Ice streams drain large portions of ice sheets and play a fundamental role in governing their response to atmospheric and oceanic forcing, with implications for sea-level change. The mechanisms that generate ice stream flow remain elusive. Basal sliding and/or bed deformation have been hypothesized, but ice stream beds are largely inaccessible. Here we present a comprehensive, multi-scale study of the internal structure of mega-scale glacial lineations (MSGLs) formed at the bed of a palaeo ice stream. Analyses were undertaken at macro- and microscales, using multiple techniques including X-ray tomography, thin sections and ground penetrating radar (GPR) acquisitions. Results reveal homogeneity in stratigraphy, kinematics, granulometry and petrography. The consistency of the physical and geological properties demonstrates a continuously accreting, shallow-deforming, bed and invariant basal conditions. This implies that ice stream basal motion on soft sediment beds during MSGL formation is accommodated by plastic deformation, facilitated by continuous sediment supply and an inefficient drainage system. Ice streams are fundamental to ice sheet dynamics, but the mechanisms controlling their flow remain elusive. Here, the authors perform macro- and microscale analyses of mega-scale glacial lineations, which indicate a continuously accreting, shallow-deforming bed during ice stream flow. Ice streams play a fundamental role in the mass balance of ice sheets25810When ice stream beds are associated with the presence of soft sediments, they are typically organized into corrugations known as mega-scale glacial lineations (MSGLs)1618192 in the Wielkopolska Lowland, is underlain by a thick (∼30 m) sequence of Quaternary sediments and represents one of the few regions in onshore Europe to show a well-preserved assemblage of MSGLs.During the last glaciation, the SE sector of the Scandinavian Ice Sheet covered much of the Baltic region and was drained by a series of ice streams1622The OPIS MSGLs are characterized by the same long axis orientation (∼130°N), a regular spacing (crest-to-crest distance) of 500–700 m, and a generally low relief of 2–4 m , which iHere we present a suite of detailed sedimentological analyses from ten sites located across the best-preserved part of the OPIS MSGL field, including ridge crests and flanks. Results reveal, at all sites and depths, that the sediment has near-identical granulometry, strong and consistent macro- and microfabric, and similar petrography, whereas the stratigraphy is represented by a single massive unit of silty–sandy diamicton. The homogenization of the OPIS bed and the fine-grained nature of the sediment indicate ice stream basal conditions dominated by continuous sediment accretion and shallow, but pervasive, deformation during the formation of MSGLs.2. Gravel-sized clasts (2–64 mm) are rare and cobbles (>64 mm) are extremely rare. At no site was any other sedimentary unit exposed.With the exception of two relatively small structures (one ice wedge cast at site T and some rootlets at site D), all ten sites present an identical stratigraphy, with only slight variations in the modern soil depth, typically 0.3–0.5 m from the ground surface . The sedThe ground penetrating radar (GPR) reached the water table  and, with the exception of a few infilled palaeo-channels and small oblique and discordant reflections, interpreted as ice wedge casts, the >10 km of GPR lines revealed a uniform radar stratigraphy. This indicates that the trench data are representative of the MSGL field. All 200 MHz acquisitions, an example of which can be seen in a-axis macrofabric was measured at multiple depths in ten sites across the OPIS bed, including some that are kilometres apart along the same MSGL crest  and others that are distributed across the same MSGL flank, from near the trough to the crest . The azimuth and dip of a minimum of 30 clasts, with an elongation ratio ≥2:1 (a:b axes) and typically a-axis in the range of 5–20 mm, were measured from an area of ∼30 × 10 cm2 at each depth interval. Clast macrofabric along the crest of, or across, the same MSGL was found to be similar, with no evidence of any systematic variation horizontally and vertically (such as a herringbone pattern) (S1 eigenvectors are within 121.5(301.5)±11.5°N. The normalized eigenvalues of the macrofabric data are very high, with a mean value of 0.75. The vast majority of fabric shapes, derived from the ratios between the three main eigenvalues plotted on an equilateral ternary diagramClast pattern) . Macrofapattern) . Eighty Microscale analysis of orientated thin sections of the diamicton within the MSGLs reveals a complex, but systematic, array of deformation fabrics, which can be interpreted as having formed by the passive rotation of sand-grade particles, into the planes of the foliations, defining a number of clast two-dimensional (2D) microfabrics26A prominent, subvertical foliation present within the lower part of the diamicton sequence locally overprints the earlier shear fabrics and is interpreted as recording the subsequent dewatering of the sediments within the core of the MSGL. Dewatering and consolidation would have been driven by the ice overburden pressure. This may have occurred penecontemporaneous with landform development, or shortly after the cessation of fast ice flow when the diamicton was unconsolidated and still able to respond to the dewatering.a-axes dipping apparently up-glacier at ∼24° relative to the horizontal, whereas the second geometry has a more variable  down-ice dip (b-axis <500 μm). Low resulting V1 dip angles are a statistical artifact of eigenvector analysis of samples with multiple fabric modes. Derived eigenvalues remain broadly consistent between samples, representing a strong girdle fabric shape in all samples. The geometry and kinematics recorded by the μCT data sets are spatially consistent: vertically within the sample, vertically within each trench and between sites, and record a sense of shear that is parallel to the orientation of the MSGLs and inferred ice-flow direction.The three-dimensional (3D) visualization of the particle bulk phase of all X-ray microtomography (μCT) scanned samples highlights that within a complex overall fabric signature, two distinctive geometries are represented by chains of particles . The dom-ice dip . These c-ice dip  illustraThe clast (2–4 mm) petrography was determined on a minimum of 300 grains per depth interval, with a distinction made between weathering-resistant components including sedimentary, flint, quartz and  crystalline lithologies, and components susceptible to postdepositional weathering. Overall, the composition of lithologies is consistent across all samples . Within 2829The <2 mm fraction of all diamicton samples is largely composed of sand (62–71%) with a minor component of silt (29–38%) and low clay contents (4–9%) . Grain sIn summary, ten sites were investigated and sampled at a high vertical resolution, within the same MSGL and across multiple MSGLs, and there are no obvious differences in clast macro- or microfabric (orientation and strength), petrography and granulometry. Sediment homogeneity might be responsible for the lack of visible evidence of outcrop-scale thrusting or folding, as these are difficult to identify when they do not involve deformation of distinctively different materials. However, given the density of sampling, a variation in fabric should have been evident had faulting or thrusting been present. Moreover, based on observations from extant9The preservation of the OPIS MSGLs, coupled with the homogenous and massive architecture of the diamicton, and the rare presence of postformational periglacial, glaciofluvial and fluvial disturbances demonstrates that these landforms and their internal structure reflect basal processes occurring beneath the active ice rather than in ice-marginal or proglacial settings. The vertical and horizontal consistency of the clast macro- and microfabrics indicates that the diamicton has experienced pervasive shearing. Theoretical3371136Two possible scenarios might be envisaged to link the sedimentary processes to the formation of the MSGLs. One scenario is that pervasive deformation of the bed was concomitant with the formation of the MSGLs, with the implication that the origin of these landforms is constructional during bed accretion. The other scenario is that the strain signature was previously imposed on the sediment and a later phase of ice stream flow generated the MSGLs by erosion. The sedimentological characteristics  found on at least one palaeo ice stream bed21Although the actual process of moulding the bed into the ice-flow parallel ridges and troughs (MSGLs) remains elusive, the data presented here indicate that they were formed through continuous sediment accumulation. Thus, to generate an uneven topography, a higher rate of accretion must have selectively occurred towards the crests of the MSGLs. Significantly, this study demonstrates that the OPIS MSGLs record ice stream flow via thin-skinned deformation, under largely invariant sedimentary and hydrological basal conditions. Specifically, indicative of an inefficient, distributed drainage system are the continuity of the MSGLs with a lack of evidence for a major meltwater drainage network; the homogeneity of the diamicton, that is, no depletion of fines; and the absence of deformational structures related to water pressure fluctuations and eluviationThe MSGLs analysed here are morphologically similar to those of many other settings worldwide32a axis macrofabric measurements were carried out on clasts with an elongation ratio of at least 2:1 (a:b). All visible elongated clasts were measured, typically with an a axis length range of 5–20 mm. Clasts were measured at each depth interval across an exposed surface of about 30 × 10 cm. The fabric measurements were undertaken by multiple operators at each site, with usually one operator per sampled interval. Samples were also collected for quantitative petrographic and granulometric characterization, which was carried out at the sedimentological laboratory of the Department of Geoscience, Aarhus University, Denmark. In addition, samples were collected for micromorphology and X-ray tomography analyses. Morphometric analysis of the MSGLs was carried out with ArcGIS on a 5-m resolution digital terrain model, using standard techniquesDetailed investigations were focussed on ten sites located across the best-preserved part of the OPIS MSGL field, including ridge crests , enabling systematic, objective and robust identification of all particles with b axis >250 μm . Acquisitions were made with a monostatic transmitting and receiving 40 MHz  unshielded antennae and 200 MHz shielded antennae. A total of 12 GPR profiles were acquired, some as long as 1,200 m, covering a total surveyed length of 10,300 m; most profiles were repeated with both antennas. Data for the 40-MHz survey were captured in step collection mode with a step length of 1 m, whereas a continuous mode acquisition was adopted for the 200 MHz survey, with a step length of 0.25 m. Configuration for both data acquisitions provided 1,024 samples per scan in a time window of 200 ns. A standard processing sequence was applied to the raw data to adjust GPR traces to a common time-zero position, filter out noise and gain attenuated GPR signals. Specifically, a horizontal running average filter was applied to remove the saturation effect caused by Tx–Rx direct coupling. A subtraction of the mean trace to the data set  was applied to filter out continuous flat reflections caused by multiple reflections between the antenna, the operators and the ground surface. This filter was applied to the data from the 40 MHz survey and limited to the first 10 ns, to avoid disrupting reflections from continuous flat layers below the surface. Following a spectral analysis of measured signals, a band-pass filter (21-38-110-156) was applied, to remove undesired frequencies coming from instrumental and environmental noise. To enhance the visibility of deeper reflections due to signal attenuation, a gain function, increasing linearly with depth, was applied. The availability of water table depths along the GPR profile (measured by augering) allowed calibration and to convert the arrival times of reflected radar waves to depth below surface. Calibrations based on each auger sample, with an instrumental accuracy of ±15 cm, were consistent with each other and indicated an EM wave velocity of ∼6 cm ns−1, in line with the velocity usually defined for this type of sediment in unsaturated conditions. This value was used for the time-to-depth conversion for all 40 MHz radargrams. The existence, in the 200-MHz acquisitions, of some diffraction hyperbola allowed adoption of the synthetic hyperbola method. An estimated EM wave velocity of about 8 cm ns−1 was consistently found and applied to all 200 MHz profiles.GPR acquisitions were carried out along and across the MSGLs long axes, with profiles usually located near the trenches used for the sedimentological analyses, using an IDS Radar System .Supplementary Figure 1 and Supplementary Tables 1-2.


The aerobic synthesis involves reduction of the centrally chelated cobalt metal ion of the corrin ring from Co(II) to Co(I) before adenosylation can take place. A corrin reductase (CobR) enzyme has been identified as the likely agent to catalyse this reduction of the metal ion. Herein, we reveal how Brucella melitensis CobR binds its coenzyme FAD (flavin dinucleotide) and we also show that the enzyme can bind a corrin substrate consistent with its role in reduction of the cobalt of the corrin ring. Stopped-flow kinetics and EPR reveal a mechanistic asymmetry in CobR dimer that provides a potential link between the two electron reduction by NADH to the single electron reduction of Co(II) to Co(I).Adenosylcobalamin, the coenzyme form of vitamin B CobR is a corrin reductase involved in the biosynthesis of adenosylcobalamin. Using a range of biophysical techniques, we have obtained molecular details on substrate binding and investigated protein stability. Kinetic experiments have identified half-site reactivity. It functions as a coenzyme or cofactor in a number of important processes, and is particularly associated with isomerization, methylation and dehalogenation reactions NADD or [(4S)-2H)]NADD. The observed rate constant for the first phase (kobs1) against NADH concentration was fitted to a hyperbolic function (K1) of 24.8±2.6 μM and a limiting maximal rate of reduction (kmax) of 41.3±1.1 s−1. This suggests that following the rapid binding of NADH to CobR, a rearrangement or conformational change is required before hydride transfer from the pyridine nucleotide. This conformational change is the rate limiting step in the reduction of the flavin and precedes a faster hydride transfer reaction. The reduction of the first flavin in the dimer, together with the associated change in conformation, greatly affects the rate of reduction at the second site. This implies communication across the dimer interface and half site reactivity.The reaction mechanism of CobR involves the reduction of the bound flavin prosthetic group by NADH. This reductive half reaction was investigated under anaerobic conditions using the stopped-flow apparatus. Absorption transients at 455 nm were recorded following the mixing of CobR (5 μM) with NADH (10–250 μM) at 25°C. These time-dependent data were best fitted to a double exponential function representing two distinct phases of the reaction. The first phase . DSC studies of the flavin reductase from S. meliloti revealed an apparent Tm of 72°C (results not shown), which raises the question of whether enhanced thermostability or structural rigidity is an evolutionary adaption that is required for the function of the CobR enzyme.Stability studies reveal CobR to be a thermostable protein with a melting temperature of 70°C, which is enhanced in the presence of the cofactor FAD to 80°C. Cofactors can aid protein stability, of course, through preferential binding to the native state ,34. AlsoG of denaturation (ΔGurea−ΔGGdnHCl) of 17.4 kJ mol−1 supporting a significant electrostatic contribution to conformational stability.Despite the protein exhibiting thermal stable properties the chemical denaturation study confirmed the protein can be unfolded at relatively low concentrations of denaturant. Unfolding curves show a two-state folding mechanism, once unfolding occurs to the dimer it precedes to an unfolded state. Differences between the chemical stability estimations via urea and GdnHCl are widespread through the literature. For example, Human Placental Cystatin, a thiol proteinase inhibitor, has a midpoint of transition of 1.5 M in GdnHCl and 3 M in urea . A complIt has been suggested that for an enzyme to be designated a CobR there should be evidence not only for reduction of the cobalt but also corrin binding . Herein,Interestingly, CobR seems to provide an axial ligand for the cobalt ion. This is similar to what is observed with the binding of cobalamin to PduS , the cobin vivo is currently unknown. It is possible that CobR transfers electrons to the adenosyltransferase or alternatively the corrin is reduced while bound to CobR and then passed on the adenosyltransferase enzyme. The fact that CobR has been shown to bind and reduce a range of corrins to the Co1+ state even in the absence of an adenosyl transferase [2+-corrin to the adenosyltransferase enzyme appears to induce a 4-coordinate conformation which reduces the thermodynamic barrier for reduction. In this state, a number of flavoproteins and even free reduced flavin have been shown to catalyse the reduction of the corrin [B. melitensis, the rate of reduction was significantly slower than the PduO type [The site of corrin reduction nsferase  perhaps e corrin . Adenosye corrin , and theduO type . This maReduction of the FAD coenzyme by NADH appears to be at least a three-step process, where a conformational change is required before hydride transfer. The protein displays half site reactivity, where reduction of the flavin in one active site affects the rate of reduction at the second active site in the dimer. This observed conformation change associated with the binding of NADH at one active site is similar to the phenomenon that was observed in the EPR studies looking at cobinamide binding, where two different coordination states were detected depending on the whether one or both of the active sites in the dimer were occupied.The functional importance and structural basis for the observed half site reactivity are unclear. The two available CobR structures (with either 1 or 4 molecules of flavin bound per homodimer) are essentially identical in structure. Comparison of the structures of Phea2 and TftC with and without NAD similarly also shows very little structural change. Consequently, any structural or dynamic changes occurring upon substrate binding or flavin reduction are subtle. Clearly, the binding of flavin or substrate to one active centre will affect the electrostatic potential at the other. Additionally, subtle changes in dynamics at the second active centre may accompany binding at the first. The two active sites of the CobR homodimer are connected through a water containing channel and it is possible that communication is mediated through this channel.The half site reactivity will result in one subunit reacting at a time. This may be beneficial to the catalytic process, since the enzyme links the one electron reduction of the corrin with the two electrons obtained from NADH. The fate of the second electron is currently unknown; however, it is known to disproportionate very quickly and this could be intramolecular rather than an intermolecular process. It is also possible that NADH binds to one active site while the corrin substrate docks at the active site on the opposite side of the dimer. Such a process could allow for regulation of the enzyme thereby controlling adenosylcobalamin synthesis. In this respect the functional importance of this half site reactivity requires further investigation.


Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects  The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently deregulated in a majority of human cancers . This paPRAS40, first reported as substrate for AKT , is an iRapamycin and its analogs  are allosteric inhibitors of mTORC1 via their binding to FKBP12, and were among the first mTOR-targeted therapeutics to enter the clinic . HoweverIn the present study, we demonstrate that the redundant phosphorylation of PRAS40 by both AKT and mTORC1 signaling is a novel mechanistic basis for the acquired resistance to rapamycin in cancer cells. Combined inhibition of AKT and mTORC1 is required for effective inhibition of PRAS40 phosphorylation on both Ser183 and Thr246 sites, which in turn increases the ability of PRAS40 to inhibit mTORC1-mediated 4E-BP1 phosphorylation and translation concomitant with suppression of tumor growth and cell motility. Our data uncover an important role of PRAS40 in the translational control of tumor progression and therapeutic response to mTORC1 inhibitors.PIK3CA), rapamycin at 50 nM effectively inhibited phosphorylation of the mTORC1 substrate p70S6 kinase (p70S6K) and its downstream target S6  and AKT inhibitors results in additive or synergistic antitumor effects -38. HoweThe p70S6K is another well-known substrate of mTORC1. Although p70S6K and its substrate ribosomal protein S6 are also known as regulators of mRNA biogenesis and translation, a growing body of evidence, including our studies, indicate that the deregulation of cap-dependent translation downstream of mTORC1 at the level of 4E-BP1/eIF4E plays a central role in tumor formation and metastatic progression; the contribution of p70S6K1 and S6 to the oncogenic action of the mTORC1 upstream activators, AKT and/or extracellular signal-regulated kinase, appears limited , 39, 40.et al. suggests that the sequence composition of an mTORC1 phosphorylation site is one of the key determinants of whether the site is good or poor mTORC1 substrate within cells [in vitro and in vivo. Our data suggest that AKT inhibition can reverse rapamycin-resistant mTORC1 substrates being sensitive to rapamycin. Mechanistically, we found that this reversal is likely due to a more complete dephosphorylation of PRAS40, which leads to an increase in the formation of PRAS40-raptor complex by enhancing inhibition of mTORC1 activity for phosphorylation of 4E-BP1. We demonstrated that a combination of AKT and mTORC1 inhibitors is required to effectively inhibit phosphorylation of PRAS40 on both Ser183 and Thr246 sites and increases PRAS40 binding to raptor  and PTEN-deficient (MDA-MB-468) breast cancer cell lines [KRAS-mutant SW620 colon cancer cells are particularly resistant to PP242, and PP242 cannot effectively inhibit 4E-BP1 phosphorylation. Intriguingly, we found that AKT inhibition by MK2206 in combination with PP242 could elicit more profound inhibition of 4E-BP1 phosphorylation compared to the responses to PP242 alone (The implications of our findings suggest that the ATP-competitive mTOR kinase inhibitors (mTORKIs) that inhibit both mTORC1 and mTORC2 may provide better therapeutic outcomes when compared with rapamycin in the clinic. mTORC2 directly phosphorylates AKT at its hydrophobic motif Ser473), which enhances the catalytic activity of AKT already phosphorylated on Thr308 . In cont3, which ll lines . Moreovell lines . Consistll lines . Furtherll lines , 49. The42 alone . Taken tIn summary, our study provides new insight into the biologic and therapeutic relevance of PRAS40 in translational regulation of tumor cell proliferation, survival and motility. Our findings reveal that regulation of PRAS40 activity through cooperative AKT and mTORC1 phosphorylation of both Ser183 and Thr246 is a key process to alter the mTORC1 substrate (e.g. 4E-BP1) specificity. In addition to the phosphorylation of 4E-BP1, PRAS40 phosphorylation may also serve as a surrogate marker to evaluate the response to the PI3K/AKT/mTOR pathway inhibitors in clinic. These findings could have high translational significance and enhance our understanding of the involvement of PRAS40 and 4E-BP1 in the regulation of cancer progression and therapeutics.Human breast and colon cancer cell lines were obtained from the American Type Culture Collection  and maintained in the appropriate medium with supplements as suggested by ATCC. Rapamycin and MK2206 were obtained from LC Laboratories  and Selleck , respectively. The PRK5-PRAS40 construct was from Addgene . The PRAS40 cDNA was amplified by PCR using PRK5-PRAS40 as a template and the product subcloned into the Sgf I and Mlu I sites of pCMV6-Myc-Flag . The insert was mutated using a QuikChange XLII mutagenesis kit . Mutants with Asp at Ser183 (S183D), Thr246 (T246D) and both Ser183 and Thr246 (S183D/T246D) were generated in pCMV6-Myc-Flag. All constructs were verified by DNA sequencing. Lentiviral shRNAs to human 4E-BP1 were from Open Biosystems  and the specificity of the targeting sequences has been verified in our previous study . LentiviCell proliferation was assessed by counting the number of viable cells in response to the treatment with the indicated drugs as described previously .Cells were plated in 100-mm dishes, grown overnight, and treated as indicated in figure legends. Both adherent and floating cells were harvested. For cell-cycle analysis, cell nuclei were prepared by the method of Nusse  and staiMigration and invasion assays were performed in Boyden chambers with coated collagen or Matrigel, respectively, as instructed by the manufacturer (BD Biosciences) and described previously .Cells were lysed in NP-40 lysis buffer, and analyzed by Western blot using equal total protein loading as described previously . Phosphoet al [PRAS40 complexes were immunoprecipitated with PRAS40 antibody  or anti-Flag antibody (Sigma) according to the procedure described by Sancak et al . At the 7GTP Sepharose beads  to capture eIF4E and its binding partners. Precipitates were washed three times with lysis buffer and resuspended in 2× Laemmli sample buffer followed by Western blot analysis.Cell lysates as prepared above were incubated with mRenilla luciferase gene and cap-independent Polio IRES-mediated translation of the firefly luciferase gene [Cells  were transfected with a bicistronic luciferase reporter plasmid (0.2 μg), pcDNA3-rLuc-PolioIRES-fLuc, which directs cap-dependent translation of the ase gene , in 12-wase gene , 12. Cap6 cells suspended 1:1 (volume) with growth factor-reduced Matrigel (BD Biosciences) on the opposite side. For efficacy studies, mice were randomized among control and treated groups (n = 5 per group) when tumors were well-established (~150-180 mm3). Rapamycin was prepared in absolute ethanol at 10 mg/ml and diluted in 5% Tween-80 and 5% PEG-400 before injection. Rapamycin was administered by intraperitoneal injection at 4 mg/kg once per day, Mon-Fri as previously reported [3 = π/6 x larger diameter x 2. Tumors were excised and snap frozen in liquid nitrogen, homogenized in 2% SDS lysis buffer and then processed for Western blot analysis as described previously [Female athymic nude mice (5-6 weeks old) were purchased from Taconic . Experiments were carried out under a protocol approved by the University of Kentucky Institutional Animal Care and Use Committee. MCF7 xenograft tumors were established by subcutaneously implanting 0.72 mg sustained release 17β-estradiol pellets into one flank at least 3 days before injecting 5 × 10reported . MK2206 reported . Controleviously , 12.t-test was used to compare the intergroup. Differences between groups were considered statistically significant at P < 0.05.All experiments were performed at least twice. Results are expressed as mean ± S.E.M. where applicable. A two-tailed Student's


Introduction. Polyaxial screws had been only tested according to the ASTM standards (when they were perpendicularly positioned to the rod). In this study, effects of the pedicle screws angled fixation to the rod on the mechanical properties of fixation were investigated. Materials and Method. 30 vertically fixed screws and 30 screws fixed with angle were used in the study. Screws were used in three different diameters which were 6.5 mm, 7.0 mm, and 7.5 mm, in equal numbers. Axial pull-out and flexion moment tests were performed. Test results compared with each other using appropriate statistical methods.  Results. In pull-out test, vertically fixed screws, in 6.5 mm and 7.0 mm diameter, had significantly higher maximum load values than angled fixed screws with the same diameters (P < 0.01). Additionally, vertically fixed screws, in all diameters, had significantly greater stiffness according to corresponding size fixed with angle (P < 0.005).  Conclusion. Fixing the pedicle screw to the rod with angle significantly decreased the pull-out stiffness in all diameters. Similarly, pedicle screw instrumentation fixed with angle decreased the minimum sagittal angle between the rod and the screw in all diameters for flexion moment test but the differences were not significant. Pedicle screw-rod systems have almost been the standard method of fixation in the treatment of various spinal disorders following advancement by Cotrel and Dubousset. Studies reported that use of pedicle screw fixation allowed surgeons to apply enhanced corrective forces to spine and advantages of the pedicle screw fixation compared with hook or hybrid systems –4. AscenStability of the pedicle screw constructs have been evaluated mainly by pullout, insertional torque, and cyclic loading tests . NumerouUnderstanding the degree of freedom's importance on the pedicle screw head was a significant milestone in development of the pedicle screw fixation. Monoaxial pedicle screws were firstly used in spinal fixation and provided effective correction for the deformity in coronal, sagittal, and axial planes. However, difficulties in achieving lodgement of the rod into the screw head for monoaxial pedicle screws in regions such as lumbosacral and sacropelvic and arrangement in the insertion depth of the screw compelled the surgeons during the operations. Additionally, following the operation, the increased stress could occur in interface of instrumentation and fixed segments, and loss of fixation could also occur. To overcome the disadvantages of the monoaxial screws, polyaxial pedicle screws were then used. Polyaxial pedicle screw fixation eased the achieving appropriate contact between the instrumentation and fixed segments in the surgical operations due to its degree of freedom capacity. There were many studies which compare the biomechanical effects of the monoaxial and polyaxial pedicle screw fixations –36. In tAlthough polyaxial screw was commonly used due to its advantage of easy rod connection, the screws are tested only in the perpendicular position  accordinStiffness can be described as the rigidity of a structure. It can be calculated by the load versus displacement curve. The slope of the linear elastic portion of load versus displacement curve gives the stiffness.This study was raised from the senior authors' observations, polyaxial screw failures  mostly during or following deformity correction procedures. In the present study it is hypothesized that angled fixed pedicle screws to the rods, as similar as surgical applications, have lower stiffness. Screws with larger core diameters may be of disadvantage in this respect. The purpose of this study is to assess the effect of pedicle screw head-shaft fixation angles on the mechanical properties of pedicle screws.Axial pullout test and flexion moment test were performed to investigate the polyaxial pedicle screw failure in the present study. Sixty titanium polyaxial pedicle screws , with 45 mm length, were used. The screws with three different outer diameters, 6.5 mm, 7.0 mm, and 7.5 mm, were used and each group had 20 screws.Screws, in equal numbers, were fully inserted perpendicularly into ultrahigh molecular weight polyethylene (UHMWPE) blocks and fixed to 6.0 mm titanium rods with 0 or 15 degrees of screw head/shaft angles on sagittal plane as can be seen in Remaining screws were sustained to flexion moment test. The test frame and crosshead speed were the same with the axial pullout test. Polyethylene blocks holding the screws and a metal block holding the rods were fixed into apparatuses allowing the flexion and downward vertical force was applied to system until the screw head cut-off. The test setup for flexion test can be seen on t-tests were performed to compare the results for different groups.Study groups were formed by screw diameter , fixation angle of screw head to rod , and the applied test (pullout and flexion moment); thus 12 groups, each group containing 5 samples, were obtained. Data were statistically analysed using Microsoft Excel 2010; two-tailed Student's P < 0.01) as given in P < 0.0001). Similarly, 7.5 mm screws had significantly higher maximum load than 6.5 mm screws with the P value of 0.0003 when they fixed vertically. 6.5 mm screws fixed with angle had significantly lower maximum load than 7.0 mm screws fixed with angle (P = 0.0002) and 7.5 mm screws fixed with angle (P = 0.0074). There were no other significant differences for maximum load. Vertically fixed screws, in all diameters, had significantly greater stiffness according to corresponding size fixed with angle (P < 0.005). Although 7.5 mm vertically fixed screws had higher pullout strength than 6.5 mm vertically fixed screws; the latter was stiffer (P = 0.0498); others showed no significant stiffness difference according to the screw diameter.7.0 mm vertically fixed screws had the highest maximum load values (pull-out strength) followed by 7.5 mm and 6.5 mm vertically fixed screws. 7.0 mm vertically fixed screws had also the highest stiffness values followed by 6.5 mm and 7.5 mm vertically fixed screws as can be seen on P < 0.02). 6.5 mm screws had greater minimum sagittal angle values than 7.5 mm screws when they were fixed vertically (P = 0.048). 6.5 mm screws had greater minimum sagittal angle values than 7.0 mm screws when they were fixed with angle (P = 0.006).7.0 mm pedicle screws fixed with angle had the highest moment at maximum load and the lowest minimum sagittal angle values as can be seen in Many studies have been conducted about the factors which affect the stability of pedicle screws. Vertebral bone mineral density, anatomy of the pedicles and insertion techniques, and pedicle screw characteristics are directly related to pullout and fatigue strength –18, 25. Polyaxial screw's mobile head design facilitates easier rod connection and provides solution for screw placement problems. Studies demonstrate that polyaxial screws are as effective as monoaxial screws in the coronal and sagittal plane correction but the latter provides greater correction of rotational deformity , 34. AltIn pullout test, all screw heads were pulled out from the screw in both vertically fixed and angled groups which can be seen in There are not any other studies in the literature evaluating stability of polyaxial screws in this manner. Although polyaxial screws have been found to have more compression and flexion stiffness than the monoaxial screws with an anterior cage support at lumbosacral spine  and instPolyaxial screw head coupling the shaft has been shown as the first part failing against load and this was suggested as a protective feature of the screw preventing screw or rod failure . It was Increasing diameters are positively correlated with pullout strength and moment at maximum load values of the screws . In the Minimum sagittal angle of 6.5 mm screws was higher than 7.5 mm when they fixed vertically and higher than 7.0 mm screws when they fixed with angle. Fixing the screw with angle decreased the minimum sagittal angle in all diameters but the differences were not statistically significant.In the present study, the effects of angled fixation of pedicle screw to the rod on the mechanical properties of fixation were investigated. Fixing the pedicle screw to the rod with angle significantly decreased the pullout stiffness in all diameters for axial pullout test. Similarly, pedicle screw instrumentation fixed with angle decreased the minimum sagittal angle between the rod and the screw in all diameters for flexion moment test, but the differences were not significant.


Cynomys ludovicianus). Our study aimed to compare the vegetation composition of an urban and exurban rangeland, and explore the role that prairie dogs play in these systems. The percent absolute canopy cover of graminoids (grasses and grass-likes), forbs, shrubs, litter, and bare ground were estimated at sampling areas located on and off prairie dog colonies at an urban and an exurban site. Herbaceous forage quality and quantity were determined on plant material collected from exclosure cages located on the colony during the entire growing season, while a relative estimate of prairie dog density was calculated using maximum counts. The exurban site had more litter and plant cover and less bare ground than the urban site. Graminoids were the dominant vegetation at the exurban plots. In contrast, mostly introduced forbs were found on the urban prairie dog colony. However, the forage quality and quantity tests demonstrated no difference between the two colonies. The relative prairie dog density was greater at the urban colony, which has the potential to drive greater vegetation utilization and reduced cover. Exurban rangeland showed lower levels of impact and retained all of the plant functional groups both on- and off-colony. These results suggest that activities of prairie dogs might further exacerbate the impacts of humans in fragmented urban rangeland habitats. Greater understanding of the drivers of these impacts and the spatial scales at which they occur are likely to prove valuable in the management and conservation of rangelands in and around urban areas.Rapid human population growth and habitat modification in the western United States has led to the formation of urban and exurban rangelands. Many of these rangelands are also home to populations of black-tailed prairie dogs ( The North American Great Plains region is a large dynamic ecosystem that is inhabited by a diverse variety of plants and animals, which have generated a heterogeneous landscape made up of three major prairie types—shortgrass, mixed, and tallgrass . Since ECynomys ludovicianus), which has faced widespread decline across its historic range (Rangelands (including prairies) provide a wide range of ecosystem goods and services, which are strongly influenced by the structure, and dynamics of the vegetation . Often, ic range . The decic range . Remainiic range .Black-tailed prairie dogs (referred to herein as prairie dogs) are herbivorous, consuming a wide range of available plant material from grasses to prickly pear cactus . They arThe aim of our study was to compare the vegetation of prairie dog habitats in an urban and an exurban rangeland, to explore whether the associated difference in human disturbance may lead to differences in vegetation abundance and composition on and off prairie dog colonies. The predictions were: (1) The presence of a prairie dog colony would reduce the abundance of vegetation and litter and increase the amount of bare ground at both sites, as a function of prairie dog foraging behavior and burrowing activity. (2) The exurban site would support a greater abundance of graminoids (grasses and grass-like plants) and forbs, and have greater quantities of litter and less bare ground than urban site, due to less human disturbance enabling prairie dog foraging and burrowing to be distributed over a larger area. (3) The urban site would have lower native plant cover due to a greater probability of non-native plant species being introduced from nearby developments. (4) The quantity and quality of forage at the exurban prairie dog colony would be greater than at the urban prairie dog colony. (5) Prairie dog density would be greater at the urban colony compared with the exurban colony due to the limited available range and lack of habitat connectivity for animals to disperse to nearby rangelands.The research was conducted in Fort Collins, Colorado from June to August 2013. Two rangeland study sites (urban and exurban) were selected according to their location relative to the city, proximity to infrastructure and in conjunction with the definitions of urban and exurban developments given in Elymus smithii (Rydb.) Gould). The species observed on- and off-colony at the urban site were different and there was no overlap in dominants. There was more similarity in the vegetation on- and off-colony at the exurban site with two common dominant species; western wheatgrass and Japanese brome (Bromus japonicus Thunb.). There were more introduced species observed at the exurban site than at the urban site.The vegetation composition varied across the sampling areas . OverallData were collected on and off of the prairie dog colonies at each of the two study sites. On-colony sampling was conducted by locating the approximate center of the colony using prairie dog burrow distribution and animal density as indicators. Once the center point was identified, a thirty-five meter transect was positioned across the colony, with the center point of the transect corresponding to the center of the colony. Two more thirty-five meter transects were established; one on each side of the central transect, running parallel and separated by a distance of 15 m. Selection of the off-colony sampling areas was dependent on the absence of burrows and a minimum buffer of 20 m from the nearest observed evidence of prairie dog activity . Once a suitable area was demarcated, three thirty-five meter transects were laid out following the same approach used for the on-colony plots. The methods described above to locate transects in the sampling areas resulted in thorough coverage of the prairie dog colonies and similar sized neighboring off-colony areas. The data used in the analyses are available in 2 frame locations (subsamples) along each transect (transect = observation). Canopy cover was estimated once each month during the summer (June–August) to track changes in vegetation composition through time. The three data collection periods were classified as early summer (June), peak standing crop (July) and late summer (August). Cover was categorized into four distinct functional groups: (1) graminoids, (2) forbs, (3) litter, and (4) bare ground. In mid-July (peak standing crop), the graminoid and forb functional groups were further subdivided according to their duration , origin (native or introduced), and growing season (cool or warm).Canopy cover and vegetation composition were determined using an extended Daubenmire frame see  placed eThe quality and quantity of forage available to the prairie dogs on the colony was estimated from three exclosure cages (dimensions (W × L × H): 30 cm × 60 cm × 75 cm) made out of 0.5 cm × 0.5 cm hardware cloth over wire panels placed on each colony (urban and exurban). The exclosure cages were positioned 15 m apart on a line that ran through the center of the sampling area perpendicular to the transects, with the first cage located at the mid-point between transects 1 and 2. In August, all herbaceous material in each cage was clipped to ground level, bagged and placed in drying ovens at 55 °C for one week. The dried material was weighed and then sent for laboratory analysis  to determine the percentage of total digestible nutrients, crude protein, acid detergent fiber, neutral detergent fiber, and a relative feed value index.2). Five repeat counts were performed at each site from the same observation point, which was approximately 150 m away from the sampling area. The data were collected from August 15 to August 31, with a minimum of 24 h between repeat counts. The counts were conducted between 7:00 am and 11:00 am and lasted for 90 min with the total number of aboveground animals within the marked observation area recorded every 10 min. A standardized settling time of 30 min was initiated prior to data collection, allowing the prairie dogs sufficient time to return to their normal behavior after the disturbance of the observer’s arrival  to test for the effects of site, presence or absence of a prairie dog colony, season, and all possible interactions. Peak standing crop cover data were analyzed separately to test for the effects of site, presence or absence of a prairie dog colony, and all possible interactions. The data were analyzed using analysis of variance in SAS 9.3 . Where F = 0.15–2.54, P = 0.100–0.863), the season by site interaction , the season by colony interaction , or the interaction of all three factors .Cover values for the plant functional groups, litter and bare ground were not affected by season  and also greater off-colony than on-colony . The overall percentage of bare ground was greater at the urban site compared to the exurban site .Absolute litter cover was greater at the exurban site than at the urban site , presence of a prairie dog colony , and the two factors simultaneously . The absolute cover of graminoids ranged from 46% off-colony at the exurban site to 0% on-colony at the urban site, while cover values off-colony at the urban site and on-colony at the exurban site were similar and around 25% , but was affected by the presence of a prairie dog colony , and the two factors simultaneously . Absolute forb cover was greatest (40%) on-colony at the urban site and least (5%) off-colony at the urban site , but had lower cover values on prairie dog colonies compared to off . The interaction between colony and site was not significant . Cover of perennial native cool season graminoids was affected by a significant interaction between site and presence of a prairie dog colony , while the presence of a prairie dog colony did not affect cover  nor did the two factors interact . Perennial introduced cool season graminoids were affected by the two-way interaction between site and colony  or the presence or absence of a prairie dog colony . The interaction between site and colony was also non-significant . Bienneial native forbs were only observed off-colony at the urban site.Perennial native forb species cover did not vary significantly by site  but was affected by the presence of a prairie dog colony . The interaction between site and colony was also significant  (Convolvulus arvensis L.) at three of the four sampled areas (see Absolute (%) cover of perennial introduced forbs was not affected by site ( = 0.01) . The grereas see . Cover oThe forage testing analysis and data gathered from weighing the dried biomass revealed no significance differences between sites for the six measures of forage quality and quantity see .F = 10.20, P = 0.02). The mean relative density of prairie dogs at the exurban colony was 14 (±2 SE) individuals per hectare, while the density at the urban colony was 19 (±3 SE) individuals per hectare. The dataset comprised nine separate counts, so caution is required with the interpretation of these results.The prairie dog population observations taken from each colony indicated that the relative measures of prairie dog densities differed significantly between the exurban and urban colonies  Repeated measures analyses of variance on absolute canopy cover of plant functional groups, litter and bare ground on- and off-colony (prairie dog) at urban and exurban sites in Fort Collins, Colorado, USA. Data were transformed using the arc-sine square root functions to meet assumptions of AOV. (B) Analyses of variance on absolute canopy cover of plant functional groups at peak standing crop on- and off-colony (prairie dog) at urban and exurban sites in Fort Collins, Colorado, USA. Data were transformed using the arc-sine square root functions to meet assumptions of AOV.Click here for additional data file.


Staphylococcus aureus enterotoxins (SEs) in the serum from patients with chronic rhinosinusitis (CRS) and its involvement in the condition. Thirty CRS patients without nasal polyps (CRSsNP), 40 CRS patients with nasal polyps (CRSwNP), and 30 healthy controls were enrolled in this study. Peripheral blood was obtained and analyzed to measure the serum levels of total IgE, specific IgE to SEA, SEB and SEC, and eosinophil cationic protein (ECP) using ImmunoCAP assays. The positive rate and level of serum specific IgE to SEB, but not to SEA or SEC, were significantly higher in CRSwNP patients compared with the controls . No significant differences were found between CRSsNP patients and controls, or between CRSsNP and CRSwNP patients. Serum total IgE was significantly elevated and positively correlated with SEB-specific IgE in the CRSsNP  and CRSwNP  groups. ECP was also significantly increased in the CRSsNP (P=0.002) and CRSwNP (P<0.001) groups, but not correlated with specific IgE to SEs in either CRS group. The results suggest that SEB may play a role in the pathogenesis of CRSwNP.The aim of this study was to determine the prevalence of  Chronic rhinosinusitis (CRS) is characterized by mucosal inflammation of the nose and paranasal sinuses and often taken as an umbrella term for a heterogeneous group of sinus diseases. CRS has been divided into CRS without nasal polyps (CRSsNP) and with nasal polyps (CRSwNP) on the basis of clinical presentation ,2. HowevStaphylococcus aureus enterotoxins (SEs), secreted by Staphylococcus aureus (S. aureus), the most common colonizer of nasal passages and sinuses, are broadly classified as superantigens . The diagnosis of CRS was based on medical history, clinical symptoms, endoscopic examination, and sinus CT scanning according to the European Position Paper on Rhinosinusitis and Nasal Polyps 2007 as well as the Chinese CRS guidelines ,2. ThirtPeripheral blood (3 ml) was collected from each subject. After centrifugation at 100 × g for 10 min, the serum was separated and stored at −70°C until further analysis. The levels of total IgE, specific IgE to SEA, SEB and SEC, and ECP in sera were measured using ImmunoCAP assays (Phadia AB) according to the manufacture’s recommendations. The detection limits were set at <2 kU/l for total IgE and 2 μg/l for ECP. A concentration of specific IgE ≥0.35 kUA/l was considered as positive, and the levels were expressed as the following grades: 0, <0.35 kUA/l; I, 0.35–0.69 kUA/l; II, 0.7–3.49 kUA/l; III, 3.5–17.49 kUA/l. IV, 17.5–49.9 kUA/l; V, 50–100 kUA/l; VI, >100 kUA/l.2 test or Fishers’ exact test. Correlations were calculated using the Spearman test. A P-value <0.05 was considered statistically significant.Statistical analyses were performed using SAS software version 9.1.3 . Specific IgE to SEA, SEB, and SEC were analyzed as ordinal data. Total IgE and ECP are presented as median and interquartile range. Data were first compared within different groups by the Kruskal-Wallis H test. The Mann-Whitney U test with the Bonferroni’s post hoc test was then applied to evaluate the statistical differences between-group comparison. Differences in proportions among groups were compared with the χAs shown in As shown in In the CRSsNP group, serum total IgE was positively correlated with specific IgE to SEA , to SEB  and to SEC . In the CRSwNP group, serum total IgE was also positively correlated with specific IgE to SEB  and to SEC , but not to SEA. There was no correlation between the serum levels of ECP and specific IgE to SEs in either group of CRS. The results are summarized in The SEs have been described as superantigens due to their ability to bridge major histocompatibility (MHC) class II molecules and directly activate polyclonal T cells and B cells in a nonspecific manner ,19. To din vivo study in mice by Hamad et al  were detected in patients with CRSsNP and CRSwNP, and healthy controls. The results demonstrated that the positive rate and level of serum specific IgE to SEB in CRSwNP patients were significantly higher in comparison with those in the controls. In line with earlier findings –22, the ad et al , SEB is Previous studies have suggested that SEB stimulation highly increases the expression of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-13, and IL-17 ,26. Theset al  in the development and severity of CRS comprehensively.In summary, the positive rate and level of SEB-specific IgE were significantly higher in the serum from the Chinese CRSwNP patients than in that from the healthy controls. In addition, the presence of total IgE correlated positively with that of SEB-specific IgE. It is suggested that SEB may play a role in the pathogenesis of CRSwNP in Chinese patients.


MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The mK and maxV values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH  Depression is a severe and recurrent mental disorder that often leads to a significant impairment of daily functions et al reported that MH could increase the amplitude of contractility in the longitudinal and circular jejunum muscles in a dose-dependent manner et al reported that MH significantly accelerated the gastric emptying and intestinal transit in rats Common antidepressants were discontinued because they inhibited gastrointestinal kinetics in vitro.Although MH showed anti-depression and gastrointestinal prokinetic effects in animal experiments, several questions were unanswered. In addition to the therapeutic effect, the safety of MH was one of the primary concerns. According to various reports, drug interactions could lead to serious adverse events or decreased drug efficacy. These interactions might occur through the inhibition or induction of hepatic and intestinal drug-metabolizing enzymes  and transporters  15H18O5, MW: 278.30, assay≥98%) was obtained from Sigma-Aldrich . Furafylline, trans-2-phenylcyclopropylamine hydrochloride, ketoconazole, sulfaphenazole, quinidine, chlormethiazole hydrochloride and ticlopidine hydrochloride were purchased from the National Institutes for Food and Drug Control . Phenacetin, coumarin, midazolam, tolbutamide, S-Mephenytoin, metoprolol, chlorzoxazone and the standards for their metabolites, including acetaminophen, 7-hydroxyl coumarin, 1-hydroxyl midazolam, 4-hydroxyl tolbutamide, 4-hydroxyl mephenytoin, α-hydroxyl metoprolol, 6-hydroxyl chlorzoxazone and irbesartan  were purchased from Sigma-Aldrich . All other chemicals and solvents were of high-performance liquid chromatography (HPLC) grade.Recombinant human CYP enzymes (Bactosomes), pooled human liver microsomes (HLMs) from ten individual donors (Bactosomes) and NADPH were purchased from Cypex Ltd. (U.K.) and stored at −80°C until use. Meranzin hydrate  with a C18 security guard column . The mobile phases consisted of 20 mM ammonium formate and acetonitrile at a ratio of 60∶40. Aliquots of 20 µL were injected at a mobile phase flow rate of 0.3 mL/min. Multiple reaction monitoring was performed in the positive mode. The transitions are listed in The concentrations of the CYP substrates and their metabolites were quantified using a Waters 2695 separation module HPLC system  coupled to a Quattro micro API triple quadrupole tandem mass spectrometer  with an electrospray ionization source. The samples were separated on a HyPURITY CThe CYP isoform-specific probe reactions used were phenacetin O-deethylation (for CYP1A2), coumarin 7-hydroxylation (for CYP2A6), tolbutamide 4-hydroxylation (for CYP2C9), metoprolol α-hydroxylation (for CYP2D6), chlorzoxazone 6-hydroxylation (for CYP2E1), S-Mephenytoin 4-hydroxylation (for CYP2C19) and midazolam 1-hydroxylation (for CYP3A4). The kinetic study of MH was studied with HLMs, an incubation mixture that consisted of MH (as a substrate), the HLMs (0.5 mg/mL) or CYP isoforms (10 pmol) and 0.1 M sodium phosphate buffer (pH 7.4) in a total volume of 0.2 mL was pre-warmed for 5 min at 37°C without (control). The inhibitory effects of MH on the activities of seven different CYP isoforms were studied with the HLMs , multiple concentrations of MH (as an inhibitor) were included in the above incubation mixture system. The reaction was initiated by the addition of 1 mg/mL triphosphopyridine nucleotide (NADPH). The final incubations were performed in a shaking water bath (37°C) for 30 min. The incubations were performed in triplicates, and the incubation conditions specific to each CYP isoform were within the linear range for the velocity of the reaction (the incubation time as well as the substrate and protein concentrations). All the reagents were dissolved in methanol, and the final solvent concentration in all incubations (including controls) was 1%. The reactions were stopped by adding 0.2 mL ice-cold acetonitrile containing irbesartan (114.9 ng/mL) as the internal standard. The samples were vortexed for 5 min. After centrifugation (12000×g for 10 min), the supernatants were transferred and aliquots of 20 µL were injected into the HPLC-MS/MS system for analysis.V) in the HLMs or CYP isoforms was expressed as 0–Ct)/V =  and Cp was the protein concentration (mg/mL or nmol). All values were expressed as the mean±standard deviation (SD). The mean intrinsic clearance rate (intCL) for the in vitro incubation was estimated using max/KmV.Kinetic analysis was performed for MH, and the data generated were used as a guide for selecting the appropriate concentrations of MH in the subsequent inhibition experiments. Thus, the kinetic parameters for the metabolism of MH was determined by incubating increasing concentrations of MH (0.5–100 µM) (without the inhibitor) at 37°C with the HLMs and NADPH under the incubation conditions. The equation of MH reaction velocity , trans-2-phenylcyclopropylamine , sulfaphenazole , quinidine , chlormethiazole , ticlopidine  and ketoconazole  were separately incubated with MH (10 µM), the HLMs and NADPH under the same incubation conditions as mentioned above. The concentrations of the inhibitors used were approximately at their respective mK value) were used to determine the inhibitory effect of MH on specific CYP isoforms. Substrates including phenacetin, coumarin, tolbutamide, metoprolol, chlorzoxazone, S-Mephenytoin and midazolam were employed at concentrations of 10, 5, 100, 7.5, 40, 100 and 5 µM, respectively 50IC values of MH were determined and compared with those of the specific inhibitors mentioned above  and a single CYP isoform-specific substrate (concentration at about the respective bove see .50IC determination), we noted that MH markedly inhibits CYP1A2 and CYP2C19, whereas its effect on the remaining CYPs  was minimal. Therefore, Dixon plots for the inhibition of CYP1A2 and CYP2C19 were determined by incubating the substrate probe at multiple concentrations with or without the test inhibitor at multiple concentrations with the HLMs and cofactors. The inhibition data obtained from the pilot experiments were used as a guide to generate appropriate probe substrate and test inhibitor concentrations for the determination of the iK values for each CYP isoform. The isoform-specific probe substrate concentrations used were 3 to 100 µM phenacetin for CYP1A2 and 15 to 120 µM S-mephenytoin for CYP2C19. The MH concentrations used were 0 to 100 µM.In pilot experiments (mK and maxV) were determined by fitting the Michaelis-Menten equation using the program GraphPad Prism Enzyme Kinetic 5 Demo . The equation was expressed as max[S]/(Km+[S])V = V. mK is the substrate concentration at which the reaction velocity is 50% of maxV. To determine the inhibition of CYP isoforms, the activity of each CYP isoform was calculated using the metabolic clearance rate of its corresponding probe substrate. The metabolic clearance rate of the probe substrate was considered to be 100% when no specific inhibitor and MH were added in the incubation assay. The 50sIC were determined by analyzing the plot of the logarithm of the inhibitor concentration versus the percentage of activity remaining after inhibition, using the SPSS software for Windows . To calculate the iK values, the inhibition data were fit to different models of enzyme inhibition  by nonlinear least-squares regression analysis using the GraphPad Prism 5 software (GraphPad Co. Ltd).To determine the major enzymes responsible for MH metabolism in HLMs, the metabolic clearance rate of the incubation solution without any specific inhibitor for MH was considered as 100%. The effects of the specific inhibitors on the metabolic clearance rate of MH were evaluated with SPSS one-way analysis of variance . P<0.05 denoted significance in all cases. The apparent kinetic parameters of MH  and TIC (1 µM), the metabolic clearance rate (MCR) of MH decreased to 29.1±7.2% and 41.3±11.1% of that of the control, respectively  on the activity of each CYP isoform determined by the metabolism of a single concentration of isoform-specific probe were tested with the HLMs . MH showed potent inhibition of CYP1A2 (phenacetin O-deethylation) and CYP2C19 (S-mephenytoin 4-hydroxylation), with gligible .50ICs were qualitatively informative and help to address whether inhibition had occurred, their values were of limited use because they could be influenced by the substrate concentration selected, and it might not be accurate to use these parameters for the quantitative prediction of drug interactions in vivo. Therefore, we performed additional experiments designed to estimate the iK values. The preliminary inhibition data generated from a single probe substrate reaction were used to simulate the appropriate range of substrate and inhibitor concentrations for use in the construction of Dixon plots for the inhibition of the CYP isoforms by MH in the HLMs, from which precise iK values were estimated.While the iK values were determined using phenacetin as the probe substrate. Of all CYPs tested, CYP1A2 was the most sensitive to the inhibition by MH , and CYP2C19 was less sensitive to the inhibition by MH (50IC = 10.91 µM). CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 were hardly sensitive to the inhibition of MH  , by the co-incubation of phenacetin  with MH  for 30 min. The iK values were evaluated using the Dixon plot method  . The IC5t method . The res42.65 µM . AccordiDrug interactions associated with the induction or inhibition of CYP enzymes was among the most important causes of side effects in humans, and the inhibition effect was considered as the most common mechanism involved in CYP-associated drug-drug interactions. The results of the present work indicated that MH had the potential to interact with a wide range of xenobiotics or endogenous chemicals that were CYP1A2 or CYP2C19 substrates during clinical treatment. Because these two enzymes are involved in the clearance of 6–7% of currently marketed drugs, there is a strong possibility that MH would be co-prescribed with drugs that interact and thus elicit frequent and severe adverse drug interactions in the population. However, it is important to note that the extent of drug interactions with MH might highly vary among individuals due to genetic polymorphisms of CYP1A2 and CYP2C19. In previous studies, allelic variants in the genes encoding CYP1A2 and CYP2C19 had been shown to affect enzyme activitiesIn conclusion, our results show that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates. Because CYP1A2 and CYP2C19 are polymorphic enzymes, the drug interactions between MH and the substrates of these enzymes should be considered during future experiments.


IgE may be considered the hallmark of allergic disorders. It is easily detected in serum and can be measured as total IgE and as allergen-specific IgE. In fact, the serum IgE assay is used to diagnose an allergy.The aim of this study is to evaluate, investigate and present the distribution of total serum IgE levels, determined with UniCap system, in food-allergy suspected patients in a Republic of Macedonia.In this study we analyzed retrospectively 8898 consecutive patients that were admitted for allergy testing at the Institute of Immunobiology and Human Genetics during the ten year period between 01.01.2001 and 01.01.2011. Total IgE levels in patient sera were detected with the in vitro system UniCAP100 .When we analyzed the number of patients according to the total IgE groups, we noted that most of the patients have normal levels of total IgE in serum. However, we also discovered a group of patients with elevated levels of total IgE that are greater than 200 kU/L. The average concentration of total serum IgE is higher in women in the age group 6 (6-7 years), followed by a steep decrease in the age group 9 (9-10 years), and after that the average concentrations of total IgE were mostly constant with the exception of a partial increase in the age group 21 (65-69 years). For men, the average serum concentrations of total IgE were highest in the age group of 6 (6-7 years), which was significantly higher than the average concentrations of total IgE in all other age groups.The large number of enrolled patients, a particular strength of this study, revealed that average concentrations of total IgE in men are higher than in women and that total IgE did not decrease with age. On the contrary, increased total IgE levels were found in patients aged 65 and 69 of both genders. We continue our work with analyses of the specific IgE antibodies values toward food and the correlation with total IgE values. The prevalence of food allergies is rising worldwide. In Asia, it has been reported that 4-5% of school age children in Singapore have a food allergy . AustralIgE-mediated reactions are characterized by the acute onset of symptoms, usually within two hours after intake or exposure to food and symptoms frequently involve abnormalities of the skin, gastrointestinal tract and/or the respiratory tract. Even though any food may bear the potential for mediating an allergic reaction and more than 170 foods have been reported to cause IgE-mediated immune responses, it is a rather small number of foods that have been found to cause the majority of allergic reactions. They are described in the literature as “major food allergens’’, and include: peanuts, nuts, eggs, milk, fish, crabs, shellfish, wheat and soy [The aim of this study is to evaluate, investigate and present the distribution of total serum IgE levels, determined with UniCap system, in food-allergy suspected patients addressed to Institute of Immunobiology and Human Genetics in a Republic of Macedonia within a 10 year period (2001-2011).In this study we analyzed retrospectively 8898 consecutive patients that were admitted for allergy testing at the Institute of Immunobiology and Human Genetics during the ten year period between 01.01.2001 and 01.01.2011. First, the patients were subjected to a selection algorithm, whose inclusion and exclusion criteria are shown in After the implementation of the inclusion and exclusion criteria the analyses were conducted on the remaining 3312 patients for which we have accurate information regarding their origin, age, values for total serum IgE and values for specific IgE antibodies directed to food allergens. 1407 patients were women and 1905 men, aged between 1 month and 83 years. For the data analysis patients were organized into age groups as outlined in in vitro system UniCAP100  [Total IgE levels in patient sera were detected with the  Sweden) . Anti-IgSerum concentrations of total IgE are associated with age. They increase during childhood and around 10 years of age reach levels that are maintained throughout life. P ≤ 0.05 were considered to indicate statistical significance. Differences in total IgE levels between men and women were investigated through application of the t-test.Results were grouped by sex, town of origin and age. The statistical analysis of the various frequency distributions and correlations were made with SPSS 20.0. Since the total IgE frequencies deviated from the normal distribution, analogue non-parametric tests independent of the distribution were applied. To lower the peak of the graphs for the total IgE levels, we applied logarithmic transformations to the dataset after which the same non-parametric tests were repeated. Descriptive statistics are expressed as means and standard error of the mean. The non-parametric Kruskal-Wallis rank test and Welch’s ANOVA test were performed. Values of First, we analyzed the frequency of patients addressed to the Institute of Immunobiology and Human Genetics for allergy testing  and we nIn further analyses, we ranked the admitted patients according to their town of origin . Figure The number of admitted patients did not correlate with the total IgE levels per group. The total IgE concentration showed an increase in age group 6, followed by a decrease in group 7 and an increase in age group 8. The concentrations of total IgE remain constant over the next groups. However, in group 21 (patients aged 65 to 70 years) we observed an increase in total IgE levels.The Kruskal-Wallis test result was: H (24) = 466.944 p <0.01 <0.05; which means that there is a statistically significant difference in the values of total IgE among specific age groups. Therefore, the null hypothesis  had to be rejected in favor of the alternative hypothesis.The results from the ANOVA test were F (24.3287) = 8.834, p < 0.001, which means that there is a statistically significant difference between the arithmetic means of total IgE between the different age groups. But since the Levenov test showed a difference in the variances, we could not use the basic ANOVA test (and there is no Gaussian distribution). Therefore, the Welch and Brown-Forsythe test were performed. They showed a statistically significant difference (in both cases) between the variances, and the result W (24) = 7.797, p < 0.001 and BF (24) = 5.147, p < 0.002.Further we examined the concentration of total IgE in men and women  and 6B, The average concentration of total serum IgE is higher in women in the age group 6, followed by a steep decrease in the age group 9, and after that the average concentrations of total IgE were mostly constant with the exception of a partial increase in the age group 21. For men, the average serum concentrations of total IgE were highest in the age group of 6, which was significantly higher than the average concentrations of total IgE in all other age groups. In both sexes we saw repeated increase in the concentration of total IgE in the age group 21, which is lower in men compared with women in the same age group.A t-test was performed to determine the significance of the difference of total IgE in both sexes. The obtained results are shown in Food allergy usually manifests in early childhood at one year of age. In children, over 80% of the reactions to food result from milk, eggs, soy, wheat, peanuts, nuts, while in adults the most due to peanuts, nuts, shellfish and fish .Analyses of admitted patients at the Institute of Immunobiology and Human Genetics in the period 01.01.2001 until 01.01.2011  showed tAccording to a number of studies the heterogeneity of the allergies depends on the geographical position -16. FiguAccording to another research in Republic of Macedonia from 2006, the most of the patients with hypersensitivity to food came from Prilep and Dojran . HoweverDifferences in admitted patients can be explained by different awareness/unawareness of family doctors for the analyses performed at the Institute and the geographical distance of the towns. Our findings reveal a clear need for the establishing of new allergy testing laboratories in additional towns throughout Macedonia.When we analyzed the number of patients  accordinIf we analyze the frequency of patients in each age group , we can Analyses of total IgE distribution by age group showed significant differences matching those published in literature . In partFrom the presented results we can interpret that the values for total IgE did not decrease with aging. One possible explanation for this may be that during the aging there is damage the regulatory function of the immune system .We also found different data in the literature for the question of whether the levels of total IgE decline over the life. Most authors have published a decrease of the levels of IgE with aging in both men and women , 25, 30,In conclusion, the large number of enrolled patients, a particular strength of this study, revealed that average concentrations of total IgE in men are higher than in women and that total IgE did not decrease with age. On the contrary, increased total IgE levels were found in patients aged 65 and 69 of both genders. We continue our work with analyses of the specific IgE antibodies values toward food and the correlation with total IgE values.


Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. Human platelets play pivotal roles in primary haemostasis and repairs of vascular injury . PlateleHeat shock proteins (HSPs) are expressed in response to a variety of biological stresses such as heat and chemicals . HSPs faIn the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. We herein show that FXa inhibitor suppresses the collagen-induced phosphorylation of HSP27 via p44/p42 MAP kinase, resulting in the inhibition of the phosphorylated HSP27 release.Collagen was purchased from Takeda Austria GmbH . Rivaroxaban and edoxaban were kindly provided by Bayer Vital GmbH  and Daiichi Sankyo Co., Ltd. , respectively. Phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific HSP27 antibodies (Ser-78) and phospho-specific HSP27 antibodies (Ser-82) were purchased from Cell Signaling Technology, Inc. . HSP27 antibodies and GAPDH antibodies were obtained from Santa Cruz Biotechnology, Inc. . The PDGF-AB enzyme-linked immunosorbent assay (ELISA) kit was obtained from R&D System, Inc. . The phosphorylated HSP27 ELISA kit was purchased from Enzo Life Science INC. . ECL Western blotting detection system was obtained from GE Healthcare . Other materials were obtained from commercial sources.Fifteen healthy volunteers  and 5 patients  who have not been taking any antiplatelet medicines, were enrolled in this study . Written informed consent was obtained from all of the healthy donors and patients after a detailed explanation. The study was approved by the Committee of Ethics of the Gifu University Graduate School of Medicine.Blood samples from healthy volunteers were donated and mixed into a 1/10 volume of 3.2% sodium citrate. Blood samples from patients were collected twice; once before administration of rivaroxaban and once after 2 days of daily administration of rivaroxaban, and then mixed into a 1/10 volume of 3.2% sodium citrate. Platelet-rich plasma (PRP) was prepared from blood samples by centrifugation at 155 x g for 12 min at room temperature. Platelet-poor plasma (PPP) was obtained from residual blood by centrifugation at 1,400 x g for 5 min.Platelet aggregation using citrated PRP was carried out in an aggregometer with laser scattering system , which can present the size of platelet aggregates based up particle counting , as described in detail previously ,23–25. BAfter the stimulation with collagen, platelet aggregation was terminated by the addition of ice-cold EDTA (10 mM) solution. The conditional mixture was centrifuged at 10,000 x g at 4°C for 2 min. To measure PDGF-AB and phosphorylated HSP27 as described below, the supernatant was isolated and stored at -20°C. For Western blot analysis, the pellet was washed twice with phosphate-buffered saline, and then lysed and immediately boiled in a lysis buffer containing 62.5 mM Tris/Cl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol, and 10% glycerol.Western blot analysis was performed as described in detail previously . BrieflyPeroxidase-labeled anti-mouse IgG or anti-rabbit IgG antibodies were used as the secondary antibodies. The primary and secondary antibodies were diluted to the optimum concentrations with 5% fat-free dry milk in TBS-T. Peroxidase activity on PVDF membrane was visualized on X-ray film by means of an ECL Western blotting detection system according to the manufacturer's protocol.The level of PDGF-AB and phosphorylated HSP27 in the supernatant of the conditional mixture were determined using each ELISA kit according to the manufacturer's instructions.The data are presented as the means ± SEM. Comparisons of continuous variables were analyzed by using Wilcoxon signed-rank test because of their non-normal distribution. A probability value of <0.05 was considered statistically significant. All statistical analyses were performed using PASW Statistics software, version 18 .We first examined the effects of FXa inhibitors, rivaroxaban on platelet aggregation induced by 1.0 μg/ml of collagen. Rivaroxaban had little effect on the collagen-induced platelet aggregation in terms of maximum transmittance up to 1000 ng/ml . AccordiIn order to investigate whether FXa inhibitor could affect the activation of p44/p42 MAP kinase induced by collagen or not, we examined the effects of FXa inhibitors on the collagen-induced phosphorylation of p44/p42 MAP kinase in human platelets. Rivaroxaban significantly suppressed the collagen-induced phosphorylation of p44/p42 MAP kinase in a dose-dependent manner in the range between 500 and 1000 ng/ml . The inhWe next examined the effects of FXa inhibitors on the phosphorylation of HSP27 induced by collagen in human platelets. Rivaroxaban significantly reduced the collagen-induced phosphorylation of HSP27 at ser-78 and ser-82 in a dose-dependent manner in the range between 500 and 1000 ng/ml . RivaroxIn our previous study , we haveWe examined the effects of FXa inhibitors on the PDGF-AB secretion induced by collagen. The secretion of PDGF-AB from collagen-stimulated platelets was not affected by 1000 ng/ml of rivaroxaban compared to collagen alone .We have recently reported that HSP27 is released from human platelets accompanied with the collagen-stimulated phosphorylation of HSP27 . TherefoWe further examined the effect of collagen on HSP27 phosphorylation (Ser-78) using platelets from non-valvular atrial fibrillation or deep venous thrombosis patients after rivaroxaban administration. Representative result before  and 2-dain vitro. In addition, we examined the effect of collagen on platelet aggregation in patients with non-valvular atrial fibrillation or deep venous thrombosis before and after rivaroxaban administration, and showed that rivaroxaban had little effect on platelet aggregation using laser scattering system. Based on our findings, which is generally consistent with the previous reports [In the present study, we investigated the direct effects of FXa inhibitors such as rivaroxaban and edoxaban on collagen-induced activation of human platelets. It has previously been reported that FXa inhibitors have no effect on platelet aggregation induced by several agonists such as collagen, ADP, ristocetin or TX agonist using light transmittance aggregometry ,13,29. I reports ,13, it iin vitro. We demonstrated that rivaroxaban and edoxaban significantly attenuated the phosphorylation levels of p44/p42 MAP kinase and those of HSP27 at two serine residues, Ser-78 and Ser-82. Additionally, we examined the effect of collagen on the phosphorylation of HSP27 at Ser-78 in patients before and after rivaroxaban administration, and found that the administration of rivaroxaban for 2 days remarkably attenuated the phosphorylation of HSP27 (Ser-78) in the collagen-stimulated platelets. It has been shown that the phosphorylation of p38 MAP kinase is correlated with the phosphorylation of HSP27 [We have previously reported that the collagen-induced phosphorylation of HSP27 is positively regulated by the activation of Rac-dependent p44/p42 MAP kinase in human platelets ,24. Therof HSP27 . We haveof HSP27 . In the The phosphorylation of HSP27 is reportedly associated with the cytoskeleton and actin polymerization in activated human platelets ,31. We nAccumulating evidence indicates that HSP27 possess extracellular effects besides intracellularly functions as a molecular chaperone ,32. HSP2In conclusion, our present findings strongly suggest that FXa inhibitor suppresses the collagen-stimulated phosphorylation of HSP27 via p44/p42 MAP kinase in human platelets, eventually resulting in the inhibition of the release of phosphorylated HSP27 from human platelets.


IgE has long been known as a therapeutic target for allergic disease, but the difficulty has been in selecting agents that don't trigger cross linkage of IgE when bound to its high affinity receptor (FceR1) on mast cells and basophils. By “designing” a monoclonal antibody (mAb) which targets that part of IgE that binds to that binds to the a-chain of FceR1, the allergic cascade can be effectively interrupted and diseases such as asthma greatly improved, providing a substantial part of their phenotype engages IgE. Clinical trials and real life studies confirm this. Beyond asthma, a whole range of other diseases dependent upon IgE initiation and triggering are being identified. These diseases are now being explored as being amenable to anti-IgE therapy some of which are comorbidities of asthma and others not. The advent of an even more potent anti-IgE mAb - QGE031 – is creating further opportunities for anti-IgE therapy to improve the lives of so many people with IgE-related diseases. It took a further 45 years before Kimishige Ishizaka, Gunnar Johansson and Hans Bennich identified reagin as the 5th immunoglobulin class, immunoglobulin (Ig)E . So. So24]. An exciting new development is the production of a much more potent form of anti-IgE, QGE031 (Novartis), which is at least 12 times more potent than omalizumab for the treatment of IgE-driven diseases where a significant unmet need exists such as severe uncontrolled asthma, atopic dermatitis and food allergies . BullousIn conclusion:– Clinical trials have repeatedly shown that anti-IgE is highly effective in reducing exacerbations and reducing corticosteroid use, as well as baseline control of asthma in adults and children.– Reduced use of health services such as hospital admission, ICU etc. makes the economic case for omalizumab in managing high risk severe asthma.– Omalizumab is active on asthma comorbidities as well as other conditions, presumably related to allergy and/or IgE.– There appear to be some long term benefits of omalizumb on airway wall remodelling.– A more active anti-IgE mAb is in development.Stephen Holgate has received honoraria from Novartis for lecturing on anti-IgE treatment and chaired the 2014 Novartis International Respiratory Advisory Council in Amsterdam. He has also contributed to other reviews on anti-IgE therapy.