screw speed changed the residence time distribution, and the screw speed influenced the mixing ability of the single-screw extruder.


Dipeptidyl peptidase-4 (DPP-4) is a target to treat type II diabetes mellitus. Therefore, it is important to understand the structural aspects of this enzyme and its interaction with drug candidates. This study involved molecular dynamics simulations, normal mode analysis, binding site detection and analysis of molecular interactions to understand the protein dynamics. We identified some DPP-4 functional motions contributing to the exposure of the binding sites and twist movements revealing how the two enzyme chains are interconnected in their bioactive form, which are defined as chains A (residues 40–767) and B (residues 40–767). By understanding the enzyme structure, its motions and the regions of its binding sites, it will be possible to contribute to the design of new DPP-4 inhibitors as drug candidates to treat diabetes. Type II diabetes mellitus, a chronic metabolic disease related to hyperglycemia, has a global estimate of reaching 642 million cases by the year 2040 . The inhThe enzyme DPP-4 is classified as a glycoprotein, composed of three distinct regions: (i) cytoplasmic-residues from 1 to 6; (ii) transmembrane-residues 7 to 29 and (iii) extracellular-residues 30 to 766. Its most important catalytic function is the degradation of incretins such as glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP), in which metabolic regulation contributes to the control of glucose levels in the blood ,9,10,11.S)-3-aminopiperidin-1-yl]-5-benzyl-4-oxo-3-(quinolin-4-ylmethyl)-4,5-dihydro-3H-pyrrolopyrimidine-7-carbonitrile (N7F), co-crystallized with the PDB code 4A5S [In this study, we applied normal mode analysis to study the functional motions of DPP-4 in four cases: (i) a system containing an inhibitor only in the A chain (residues 40–767) of the protein; (ii) containing an inhibitor only in the B chain (residues 40–767) of the protein; (iii) with an inhibitor in both chains, and (iv) without an inhibitor in both chains. We used 6-[ and an opening motion that exposes the active site, both movements being present in all systems (with or without an inhibitor). It was found that some residues located at certain regions are involved in the movement of exposure of the active site, the β chain region  and α-helix region , while in the torsion movement between the chains, in addition to the regions already mentioned, there is still the α-helix region formed by Phe713, Gln714, Gln715, Ser716, Ala717, Gln718, Ile719, Ser720, Lys721, Ala722, Leu723, Val724, Asp725, and Val726. The vectors indicative of torsional motions between the chains and the active exposure of the sites are shown in By analyzing the overlapping graphs of Cα atom fluctuations of the chains A and B , we deteWe used BINANA  to analyIn the A chain of DPP-4, we found a greater number of hydrogen bond interactions than in the B chain, with the presence of π-π T-shaped-like (PIT-like )interactions only in the A chain. On the other hand, the hydrophobic contacts, π-π stacking (PIS) interactions and salt bridges were more numerous in the B chain. Thus, it can be speculated that new inhibitors have to contain functional groups that will allow for establishing interactions with a similar profile, since they are supposed to be coupled in both DPP-4 active sites (chains A and B).We used the BINANA  tool to The structure of DPP-4  was In chain B, three similar sites were predicted with the addition of Gly549 at site 1. Residues such as Arg125, Phe357, Arg358, Tyr547, Ser630, Tyr631, and His740 were detected as an integral part of more than one binding site. Most of the residues detected at sites 1 and 2 have already been described in the literature as being present in the region of the active site ,11. The Analyzing the results obtained from FTSite and FTMap, using the standard probe molecules (mentioned in the methodology), it was possible to determine those that present the greatest affinity for the different binding sites, as follows: (1) in site 1, 16 probes  energy gradient convergence criterion of 10−5 kcal mol−1 Å−1. We calculated 81 modes using the CHARMM 40b1 [Firstly the systems were minimized with the conjugate gradient (CG) methods followed by the Adopted Basis Newton-Raphson algorithm (ABNR). The harmonic constraints were applied during the CG stages, progressively decreasing from 250 to 0 kcal molRMM 40b1  program RMM 40b1  force fiThe algorithm BINANA  1.2.0 idThe ligand and receptor files were prepared in PDBQT format by adding hydrogens and Gasteiger partial charges through the tools of AutoDockTools ; the calThe cutoff radius values adopted correspond to the default values for each interaction: (i) π-π interaction distance cutoff: 7.5Å; (ii) cation-π distance cutoff: 6.0Å; (iii) close contacts distance cutoff: 2.5Å; (iv) active site flexibility distance cutoff: 4.0Å; (v) π-padding distance: 0.75Å; (vi) hydrophobic distance cutoff: 4.0 Å; (vii) T-stacking closest distance cutoff: 5.0Å; (viii) T-stacking angle tolerance: 30.0 Å; (ix) close contacts distance cutoff: 4.0Å; (x) π-stacking angle tolerance: 30.0Å; (xi) hydrogen bond angle cutoff: 40.0Å; (xii) hydrogen bond distance cutoff: 4.0Å; (xiii) salt bridge distance cutoff: 5.5Å; and (xiv) electrostatic distance cutoff: 4.0Å.In the search for possible binding sites of DPP-4, we used FTSite ,19,20 anIn this study, we observed a small difference in the flexibility profiles of the two DPP-4 chains, suggesting that chain A is more flexible and that there are fewer interactions between chain B and the inhibitors. The most significant movements observed involve the exposure of the active site and the twist between the two chains. Considering the N7F inhibitor, we detected a greater number of interactions of the hydrogen bonding and T-stacking types in the A chain of the DPP-4 enzyme.Regarding the three sites identified here, it was noted that with the exception of the residues Phe357 and Arg358, the other residues detected at the third binding site have not yet been exploited for identification of new bioactive ligands, noting that the third site did not correspond to the non-catalytic binding sites of DPP-4 . Therefore, it might be possible that the region of the third binding site could be occupied without altering its non-catalytic functions.In addition to structural information on the enzyme, our study contributes to understand protein dynamics in order to use this enzyme for drug design more efficiently. We proposed that the design of new compounds sharing physicochemical characteristics similar to the 16 probe molecules can be a good approach to plan efficient inhibitors of DPP-4. For site 2, it was observed that compounds containing groups with a physicochemical profile similar to the probe molecules related to acetaldehyde, isopropanol and urea were not the best compositional options for coupling in this region. In the case of site 3, molecules with structural similarity containing ethanol, isopropanol, methanamide, tert-butanol would not be suitable. The binding site 3 see  should b


Phil. Trans. R. Soc. A369, 2942–2956 (doi:10.1098/rsta.2011.0104)). The fluid is assumed inviscid and incompressible, and the flow irrotational. A numerical method based on boundary integral equation techniques is used to compute solitary waves and forced waves to Euler's equations.The focus of this work is on three-dimensional nonlinear flexural–gravity waves, propagating at the interface between a fluid and an ice sheet. The ice sheet is modelled using the special Cosserat theory of hyperelastic shells satisfying Kirchhoff's hypothesis, presented in (Plotnikov & Toland. 2011 This article is part of the theme issue ‘Modelling of sea-ice phenomena’. Under certain conditions, the floating ice sheets can be modelled as an elastic medium, and their interaction with the fluid makes the resulting hydroelastic problem mathematically challenging . A renewThere are a variety of models for an ice sheet floating on top of a body of water, starting with the linear model used by Greenhill  or the KMost of the analytical and numerical studies of nonlinear flexural–gravity waves (or hydroelastic waves) concentrate on two-dimensional problems. Weakly nonlinear models, such as the nonlinear Schrödinger equation or the fifth-order Korteweg–de Vries equation, have been derived to analyse flexural–gravity waves ,13. The Steady and unsteady solitary waves have been investigated numerically in a variety of configurations by a number of authors, using boundary integral methods and high-order spectral methods ,20, usinIn most cases, the elastic shell is assumed to be without mass, but very recently theoretical and numerical works have been published that consider the case of heavy hydroelastic waves –30. Otheet al. [There are fewer three-dimensional studies of flexural–gravity waves, owing to the complexity of the problem. Linearized problem patterns of flexural–gravity waves generated by moving loads have been presented by Davys et al.  by inveset al. .Solitary waves in three dimensions have also been studied by deriving a Benney–Roskes–Davey–Stewartson model for a fluid of arbitrary depth covered by an elastic sheet and considering small-amplitude waves . This moThe focus of this work is on waves generated by moving pressures and on fully localized solitary flexural–gravity waves. These are computed in three dimensions for water of finite or infinite depth, using the nonlinear model formulated in Plotnikov & Toland  for the The layout of this paper is follows. The next section includes a description of the problem and its reformulation, with the following section describing the numerical method used. The numerical results section follows, including sample wave profiles. The paper ends with conclusions and discussions of the results.2.Φ and variable surface ζ under a sheet of ice in three dimensions are given byρ is the density of the water, D is the flexural rigidity of ice, g is gravitational acceleration, h is the depth of the water (which could be taken to be infinite), p is an external pressure exerted on the ice and Pflex is a term describing the effect of the ice on the surface of water. The schematic for the domain of interest is shown in Oxyz are defined such that the z-axis is oriented vertically upward and the waves propagate along the x-axis.The Euler equations describing an incompressible, inviscid, irrotational fluid with velocity potential D is given byE is Young's modulus (i.e. the modulus of elasticity), ν is the Poisson ratio describing the effects of transverse strain relative to axial strain and d is thickness of the ice.The term describing the effects due to the presence of ice is modelled using the Cosserat theory of hyperelastic shells . It assuPflex in Cartesian coordinates asH is the mean curvature and K is the Gauss curvature of the ice–water interface, given byPflex has a much simpler form asAfter some algebra, we can express ty waves ,45 a link1x+k2y−ωt)i, hence no waves are generated in the far field. However, for U > cmin, there are two wavenumbers for which U = c(k) and a more complicated pattern emerges. This analysis generalizes to three dimensions and, in this work, we focus on the case of U < cmin and we will look for symmetric solutions in x and y.There are very different wave patterns expected when we consider a pressure travelling at a speed min from . In figu we show  for infi we show  for finiU, it is convenient to use a reference frame moving with the wave by setting U as the unit of velocity and introduce a unit of length L. This allows us to rewrite the Bernoulli condition at the surface  in termP =  and P* =  isg = G and f = Φ − x, which satisfies Laplace's equation. After the proper substitutions and some manipulation, we obtainn is the normal to the ice–water interface S pointing into the fluid, and P* is a point from S.To solve equations , we use We defineζ = ζ, ζ* = ζ and ϕ* = ϕ.The final form of equations to solve for flexural–gravity waves in infinite depth isy-direction with ζ = ζ and ϕ = ϕ implies that we can solve the set of equations . Thus, 3.xi and yj to be equally spaced points such that i = 1, …, N and j = 1, …, M. Let the vector of unknowns be ϕxi,j) and use finite differences for the derivatives. This gives 2(N − 2)M equations. We obtain 2M equations from symmetry about the y-axis and 2M more equations from decay at the boundaries. To obtain ζ and ϕ, we use a trapezoid rule. Derivatives are computed using central difference, second-order accurate schemes, except at the border where one-sided schemes are needed. This gives 2NM equations, which can be written asThe equations given by  and 2.1 with theu0(i) set up an initial guess (ii) until convergenceJ(un)δn =  − G(un)(a) solve un+1 = un + δn(b) set (c) test for convergence and repeat steps if not converged.To solve the system, we use Newton's method ,47, whicu0 and the Jacobian matrix J. To compute non-trivial solitary waves (ones that are non-zero), we need a good initial guess in order for the numerical method to converge. However, since the set of equations is complicated and very nonlinear, there is no analytical expression for it. In order to obtain solitary waves, the numerical procedure is to compute a branch of forced waves, reduce the forcing, and use that as an initial guess. The particular form of the forcing pressure is not important. The main requirement is that it has a bounded support. Here we chooseL0 = 1 and p0 to be small, positive or negative. To obtain fully nonlinear waves, we use a continuation method. We start with a small amplitude, using the flat water as an initial guess with F > Fmin, where Fmin corresponds to the minimum of  problem. We expect bifurcation of solitary waves to start from a point on the curve shown in h considered, and solitary waves to exist for F > Fmin. Similar approaches were used before for gravity–capillary waves for the two-dimensional problem [This method relies on an initial guess nimum of . We use  problem  and for  problem ,43.J. Owing to the form of the equations we are solving, . Figures U < cmin, and solid lines are the solitary waves without a forcing.In this section, we present the numerical results for forced and free surface waves. For what follows, we use the doubly symmetric equations on a quarter of the domain with ing term  had p0 =Fmin in each case. The dashed line is for infinite depth and the solid line is for h = 1. For infinite depth, it can be shown that in non-dimensional units Fmin ≈ 0.4725 and for h = 1, Fmin ≈ 1.0272. These are the values used to normalize the horizontal axis. If these solitary branches bifurcated from Fmin, then they would be shown to originate from F) from the bifurcation point. This implies that the solitary waves' existence requires smaller nonlinear effects for finite depth than those required for infinite depth. This is numerical evidence that, for h = 1, branches of solitary wave solutions bifurcate from zero amplitude, a conclusion that cannot be drawn for waves in infinite depth. This agrees with asymptotic results [ results  that sugIt becomes difficult to follow the branches numerically for large-amplitude solutions. In particular, the elevation branch may have turning points as in two dimensions , but morh = 1. The full wave profiles are shown on the left and the cross-sections on the right. As anticipated from x-direction, but finite depth waves are less compact in the y-direction. On the elevation branches, the elevation of the wave at the centre point  =  becomes smaller in magnitude than the depressions which are adjacent to it, similar to the gravity–capillary wave problem [Solitary waves are shown in figures  problem .5.In this work, we employ the boundary integral equations method ,45 to coF. In shallow water, such as here with h = 1, the numerical results suggest that the branch starts from zero amplitude, as predicted by the weakly nonlinear model [One issue is still left to resolve, and that is the asymptotic predictions for the start of the bifurcation branch. It was shown in  that flear model . There har model , but morIt is worth noting that some large-amplitude solutions calculated here may become unphysical, as the strain of the ice plate may be higher than the yield strain of ice. In this case, the elastic model for the sheet will become unrealistic and different models should be used (see  for a mo


Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic  and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field. Infectious bronchitis virus (IBV) infects ciliated epithelial cells in the chicken respiratory tract. While some IBV strains replicate locally, others can disseminate to various organs, including the kidney. Here, we elucidate the determinants for kidney tropism by studying interactions between the receptor-binding domain (RBD) of the viral attachment protein spike from two IBV strains with different tropisms. Recombinantly produced RBDs from the nephropathogenic IBV strain QX and from the nonnephropathogenic strain M41 bound to the epithelial cells of the trachea. In contrast, only QX-RBD binds more extensively to cells of the digestive tract, urogenital tract, and kidneys. While removal of sialic acids from tissues prevented binding of all proteins to all tissues, binding of QX-RBD to trachea and kidney could not be blocked by preincubation with synthetic alpha-2,3-linked sialic acids. The lack of binding of QX-RBD to a previously identified IBV-M41 receptor was confirmed by enzyme-linked immunosorbent assay (ELISA), demonstrating that tissue binding of QX-RBD is dependent on a different sialylated glycan receptor. Using chimeric RBD proteins, we discovered that the region encompassing amino acids 99 to 159 of QX-RBD was required to establish kidney binding. In particular, QX-RBD amino acids 110 to 112 (KIP) were sufficient to render IBV-M41 with the ability to bind to kidney, while the reciprocal mutations in IBV-QX abolished kidney binding completely. Structural analysis of both RBDs suggests that the receptor-binding site for QX is located at a different location on the spike than that of M41.IMPORTANCE Infectious bronchitis virus is the causative agent of infectious bronchitis in chickens. Upon infection of chicken flocks, the poultry industry faces substantial economic losses by diminished egg quality and increased morbidity and mortality of infected animals. While all IBV strains infect the chicken respiratory tract via the ciliated epithelial layer of the trachea, some strains can also replicate in the kidneys, dividing IBV into the following two pathotypes: nonnephropathogenic  and nephropathogenic viruses (including IBV-QX). Here, we set out to identify the determinants for the extended nephropathogenic tropism of IBV-QX. Our data reveal that each pathotype makes use of a different sialylated glycan ligand, with binding sites on opposite sides of the attachment protein. This knowledge should facilitate the design of antivirals to prevent coronavirus infections in the field. Escherichia coli (Infectious bronchitis is a disease in chickens caused by infectious bronchitis virus (IBV). In the poultry industry, infection of chicken flocks with IBV causes economic losses by reducing egg quantity and quality. In addition, animals become more susceptible to secondary bacterial infections like hia coli . The sevhia coli . Phylogehia coli , of whichia coli . The morhia coli , 3, whichia coli .IBV primarily infects the respiratory tract, where the virus can bind and infect the ciliated epithelial lining of the trachea , 5. Upon7–Binding to host tissues is the first step in the viral life cycle of IBV and therefore a critical factor in determining tissue tropism. Tissue tropism differs based on the amino acid composition of the spike protein as shown by recombinantly produced proteins –17 and i–In this study, we set out to identify how genetic variations in IBV spike proteins have contributed to different host tropisms. We demonstrate that QX-RBD binding to trachea and kidney is dependent on a different sialylated glycan ligand compared to M41-RBD. In particular, introduction of amino acids 110 to 112 (KIP) of the QX spike into M41-RBD was sufficient to extend its tropism toward the kidney. Previous docking experiments  and struEighty-five percent of the amino acids between the sequences of the first 257 amino acids of IBV-QX and IBV-M41 are either identical or similar. Here, we set out to determine which of the dissimilar amino acids are the determinants for the difference in tissue tropism.In previous work, we demonstrated the M41-RBD was sufficient to bind to chicken trachea . To veriin vivo, we first determined if the potential RBD of QX behaved like that of M41 (N-glycosidase F (PNGase F) to remove posttranslational glycosylation. QX- and M41-RBD migrated comparably at around 55 kDa (including glycosylation) and had a backbone of around 32 kDa as expected after PNGase F treatment  of M41 and a comparable size fragment of the QX spike displayed a sequence identity of 73.6% , with tht of M41  and thatt of M41 . Both prreatment . Circulareatment  presentereatment . Finallyreatment , indicatin vivo for replication of both genotypes. Detailed analysis of staining present in the kidney showed that binding of QX-RBD was restricted to the parietal epithelium of Bowman’s capsule in the glomerulus  on chicken tissues that differs from the receptor elucidated for IBV-M41. Using chimeric proteins and The N-terminal domain of IBV-QX spike protein comprises, like previously shown for M41 , a recepTo elucidate the specific ligand used by QX-RBD, we performed several binding studies using previously developed glycan arrays , 31 contComparison of the spikes of various IBV strains with reported nephropathogenicity, including IBV clade GI-14 (including strain B1648 ) and clain silico predictions of the interaction with alpha-2,3-linked sialic acid ligands in M41-RBD pointed toward three amino acids, S87, N144, and T162, which are in close proximity to four essential N-glycosylation sites  (N-glycosylation sites is at a different position (N59 in M41 and N58 in QX). This may result in a different conformation of the ligand-binding site, thereby preventing QX-RBD wild-type binding to the M41 ligand, which was supported by experimental evidence using the chimeric M41-RBD protein where this glycosylation site was replaced, resulting in loss of binding to trachea tissue (data not shown). Furthermore, in the publication where the cryo-EM structure of M41 was resolved, the loop consisting of amino acids present in HVR 2 of the spike was proposed to be required for receptor binding  . Althoug binding . Our dat binding . In Fig.in vivo and may contribute to designing new antivirals to prevent coronavirus infections in the field.In conclusion, we demonstrated that IBV-QX binding to chicken trachea and kidney tissue is dependent on a sialylated glycan receptor and that amino acids in HVR 2 of the QX-RBD are critical for this receptor-binding profile. This knowledge adds to our understanding of differences in tissue tropism between IBV strains AY851295) sequences followed by a trimerization domain (GCN4) and strep-tag (ST) were described previously (AFJ11176), containing upstream NheI and downstream PacI restriction sites, was obtained from GenScript and cloned into the pCD5 expression vector by restriction digestion as previously described (The expression plasmids containing the codon-optimized M41-ED (amino acids 19 to 1091 ), M41-S1eviously . The codRecombinant RBD proteins were produced in human embryonic kidney (HEK293T) cells. In short, cells were transfected with pCD5 expression vectors using polyethylenimine (PEI) at a 1:12 (wt/wt) ratio. Cell culture supernatants were harvested after 6 days. The recombinant proteins were purified using Strep-Tactin Sepharose beads as previously described . ProteinRecombinant IBV RBD proteins were exchanged into buffer containing 10 mM sodium phosphate, pH 7.75, and diluted to 0.06 mg/ml. CD spectra were collected on a Jasco J-810 spectropolarimeter with a Peltier thermostatted fluorescence temperature controller module by accumulating 4 scans from 285 to 190 nm with a scanning speed of 10 nm/min, digital integrated time of 1 s, bandwidth of 1 nm, and standard sensitivity of 25°C. A thermal melt was done from 25°C to 95°C with a ramp rate of 1°C per minute. A full CD scan was collected at 95°C. After lowering the temperature to 25°C, the protein was allowed to refold for 20 min at 25°C, and a third CD scan was taken at 25°C to measure recovery. Secondary structure calculations for the CD data collected at 25°C before the thermal melt were processed by DichroWeb  using th2SO4. The optical density at 450 nm was measured in a FLUOstar Omega instrument (BMG Labtech), and MARS data analysis software was used for data analysis. Statistical analysis was performed using a two-way analysis of variance (ANOVA).Neu5Acα2-3Galβ1-3GlcNAc-PAA  was coated in a 96-well Nunc MaxiSorp plate  at 0.5 μg/well overnight at 4°C, followed by blocking with 3% bovine serum albumin (BSA)  in phosphate-buffered saline (PBS)-0.1% Tween 20 overnight. RBD proteins were preincubated with Strep-Tactin HRP antibody  (1:200) for 30 min on ice. Indicated protein amounts were diluted in PBS and applied onto the coated well, followed by incubation for 2 h at room temperature. TMB  substrate was used to visualize binding, after which the reaction was terminated using 1 M HArthrobacter ureafaciens (AUNA)  in 10 mM potassium acetate and 2.5 mg/ml Triton X-100, pH 4.2, and incubated at 37°C overnight before protein application.Protein histochemistry was performed as previously described . Recombi


It has been demonstrated that their selective inhibition alters the equilibrium of the metabolites above affecting the biosynthesis and energy metabolism of the organism. In this context, our interest has been focalized on the fungus Malassezia restricta, which may trigger dandruff and seborrheic dermatitis altering the complex bacterial and fungal equilibrium of the human scalp. We investigated a rather large number of inorganic metal-complexing anions (a well-known class of CA inhibitors) for their interaction with the β-CA (MreCA) encoded by the M. restricta genome. The results were compared with those obtained for the two human α-CA isoforms (hCAI and hCAII) and the β-CA from Malassezia globosa. The most effective MreCA inhibitors were diethyldithiocarbamate, sulfamide, phenyl arsenic acid, stannate, tellurate, tetraborate, selenocyanate, trithiocarbonate, and bicarbonate. The different KI values obtained for the four proteins investigated might be attributed to the architectural features of their catalytic site. The anion inhibition profile is essential for better understanding the inhibition/catalytic mechanisms of these enzymes and for designing novel types of inhibitors, which may have clinical applications for the management of dandruff and seborrheic dermatitis.Carbonic anhydrases  are ubiquitous metalloenzymes, which catalyze the crucial physiological CO Recently, an extensive study concerning the inhibition profile of MgCA has been reported using CAIs, such as sulfonamides, anions, a series of 6-substituted benzoxaboroles, monothiocarbamates, dithiocarbamates, famotidine, natural polyphenols, and phenols. The results identified efficient and selective inhibitors [Malassezia restricta represents the larger proportion of Malassezia sp. on the scalp in various populations and may also trigger dandruff and seborrheic dermatitis, in conjunction with the complex bacterial and fungal equilibrium of the human scalp [M. restricta (MreCA) is a 230 amino acid residues protein, which showed high catalytic activity for the hydration of CO2 to bicarbonate and protons, with a kcat of 1.06 × 106 s−1 and kcat/KM of 1.07 × 108 M−1 s−1 that is also sensitive to inhibition by the sulfonamide acetazolamide [Malassezia globosa in order to better understand the inhibition/catalytic mechanisms of these enzymes fundamental for many physiologic processes, and for designing novel types of inhibitors, which may have clinical applications for the management of dandruff and seborrheic dermatitis. Here, we show that MreCA exhibits a unique anion inhibition profile.In recent years, the pharmaceutical industry has focused its attention on inorganic drugs in addition to organic compounds ,2. This 3− + H+) ,23,24,25 CO32−) ,27,28,29 (CO32−) . The met (CO32−) . CAs areirulence . Generalirulence . Fascinaathology . For exa− ,27,28,hibitors ,36,37,38an scalp ,40,41,42zolamide . In the 2 sensing by producing the bicarbonate for the activation of adenylyl cyclase (cAMP), which regulates the fungal capsule biosynthesis or filamentation [M. restricta will be fundamental for a better comprehension of the biological functions of these enzymes, which can interfere with the life cycle of the fungus. In the present study, the recombinant β-CA (MreCA) encoded in the genome of the fungus M. restricta was prepared with a tail of six histidines at the C-terminus as reported previously [CAs have a crucial role in fungal biology since they sustain the bicarbonate-dependent carboxylation reactions, are necessary for the sexual reproduction in basidiomycetes and filamentous ascomycetes, and, are involved in COentation ,44,45. Teviously . The pur2 molecules to bicarbonate per second (kcat =1.06 × 106 s−1) and was efficiently inhibited by the classical sulfonamide inhibitor acetazolamide (KI of 50.7 nM) [M. globosa are phylogenetically grouped with the β-CA encoded by the genome of Ustilago maydis, a pathogenic fungus responsible for the plant disease known as corn smut [Additionally, the stopped-flow technique confirmed that the present enzyme showed a high conversion of CO50.7 nM) . Intriguorn smut .U. maydis  from . maydis . The two. maydis . Indeed,. maydis . Thus, t(i)I > 50 mM). It is interesting to note that MreCA has a very intriguingly inhibition pattern versus these anions when compared with its ortholog MgCA, or the two human isoforms (hCA I and hCA II), MrCA being less or not inhibited by most of the anions tested. The enzyme from M. globosa showed a KI in the range of 4.06–21.4 mM for most of these inhibitors, apart from carbonate, bisulfite, and peroxydisulfate. Indeed, the two human isoforms belonging to the α-CA class were well inhibited by the ‘‘metal ion poisons”, such as cyanide and azide. The behavior of MreCA is somewhat difficult to explain observing the different inhibition profile compared to MgCA, hCA I, and hCA II. However, it fortifies the thesis that the synthesis of new drugs capable of interfering selectively with MreCA and MgCA activity can avoid the inhibition of the human CAs (α-class enzymes), leading to the inactivation of the CAs encoded by the scalp microbes necessary for the integrity of the human skin.Anions, which generally complex many cations, including Zn(II), such as fluoride, chloride, bromide, cyanate, thiocyanate, cyanide, nitrite, carbonate, bisulfite, sulfate, hydrogen sulfate, pyrophosphate, divanadate, perrhenate, peroxydisulfate, iminodisulfite, and fluorosulfonate, did not show any inhibitory action against MreCA (K(ii)I ranging between 3.9 and 9.0 mM. Most of these anions showed a similar KI for the homologous enzyme MgCA, with the exception of sulfamic acid and phenylboronic acid, which are very effective inhibitors of MgCA, with Ki of 0.083 and 0.089 mM, respectively. Interesting, the human isoforms (hCAI and hCAII) are not well inhibited by phenyl boronic acid.Simple and complex anions investigated here, including iodide, nitrate, sulfamic acid, phenylboronic acid, perchlorate, osmate, perruthenate, esafluorofosfato, and trifalate, showed an interesting inhibition profile for MreCA, with K(iii)I of 0.075–0.86 mM). These small molecules/anions are in fact well known to effectively inhibit many CAs belonging to all genetic families, as well as the two human isoforms (hCA I and hCA II).The most effective MreCA inhibitors identified in this study were bicarbonate, sulfamide, phenyl arsenic acid, stannate, tellurate, tetraborate, selenocyanate, trithiocarbonate, and diethyldithiocarbamate , due to their affinity for metal ions in solution or when bound within metalloenzyme active sites . The aniMalassezia restricta corresponding to a β-CA was back-translated into the nucleotide sequence and optimized for the codon usage to increase its expression in Escherichia coli cells. The synthetic M. restricta gene, as obtained from GeneArt Company  was cloned into the expression vector to obtain the pET100D-Topo/MreCA. Competent Escherichia coli BL21 (DE3) codon plus cells (Agilent) were transformed with pET100D-Topo/MreCA and induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG). After growth, the cells were harvested and disrupted by sonication. The cellular extract was loaded and purified onto a His-select High-Flow (HF) Nickel affinity column. After the column, the resulting enzyme was 80% pure.The amino acid sequence of w/v) SDS-PAGE, followed by electrophoretic transfer to a PVDF membrane with transfer buffer  using Trans-Plot SD Cell . His-Tag Western blot was carried out using the Pierce Fast Western Blot Kit . Blotted membrane had been placed in the wash blot solution Fast Western 1 Wash Buffer to remove transfer buffer. Primary Antibody Working Dilution was added to the blot and incubated for 30 min at room temperature (RT) with shaking. Afterwards, the blot was removed from the primary antibody solution and incubated for 10 min with the Fast Western Optimised HRP Reagent Working Dilution. Subsequently, the membrane was washed two times in about 20 mL of Fast Western 1 Wash Buffer. Finally, the membrane was incubated with the detection reagent working solution and incubated for 1 min at room temperature and then developed with X-ray film.MreCA was subjected to a 12% (2 hydration activity [2SO4 for maintaining constant ionic strength (this anion is not inhibitory and has a KI > 200 mM against this enzyme), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each measurement, at least six traces of the initial 5–10% of the reaction were used for determining the initial velocity, working with 10-fold decreasing inhibitor concentrations ranging between 1 nM and 10–100 µM . The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 1 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature before assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using the Cheng–Prusoff equation, and represent the mean from at least three different determinations. The human isoforms hCA I and II and the fungus MgCA were assayed in the same conditions as above, with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and phenol red as an indicator [An Applied Photophysics stopped-flow instrument was used for assaying the CA catalyzed COactivity . Bromothndicator . All enzndicator ,51,52,53Malassezia restricta is one of the micro-organisms involved in the disequilibrium between various commensals, such as M. globosa, Cutibacterium acnes (formerly named Propionibacterium acnes) and Staphylococcus sp., which contribute to dandruff and seborrheic dermatitis symptoms [M. restricta genome. Indeed, a full inhibition profile of MreCA, as well as the ortholog enzyme MgCA, and the possible off-targets hCA I and hCA II, was carried out with the known CA inorganic metal-complexing anions. MreCA activity, diversely from its homologous MgCA, is not readily inhibited by anions, which generally complex effectively cations . The most effective MreCA inhibitors were diethyldithiocarbamate, sulfamide, phenyl arsenic acid, stannate, tellurate, tetraborate, selenocyanate, trithiocarbonate, and bicarbonate. As described in the literature, the compounds investigated bind to the Zn(II) ion in the enzyme active site either by substituting the nucleophile (water molecule/hydroxide ion) as the fourth zinc ligand or by adding to the zinc coordination sphere to generate a trigonal bipyramidal geometry relative to the metal ion. The final result is a possible inactivation of the enzyme due to the degree of the structural distortion caused by the inhibitor. Thus, although CAs have a similar catalytic site, each biocatalyst has unusual architectural features, which must occur in the interaction between the protein and the anions investigated, explaining the differences in the degree of structural distortion and the different KI values obtained for the four proteins studied. In conclusion, the investigation of the anion inhibition profiles of the fungal CA enzymes represents a significant starting point for designing novel metal-based antifungal treatments, which need to be further investigated using structural analysis of MrCAsymptoms ,41,42,53


Bisperoxovanadium (pyridine-2-carboxyl) [bpV(pic)] is a commercially available PTEN inhibitor. Previous studies from us and others have shown that bpV(pic) confers neuroprotection in cerebral ischemia injury. We set up to determine whether ERK 1/2 activation plays a role in bpV(pic)-induced neuroprotective effect in cerebral ischemia injury. We found that the phosphorylation levels of Akt (p-AKT) and ERK1/2 (p-ERK 1/2) were down-regulated after cerebral ischemia–reperfusion injury. The injection of bpV(pic) after injury not only increased the level of p-AKT but also the level of p-ERK 1/2. While the inhibition of PTEN mediated the up-regulatation of p-AKT and p-ERK 1/2 by bpV(pic). Interestingly, the ERK 1/2 activation induced by bpV(pic) was also independent of the inhibition of PTEN. Our results indicate that bpV(pic) protects against OGD-induced neuronal death and promotes the functional recovery of stroke animals through PTEN inhibition and ERK 1/2 activation, respectively. This study suggests that the effect of bpV(pic) on ERK 1/2 signaling should be considered while using bpV(pic) as a PTEN inhibitor.The online version of this article (10.1007/s11064-018-2558-z) contains supplementary material, which is available to authorized users. Ischemic stroke is induced by arterial embolism, microangiopathy or macroaniopathy, results in oxygen and glucose deprivation in brain, leading to brain damage/injury and neurologic deficit . OxidatiPTEN (phosphatase and tensin homolog deleted on chromosome 10) a tumor suppressor, associate with several neoplastic diseases . PTEN plBisperoxovanadium (pyridine-2-carboxyl) [bpV(pic)] is a commercially available PTEN inhibitor . PreviouMitogen-activated protein kinase (MAPK) is a family of serine/threonine protein kinases that are widely expressed in all eukaryotic cells . ExtraceIn this study, we aimed to investigate the relationship of bpV(pic) and ERK 1/2, and confirm the bpV(pic)-induced neuroprotect whether connect with ERK 1/2. Our results show that bpV(pic) confers neuroprotection after cerebral ischemia–reperfusion injury through inhibition of PTEN lipid phosphatase activity as well as ERK 1/2 activation.Adult male Sprague-Dawley (SD) rats were housed with three rats per cage on a 12 h light/dark cycle in a temperature-controlled room (23–25 °C) with free access to water and food. Animals were allowed at least 3 days to acclimatize before experimentation. We used total 248 male rats in our in vivo expriments, 56 male rats were discarded during the operational procedures. 12 adult pregnant female rats and 45 embryos were used in our expriments for cortical neuronal cultures. All animal use and experimental protocols were approved and carried out in compliance with the IACUC guidelines and the Animal Care and Ethics Committee of Wuhan University School of Medicine. Samples to the experimental groups and to collect and process data was assigned by randomization. The experiments were performed by investigators blinded to the groups for which each animal was assigned.2O and 30% O2 by using a mask. The rectal temperature was maintained at 37.0 ± 0.5 °C using a homoeothermic blanket. A midline incision was made in the neck, the right external carotid artery (ECA) was carefully exposed and dissected and a 3–0 monofilament nylon suture was inserted from the ECA into the right internal carotid artery to occlude the origin of the rightmiddle cerebral artery (approximately 22 mm). After 90 min of occlusion, the suture was removed to allow reperfusion, the ECA was ligated and the wound was closed. Sham-operated rats underwent identical surgery and/or i.c.v. injections except that the suture was inserted and withdrawn immediately. At 24 h after middle cerebral artery occlusion (MCAO), rats (n = 24) were reperfused with ice-cold 0.9% saline after anaesthetized with 4% isoflurane in 70% N2O and 30% O2, and the brains were rapidly removed for western blot analysis and 2,3,5-triphenyltetrazolium chloride (TTC) staining.Transient focal cerebral ischaemia was induced using the suture occlusion technique , 21. TheThe brain was placed in a cooled matrix and 2 mm coronal sections were cut. Individual sections were placed in 10 cm petri dishes and incubated for 30 min in a solution of 2% TTC in phosphate buffered saline at 37 °C. The slices were fixed in 4% paraformaldehyde at 4 °C for 24 h. All image collection, processing and analysis were performed in a blind manner and under controlled environmental lighting. The scanned images were analyzed using image analysis software . The infarct volume was calculated to correct for edema. The normal volume of contralateral hemisphere and the normal volume of ipsilateral hemisphere were measured, and the infarct percentage was calculated as % contralateral structure to avoid mismeasurement secondary to edema , 24.2 and 70% N2O in a sealed perspective box. When the rats were deeply anaesthetized, we would use the ear bars and upper incisor bar to secure the rat’s head in a stereotaxic frame, and the rats were continuously under anaesthesia with 4% isoflurane using a mask. Next, making a small sagittal incision, and bregma was located as the anatomical reference point. The cerebral ventricle  was performed using a 23 gauge needle attached via polyethylene tubing to a Hamilton microsyringe, and drug infusion at a rate of 1.0 µL/min. Proper needle placement was verified via withdrawing a few microlitres of clear cerebrospinal fluid into the Hamilton microsyringe.Rats were anaesthetized with a mixture of 4% isoflurane in 30% O2O and 30% O2. The embryos were sprayed with 70% ethanol that were removed after the rats. The embryos were rapidly excised, the embryo skull was removed, and the brain tissue was compeletely removed. The cerebral cortex tissue was separated under a microscope and placed in a fresh frozen liquid and placed in ice-cold plating medium . The cortical neurons were plated on Petri dishes coated with poly-d-lysine (PDL) and suspended in plating medium. Half of the plating medium was removed and replaced with maintenance medium  in the same manner every 3 days. The cultured neurons were used for the experiments after 12 days  and AKT Inhibitor IV (ChemBridge 5233705) were purchased from Santa Cruz Biotechnology . ERK 1/2 inhibitor U0126 was purchased from Delleckchem .473 [202/Tyr204 [Ischemic stroke results from thrombotic blockage, which in brain causes the oxygen and glucose deprivation, leading to brain damage and neurological deficit. We generated an experimental stroke model induced by MCAO , 35 for 473  and the 2/Tyr204 . ImmunofWestern boltting analysis of ipsilateral brain homogenates at the indicated time point after I/R revealed a down-regulate p-AKT and p-ERK 1/2 levels as time goes on  can activate AKT through inhibition of PTEN. Previous studies have shown that bpV(pic) confers neuroprotection in cerebral ischemia injury . In the To further investigate the effect of bpV(pic). We used the human neuroblastoma SH-SY5Y cells, which were treated with different concentrations of bpV(pic) . Results show that bpV(pic) increased the level of p-AKT in SH-SY5Y cells Fig. a. BpVpib to furtWe know from a previous study that PTEN can negatively regulate AKT activation through PI3K signal pathway because of it’s lipid phosphatase activity. To further reveal the mechanism of bpV(pic) inhibition function in PTEN, we established the PTEN WT and phosphatase domain mutant: PTEN G129E, in which Glu replaces Gly129, which lacks lipid phosphatase activity . BecauseFrom Fig. We set up to determine whether bpV(pic) directly activates of ERK 1/2, so we used the recombinant ERK 1/2 to test the activation effect of bpV(pic) in it . Our resFrom Fig. On the basis of Fig. PTEN is widely known as a tumor suppressor that is mostly associated with several neoplastic diseases. PTEN also plays a pivotal role in neuropathic diseases , 40. PreBpV(pic) is a commercially available PTEN inhibitor, and previous studies from us and others have shown that bpV(pic) confers neuroprotection in cerebral ischemia injury , 48. We Previous studies have demonstrated that the AKT activity can be increased by bpV(pic) . Our finOur finding demonstrates that bpV(pic) can activate AKT activity through the inhibition of PTEN lipid phosphatase activity. Interestingly, we show that ERK 1/2 can be also activated by bpV(pic). The bpV(pic)-induced ERK 1/2 activation is not only through the inhibition of PTEN lipid phosphatase activity, but is also independent of PTEN inhibition. Consequently, bpV(pic)-induced neuroprotection in ischemic stroke is depend on the inhibition of PTEN lipid phosphatase activity and the enhancement of ERK 1/2 activity that is independent of PTEN inhibition. Therefore, the effect of bpV(pic) on ERK 1/2 signaling should be considered while using bpV(pic) as a PTEN inhibitor in experimental conditions.Below is the link to the electronic supplementary material.Supplementary material 1 (TIF 1285 KB)Supplementary material 2 (TIF 653 KB)Supplementary material 3 (TIF 861 KB)


Tau is a neuronal microtubule-associated protein (MAP) that is involved in the regulation of axonal microtubule assembly. However, as a protein with intrinsically disordered regions (IDRs), tau also interacts with many other partners in addition to microtubules. Phosphorylation at selected sites modulates tau’s various intracellular interactions and regulates the properties of IDRs. In Alzheimer’s disease (AD) and other tauopathies, tau exhibits pathologically increased phosphorylation (hyperphosphorylation) at selected sites and aggregates into neurofibrillary tangles (NFTs). By bioinformatics means, we tested the hypothesis that the sequence of tau has changed during the vertebrate evolution in a way that novel interactions developed and also the phosphorylation pattern was affected, which made tau prone to the development of tauopathies. We report that distinct regions of tau show functional specialization in their molecular interactions. We found that tau’s amino-terminal region, which is involved in biological processes related to “membrane organization” and “regulation of apoptosis,” exhibited a strong evolutionary increase in protein disorder providing the basis for the development of novel interactions. We observed that the predicted phosphorylation sites have changed during evolution in a region-specific manner, and in some cases the overall number of phosphorylation sites increased owing to the formation of clusters of phosphorylatable residues. In contrast, disease-specific hyperphosphorylated sites remained highly conserved. The data indicate that novel, non-microtubule related tau interactions developed during evolution and suggest that the biological processes, which are mediated by these interactions, are of pathological relevance. Furthermore, the data indicate that predicted phosphorylation sites in some regions of tau, including a cluster of phosphorylatable residues in the alternatively spliced exon 2, have changed during evolution. In view of the “antagonistic pleiotropy hypothesis” it may be worth to take disease-associated phosphosites with low evolutionary conservation as relevant biomarkers into consideration. The microtubule-associated protein (MAP) tau is involved in the regulation of neuronal microtubule (MT) assembly. Together with the closely related MAP2, it has arisen in evolution after a duplication of an ancestral gene of ancient cyclostomes , which are a hallmark of several neurodegenerative diseases collectively called “tauopathies” , which are known to interact with many binding partners and are considered to be involved in various signaling and regulation processes  was generated by the Random Coil Generator (RCG) software  and lamprey (Petromyzon marinus), cartilaginous fish , coelacanth  and amphibian . The selected organisms were grouped according to higher taxons  and were color-coded from light yellow to orange in the respective order. The following estimated divergence times between mammals and other groups of vertebrates were obtained from TimeTreeMAPT sequences were retrieved from Uniprot® ; mostly neural networks that were trained on different datasets of ordered and disordered proteins to predict disorder for analyzed sequences  IUPred2A ; predicts structural disorder based on a biophysical model and optimized for different length disordered regions  PhosphoSitePlusAlignment of selected sequences was performed with Tcoffee . However, experiments using recombinant tau fragments have provided evidence that segments encoded by exons 2 and 10 promoted tau aggregation, whereas the segment encoded by exon 3 depressed it  and the relation of predicted disorder values and divergence times were analyzed with linear regression model. An example of a respective representation of the change in the level of disorder of different tau regions through vertebrate evolution with one disorder predicting algorithm is shown in Thus, the data indicate that in particular tau’s amino-terminal region (NTR and PRR) with a noticeable contribution of the alternatively spliced exon 2 exhibits a strong evolutionary increase in disorder, which suggests that novel interactions in addition to tau’s microtubule-related activities developed during evolution. As discussed before, this may, in particular, involve biological processes related to “membrane organization” and “regulation of apoptotic processes” which are both mediated by this region.Phosphorylation is a common PTM, which regulates the function of many proteins and is also thought to modulate the conformational flexibility and interactions of IDRs. Tau is subject to many phosphorylation events and changes in the pattern, the stoichiometry and the dynamics of phosphorylation have been associated with functional changes of tau during physiological and pathological conditions.Xenopus tropicalis and simpler organisms , while the number of phosphorylatable residues in the C-terminus (MBR and CTR) remained unaltered. This is in line with the observation that the C-terminus, in general, is evolutionary much more conserved. Interestingly, the NTR and PRR showed opposite changes. While the number of phosphorylation sites in the NTR decreased, which was mostly due to decreased number of serine residues, the number of phosphosites in the PRR showed a strong increase with divergence time.Pathological changes in tau phosphorylation are thought to induce tau-mediated toxicity and affect tau aggregation  are present in these two regions. In general, the disease-associated phosphorylation sites with low conservation are present in the NTR and PRR. All phosphorylation sites in regions of low disorder are highly conserved, whereas about 40% of phosphorylation sites in high disorder regions displayed lower conservation , which iSpecific phosphosites of tau may be of particular importance during disease development and may be different from those, which have been identified in tau isolated from PHFs. Recent profiling of phosphorylated tau peptides in cerebrospinal fluid (CSF) by mass spectrometry has revealed 11 sites that are increased by at least 40% in the CSF of patients with AD compared to controls  and which exhibit moderate conservation.We tested the hypothesis that the sequence of tau has changed during vertebrate evolution in a way that novel interactions developed. We also tested the hypothesis that tau phosphorylation may have been affected by evolutionary changes, which made tau prone for the development of tauopathies. We show that: (1) distinct regions of tau show functional specialization in their molecular interactions; (2) tau’s amino-terminal, non-microtubule binding region exhibits a strong evolutionary increase in protein disorder; and (3) the number of predicted phosphorylation sites changes during evolution in a region-specific manner, while disease-specific hyperphosphorylated sites are highly conserved.Our data indicate that the highly conserved MBR on one side and the much less conserved N-terminal region on the other side show specific functional specialization. Since the MT-associated proteins tau, MAP2 and MAP4 contain a similar CTR with multiple highly conserved microtubule-binding repeats, tau’s N-terminal region may provide the tau-specific interactions, which may also be involved in its unique subcellular localization with an enrichment in the axonal compartment, which occurs early during neuronal development . Interestingly, however, the NTR and PRR showed opposite changes in the number of predicted phosphorylation sites during evolution. While the number of predicted phosphorylation sites in the NTR decreased, the PRR showed a strong increase with divergence time and also when fish, birds and mammals were compared. Thus, the data suggest that the propensity of regulation by phosphorylation evolved in an opposite manner in the PRR and the NTR. While it increased in the PRR, it decreased in the NTR. This could also be of relevance for the regulation of apoptotic processes, where the PRR and NTR had an antagonistic effect, which might be differentially regulated by region-specific phosphorylation. Of note, changes in tau phosphorylation during evolution can also be caused by changes in the number and regulation of kinases and phosphatases. Eukaryotic protein kinases regulate almost every biological process and have evolved as dynamic molecular switches , and domestic cat (Felis catus) develop NFTs, while closely related species such as rat (Rattus norvegicus) or the domestic dog (Canis lupus familiaris) do not  were present in these two regions. The sequence comparison revealed that all of the hyperphosphorylated sites are highly conserved and thus have the potential to be phosphorylated in most species. Comparison of the occurrence of NFTs in various animal species indicates that certain species such as degu . These evolutionary novel interactions relate to regulation of localization to membranes and regulation of apoptosis, i.e., biological processes, which may be of pathological relevance with respect to tau localization and cell survival. Furthermore, the data indicate that the number of predicted phosphorylation sites in the PRR showed a strong increase with divergence time and show that novel clusters of phosphorylatable residues developed during evolution in the PRR and in an alternatively spliced exon in the NTR. Thus, the data suggest that the propensity of regulation of tau function by phosphorylation  developed during evolution and may point to isoform-specific regulatory mechanisms of tau isoforms containing N-terminal inserts. In view of the “antagonistic pleiotropy hypothesis,” an increase in phosphorylatable residues in the PRR during evolution could be beneficial for the organism’s fitness by providing increased ability for regulated interactions, while it would be detrimental at higher age due to increased propensity for miss-regulation and aggregation.All datasets generated for this study are included in the manuscript and/or the NT, AM and RB designed the research. NT performed the research. NT, LB, AM and RB analyzed, interpreted the data and wrote the article.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.


Gonadotropin Regulated Testicular Helicase (GRTH/DDX25), expressed in the male gonad, is essential for the completion of spermatogenesis. Our early studies revealed a missense mutation (R242H) of GRTH in 5.8% of Japanese patient population with azoospermia. Transfection of the mutant GRTH construct in COS-1 cells leads to loss of the 61 kDa cytoplasmic phospho-species. Mice with knock-in of the human GRTH mutation are sterile and lack sperm with normal androgen and mating behavior. These findings provide an avenue for the development of a non-hormonal male contraceptive. Using site directed mutagenesis and a site-specific phospho-antibody, we have identified T239, structurally adjacent to the patient’s mutant site as the GRTH phospho-site. Molecular modelling provided structural basis for the role of R242 and other critical solvent-exposed residues at the GRTH/PKA interface (E165/K240/D237), on the control of GRTH phosphorylation at T239. Single or double mutations of these residues caused marked reduction or abolition of the phospho-form. These effects can be ascribed to critical disruptions of intramolecular H-bonds at the GRTH/PKA interface, which leads to modest but consequential structural changes that can affect PKA catalytic efficiency. Inhibition of phosphorylation may be achieved by small, drug-like molecules that bind to GRTH and reconfigure the GRTH/PKA interface. This testis-specific protein expressed in Leydig and germ cells of the male gonad is transcriptionally upregulated by gonadotropin through direct and indirect actions of androgen via androgen receptor, respectively5. In Leydig cells, GRTH regulates cholesterol homeostasis via its action on StAR expression6. In germ cells, it is expressed predominantly in meiotic spermatocytes and round spermatids where is essential for spermatid development and completion of spermatogenesis. GRTH knockout mice lack sperm due to failure of round spermatids to elongate at step 8 of spermiogenesis7. This multifunctional protein participates in the export of relevant mRNAs from nucleus to cytoplasmic sites of germ cells for storage/degradation in chromatoid bodies of round spermatids and translation, as indicated through its association with actively translating polyribosomes in germ cells9. It was also found to be a regulator of miRNA biogenesis10 and an inhibitor of germ cell apoptosis11.Gonadotropin Regulated Testicular Helicase (GRTH/DDX25) is a member of the DEAD-box family of RNA helicases identified and isolated from rat, mouse, and human testes8. GRTH phosphorylation was induced by cAMP in COS-1 cells transfected with GRTH. The phospho-species was significantly increased with overexpression of Protein Kinase A catalytic subunit (PKA-Cα) and prevented by co-expression of rabbit protein kinase inhibitor (PKI). The 61-kDa species was detected in Western blot by phospho-Thr antibody but not by Ser or Tyr antiserum8.In germ cells of mouse and rat, there are two species of GRTH, a 56 kDa non-phosphorylated form, predominantly found in the nucleus, where it interacts with CREM and participates in mRNA transport, and the 61 kDa phosphorylated form (pGRTH), present exclusively in the cytosol and found to be associated with polyribosomes. The non-phospho form was also found in the cytoplasm of germ cells. In COS-1 cells transfected with GRTH cDNA, a similar pattern of expression of these forms was observed12. Transfection of this mutant in COS-1 cells causes loss of the 61 kDa cytoplasmic phospho- species with preservation of the non-phospho GRTH form12. Recently, a knock-in (KI) model created in our laboratory based on the R242 to H mutant revealed the functional relevance of this mutation. KI mice are sterile with reduction of testicular size, lacking sperm due to arrest at step 8 of round spermatids and complete loss of the pGRTH with unchanged non-phospho form. The lack of pGRTH does not affect the levels of plasma testosterone or mating behavior but solely inhibits sperm formation13.Our previous studies revealed the presence of a missense mutation of arginine to histidine at position 242 (R242H) in exon 8 of GRTH in 5.8% of Japanese patients with azoospermiaIn this study, using site directed mutagenesis, we identified the GRTH/DDX25 phosphorylation site and the critical residues at the GRTH/PKA interface that control the phosphorylation status of the protein. Changes in basicity in the environment of position 242 upon R to H mutation or local conformational changes induced by this structural perturbation can affect phosphorylation by PKA at an adjacent putative site. Molecular modeling provided a structural rationale for these effects and guided the selection of residues targeted for mutations that abolish GRTH phosphorylation. The partial mapping of the phospho-site and the GRTH/PKA interface allowed us to identify critical structural motifs that are necessary for cAMP/PKA binding and/or catalysis; local perturbation of these motifs can lead to complete abolition of phosphorylation, hence spermatogenic arrest. Collectively, these findings provide an avenue for the development of a male contraceptive through compounds that mimic the human mutation or that directly or indirectly block the action of PKA on the phosphorylation site selectively and specifically.8, scanning analysis of consensus PKAα phosphorylation motifs identified five putative phosphorylation sites at positions 38, 212, 239, 355 and 40812. Site directed mutagenesis and expression of the corresponding constructs in COS-1 demonstrated that the mutation T239A and R242H completely abolished the 61 kDa phospho-band with preservation of the non-phospho species when compared with wild-type construct  is common in testis15. These findings lay the foundation for further probing and structural analysis of the GRTH/PKA binding interface with the goal to identify potential binding sites for small drug-like molecules that could elicit similar deleterious effect on T239 phosphorylation as does H242.Based on our initial studies indicating that the cytoplasmic form of GRTH was phosphorylated at a threonine residueuct Fig. . Only thuct Fig. . In contuct Fig. , whereasuct Fig. . Thus, t18. The crystal structure of DDX25 has not been resolved, so it was here modeled based on the structure of DDX19, the member of the family with the closest amino acid sequence identity (~65%) to DDX25 X25 Fig. , perturbResidue E165 interacts with R242 through two persistent H-bonds; this strong interaction appears to be critical for GRTH phosphorylation since disengagement of R242 by a single E165A mutation highly reduced T239 phosphorylation. Changes in the cytosolic form of GRTH with preservation of the non-phospho form are shown in Western Blots of cytoplasmic extracts of COS-1 cells transfected with wild-type and mutants GRTH using a polyclonal antibody to GRTH Fig. . This inCo-expression in COS-1 cells of the catalytic subunit of PKA with either wild-type GRTH or with the mutants relevant for phosphorylation by endogenous PKA, revealed that overexpression of PKA-Cα minimally relieved the inhibition of phosphorylation caused by E165A, K240A and D237A to only a small fraction when compared to its effect for wild-type GRTH Fig. . In contSubsequently, the association of PKA-Cα with GRTH and mutants was examined using Immuno-Precipitation (IP) of COS-1 cells extracts that were transfected with the GRTH-V5 construct alone or co-transfected with the PKA-Cα using either V5 and PKA-Cα antibodies. We observed co-IP of GRTH and PKA-Cα using either of these antibodies Fig. . The int239TKIR conforms to the PKA recognition motif pS-X-X-R more common in testis than in other tissues15. Phosphorylation of GRTH was also observed in the mutant S239, although it was found to be less efficient.In this study we have identified residue T239 as the phosphorylation site of GRTH. Mutation of T239 abolished the pGRTH cytoplasmic 61 kDa species, whereas the non-phospho 56 kDa form was present in the nucleus and in the cytoplasm of transfected COS-1 cells. This was further confirmed with a GRTH peptide antiserum and by a highly site-specific phospho-peptide antibody. The GRTH phosphorylation sequence 19. GRTH/DDX25 phosphorylation is relegated exclusively to T239 in the core region and not within any of the conserved domains of the DDX family. The GRTH phospho-species is essential for the cytoplasmic functions of this helicase, including shuttling of messages to the chromatoid body and polyribosomes, maintenance of the structure of the chromatoid body of the round spermatid, and the expression of messages relevant for completion spermatogenesis20. Moreover, the phospho-species differs from the non-phospho form whose functions are concerned with the transport of messages from nuclear to cytoplasmic sites through a CREM related mechanism. The phospho-site of GRTH resides in the structure adjacent to R242 found as a heterozygous missense mutation to histidine in a Japanese population of infertile men12. This mutation abolishes phosphorylation of T239, and we have also identified in this study other single and double mutants, not within the PKA consensus sequence, that are essential for phosphorylation. Molecular modelling based on the crystal structure of the RecA domain 1 of DDX19, which shares overall 65% amino acid identity with GRTH, has identified relevant structural motifs on the GRTH surface required for productive association with PKA. The binding site is rich in basic and acidic residues that engage in a complex network of intramolecular H-bonds unique to DDX25. The loop containing the PKA-targeting motif is amid such arrangement and then susceptible to structural changes of the local environment. The mutual proximity of several charged/H-bond-prone residues renders the binding pocket unstable, leading to topographic reconfiguration upon local perturbations with significant inhibition or abolition of phosphorylation by PKA.There is growing evidence that post-transcriptional modifications, including phosphorylation, of members of the DDX family regulate protein function. These modifications are mostly present in the divergent flanking sequences , presumably with specific inherent functions; some of these, however, are also present in core region21.Molecular modeling and mutagenesis analysis led us to conclude that disruption of strong intramolecular H-bonds  is the reason for the major reduction of T239 phosphorylation by single mutations (E165A or K240A). Complete abolition was achieved only by the double mutation (E165A and K240A). Overexpression of the PKA catalytic subunit (PKAc) or its endogenous generation/activation by cAMP partially relieved the reduction induced by the single mutations but was ineffective in the case of the double mutants, in which case complete abolition was retained  and in vivo, leading to abrogation of spermatogenesis in mice13. The local topography of GRTH which is relatively shallow with no deep crevices  oral male contraceptive. The results of this study suggest that a small, drug-like molecule that bind to GRTH at the GRTH/PKA interface could trigger modest by consequential local perturbations with similar effects to those revealed by mutations ces Fig. , may at PfuUltra HF DNA polymerase with the oligonucleotide primers containing the required point mutation (see Table Point mutations of human GRTH-plasmids were prepared using the QuikChange II Site-Directed Mutagenesis Kit (Agilent technologies) as per the protocol. PCR reaction was setup using mutagenesis-grade 3. The plasmid DNA was sequenced and confirmed by the dideoxy-nucleotides chain termination method. COS-1 (ATCC® CRL-1650™) cells were cultured in T75 flask at 37 °C with 5% CO2 containing Dulbecco Modified Eagle Medium (DMEM) high glucose, GlutaMaxTM Supplement, HEPES  supplemented with 10% fetal bovine serum and 1X Antibiotic-Antimycotic . Human pGRTH-Sport constructs of wild type (WT) and mutants  was used in the present studyTo evaluate the effect of protein kinase A (PKA) on phosphorylation of GRTH-WT and mutants, COS-1 cells were transfected with plasmids (15 μg) expressing full-length GRTH-WT and mutants  alone or co-transfected with plasmid (15 µg) expressing the PKA α catalytic subunit (PKAα) in 10 cm culture dish and empty plasmid was used for equalization, and cultured further for 24 hr after transfection. Cytoplasmic protein was prepared as described below for analysis by Western Blots. In other studies, 8-bromo, 0.05 µM , was added to COS-1 cells transfected with full length GRTH cDNA and further incubated for 24 h at 37 °C.1 or custom made affinity purified phospho-site-specific GRTH polyclonal antibody raised in rabbit to the peptide sequence (CKLIDL[pT239]KIRV) of GRTH (1:2000). Goat anti-Rabbit IgG (H + L) Poly-HRP (1:5000) was used as the secondary antibody and the immunosignals were detected by the FluorChem E system . PKA and pCREB was detected using PKA-Cα and pCREB specific antibodies , respectively. The 61 kDa pGRTH band intensity was measured using ImageJ software and normalized with β-actin.Nuclear and cytoplasmic protein extracts were prepared from COS-1 cells using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents , containing 1 × protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) following the manufacturer’s protocol. Cytoplasmic extraction reagents (CER I and II) were added to the cell pellet and centrifuged at ~16,000 × g to obtain the supernatant (cytoplasmic fraction) leaving the insoluble pellet (nuclear fraction) which was then suspended in ice-cold nuclear extraction reagent (NER). The pellet was vortex for 15 secs for every 10 mins, for a total of 40 mins to extract the nuclear fraction. Concentration of protein for each extract was determined using Quick Start™ Bradford Protein Assay . Protein (30 µg) separated by 4–12% Bis-Tris Protein Gels was transferred to nitrocellulose membranes and incubated with a specific affinity-purified anti-GRTH rabbit polyclonal antibodyCOS-1 cells were transfected with GRTH-V5-His construct alone or co-transfected with the PKA-Cα construct, were lysed using RIPA lysis buffer containing halt protease and phosphatase inhibitor cocktail (ThermoScientific). Total lysates (0.5 mg) were initially subjected to preclearing by incubation with 50 μl of protein A/G-agarose beads and 1 μg of rabbit IgG in IP binding buffer (ThermoScientific) with gentle agitation for 30 mins at 4 °C. Then the supernatant was incubated with either 4 μg of V5 antibody or PKA-Cα antibody overnight at 4 °C to co-immunoprecipitate PKA-Cα or GRTH-V5, respectively. Protein A-agarose beads (50 μl) was added and incubated for 4 h at 4 °C. The IP complex bound to protein A-agarose was washed four times with IP binding buffer and later eluted from beads using 1x LDS sample buffer (ThermoScientific) at 100 °C for 5 mins. The eluted samples were subjected to Western blot analyses for detection GRTH using their respective antibodies.24. DDX19 shares about 65% sequence identity with DDX2525. Only the domain 1, which contains the DEAD-box and R242 was considered, comprising a total of 233 residues (H80 through L302). Upon removal of ATP and RNA, the wild-type DDX25 protein was subjected to a 20-ns molecular dynamics (MD) simulation to obtain a relaxed conformation, which was taken as the initial structure for all subsequent simulations. Single or double mutants  CHARMM force field (c42 version) with PMAP corrections26 in a cubic cell of ~9 nm of side length, filled with TIP3P water, at constant temperature (35 °C) and pressure (1 atm). Cubic PMC and PME were for the treatment of electrostatics. . Upon gradual heating and equilibration, the simulations were extended up to 50 ns and the last 30 ns were used to collect sufficient statistics for analysis.The structure of DDX25 was modeled by threading its amino acid sequence on the structure of DDX19, which was co-crystalized with an ATP analog and a short RNA fragment (PDB ID: 3G0H)Data are presented as the mean ± SEM of three independent experiments. Mean values of the data was compared and analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using the Prism software program . A probability of P < 0.05 was considered statistically significant.Supplimentary Figures [Full Blots]