Since both peptides exhibited inhibitory capability of the p38α kinase mediated phosphorylation of MEF2A, in a subsequent step we pursued the discovery of small molecule peptidomimetics. For this purpose we characterized in detail the peptide-p38α interaction using molecular dynamics simulations, leading to the definition of a pharmacophore for the peptide-protein interaction. This hypothesis was used as query for a in silico screening, leading to the discovery of a fused ring compound with micromolar inhibitory activity. Site-directed mutagenesis studies support that the compound binds to the putative novel allosteric site in p38α.In the present study, we report the discovery of a novel allosteric inhibitory site for p38α, a subclass of the mitogen-activated protein kinases (MAPK) family. The putative site was discovered after inspection of the crystallographic structure of the p38α-MK2 complex. MK2 (MAPK-activated protein kinase 2) is an interesting protein playing a dual role as modulator and substrate of p38α. This intriguing behavior is due to the ability of the two proteins to form distinctive heterodimers when p38α is phosphorylated or not. We hypothesized that the regulatory action of MK2 is due to its capability to keep p38α in an inactive conformation and consequently, we investigated the atomic structure of the p38α-MK2 complex to understand such regulatory behavior at the molecular level. After inspection of the complex structure, two peptides designed from the MK2 regulatory loop in contact with p38α with sequences Tyr Protein phosphorylation is the most widespread post-translational modification used in signal transduction. This process is catalyzed by protein kinases, a large family of highly related enzymes covering about 2% of the human genome . ProteinDysregulation of kinase activity can result in dramatic changes directly affecting the control of the above mentioned processes, being responsible for the onset and/or progression of various human diseases including inflammatory, cardiovascular, metabolic, neurodegenerative and cancer . AccordiIn contrast to type I kinase inhibitors, allosteric inhibitors induce a redistribution of the kinase conformational ensemble, increasing the population of inactive conformations through the displacement of specific motifs that are key for the catalytic activity from their optimal positions. Moreover, unlike the specific requirements of the active conformation responding to a set of highly conserved features, impairing activation allosterically can be done in diverse ways. Thus, type II and III kinase inhibitors bind to allosteric subsites next to the ATP binding site that emerge through the acquisition of the inactive conformations DFG-out and αC-out. Interestingly, in contrast to the latter, the former type of inhibitors are ATP competitive. Some of the allosteric inhibitors described in the literature are exceptionally selective while others are not, stressing the point that allosteric inhibitors are not necessarily selective. Actually, inhibitors that bind to remote areas from the ATP-binding site typically present a great degree of selectivity, due to the fact that these sites are generally less conserved in sequence and structure. This kind of inhibitors are known as type IV kinase inhibitors, and they mainly exert their function allosterically by stabilizing inactive conformations or through the blockade of interactions with other proteins .D = 2.5 nM) where the ATP-binding sites of both kinases are buried in the heterodimer interface, preventing them from phosphorylating their respective substrates -peptide-1 (peptide-2), in their zwitterionic form. We further analyzed the potential of this site for the design of allosteric peptidomimetic inhibitors by means of a detailed study of the structural features of the peptide-p38α interaction, leading to the development of a pharmacophore that was subsequently used for the discovery of small molecule hits by virtual screening.Accordingly, we investigated the 2) during the subsequent 70ns at a constant volume. At this point restraints were lifted and the system was allowed to run for another 100ns. All the simulations were carried out using the program pmemd of the AMBER12 software -peptide-1 (peptide-2) in their zwitterionic forms were selected to test their phosphorylation inhibitory capability mediated by p38α in vitro. Despite peptides did not show any phosphorylation inhibition capability using the p38α peptide substrate, they exhibited inhibitory capability of the p38α mediated MEF2A phosphorylation in a concentration dependent manner. Two fragments of the MK2 regulatory loop in contact with p38α, with sequences: TyrIn a subsequent step, aimed at discovering small molecule mimics of the peptides with inhibitory capability of p38α, we focused on the characterization of the structural features of the peptide-p38α interaction. For this purpose we run a 200ns molecular dynamics simulation of peptide-1 bound to p38α to allow the peptide to adapt to the receptor. First, since the p38α activation loop is not complete in the crystallographic structure, it was modelled as described in the methods section and its structure refined in the dimer complex environment by means of a 10ns MD simulation. As shown in 2 with the amide nitrogen of Gly5, the nitrogen-delta of the imidazole ring of His4 and the amide nitrogen of the same residue; an interaction between the nitrogen-delta of the imidazole ring and the amide nitrogen of His4; and an interaction between one of the oxygen atoms of the carboxyl terminal group of Leu6 and the amide hydrogen of Ser2.Since the conformation of the peptide remains stable along the MD run, we computed the minimized average structure of the last 40ns of the MD simulation and used as the bound conformation of peptide-1 to p38α . In the 3 with the polar hydrogens of the Arg189 guanidine group in p38α, which also interacts with the oxygen carbonyl atom of the peptide-1 Asn3 backbone. On the other hand, the hydrogens of the amide group located on the side chain of peptide-1 Asn3 interact with the carbonyl oxygen of p38α Thr180 backbone. Moreover, a fourth interaction involves the nitrogen-epsilon of the imidazole ring of peptide-1 His4 side chain with the carbonyl oxygen of p38α Asp177 backbone and the carboxylic atoms of its side chain. Finally, there is a hydrogen bond between the carboxylate oxygens of Leu6 and the polar hydrogens of p38α Arg186 guanidine group.Analysis of the hydrogen bond interactions between peptide-1 and p38α along the molecular dynamics trajectory reveals five relevant interactions as shown in 3 and His4 are those that contribute the most to the binding free energy, and together Tyr1 exhibit the largest van der Waals contribution  is also a hydrophobic/aromatic moiety situated on the centroid of the of the His4 side chain; point three (P3) is a hydrogen bond acceptor located on the carbonyl oxygen of Asn3 side chain; point four (P4) is a hydrogen bond acceptor corresponding to the carboxylic oxygen of Leu6 interacting with Arg186; and point five (P5) is a hydrogen donor point, mimicking the hydrogen bond interactions observed between the NH group of the imidazole ring of His4 and the backbone carbonyl oxygen and side chain oxygen atoms of Asp177. These points fulfill specific geometrical constraints that are defined by spheres centered on each of the points with various radii to account for the tolerance in the fulfillment of each of the points. The coordinates of the points referred to an arbitrary origin and the radius (r) of the spheres in Angstroms are: P1 , r = 1.5; P2 , r = 1.5; P3 , r = 1.2; P4 , r = 1.2; P5 , r = 1.2.Based on the analysis of the molecular dynamics trajectory, we could characterize the interaction between peptide-1 and p38α through a five point pharmacophore defined on the peptide that includes the most relevant interactions identified : Point oThe pharmacophore described above was subsequently used for virtual screening as described in the methods section. The screening study permitted to identify around 1300 compounds fulfilling the pharmacophore requirements in ca. 4700 conformations. These molecules were subjected to a docking study that permitted to reduce the set to 452 compounds. In order to reduce further the number of hits, molecules were subjected to a diversity analysis . As a re50 of 16 μM.The chemical structure of the most active compound discovered (UPC-K-005) is shown in In order to confirm that its phosphorylation inhibitory capability is produced through the novel site proposed, we first carried out competition experiments with ATP to demonstrate the compound behaves as a non-competitive inhibitor and second, we measured its binding affinity to two p38α mutants.ATP competition experiments were carried out by measuring the inhibitory capability of UPC-K-005 at different ATP concentrations  the tertiary tetrahydropyridine ring nitrogen will be protonated in aqueous solution [177, as required to fulfill pharmacophore point P5.To understand the features of the ligand-protein interaction we carried out a docking study of UPC-K-005 to p38α. solution , so that366-Ala390, bound to the hydrophobic docking groove of p38α, the CD domain involving residues Asp313, Asp315, and Asp316 and the ED site involving residues Glu160-Asp161. As mentioned above, the peptide corresponding to the fragment 370–400 of MK2 has been shown to be a potent inhibitor of p38α-dependent phosphorylation of MK2 and ATF-2, suggesting that the inhibitory capability of these D-motifs is not limited to block a specific substrate only [There are numerous examples in the literature where protein-protein interactions can be effectively inhibited using peptide sequences inspired by contact regions of the protein complex . This isate only .in vitro assay and furthermore, show evidence that the compound is not an ATP-competitive inhibitor and that binds to the same regulatory phosphorylation region of p38α, based on the decrease of its inhibitory potency shown with the Arg186Ala and Arg189Ala p38α mutants.Present results show evidence of the inhibitory profile of peptide-1 and -2 in the phosphorylation of MEF2A. As mentioned before, both peptides are segments of the MK2 regulatory loop in contact with p38α and identified after the analysis of the crystallographic structure of the MK2-p38α complex . Based oDespite the small molecules discovered were designed to mimic peptide-1, experimental results show a differential inhibitory behavior between the peptides and UPC-K-005. Whereas peptides inhibit p38α phosphorylation in a dose-response form when MEF2A is used as substrate, they fail to inhibit p38α phosphorylation when the p38α peptide substrate is used. In contrast, UPC-K-005 inhibits both the p38α peptide substrate as well as ATF2 protein substrate in a dose-response manner, suggesting that they are more universal inhibitors. This intriguing behavior suggests that peptides and small molecules, in spite of binding to the same putative binding site they may inhibit phosphorylation through different mechanisms. A plausible explanation for this differential behavior is that peptides act by blocking the binding of a substrate protein, whereas small molecules may induce a conformational change in p38α forcing the protein to be in an inactive state. In other words, the former inhibit phosphorylation through a protein-protein inhibition disruption mechanism, whereas the small molecules may inhibit phosphorylation through an allosteric mechanism that stabilizes p38α in an inactive conformation. This is an interesting hypothesis that should be considered as preliminary and requires further investigation currently being carried out in our laboratory.50 of 13 μM and is disclosed in the present work. In order to test that the compound binds to the new proposed site, we measured the phosphorylation inhibitory capability of UPC-K-005 on the p38α mutants Arg186Ala and Arg189Ala. The results clearly show a lower inhibitory capability in comparison to the wild type. In parallel the inhibitory capability of SB203580, known to bind to a different site [We describe in this work a novel allosteric binding site on p38α that corresponds to one of the contact regions of the p38α-MK2 heterodimer where p38α is not activated. The two proteins interact at this site through two interconnected loops stabilized by several hydrogen bonds including an anti-parallel β-sheet. In order to check the functional profile of the site, we first tested the inhibitory capability of two amino acid fragments of the MK2 sequence. The results of the test show the peptides with an inhibitory capability of p38α at the micromolar range. We further identified the key interactions between the two segments through a short molecular dynamics and defined a pharmacophore for the interaction defined by five points: two proton accepting groups, a hydrogen donor group and two aromatic/hydrophobic groups. This pharmacophore was used to carry out a virtual screening process using the MOE lead-like database. The results of these studies permitted to identify a few small molecule inhibitors of p38α. The most active (UPC-K-005) exhibits a ICent site  was measS1 Tablep38α activity measurements and normalization at diverse concentrations of ATP in the presence of compound UPC-K-005. SB203580 is also studied for comparison purposes.(DOCX)Click here for additional data file.


Calotropis procera leaf, as well as its possible mode of inhibiting these enzymes. Acetone, aqueous and ethanolic extracts of C. procera leaf was subjected to standard enzymes’ inhibitory assay in-vitro using porcine pancreatic α-amylase and rat intestinal α-glucosidase. Results obtained showed that out of all the extracts tested, ethanolic and aqueous extracts possessed the best inhibition of α-amylase (IC50 7.80 mg/mL) and α-glucosidase (3.25 mg/mL) respectively. The kinetic analysis of the mode of inhibition of these enzymes by the leaf extracts of C. procera, revealed that these extracts inhibited both enzymes in a non-competitive manner. It is speculated that the α-amylase and α-glucosidase inhibitory properties of leaf extracts of C. procera may be due to the presence of some phytochemicals such as flavonoids, tannins and saponins in the plant. It can be concluded from this study that the Calotropis procera extracts could serve as source of antidiabetic agents which may act through the inhibition of carbohydrate hydrolyzing enzymes, α-amylase and α-glucosidase.The side effects associated with the usage of synthetic antidiabetic drugs make it imperative to search for alternative drugs from medicinal plants. Therefore, this study was aimed at evaluating the α-amylase and α-glucosidase inhibitory potential of  Blighia sapida, Morus alba, Spondias monbin, Treculia africana and Calotropis procera  is a perennial, greyish-green, woody shrub with broad fleshy leaves that grows wild in the tropics and in warm temperate regions , wh, whC. protective  and antiotective .C. procera  was deposited in the University herbarium.The plant Chemicals Porcine pancreatic α-amylase, rat intestinal α-glucosidase and paranitrophenyl-glucopyranoside (PNPG) were products of Sigma-Adrich Co., St Louis, USA while starch soluble (extra pure) was obtained from J. T. Baker Inc., Phillipsburg, USA. Other chemicals and reagents were of analytical grade and water used was glass-distilled.Preparation of plant extractsCalotropis procera were cut and washed with water to remove all contaminants; they were dried under room temperature and grounded to powder. The powdered leaves were divided into three portions and each portion was dissolved in either acetone, ethanol or water. They were all left to steep in covered containers for 24 h; the resulting infusions were decanted and filtered. Acetone and ethanolic extracts were evaporated in a rotary evaporator , while aqueous extract was freeze-dried using Virtis Bench Top  freeze dryer. Dried extracts were weighed and dissolved in 10% dimethylsulphoxide (DMSO) to yield a stock solution from which lower concentrations were prepared.Fresh leaves of Phytochemical screening Phytochemical compositions of the leaves were determined using the methods variously described by Trease and Evans  and Sofoα-Amylase inhibitory assayoC for 10 min, after which 250 µL of 1% starch solution in 0.02 M sodium phosphate buffer (pH 6.9) was added at timed intervals and then further incubated at 25 °C for 10 min. The reaction was terminated after incubation by adding 500 µL of dinitrosalicylic acid (DNS) reagent. The tubes were then incubated in boiling water for 5 min and cooled to room temperature. The reaction mixture was diluted with 5 mL distilled water and the absorbance was measured at 540 nm using spectrophotometer. A control was prepared using the same procedure replacing the extract with 10% DMSO. The α-amylase inhibitory activity was calculated as percentage inhibition.This assay was carried using a modified procedure of McCue and Shetty  and the control-Absextracts)/Abscontrol] x 100% Inhibition = [(Abs50) were determined graphically.Concentrations of extracts resulting in 50% inhibition of enzyme activity  of inhibition of alpha amylase activity by the extract was determined by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis-Menten kinetics.The mode of inhibition of the enzyme by the leaf extract was conducted using the extract with the lowest ICi et al. . Brieflyα-Glucosidase inhibitory assayet al. /Abscontrol] x 100% inhibition = [(Abs50) were determined graphically.Concentrations of extracts resulting in 50% inhibition of enzyme activity  where v is reaction velocity and [S] is substrate concentration was plotted. The mode of inhibition of α-glucosidase activity by the extract was determined by analysis of the double reciprocal (Lineweaver-Burk) plot using Michaelis-Menten kinetics.The mode of inhibition of the enzyme by the leaf extract was conducted using the extract with the lowest ICi et al. . BrieflyStatistical AnalysisStatistical analysis was performed using GraphPad Prism 5 statistical package . The data were analyzed by one way analysis of variance (ANOVA) followed by Bonferroni test. All the results were expressed as mean ± SE for triplicate determinations.Calotropis procera leaves. It shows that flavonoids, reducing sugars and steroids are present in both the ethanolic and aqueous extracts while tannins was detected in acetone and aqueous extracts. However, saponin was present in the aqueous extract only. Calotropis procera. At all concentrations tested, there were no significant differences (p > 0.05) among the values obtained for acetone, ethanolic and aqueous extracts of the plant except at 10.00 mg/mL where the percentage inhibition value for ethanolic extract was significantly different (p < 0.05) compared to the other extracts. Extrapolation of α-amylase inhibition effectiveness from the dose – response curve showed that ethanolic extract contained the most potent α-amylase inhibitor with an IC50 value of 7.80 mg/mL . α-glucosidase inhibition by the different extracts was found to be dose dependent. At lower concentrations (0.63 and 1.25 mg/mL), there were significant differences (p < 0.05) between the ethanolic and aqueous extract in the inhibition of the enzyme but no significant difference (p > 0.05) between acetone and ethanolic nor between acetone and aqueous extracts. At higher concentrations, significant differences (p < 0.05) were noted between ethanolic extract when compared to either acetone or aqueous extract but there was no significant difference between acetone and aqueous extracts. Aqueous extract of this plant displayed the lowest IC50 (3.25 mg/mL) for the inhibition of α-glucosidase  of the extract is required to effectively inhibit α-amylase compared to the α-glucosidase which was obtained at 3.25 mg/mL. This is attested to by the respective IC50 generated for the inhibition of the enzymes by the extracts. This is in agreement with previous report that any medicinal plants that may be used as antidiabetic agents should be a mild inhibitor of α-amylase and strong inhibitor of α-glucosidase , a major mediator of glucose removal from the circulation and a key regulator of whole-body glucose homeostasis. It also helps in the repression of the key gene responsible for adipogenesis thereby helping to reduce blood glucose level without increasing the adiposity (The inhibitory effect of the ethanolic and aqueous extracts of diposity . FlavonoCalotropis procera tested, the ethanolic extract displayed the most effective inhibition of α-amylase while aqueous extract displayed the most effective inhibition of α-glucosidase in-vitro and the mode of inhibition of both enzymes is the non-competitive one. The potent inhibitory activities of this plant may be due to the synergistic effect of phytochemicals present in it.  It can be concluded from this study that out of the three extracts of


Paper focuses on biomechanics, specifically on locking cortical bone screws in angularly stable plates used for the treatment of bone fractures in the medical fields of traumatology and orthopaedics. During extraction of titanium-alloy implants, problems are encountered in an effort to loosen some locking bone screws from the locking holes of an angularly stable plate and the subsequent stripping of the internal hexagon of the screw head. The self-locking of the screw-plate threaded joint was verified by calculation and the effect of the angle of the thread on the head of the locking cortical bone screw on self-locking was evaluated. The magnitude of the torque, causing the stripping of the internal hexagon (the Inbus type head) of a locking cortical bone screw with a shank diameter of 3.5 mm from Ti6Al4 V titanium alloy to ISO 5832-3, was determined experimentally. Also, it was experimentally found that the rotation of the screwdriver end with a hexagonal tip inside the locking cortical bone screw head during stripping of the internal hexagon causes strain of the screw head perimeter and thereby an increase of thread friction. The effect of tightening torque on the possibility of loosening of the locking cortical bone screw from the locking hole of an angularly stable plate was assessed experimentally. From the evaluation of five alternative shapes of locking cortical bone screw heads in terms of the acting stress and generated strains, it follows that the best screw is the screw with the Torx type head, which demonstrates the lowest values of reduced stress and equivalent plastic strain. Based on experiments and simulations the authors recommend that all global producers of locking cortical bone screws for locking holes of angularly stable plates use the Torx type heads, and not heads of the Inbus type or the Square, PH, PZ types. Traumas of the skeleton are treated using trauma implants which are applied into the body permanently or until the fracture heals or requires re-operation –4. SometThe principle of angularly stable plates is based on the solid fixation of the screw conical head in the plate hole by the screw thread. The thread on the conical screw head must have the same or smaller pitch than the thread on the shank (bone thread). The thread on the screw head can be multi-thread. During screw tightening, bone fragments must not be compressed (or decompressed) against the plate (or from the plate). Angularly stable plates are produced as direct plates and also as shaped plates according to the anatomical shape of the bone surface , 3, 4 F.Fig. 1aThe system of angularly stable plates is based on the internal fixator principle. An angularly stable connection of the plate system with the bone is achieved using a fixed screw-plate connection. Thereby, part of the load of the plate system is transferred from the screw-bone connection to the screw-plate connection. Therefore, the system can be used successfully even in lesser quality and osteoporotic bones.If an angularly stable plate is used with locking screws, then the plate and screws together form a sufficiently strong and rigid system. The plate does not need to be pressed against the bone; therefore, the bone´s blood supply is not restricted. Locking screws can be applied unicortically, without compromising the strength of the system or fracture fixation, because the screws are fixed in the plate.According to the operating procedure, the locking cortical bone screws in an angularly stable plate must be tightened manually through a torque limiter  with theNevertheless, even when keeping to this procedure, during the extraction of titanium-alloy implants the problems with loosening of some locking bone screws Fig.  from theRm = (860 ÷ 1050) MPa. The modulus of elasticity of titanium alloy E = 113.5 GPa, the modulus of elasticity of stainless steel E = 210 GPa, so the titanium alloy is 145% more elastic. A comparison of properties of different materials can be made by the method specified in [Trauma implants are produced using austenitic steel 1.4441 according to ISO 5832-1  and titanium alloy Ti6Al4 V according to ISO 5832-3. For both these materials the tensile strength is the same:  = 860 ÷ 50 MPa. TFor the analysis of locking self-taping bone screws for angularly stable plates the laboratory experiments and the numerical simulations were carried out.The experiments ,α = φ … self-locking limit of threaded joint (equilibrium is achieved only through the effect of sliding friction in threads),α < φ … self-locking threaded joints ,Self-locking is achieved according to the ratio of the thread angle β = 60º (metric thread),thread peak angle on screw head: p = 0,4 mm,thread span (distance between adjacent threads): s = 2p = 0,8 mm ,thread pitch (distance for one rotation): friction angle in threads: f = 0.11–0.17,sliding coefficient: α ≤ 3.3º.thread pitch angle: In the case of the locking cortical bone screw in question Fig. , productThe result is shown in Sect. The diameter of the head of each tested locking bone screw with a shank diameter of 3.5 mm from titanium alloy Ti6Al4 V according to ISO 5832-3 was measured 10 times at various points while rotating the screw using a Mitutoyo digital micrometer with the range of (0–25) mm, accuracy 0.001 mm, made in Japan, see Fig. A KRAFTWERK torque screwdriver—model 2039, range (1–4) Nm ± 6.0%, according to ISO 6789 Fig. , which wThe magnitude of the torque causing stripping of the internal hexagon in the locking cortical bone screw head was determined experimentally in the laboratory. The locking bone screw was fixed with a strong clamping force by its shank in a bench vice to prevent its rotation during the application of the torque screwdriver. A torque screwdriver with a 2.5 mm hexagonal bit was used for the experiment , or 2 Nm (for screws 2 and 3) respectively, upwards in 0.1 Nm increments. Then the screwdriver was rotated manually with a 2.5 mm bit in the locking bone screw head. With the low torques set, the internal limiter of the screwdriver always engaged which indicated that at that particular torque setting the internal hexagon in the bone screw head was not stripped, see Fig. A comparison of the measured values of mean diameter of the Inbus type head of locking bone screw with a shank diameter of 3.5 mm from Ti6Al4 V titanium alloy according to ISO 5832-3 before and after the stripping of the internal hexagon in the screw head and the calculated mean value and the absolute error of measurement—for screws 1–5 is seen in Table The results are commented on in Sect. It is also important to determine whether the rotation of the end of the screwdriver with a 2.5 mm hexagonal bit inside the locking cortical bone screw head, during the stripping of the internal hexagon, causes greater strain of the screw head perimeter. Greater strain of the screw head leads to an undesirable increase of friction of the thread in the locking hole of the angularly stable plate. Therefore, the diameter of each locking bone screw head  Nm  according to ISO 6789. On the screwdriver dial, torque values were set from 0.1 Nm upwardly in 0.1 Nm increments. A 2.5 mm hexagonal bit was used for experiments. The plate in question was gripped in a workbench vice.First, the locking cortical bone screws with a shank diameter of 3.5 mm and with the Inbus type heads were screwed into the locking holes of an angularly stable plate by the torque screwdriver at the set torque. Subsequently the screws were unscrewed using the torque screwdriver. During loosening of the bone screws from the holes of the angularly stable plate the dial of the torque screwdriver was initially set to lower torque values than the torque necessary to screw in the given screw, and subsequently the torque was increased in 0.1 Nm increments until the screw was released and unscrewed.The measured torque values for respective screws with the Inbus type heads in specific locking holes of an angularly stable plate are shown in Tables The results are commented on in Sect. The evaluation of various head shapes of locking cortical bone screws with respect to the acting stress and produced strains was carried out by the numerical simulations.A head geometry was found for the most commonly used screw head types – Inbus, Square, PH, PZ and Torx Fig. , and thi−1 (corresponds to 5 s−1) was prescribed to the screwdriver. The screw head was defined as a real (deformable) body. Friction between both bodies was set by the Coulomb friction coefficient f = 0.05.During screwing, the rotating movement of the screwdriver  with the screw head exists. In the FEM analysis the screwdriver was defined as an absolutely rigid (non-deformable) body which is an acceptable simplification of the real state. A rotational velocity of 0.0873 rad sThe boundary condition of the absolutely rigid rotating body will, to a certain extent, distort the real values of reduced stress, nevertheless, for comparison of the various screw head shapes  the utilization of this calculation model is suitable.The screw head was fixed at its external edge, i.e. outer radius, in all directions, which corresponds to rigid fixation (screw head is fixed in plate). In all models the finite elements grid was made finer in areas of expected high values of stress and plastic strain. The finite elements grid was formed by type 11 elements (four-node element in MSC.Marc/Mentat software). All analyses  were set identically.Reduced stress according to the HMH (von Mises) theory, equivalent plastic strain and the relation between the tightening torque and screwdriver torsion angle were analysed in the screw heads.Analyses were calculated simply—as 2D calculations (planar strain).Re = 450 MPa, ultimate strength Rm = 1000 MPa, modulus of elasticity E = 110 GPa, fatigue strength (107 cycles) σf = 289 MPa, hardness to Brinell HB = 225. This material is very similar to the real material of tested screws  is fulfilled  between the rigid body (screwdriver) and deformed body (screw head).Rotation of the screwdriver by 5° generates a reduced stress in the screw head of 881.6 MPa, see Fig. Figure A discussion of the results is shown in Sect. The maximum reduced stress value again occurs in the area of the expected maximum, i.e. in the area of contact of the rigid body (screwdriver) with the deformable body (screw head).Rotation of the screwdriver by 5° generates a reduced stress in the screw head of 1041 MPa, see Fig. Figure A discussion of the results is shown in Sect. The maximum reduced stress value again occurs in the area of the expected maximum, i.e. in the area of contact of the rigid body (screwdriver) with a deformable body (screw head).Rotation of the screwdriver by 5° generates a reduced stress in the screw head of 1647 MPa, see Fig. Figure A discussion of the results is shown in Sect. The maximum reduced stress value again occurs in the area of presence of short arms of the screw, see Fig. Rotation of the screwdriver by 5° generates a reduced stress in the screw head of 2205 MPa, see Fig. Figure A discussion of the results is shown in Sect. The maximum reduced stress value again occurs in the area of the expected maximum, i.e. the area of contact of the rigid body (screwdriver) with a deformable body (screw head).Rotation of the screwdriver by 5° generates a reduced stress in the screw head of 656.4 MPa, see Fig. Figure With respect to lower values of stress and strain the Torx type head is better than the Inbus type head.A discussion of the results is shown in Sect. Verifying of the self-locking of the threaded joint by calculation and evaluation of the effect of thread pitch on the locking cortical bone screw head on self-locking was carried out  of the locking cortical bone screw with a shank diameter of 3.5 mm from titanium alloy Ti6Al4 V to ISO 5832-3 was experimentally determined  causes strain of the screw head perimeter and thereby increased thread friction ; also the equivalent plastic strain is lowest from the analysed heads (plastic strain is approximately 21.3% lower than in screws with the Inbus type head).The Inbus type head of the locking cortical bone screw was analysed experimentally and also numerically (by simulations using the Finite Elements Method). Very similar results were achieved by both approaches (arising of plastic strain in the most stressed area of the screw head when loading by torque) so it is possible to state that the results are reliable for the Inbus type head.The Inbus and the Torx type heads of the locking cortical bone screws are usually used in medical practice  and so it is not necessary to test their utilization clinically.The remaining head types  of the locking cortical bone screws are used in technical practice  but not in medicine. However the Square, PH and PZ type heads of the locking cortical bone screws are used in this paper only as alternatives for the comparison. The numerical simulations of the Square, PH and PZ type heads of the locking cortical bone screws which show worse results than the Inbus and the Torx type heads of the locking cortical bone screws support the right idea of not using them in medical practice.Because a sufficiently good sameness of the experimental and the numerical results for the Inbus type head of the locking cortical bone screw was achieved it is possible to state that the numerical simulation of other head types  are also sufficiently exact even without experiments.The highest stress and plastic strain is generated in the screw with the PZ type head.Therefore, the best choice is to use screws with the Torx type head, which reaches the lowest values of reduced stress and also the smallest equivalent plastic strain . From the results, it decidedly follows that the Torx type head is the most suitable for medical practice, the Inbus type head is acceptable for medical practice and the Square, PH and PZ type heads are unsuitable for medicine practice.Figure The effect of tightening torque on the possibility of unscrewing of a locking cortical bone screw from a locking hole of an angularly stable plate was evaluated experimentally. It was found that when the locking bone screw with a shank diameter of 3.5 mm is tightened with a tightening torque of 0.1 Nm in a locking hole of an angularly stable plate, the threaded joint is very strong—without any clearance. The results of experiments (see Sect. Based on the evaluation of five alternative head shapes of locking cortical bone screws with respect to the acting stress and generated plastic strains by simulations in MSC.Marc/Mentat software (see Sect. Based on experiments and simulations the paper´s authors recommend that all global producers of locking cortical bone screws for locking holes in angularly stable plates use the Torx type heads and not heads of the Inbus or the Square, PH, PZ types.


N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D) and two GluN3 (A–B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors  than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics. Therefore, NMDA receptors are fully activated only during concurrent binding of agonists and depolarizing membrane potential, thus acting as co-incidence detectors. Additionally, there are two variants of GluN3 (A-B) subunits that can co-assemble with GluN1 to form excitatory glycine receptors  receptor is a subtype of the ionotropic glutamate receptor (iGluR) family. NMDA receptors have been implicated in the pathogenesis of several neurological and psychiatric disorders including Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, and schizophrenia  chemical groove that is stabilized by intersubunit interactions at three different points, Site-I, II, and III , or Hellobio Ltd., Bristol, UK .pci_sepGluN2B) was purchased from Addgene, Cambridge, MA. GluN1 mutants  were generated by site-directed mutagenesis  and confirmed by DNA sequencing. Plasmids were linearized with NotI  or Xba1 (GluN2B), and transcribed in vitro with T7 , SP6 (GluN1 mutants) RNA polymerase using the mMessage mMachine transcription kits .NMDA receptor constructs: cDNA encoding the NMDAR1a subunit (GluN1a) was a generous gift of Dr. Nakanishi . cDNA encoding the GluN2B  into the oocyte cytoplasm. Oocytes were incubated in ND-96 solution at 18°C prior to electrophysiological recordings (1–3 days). Electrophysiological responses were measured using a standard two-microelectrode voltage clamp [Warner Instruments  model OC-725C] designed to provide fast clamp of large cells. The recording buffer contained 116 mM NaCl, 2 mM KCl, 0.3 mM BaCl2, and 5 mM HEPES, pH 7.4. Response magnitude was determined by the steady plateau response elicited by bath application of agonists (10 μM L-glutamate and 10 μM glycine) at a holding potential of −60 mV. Response amplitudes for functional NMDA receptors were generally between 0.1 and 2 μA. After obtaining a steady-state response to agonist application, agonist plus test compounds were applied, using 8-channel perfusion system , on the oocytes and the responses were digitized for quantification . Dose-response relationships were fit to a single-site with variable slope , using a non-linear regression to calculate IC50 or EC50 and percentage maximal inhibition. Statistical Analysis: Values given represent means (±) S.E. In order to present only highly significant results, statistical significance was determined at the alpha level p < 0.01 (*) and p < 0.001 (**) using a student's two-tailed, unpaired t-test.GluN subunit expression and electrophysiology in Xenopus oocytes: Stage IV frog oocytes were obtained from Xenopus-I, . NMDA receptor subunit cRNAs were dissolved in nuclease free sterile H(N521A), increased the potency of glycine , glutamate  and 5,7-DCKA , that is a glycine site competitive antagonist . Since the GluN1 521st position is known to be crucial for intersubunit interactions in such a way that side chain length will remain the same as wild type, but only the reactive groups will be changed. The amino (NH2) group of asparagine is replaced by the carboxylic acid (COO−) group of aspartic acid in the N521D mutant. In contrast to the N521A mutant, the N521D mutant decreased the glutamate potency  side chain in GluN1/2B interactions. Site-II mutations (K531A and Y535A) did not significantly affect the glycine or glutamate EC50. However, a remarkable more than 25-fold significant increase  in 5,7-DCKA potency was observed with the Y535A mutant. Alternatively, DL-AP5 potency was not significantly changed by the site-II mutants. These results reveal that GluN1/2B intersubunit interactions through site-II largely influence the potency of competitive antagonist binding at GluN1, but have insignificant effect on glutamate site antagonist activity. The site-III mutation (E781A) was made close to the distal end of the GluN1/2B LBD interface, a region that is physically connected with the TMD and a more dynamic site of the LBD than the other two sites, as shown in Figure E781A mutation occurring at the GluN1 subunit, the potency of glutamate (that binds with GluN2) was significantly reduced . Converse to what was observed for glutamate, E781A increased the potency of the glycine site antagonist 5,7-DCKA , however, the potency of the glutamate antagonist, DL-AP5 was unaltered. This result corroborates the observations made with site I & II.The data obtained from the two electrode voltage clamp (TEVC) electrophysiology experiments reveal that GluN1/2B LBD interactions play a crucial role in determining potency of the ligands binding at the glycine or glutamate binding cleft of NMDA receptors. The topology diagram of the GluN1/2 dimer shows the location of the LBD, NTD, and TMD, and the three LBD interaction sites are numbered Figure . The sit Figures , Table 1N52, increasN521A) made ifenprodil seven-fold less potent  compared to the wild type receptors, Figures th position contributes to translating NTD mediated negative modulatory signals to the TMD.Ifenprodil is a GluN2B selective negative allosteric modulator binding at the interface of the GluN1/2B NTD, that is located upstream to the LBD, Figure 2+ is an endogenous antagonist that blocks the NMDA receptor with varying potency based on the GluN2 subunit combination. Zn2+ inhibits GluN2A with a high affinity by binding with NTD, and inhibits other GluN2 subunit containing NMDA receptors by binding only with the voltage dependent low affinity binding site at the TMD. The results obtained from the Zn2+ dose response curves show no significant changes in Zn2+ activity with the mutant receptors compared to the wild type receptors  molecules like memantine. To test this hypothesis, we have studied the effect of memantine, that binds at the extracellular vestibule of the ion channel pore by displacing endogenous Mg2+ ions, on mutant receptors. In agreement with our hypothesis, both site-I & II LBD mutations increased the potency of memantine . Further, the site-II mutant (Y535A) significantly reduced the efficacy of memantine relative to the wild type receptor  domains. More than 50 x-ray crystallographic structures are available either as LBD alone or together with upstream (NTD) and downstream (TMD) domains. Historically, the LBD was considered a promising drug target because early chemical tools developed to study NMDA receptors were found to bind at the glutamate or glycine site  values of the different compounds studied. The length of the amino acid sidechain at site I (GluN1 521st position) plays a crucial role in the interaction with the co-assembling GluN2B subunit that determines agonist potency. Increased 5,7-DCKA but unaltered DL-AP5 potency shows site-I has a relatively minor role in the overall inactivation process, which is largely controlled by the more distal part of the LBD (site-II). This is in agreement with the previous reports that site-II influences the receptor desensitization . In Alzheimer's disease, Aβ oligomers directly interact with the extracellular domain of NMDA receptor subunits and destabilize interdomain interactions to induce excitotoxicity  from the Edward Via College of Osteopathic medicine and Virginia-Maryland College of Veterinary Medicine funded to BC and BK.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.


Schizosaccharomyces pombe has two types of TOR complex, namely TOR complex 1 (TORC1) and TORC2. It is interesting that the two TOR complexes in S. pombe have opposite roles in sexual differentiation, which is induced by nutrient starvation. TORC1, which contains Tor2 as a catalytic subunit, promotes vegetative growth and represses sexual differentiation in nutrient-rich conditions, while TORC2 is required for the initiation of sexual differentiation. Multiple targets of TORC1 have been identified. Some of these, such as S6 kinase and an autophagy regulator Atg13, are known targets in other organisms. In addition, there is a novel group of TORC1 targets involved in the regulation of sexual differentiation. Here, we review recent findings on phosphorylation targets of TORC1 in S. pombe. Furthermore, we briefly report a novel S. pombe target of TORC1.Target of rapamycin (TOR) kinase controls cell metabolism and growth in response to environmental cues such as nutrients, growth factors, and stress. TOR kinase is widely conserved across eukaryotes. As in other organisms, the fission yeast  Saccharomyces cerevisiae, in which TOR kinase was originally identified [It is a fundamental property of cells to grow and decide when to divide by sensing environmental conditions. Target of rapamycin (TOR), a highly conserved serine/threonine kinase, acts as a key factor in this process, and orchestrates various growth-related functions in response to environmental conditions . TOR exientified , TORC1 centified . While TORC1-mediated signaling is unambiguously important and indispensable for eukaryotic cells, the current knowledge regarding TORC1 targets is still restricted. The most well-known substrates of TORC1 are S6K1 , which is a protein kinase to phosphorylate ribosomal protein S6, and 4E-BP1 (eIF4E-binding protein). TORC1 promotes protein synthesis through phosphorylation of these effectors. S6K1 is a member of the AGC (protein kinase A/G/C) kinase family, which includes PKA -dependent protein kinase), AKT , SGK (serum- and glucocorticoid-induced protein kinase), and PKC (protein kinase C) . PhosphoSchizosaccharomyces pombe is a unicellular microorganism, which is one of the best model organisms for the study of molecular mechanisms underlying various eukaryotic cellular processes, as well as the budding yeast S. cerevisiae. An intriguing feature of S. pombe is that sensing of nutritional  availability is intimately linked to the change of growth mode from vegetative to reproductive. Under nutrient-rich conditions, the fission yeast normally proliferates by mitotic division in the haploid state, displaying one of two mating types, +h or −h. When starved of nitrogen, the cells begin sexual differentiation, that is, cells exit from the G1 phase of the mitotic cycle and conjugate with cells of the opposite mating type to form diploid zygotes, which then undergo meiosis and spore formation. Fission yeast S. pombe has two TOR homologs, Tor1 and Tor2 [S. pombe contains two forms of TOR complex, namely TORC1 and TORC2 [S. pombe cells [tor2 mutation for instance, stop proliferation and initiate sexual differentiation ectopically even in the presence of ample nitrogen. Conversely, TORC2 is required for sexual differentiation. Cells lacking TORC2 cannot enter sexual differentiation [S. pombe  pathway. PP2A-B55 antagonizes CDK activity in the G2 phase, resulting in increasing cell size in G2 [In addition to Psk1, two other AGC kinases, Sck1 and Sck2, are phosphorylated by TORC1 in vitro . In vivoze in G2 ,30. The ze in G2 . It has ze in G2 ,32.S. cerevisiae, the major Maf1 kinase is Sch9, which is an ortholog of S6K1/Psk1 and a target of TORC1 [S. cerevisiae TORC1 could weakly phosphorylate Maf1 in vitro [S. pombe, Maf1 phosphorylation depends on TORC1, but not TORC2, although it remains elusive whether TORC1 is directly involved [RNA polymerase III (pol III) generates small untranslated structural RNAs for protein synthesis, such as 5S rRNA and tRNA. In response to nutrient availability and cellular stress, pol III transcription is controlled by a conserved repressor Maf1, which is regulated by phosphorylation ,34,35. Dof TORC1 ,37, wherin vitro . In mammin vitro ,40,41. Iinvolved .S. cerevisiae, the conserved serine/threonine kinase Atg1 is pivotal in the regulation of autophagy, which forms a multimeric protein complex [S. cerevisiae, Atg13 is highly phosphorylated in nutrient-rich conditions, and becomes rapidly dephosphorylated upon nutrient deprivation or inhibition of TORC1 by rapamycin [S. cerevisiae.Autophagy is a highly conserved intracellular degradation mechanism in eukaryotes . TORC1 n complex ,44,45. Aapamycin . It has apamycin . TORC1 iapamycin ,46. Sincapamycin , Atg1 miS. cerevisiae, ULK1 forms a multimeric protein complex, including mammalian Atg13 [In mammals, TORC1 also negatively regulates autophagy . Mammalian Atg13 ,50,51. Wan Atg13 . In vivoan Atg13 . It has an Atg13 .S. pombe carries an autophagy system similar to that of other eukaryotes. Autophagy-deficient mutants lose their viability under conditions of prolonged nitrogen starvation [S. cerevisiae, it has been demonstrated that S. pombe Atg13 is highly phosphorylated under nutrient-rich conditions, and this phosphorylation decreases upon nitrogen starvation, which induces the inactivation of TORC1 [tor2 mutant  is an evolutionarily conserved protein in eukaryotes and is involved in many cellular processes . In S. pd rps602 . Rps6 isd rps602 ,21. The d rps602 . AGC kind rps602 . Since ttor2 mutant cells, or by the treatment with TOR inhibitors such as rapamycin and Torin1 [TORC1 negatively regulates the Greatwall-Endosulfine, Ppk18-Igo1, via the inhibition of the Greatwall kinase Ppk18 as mentioned above. The downregulation of Ppk18 has been suggested to be mediated by Sck2, which is a direct effector of TORC1, since it was observed that Sck2 overexpression inhibits Ppk18 activity . It has d Torin1 .S. cerevisiae [S. cerevisiae, TOR prevents the phosphorylation of eIF2α by Gcn2 [S. pombe, Gcn2 phosphorylates eIF2α at serine 52 under TORC1-inactive conditions such as mutation of Tor2 and nitrogen deprivation, while the precise mechanisms underlying Gcn2 activation remain unknown [S. pombe eIF2α can be detected by the commercially available anti-phospho-eIF2α antibody [The phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) causes repression of general protein synthesis in response to various forms of environmental stress. Gcn2 is known to phosphorylate eIF2α in mammals and in the budding yeast revisiae . In S. c by Gcn2 ,71. Simi unknown . Serine antibody .S. pombe TORC1 promotes the phosphorylation of the GATA transcription factor Gaf1 by inhibiting the PP2A-like phosphatase Ppe1 [isp7, and negatively regulates later nitrogen stress genes, such as ste11 [S. cerevisiae [ase Ppe1 . Phosphoas ste11 ,74. The revisiae ,76,77.S. pombe TORC1-dependent phosphorylation targets  regulates the change of growth mode in response to environmental conditions, which could be regarded as a simple model of cell differentiation. Hopefully, S. pombe research in the future will bring a new perspective to the study of TOR-mediated cellular regulation.In this review, we have overviewed the  targets . Recent gad8, which is phosphorylated and activated by TORC2, results in the same phenotype as the lack of TORC2, such as temperature sensitivity and sterility, Gad8 is thought to be the major TORC2 phosphorylation target [S. pombe will contribute to the clarification of the essence of the TOR signaling pathway.Since the disruption of n target . In cont


Fixation of radial neck fractures can be achieved with a plate and screw construct or with two screws. This study evaluated the biomechanical properties of three different fixation methods following radial neck fractures.Twenty-four fourth-generation composite radii were sawed to simulate an unstable radial neck fracture. They were then instrumented with a plate and screw construct or two different orientations  of screw fixation. Implants were tested under bending and torsional loads via a tension torsion composite test system. Bending and torsional failure loads were added to the remaining implant-radius constructs if they did not fail during the previous tests.During the bending loading test, the crossed-screw group showed the greatest stiffness, followed by the parallel-screw group, the plate group demonstrating the weakest stiffness. There was no significant difference between the crossed- and the parallel-screw groups. However, there was a significant difference between the two screw groups and the plate group. During the bending failure test, the largest stiffness was found for the crossed-screw group, while the plate group exhibited the smallest stiffness. There was a significant difference between the three groups. During the torsion loading test, the highest stiffness was observed for the crossed-screw group, while the plate group showed the lowest stiffness. In the torsion failure test, the failure torques were 11.97 ± 2.659, 8.531 ± 1.768, and 7.079 ± 1.666 N m respectively for the crossed-screw, parallel-screw, and plate groups. There was a significant difference between the crossed-screw group and the two other groups.Crossed screws and plate fixation are commonly used in clinical practice to treat simple radial neck fractures. While the present study shows that the parallel-screw method results in similar biomechanical strength as the two other techniques, it has the advantages of reaching limited wound exposure and having the implant buried. Therefore, it may be widely used in clinical practice. Radial head and neck fractures are uncommon, their reported incidence being approximately 55.4 per 100,000 persons , 3, duriA simple radial neck fracture model was used to standardize our investigation. Although this model does not reproduce radial head and neck fractures, the results of this study can still help orthopedic surgeons to develop the most reasonable internal fixation pattern. We aimed at comparing the stiffness and strength of the plate and two different screw orientations, the plate and the crossed screw being commonly used in clinical practice, and the parallel screw being specifically designed by us for this study.We used 24 identical  synbone radii . Each radius was cut at the mid-shaft level, leaving an approximately 10-cm long proximal segment. A transverse osteotomy was then made at the head-neck junction by using a micro-sagittal saw, this simulating a longitudinally unstable radial neck fracture.Three different fixation devices were tested for reconstruction after the osteotomy: a radial head plate and screws  or two different orientations  of screw fixation . The plate group included a plate construct involving five bicortical screws. In the crossed-screw group, the screws were placed approximately 60° apart, as described by Smith and Hotchkiss . In the The bending load was applied to the radial head through a custom solid cup made of PMMA. The loading orientation was posterior to anterior. Before the actual test, a preload of 10 N was applied three times at the same velocity (2 mm/min) to the radial head which had to resist to a horizontal slide. This position was regarded as the baseline to record the displacement of the head and the data were then cleared. Next, the construct was loaded in compression at a rate of 2 mm/min. The test was stopped when the displacement of the radial head reached 2 mm.After the bending load test, if the fracture models did not fail, a failure load test was performed at a rate of 2 mm/min. Bending stiffness and bending failure loads were recorded.First, the head of the radial was coupled with the actuator of the Electro Force 3510 Tension torsion composite test system with the use of an additional double gimbal fixture . Radii were then loaded for 10 cycles at 1 Hz in both the anterior and posterior torsional direction at five levels: − 0.5°, − 1.0°, − 1.5°, − 2.0°, and − 2.5°. The torsional stiffness obtained was then used to evaluate the ability of the fixed structure to resist rotation.Similarly to the bending load test procedure presented above, the fracture models underwent a failure load test at a rate of 5°/min if they did not fail after the torsional load test. Torsional stiffness and torsional failure loads were recorded.Failure of the model was defined as (1) a new fracture line appearing in the model in addition to the original fracture line; (2) an internal fixation failure, such as plate or screw bending, cutting, or fracture; (3) a lateral displacement of the radial head superior to 5 mm or a torsion displacement of the radial head that exceeded 14.5°; and (4) a flat load-displacement curve in the data acquisition image or an absence of change in the displacement of the model while the load still increased.p < 0.05.The stiffness was defined as the slope of the regression line fitted to the loading segment of the cyclic load displacement curves. Data of each group are presented as mean ± standard deviation (SD). For statistical analysis, SPSS 21.0  was used. Mechanical parameters were compared by using one-way analyses of variance. The level of statistical significance was set at p = 0.003) and crossed-screw groups (p < 0.001). All bending load data were processed as a displacement-load curve, as depicted in Fig. We compared the stiffness of the three structures from the five bending load levels Table . The stip < 0.05). Results of the torsion failure test showed that stiffness was significantly higher for the crossed-screw group than for the parallel-screw and plate groups, with no significant difference between the parallel-screw group and the plate group . Also, the parallel-screw method only requires a small wound exposure, the two screws being inserted in parallel at a 45° angle with the radial axis in order to fix the fracture site. The biomechanical results obtained in the parallel-screw group are just slightly less than those obtained for the crossed-screw group. Therefore, we can consider using two parallel screws to fix a simple radial neck fracture.Although our study provides interesting results regarding the biomechanical properties of three different fixation methods, it also has some limitations. First, since the biomechanical properties of the standard bone were investigated without any muscle and other corresponding soft tissue attachment, it cannot simulate the real human elbow joint force transmission and role. Second, our sample size was relatively small. Bending load and torsion direction of the body cannot completely simulate the real daily activities of the human body or the mechanical injury mechanism. In addition, it should be noted that there is a subtle difference in screw orientation in the coronal plane. We attempted to control for this by predrilling the screw trajectory with a custom-made jig, but our funds were limited, and we could not proceed this way. Also, the biomechanical analysis provided in this study only reports bending, torsional, and failure loads. The observed index only includes bending and torsional stiffness as a trade-off. Other biomechanical performance indicators are lacking. Finally, the use of synthetic bone models as opposed to cadaveric specimens could also be seen as a limitation of this study.Results of this biomechanical study suggest that the crossed-screw fixation is optimal for Mason II radial neck fractures among the three internal fixation strategies analyzed in this study . Alternatively, the parallel-screw method also constitutes a good internal fixation strategy. The stiffness of the plate was the weakest among the three investigated techniques and was also the one that required the largest wound exposure. However, our conclusion needs to be supported by additional large sample size studies investigating its biomechanical and clinical application.


Extensor digitorum longus (EDL) and soleus muscles from male C57BL/6 mice were isolated, divided into halves, and then incubated with ucOC with or without the pretreatment of ERK inhibitor U0126. ucOC increased muscle glucose uptake in both EDL and soleus. It also enhanced phosphorylation of ERK2 (Thr202/Tyr204) and AS160 (Thr642) in both muscle types and increased mTOR phosphorylation (Ser2481) in EDL only. ucOC had no significant effect on the phosphorylation of AMPKα (Thr172). The inhibition of ucOC-induced ERK phosphorylation had limited effect on ucOC-stimulated glucose uptake and AS160 phosphorylation in both muscle types, but appeared to inhibit the elevation in AKT phosphorylation only in EDL. Taken together, ucOC at the physiological range directly increased glucose uptake in both EDL and soleus muscles in mouse. The molecular mechanisms behind this ucOC effect on muscle glucose uptake seem to be muscle type-specific, involving enhanced phosphorylation of AS160 but limitedly modulated by ERK phosphorylation. Our study suggests that, since ucOC increases muscle glucose uptake without insulin, it could be considered as a potential agent to improve muscle glucose uptake in insulin resistant conditions.Emerging evidence suggests that undercarboxylated osteocalcin (ucOC) improves muscle glucose uptake in rodents. However, whether ucOC can directly increase glucose uptake in both glycolytic and oxidative muscles and the possible mechanisms of action still need further exploration. We tested the hypothesis that ucOC  All mice were group housed with a 12-h light/12-h dark cycle and fed standard laboratory chow  and water ad libitum until 9–12 weeks old. The study was approved by the Animal Experimentation Ethics Committee of Victoria University (AEC14/009) and conformed to the Australian National Code of Practice for the Care and Use of Animals for Scientific Purposes. The mice for each group in this study were randomly allocated.Eight-week-old male C57BL/6J mice   and evenly divided into halves longitudinally. After muscle dissection, mice were euthanized via cervical dislocation under anesthesia.Mice were fasted for 4 h before deep anaesthetization with 60 mg kgex vivo, similar to what has been performed in rat muscle in previous studies (N = 5), after 30 min preincubation, muscle samples were exposed to the ERK inhibitor U0126 (1 µM)  or dimethyl sulfoxide (DMSO) vehicle  for 30 min. Then, muscle samples were stimulated for 90 min with increasing doses  of recombinant ucOC . These doses of ucOC were chosen because they are within the physiological range in mice  and 16 mM d-[1-14C] mannitol (PerkinElmer) with or without U0126/Vehicle or ucOC. After 10 min, muscles were rapidly rinsed with ice cold KHB buffer, then immediately frozen in liquid nitrogen. On the day of sample processing, muscle samples were lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (60 µL RIPA for 1 mg sample)  with Inhibitor Cocktail  and 100 mM dithiothreitol (Sigma-Aldrich) using TissueLyser II  followed by gentle rocking at 4°C for 1 h. Half of the lysate was pipetted into vials with scintillation cocktail for scintillation counting (β-counter) with Tri-Carb 2910TR Liquid Scintillation Analyzer (PerkinElmer) and the other half was used in western blotting.The method to assess 2-Deoxy-D-glucose (2-DG) uptake has been described previously . BrieflyAfter muscle samples were homogenized using RIPA buffer, protein concentrations in the lysate were determined by Bio-Rad Protein Assay . Equal amounts of protein were subjected to electrophoresis on Criterion stain-free precast gels  and then transferred electrophoretically using Trans-Blot Turbo Transfer System (Bio-Rad) onto a polyvinylidene fluoride membrane (Bio-Rad). Then, a stain-free blot image was taken using ChemiDoc Imaging System (Bio-Rad) for total protein measurement in each sample lane. Immunoblotting was performed at optimum conditions for each antibody. Bands were identified using ChemiDoc Imaging System, using SuperSignal West Femto Maximum Sensitivity Substrate . Band densities of both stain-free blot and immunoblotting were measured using Image Lab Software (Bio-Rad). Values of immunoblotting bands were normalized using total protein values. p-ERK (Thr202/Tyr204), ERK, p-AMPKα (Thr172), AMPKα, p-mTOR (Ser2481), mTOR, p-AKT (Ser473), AKT, p-AS160 (Thr642), AS160, and p-PKCδ/θ (Ser643/676) antibodies were purchased from Cell Signaling.Two data points for AMPKα phosphorylation assessment were excluded due to western blot imaging artifacts. However, their exclusion did not alter the statistical outcome, interpretation, or conclusions of the results.−1 group and 30 ng mL−1 group were chosen for western blotting and correlation analysis as representatives of low and high doses of ucOC.Fold-changes for western blotting data were calculated by normalization to control groups within the same animals. 3 ng mLt-tests were used to analyze the effects of ucOC, for each individual concentration, on muscle glucose uptake, protein phosphorylation, protein abundance, and phospho/total ratio compared to paired control samples. This paired comparison was used to exclude individual variances.Paired post hoc test was applied.To analyze the dose–response effects of ucOC on muscle glucose uptake, basal glucose uptake data from all groups were combined, then one-way ANOVA with Tukey −1 ucOC treatment group. Rule of thumb for interpreting the size of a correlation coefficient will be applied to measure the strength of correlation between two variables .All data are reported as mean ± SEM.−1 (P < 0.05) and 30 ng mL−1 (P < 0.01) in EDL, and 0.3 ng mL−1 (P < 0.01) and 30 ng mL−1 (P < 0.01) in soleus , from 2.91 to 4.32 μmol g−1 h−1 and from 3.12 to 4.16 μmol g−1 h−1, respectively.Compared with paired controls, muscle glucose uptake was significantly higher following the treatment of ucOC at doses of 10 ng mL−1 significantly increased p-mTOR  in both EDL and soleus muscles , but not in soleus  and a high positive correlation (r = 0.85), respectively.p-ERK2 levels were not associated with glucose uptake levels Figures A,B or p-p-AMPKα levels were not associated with glucose uptake or any signaling protein phosphorylation levels in either muscle type (data not shown).−1)-induced increases in ERK2 phosphorylation in both EDL and soleus , but not in soleus  had limited effect on p-mTOR following ucOC treatment in either EDL or soleus  and soleus (oxidative muscle) muscles. Furthermore, ucOC increased the phosphorylation of ERK2, mTOR, and AS160 in EDL and enhanced the phosphorylation of ERK2 and AS160 in soleus muscle. It appears that ERK phosphorylation was not directly involved in ucOC-stimulated glucose uptake and AS160 phosphorylation in both muscle types.We report that physiological levels of ucOC per se probably only upregulates muscle glucose metabolism in oxidative muscle fibers . Therefore, ucOC may enhance AS160 phosphorylation via AKT independent mechanisms, which may be the major mechanisms underlying the ucOC-induced enhancement of glucose uptake and AS160 phosphorylation. Indeed, several other signaling proteins are also able to increase AS160 phosphorylation, including AMPK and conventional/novel (c/n) protein kinase C (PKC)  despite relatively modest increases in AKT phosphorylation (Ser473) ( C (PKC) .−1 ucOC treatment, there is a significant increase in phosphorylated ERK2 (Thr202/Yyr204) in both EDL and soleus muscles. However, there was limited change in AMPKα phosphorylation at Thr172, a phosphorylation site that has widely been reported as an indicator of AMPK activity . Consistently, a previous finding also suggested that partial ERK inhibition dampened ucOC-stimulated AKT phosphorylation in C2C12 myotubes  and conformed to the Australian National Code of Practice for the Care and Use of Animals for Scientific Purposes.XL, XZ, TB-S, and IL design the study. XL, LP, and EM performed experiments and data collection. XL and LP did the data analysis. XL wrote the paper. XL, LP, XZ, AH, GM, TB-S, and IL substantially contributed to manuscript revision and approved the final version.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.


Lysophosphatidic acid (LPA) is a pleiotropic bioactive lysophospholipid involved in inflammatory mediator synthesis. Signaling through p38MAPK, ERK, Rho kinase, and MSK-CREB contributes to LPA-mediated IL-8 production in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. The study was undertaken to investigate how LPA activates MSKs and how signaling crosstalk between TNFα and LPA contributes to the super-production of cytokines/chemokines. RAFLS pretreated or not with TNFα were stimulated with LPA. Immunoblotting with phospho-antibodies monitored MSK activation. Cytokine/chemokine production was measured using ELISA and multiplex immunoassays. LPA induced MSK activation by signaling through ERK whereas p38MAPK, Rho kinase, NF-κB or PI3K contribute to IL-8 synthesis mainly via MSK-independent pathways. Priming with TNFα enhanced LPA-mediated MSK phosphorylation and cytokine/chemokine production. After priming with TNFα, inhibition of ERK or MSK failed to attenuate LPA-mediated IL-8 synthesis even if the MSK-CREB signaling axis was completely or partially inhibited. In TNFα-primed cells, inhibition of LPA-mediated cytokine/chemokine synthesis required a specific combination of inhibitors such as p38MAPK and ERK for IL-8 and IL-6, and Rho kinase and NF-κB for MCP-1. The ability of the signaling inhibitors to block LPA induced cytokine/chemokine synthesis is dependent on the inflammatory cytokinic environment. In TNFα-primed RAFLS the super-production of IL-8 and IL-6 induced by LPA occurs mainly via MSK-independent pathways, and simultaneous inhibition of at least two MAPK signaling pathways was required to block their synthesis. Since simultaneous inhibition of both the p38MAPK and ERK-MSK-CREB pathways are required to significantly reduce LPA-mediated IL-8 and IL-6 production in TNFα-preconditioned RAFLS, drug combinations targeting these two pathways are potential new strategies to treat rheumatoid arthritis. Rheumatoid arthritis is a systemic, severe autoimmune disease associated with chronic inflammation of peripheral joints and adjacent tissues, as well as hyperplasia of synovial lining cells  along with infiltration of immune cells into the synovial cavity, forming a pannus, leading to joint deformation and pain . Synovia1-LPA6 have been identified  can be activated by either ERK or p38MAPK . MSK1 is3 expression and strongly enhances cytokine/chemokine release induced by LPA  was from Millipore Corporation . FBS and DMEM were from Wisent Inc. . Propidium iodide and Annexin V-eFluor450 were from BD Pharmingen .TNFα was purchased from PeproTech Inc. . 1-Oleoyl-sn-glycerol 3-phosphate sodium salt  was purchased from Sigma-Aldrich Canada . Antibodies against human phospho-MSK1 (Ser-376)/MSK2 (Ser-360), phospho-MSK1 (Ser-212), MSK2 and GAPDH were from R&D Systems Inc. . Antibodies against human phospho-CREB (Ser-133), phospho-MSK1 (Ser-360), phospho-MSK1 (Thr-581) and MSK1 were purchased from Cell Signaling Technology . Antibody to actin was from Sigma–Aldrich Canada . Inhibitors of p38MAPK (SB203580), ERK (PD98059), Rho kinase (Y27632), and NF-κB (Bay11-7082) were purchased from Calbiochem . The PI3K inhibitor wortmannin was from Millipore Corporation . MSK inhibitor SB-747651A was obtained from Axon Medchem . Human IL-8 ELISA kit was purchased from BioSource International Inc. . The human cytokine/chemokine Luminex multiplex immunoassay kit (Milliplex2) and grown in DMEM supplemented with 10% FBS, penicillin (100 IU), and streptomycin (100 M) as described previously  for 8 h prior to LPA treatment  according to the manufacturer’s instructions. The dynamic range of the assay is 3.2-10000 pg/ml. The assay sensitivities  were 0.9 ± 1.3, 0.4 ± 0.7, 8.6 ± 14, 1.9 ± 3.4, and 1.2 ± 1.9 for IL-6, IL-8, IP-10, MCP-1, and RANTES, respectively.Human cytokines/chemokines IL-6, IL-8, IP-10, MCP-1, and RANTES were monitored using a Luminex multiplex immunoassay (MilliplexRAFLS were lysed in boiling sample buffer  for 7–10 min. Equal amounts of protein were loaded on gels and separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were later transferred from polyacrylamide gel to methanol-soaked PVDF membranes . Primary antibody incubation was performed overnight at 4°C, 1 h at 37°C, or 2 h at room temperature according to optimization tests. The membranes were washed three times and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Membranes were when washed three times and antibody-antigen complexes were revealed using Western Lightning chemiluminescence reagent according to the manufacturer’s instructions .t-test . For studies using samples from more than one treatment, statistical significance between control and treated cells was determined by one-way ANOVA or two-way ANOVA multiple comparison test according to the context. P values less than 0.05 were considered statistically significant.Unless otherwise stated, the data are from at least three independent experiments presented as mean values ± SEM. Prism 7.0 software was used for all statistical analyses. Statistical significance of the difference between samples of two different treatments was determined by Figures 1A, 2A,B). After 8 h stimulation with TNFα phosphorylation of MSK1 at Ser-212 (p-MSK1) remained significantly elevated  when compared to the non-treated cells . Further efforts to detect MSK1 phosphorylation at Ser-360 or Thr-581 were fruitless due to the detection threshold of the antibodies and of the chemiluminescence reagent used.Our previous study has shown the key role of the MSK-CREB signaling axis in LPA-mediated IL-8 secretion in RAFLS . LPA wasFigures 2A,B). LPA stimulation for 5 min after treatment with TNFα for 8 h did not increase the levels of p-MSK1/2 . The levels of p-MSK1 were increased by ∼2-fold in response to LPA stimulation for 5 min. . TNFα treatment for 8 h significantly enhanced the levels of p-MSK1 induced by LPA . The levels of p-MSK1 were increased by 56 ± 16% (p < 0.0001) when compared to LPA-treated samples.Stimulation of RAFLS with LPA for 5 min led to a six-fold increase in the levels of p-MSK1/2 . In all subsequent experiments with TNFα the time point 8 h, when p-CREB returned to baseline levels, was selected to monitor LPA-induced signaling and functional responses in primed cells. RAFLS stimulation with LPA for 5 min led to a ∼5-fold increase in the levels of p-CREB . TNFα pretreatment for 8 h significantly enhanced LPA-mediated CREB phosphorylation at 5 min but not at the later time points tested .CREB phosphorylation in response to TNFα was maximal at 30 min (∼4.5-fold increase) and slowly declined to baseline levels by 8 h , the data would suggest that increased signaling through the MSK1/2-CREB axis may in turn enhance LPA-induced responses in TNFα-primed RAFLS.Overall the data suggest that TNFα priming for 8 h tends to increase LPA-mediated phosphorylation of MSK1/2 and CREB. Since TNFα has no effect on MSK1, MSK2, and CBEB protein expression . In contrast, the ERK inhibitor significantly decreased the levels of p-MSK1/2  , and reduced that of p-MSK1  induced by LPA .Incubation of RAFLS with inhibitors of p38MAPK, Rho kinase, or PI3K did not affect the level of p-MSK1/2 or p-MSK1 induced by LPA  and 66 ± 8% , respectively. Inhibition of PI3K in TNFα-primed RAFLS stimulated with LPA significantly decreased the levels of p-MSK1/2 by 48 ± 10%  but did not affect those of p-MSK1 .Inhibition of ERK activation after priming with TNFα for 8 h significantly reduced the levels of p-MSK1/2 and p-MSK1 induced by LPA by 54 ± 18% . Simultaneous inhibition of ERK signaling together with Rho kinase, or p38MAPK and Rho kinase, or p38MAPK and PI3K, or all of the signaling pathways together did not further reduce p-MSK1/2 . In contrast, the same combinations of inhibitors slightly attenuated p-MSK1 phosphorylated at Ser-212 when compared to cells treated with the ERK inhibitor alone . The most potent combinations were inhibition of ERK, p38MAPK, and Rho kinase, and that of ERK, Rho kinase, and PI3K signaling .The next series of experiments evaluated the effects of inhibiting ERK in combination with one or more signaling pathways. Inhibition of both ERK and p38MAPK did not further reduce MSK phosphorylation compared to cells treated with ERK inhibitor alone , we performed experiments to determine whether simultaneous inhibition of at least two pathways could further diminish MSK phosphorylation. As shown in Figure 5B, all combinations of inhibitors moderately decreased the levels of p-MSK1/2. However, inhibition of Rho kinase and p38MAPK or Rho kinase and PI3K did not decrease the levels of p-MSK1/2 compared to cells treated with the inhibitor of Rho kinase alone. Combination of p38MAPK and PI3K, or of p38MAPK, PI3K, and Rho kinase inhibitors reduced p-MSK1/2 to an extent greater than inhibition of either signaling pathway alone . In contrast, these combinations of inhibitors did not reduce p-MSK1 at Ser-212 . Taken together the data indicate that in both control and TNFα-primed RAFLS stimulation with LPA results in the phosphorylation of MSK1 at Ser-376 and Ser-212 and/or of MSK2 at Ser-360 via ERK. Although combination of p38MAPK and Rho kinase, p38MAPK and PI3K, or of p38MAPK, PI3K, and Rho kinase inhibitors reduced LPA-mediated phosphorylation event at Ser-376 of MSK1 (or Ser-360 of MSK2) in TNFα-primed cells, phosphorylation at Ser-212 of MSK1 was not inhibited.Since inhibition of p38MAPK, Rho kinase, or PI3K alone partially inhibited LPA-induced p-MSK1/2 in TNFα primed RAFLS (p < 0.01) in control and TNFα-primed cells, respectively . Whereas the inhibitor of ERK or p38MAPK alone reduced LPA-mediated CREB phosphorylation by ∼50%, their combination completely blocked CREB phosphorylation in TNFα-primed cells . The MSK inhibitor SB-747651A fully inhibited LPA-mediated CREB phosphorylation in unprimed cells and reduced the levels of phosphorylated CREB by 54 ± 4% (p < 0.01) in cells pre-treated with TNFα for 8 h . Taken together, the data suggest that in both control and TNFα-primed cells LPA-induced CREB phosphorylation relies on p38MAPK and ERK signaling.LPA-mediated CREB phosphorylation at Ser-133 is MSK-dependent in RAFLS . In the Figure 7 and as previously reported, priming of RAFLS with TNFα for 8 h strongly enhances the subsequent release of IL-8 as well as of other cytokines/chemokines in response to stimulation with LPA (1 and LPA3 receptor activation (1/3 antagonist inhibited the super-production of IL-8 (and IL-6 as well) induced by LPA . Inhibition of PI3K and NF-κB also decreased LPA-induced IL-8 secretion .As show in with LPA , 2014. Etivation . In TNFαd by LPA . TNFα-prammation . Though ammation , 2014, tFigure 8D), wortmannin , and the highest dose of Bay11-7082 , whereas the other signaling inhibitors were without effect. However, control experiments revealed that the effect of the NF-κB inhibitor (Bay11-7082) at 10 μM was cytotoxic for RAFLS whereas all other compounds had no significant impact on cell viability . Only LPA-induced IL-8 secretion remained sensitive to inhibition by wortmannin after priming with TNFα .IL-8 secretion due to TNFα priming was sensitive to inhibition by SB-747651A  significantly decreased the production of IL-8 by 61 ± 3%  when compared to TNFα-primed RAFLS stimulated with LPA. Though not statistically significant, combinations of the p38MAPK inhibitor with those of Rho kinase, PI3K, or NF-κB showed a tendency to more strongly decrease IL-8 secretion compared to other combinations of inhibitors , thereby suggesting a role for p38MAPK in exacerbating LPA-induced IL-8 release in TNFα-primed RAFLS. In contrast, combined inhibition of Rho kinase and PI3K or of Rho kinase and NF-κB increased the release of IL-8 by 72.5% (p < 0.05) and 40 ± 30%, respectively .As neither the p38MAPK, ERK, Rho kinase, nor the NF-κB inhibitors (1 μM) acting independently could inhibit IL-8 in response to LPA in TNFα-primed cells, we next tested various combinations of signaling inhibitors at concentrations that had no significant impact on cell viability , was reduced by 41 ± 3% when these two pathways were inhibited . Combination of Rho kinase and PI3K inhibitors significantly enhanced  the release of IL-6 compared to the control TNFα-primed cells stimulated with LPA. MCP-1, IP-10, and RANTES exhibited different inhibition patterns with combinations of inhibitors. The release of IP-10 and of RANTES was not affected by any of the combinations of inhibitors  whereas combinations of Rho kinase and PI3K, Rho kinase and NF-κB, or PI3K and NF-κB inhibitors decreased MCP-1 secretion to 48 ± 23%, 34 ± 21%, and 53 ± 17% of control LPA-stimulated cells, respectively .The pattern of IL-6 secretion by cells treated with the same combinations of signaling inhibitors was similar to that of IL-8. LPA-induced IL-6 secretion after TNFα priming, which was not inhibited by a pre-incubation with either the inhibitor of p38MAPK (not shown) or of ERK .Using pharmacological inhibitors or siRNA, we previously demonstrated that LPA induces MSK1 and MSK2 activation and MSK-mediated CREB phosphorylation at Ser-133, thereby promoting the synthesis of IL-8 and MCP-1 . Here weRegulation of IL-8 expression involves kinase cascades and transcription factors such as AP-1, CREB, C/EBP-1, and NF-κB that bind to the IL-8 promoter . InteresFigure 1A). Surprisingly ERK-dependent phosphorylation of MSK1 at Ser-212 remained elevated compared to unstimulated RAFLS. In TNFα-primed RAFLS, LPA consistently enhanced p-MSK1/2 and p-MSK1 at Ser-212 mainly via ERK activation. After priming with TNFα, inhibition of p38MAPK, Rho kinase, and PI3K slightly reduced LPA-mediated p-MSK1/2 without reducing MSK1 phosphorylation at Ser-212 , suggesting that p38MAPK, Rho kinase and PI3K may regulate MSK2. Furthermore, combinations of inhibitors that further decreased the levels of p-MSK1/2 had no impact on p-MSK1 at Ser-212. MSK1 activation by TNFα and IL-1β has been reported in human keratinocytes. These studies report that a non-specific inhibitor of NF-κB reduced MSK1 autophosphorylation at Ser-376, but had no impact on the sites (Ser-360 and Thr-581) directly phosphorylated by ERK or p38MAPK . We cannot totally exclude that inhibition of PI3K could interfere indirectly with Rho kinase, ERK and/or p38MAPK activation (Class I PI3Kδ expressed in the RA synovial intimal lining was reported to regulate FLS growth and TNFα signaling . The sevtivation , therebyLPA and TNFα are well known to activate NF-κB signaling in various cell types including synovial fibroblasts . Upon acFigure 10. Priming of RAFLS with TNFα for 8 h enhances LPA-mediated cell functional responses including MSK activation and cytokine/chemokine production. However, after inflammatory conditioning with TNFα, LPA-mediated cytokine/chemokine secretion was mainly MSK-independent and insensitive to inhibition by Rho-kinase, p38MAPK, or NF-κB inhibitor.In this study we show that LPA-induced IL-8 production is mediated through the ERK1/2-MSK signaling axis and MSK-independent pathways. A summary of the proposed signaling pathways involved in LPA-induced IL-8 production in TNFα-primed RAFLS has been presented in The key finding is that simultaneous inhibition of at least two signaling pathways such as ERK1/2 and p38MAPK was required to inhibit IL-8 and IL-6 production in TNFα-preconditioned RAFLS. Since both TNFα and LPA activate p38MAPK and ERK-MSK-CREB signaling, drug combinations targeting these two pathways are potential new strategies to treat RA.WH, CZ, and SB designed the experiments. WH and CZ performed the experiments and analyzed the data. WH and SB wrote the paper.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. The reviewer MP and handling Editor declared their shared affiliation.


Epidemiological studies from Sub-Saharan Africa indicate that allergies are on the rise in this region especially in urban compared to rural areas. This increase has been linked to improved hygiene, lifestyle changes, and lower exposure to pathogens in childhood. Reduced exposure to parasitic worm (helminth) infections and allergy outcomes has been the focus of a number of population studies over the years. Paradoxically, there are parallels in the immune responses to helminths and to allergies. Both conditions are associated with elevated levels of immunoglobulin E, high numbers of T helper 2 cells, eosinophils, and mast cells. These immune parallels have meant that the diagnosis of allergies in parts of the world where helminths are endemic can be hampered. The aim of this review is to examine observations from population studies conducted in Sub-Saharan Africa that demonstrate how helminth infections influence the parameters used to diagnose allergy outcomes in this region. We explore specifically how helminth infections hinder the in vitro diagnosis of allergic sensitization, influence the clinical manifestations of allergy, and also the effect of anthelmintic treatment on allergy outcomes. Advancing our understanding of how helminths influence allergy diagnosis is imperative for the development of improved tools to assess, diagnose, and treat allergic disorders in both helminth-endemic and non-endemic countries worldwide. Time trends indicate a sharp rise in the incidence of allergic disease worldwide especially among children . This inHelminth infections remain highly prevalent in tropical areas with over 1.5 billion people worldwide thought to be chronically infected with at least one soil-transmitted helminth . MoreoveDespite the lack of geographical overlap in areas of the world with the highest burden of helminth infection and places most affected by allergic disease, strong parallels exist between these two conditions on the immunological level. Both are associated with the induction of strong T helper 2 (Th2) immune responses leading to elevated levels of immunoglobulin E (IgE) as well as the presence of eosinophils, mast cells, and Th2 cytokines , 24. TheIn this review, findings from population studies conducted in Sub-Saharan Africa that illustrate how helminth infections influence the parameters used to diagnose allergy outcomes are examined. We explore specifically how helminth infections hinder the in vitro diagnosis of allergic sensitization and influence the clinical manifestations of allergic disease.The clinical diagnosis of IgE-mediated allergy in many countries is based primarily on a physical examination, history of symptoms, and the determination of allergic sensitization •. AlthouAllergic sensitization—the production of serum-specific IgE against harmless antigens known as allergens—is a well-established factor in the development of allergic disease  and crucIn vitro assessment of allergic sensitization can be determined by the measurement of allergen-specific IgE by assays such as an enzyme-linked immunosorbent assay (ELISA), radioallergosorbent test (RAST), ImmunoCAP assay or more recently, microarray allergen assay .Skin prick testing is used to assess allergic sensitization to a panel of environmental or food allergens as well as to negative and positive controls. A skin prick test (SPT) involves applying an allergen in liquid form to the skin of a patient, piercing the drop with a lancet and determining whether a reaction occurs within 15 to 20 min following application . If the Ascaris lumbricoides had 28 times higher levels of total IgE [In the late 1960s, S.G.O. Johansson and colleagues published one of the first studies linking the then newly characterized IgE antibody to helminth infections. Their investigation found that a group of pre-school children in Ethiopia had up to 20 times higher levels of total IgE compared to Swedish children and that a sub-group of Ethiopian children infected with the helminth otal IgE •. Even iotal IgE •.Subsequently, several investigations from Sub-Saharan Africa have observed raised levels of allergen-specific IgE related to helminth infection that did not translate into allergy symptoms •, 32, 33Since the early 1980s, two forms of cross-reactivity linked to allergy have been described: cross-reactivity due to proteins and cross-reactivity due to the sugar structures on glycoproteins known as cross-reactive carbohydrate determinants (CCDs).Ascaris and cockroach or mite, is not always significant [Cross-reactivity between plant-derived proteins is well-characterized especially in relation to food allergy . Althougnificant .Ascaris lumbricoides and homologues from house dust mite or cockroach, there are very few studies in this area from Sub-Saharan Africa. However, published literature provides indications that IgE cross-reactivity between homologous proteins may explain some observations made in studies from Africa over the years. For example, in urban black adolescents in Cape Town, South Africa, a positive association was seen between specific IgE to Ascaris antigen and SPT reactivity to aeroallergens (house dust mite and cockroach) [Ascaris such as tropomyosin or GST and their corresponding homologues in house dust mite and cockroach.Although a number of population studies from South America have investigated IgE cross-reactivity between proteins from helminths such as ckroach) . AlthougAscaris, other helminths have been implicated in IgE cross-reactivity involving proteins. For example, cross-reactivity between the filarial nematode Onchocerca volvulus tropomyosin (OvTrop) and house dust mite tropomyosin (Der p 10) has been shown [Aside from en shown . While tN-acetylglucosamine and a β--linked xylose on a core mannose [Cross-reactive carbohydrate determinants (CCDs) on insect and plant glycoproteins are carbohydrate components that have been linked to IgE cross-reactivity . The mos mannose . Aalbers mannose . A subse mannose .S. haematobium infection was also positively linked to peanut IgE sensitization as well as a strong correlation seen between IgE to CCDs and IgE to whole peanut extract. Inhibition assays in a subset of children showed that S. haematobium soluble egg antigen and the CCD marker bromelain were strong inhibitors of IgE binding to peanut extract [The role of helminths in carbohydrate cross-reactivity was illustrated in an investigation in Ghana among 1604 school-children in which raised levels of IgE against whole peanut extract were observed that did not translate into peanut allergy symptoms •. In tha extract •. IgE to extract .Although IgE to CCDs is generally thought to be biologically inactive, the clinical relevance of IgE to some CCDs has been reported in a study that found that about one third of CCD-positive sera from individuals with tomato allergy had biologically relevant CCD-specific IgE . MoreoveIgE cross-reactivity between helminth antigens and environmental or food allergens demonstrates the limited diagnostic value of assessing IgE reactivity to whole allergen extracts in populations from helminth-endemic areas. Whole extracts are made up of multiple allergenic and non-allergenic components which often lack standardization in their allergen content . In receS. haematobium infection, CRD in a subset showed that despite these high levels, very low responses to recombinant peanut allergens Ara h 1, 2, and 3 were seen [Among Ghanaian schoolchildren with elevated IgE to whole peanut extract that was strongly associated with ere seen . Althougere seen . While 4ere seen . Both thA. lumbricoides, Strongyloides stercoralis, and hookworm, involve the migration of the larval stage through the lungs which may result in respiratory symptoms often associated with asthma such as wheeze and dyspnoea (shortness of breath) [Research has shown that in some individuals who have low exposure to helminths such as travelers or recent migrants to a helminth-endemic area, robust inflammatory allergic responses can develop to helminths . In addi breath) •.A. lumbricoides infection and symptoms of asthma such as wheeze seen in a number of population studies in Sub-Saharan Africa. For example, in South Africa, an investigation found that A. lumbricoides infection among urban and rural children was associated with increased odds of a marker of airway hyperresponsiveness: exercise-induced bronchoconstriction [A. lumbricoides infection, and reported wheeze among participants greater than 5 years of age [Th2-mediated responses to migrating larvae during helminth infection provides one explanation for positive associations between triction . More res of age . It is is of age .Onchocerca volvulus infection, and in this form, microfilariae induce a state of immune hyporesponsiveness in the host which ensures the survival of the parasite through the regulation of strong Th2 immune responses [Migrating larvae are also a feature of other helminth infections such as onchocerciasis where larvae known as microfilariae migrate to the skin, eyes, and organs . Onchoceesponses . The moresponses . In bothesponses .Onchocercavolvus and all those tested were found to be positive for onchocerciasis based on serology for the marker of active disease IgG4 or skin snip test [The effects of the reversal of immune hyporesponsiveness associated with chronic onchocerciasis have been illustrated by a small retrospective study of 27 Ethiopian immigrants in Israel who presented with pruritic skin disease simulating atopic dermatitis •. In thanip test •. Interenip test •. They anip test •.Toxocara spp. can also induce Th2 inflammatory responses in the airways or the skin [Anisakis. Marine mammals are definitive hosts of Anisakis while fish and cephalopods (octopuses/squids/cuttlefish) are intermediate hosts for its larval stages [Anisakis by consumption of the larval stage of the parasite in undercooked/raw fish or cephalopods [Anisakis infection can include gastro-allergic reactions as well as anaphylaxis while exposure through the respiratory tract or skin can result in anaphylaxis, rhinoconjunctivitis, asthma, and dermatitis [Anisakis [Anisakis infective larval stage (L3) induced strong Th2 immune responses in mice. A later study by the same group found that following epicutaneous exposure to Anisakis antigens in mice, the Th2 cytokine interleukin (IL)-13 played an important part in protein contact dermatitis while another Th2 cytokine IL-4 was key in driving systemic anaphylaxis [Aside from migrating larval stages, transient helminth infections where humans are not the definitive host like the skin . This hal stages . Humans halopods . Symptomrmatitis . NieuwenAnisakis . Their iphylaxis .Anisakis infection in humans in other parts of Sub-Saharan Africa. It is possible that this is as a result of under-reporting as many cases of the infection go undiagnosed [There are no other published studies on iagnosed .Chronic helminth infections induce immune regulatory networks in the host which are characterized by regulatory T cells, regulatory B cells, and alternatively activated macrophages . An antiRemoval of helminths by chemotherapy can reverse immune hyporesponsiveness to allergens associated with chronic helminth infections. Although observations have varied, several longitudinal studies conducted in helminth-endemic areas worldwide have observed a positive association between anthelmintic treatment and allergen skin reactivity –77. HoweSchistosoma infection and meben-dazole for intestinal helminths) or a placebo every 3 months and skin prick tested every 6 months for a 30 month follow-up period [In Gabon, a randomized, controlled trial was conducted in which 317 children aged 5–13 years living in a helminth-endemic area were given anthelmintic treatment  for soil transmitted helminths among pregnant women was strongly associated with an increased risk of doctor-diagnosed infantile eczema in their children •. Praziqnegative •. The trnegative •. Subseqnegative . By 5 yenegative .S. mansoni infection (47.2%) [Recently, Stein and colleagues examined the burden of helminth infections among 126 immigrants from Ethiopia newly arrived in Israel and related these to allergy outcomes •. The mo (47.2%) •. Among  (47.2%) •. Less t (47.2%) •. Both cStudies from helminth-endemic areas in Sub-Saharan Africa on IgE cross-reactivity between helminth antigens and allergens have shown that the specificity and diagnostic accuracy of in vitro testing of allergy can be greatly improved by the use of recombinant allergens for the evaluation of IgE responses to allergens. Future investigations in this region could greatly benefit from the use of CRD.Although allergy studies among Ethiopian immigrants in Israel have had small sample sizes, they provide pertinent examples of how changing environments due to intercontinental migration from a helminth-endemic region to a high income country can lead to clinical manifestations of allergy symptoms that are closely linked to helminth infections. With more global migration and movement of people, such observations have implications for improving allergy diagnosis not only in helminth-endemic areas but in other parts of the world.Research from Sub-Saharan Africa has shown that novel allergens exist that are relevant to this region, particularly in relation to food allergy . AdditioUnderstanding the relationship between helminth infections and allergies has advanced our knowledge of how environmental factors that influence pathogenic exposures can hamper the diagnosis of allergy outcomes. Future investigations must build on our understanding of these links to generate new tools to diagnose, treat and prevent allergies not only in high income countries but also in the developing world where allergies are emerging as conditions of public health importance.