text:
3. Materials and methods
Dictionary of Crystallography
IUPAC Gold Book
research papers\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)
Volume 1| Part 4| July 2014| Pages 240-249
doi:10.1107/S2052252514012585
Structure and function study of the complex that synthesizes S-adenosylmethionine
Ben Murray,a,b Svetlana V. Antonyuk,a Alberto Marina,b Sebastiaan M. Van Liempd,c Shelly C. Lu,d Jose M. Mato,c S. Samar Hasnaina* and Adriana L. Rojasb*
aMolecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, L69 7ZX, England, bStructural Biology Unit CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain, cMetabolomics Unit, CIC bioGUNE, CIBERehd, Parque Tecnológico de Bizkaia, 48160 Derio, Bizkaia, Spain, and dDivision of Gastroenterology and Liver Diseases, USC Research Center for Liver Diseases, USC–UCLA Research Center for ALPD and Cirrhosis, Keck School of Medicine, Los Angeles, California, CA 90033, USA
*Correspondence e-mail: s.s.hasnain@liverpool.ac.uk, arojas@cicbiogune.es
Edited by Z.-J. Liu, Chinese Academy of Sciences, China (Received 11 April 2014; accepted 30 May 2014; online 12 June 2014)
S-Adenosylmethionine (SAMe) is the principal methyl donor of the cell and is synthesized via an ATP-driven process by methionine adenosyltransferase (MAT) enzymes. It is tightly linked with cell proliferation in liver and colon cancer. In humans, there are three genes, mat1A, mat2A and mat2B, which encode MAT enzymes. mat2A and mat2B transcribe MATα2 and MATβ enzyme subunits, respectively, with catalytic and regulatory roles. The MATα2β complex is expressed in nearly all tissues and is thought to be essential in providing the necessary SAMe flux for methylation of DNA and various proteins including histones. In human hepatocellular carcinoma mat2A and mat2B genes are upregulated, highlighting the importance of the MATα2β complex in liver disease. The individual subunits have been structurally characterized but the nature of the complex has remained elusive despite its existence having been postulated for more than 20 years and the observation that MATβ is often co-localized with MATα2. Though SAMe can be produced by MAT(α2)4 alone, this paper shows that the Vmax of the MATα2β complex is three- to fourfold higher depending on the variants of MATβ that participate in complex formation. Using X-ray crystallography and solution X-ray scattering, the first structures are provided of this 258 kDa functional complex both in crystals and solution with an unexpected stoichiometry of 4α2 and 2βV2 subunits. It is demonstrated that the N-terminal regulates the activity of the complex and it is shown that complex formation takes place surprisingly via the C-terminal of MATβV2 that buries itself in a tunnel created at the interface of the MAT(α2)2. The structural data suggest a unique mechanism of regulation and provide a gateway for structure-based drug design in anticancer therapies.
Keywords: methionine adenosyltransferases; cell growth; liver cancer; X-ray scattering; methylation; drug design.
3D view: 4ktt,4ktv,4ndn
PDB references: SAMe-bound, 4ktt; ADO-bound, 4ktv; imido-triphosphate-bound, 4ndn
Transmethylation, the transfer of a methyl group between molecules, plays a central role in fundamental biological processes such as cell growth, gene expression and apoptosis (Lu & Mato, 2012 ). The S-adenosylmethionine (SAMe) molecule (Landgraf & Booker, 2013 ), which is synthesized by methionine adenosyltransferase (MAT), is the main source of methyl groups in all living organisms. MAT enzymes are conserved from bacteria to mammals, thus highlighting their essential regulatory function in maintaining the appropriate levels of SAMe.
Mammals express three MAT genes, mat1A, mat2A and mat2B; the former two encode two homologous MAT catalytic subunits, MATα1 and MATα2, and the last one encodes the regulatory subunit MATβ. Association of MATα and MATβ subunits results in the formation of the MATαβ complex. Recently, it was shown that there exist two major splicing variants of the mat2B gene that encode two proteins MATβV1 and MATβV2. Both isoforms interact with MATα2 (Yang et al., 2008 ; Xia et al., 2010 ), revealing the existence of two new complexes: MATα2βV1 and MATα2βV2 (Fig. 1 ). MATα2 expression confers a cell growth advantage and is considered increasingly important for differentiation and apoptosis (Lu & Mato, 2012 ), for instance in human hepatocellular carcinoma (HCC) (Yang et al., 2008 ), colon cancer (Chen et al., 2007 ) and leukaemic cells (Attia et al., 2008 ). MATα2β interacts with a large variety of proteins both in the nucleus and cytoplasm of mammalian cells, including HuR (Xia et al., 2010 ), an mRNA-binding protein known to stabilize the mRNA of cyclins; GIT1 (Peng et al., 2013 ), a scaffold protein that activates ERK1/2; MafK (Katoh et al., 2011 ), a Maf family transcription factor; and many others. Global histone methylation decreases upon MAT knockdown in Caenorhabditis elegans causing the release of heterochromatin from the nuclear periphery (Towbin et al., 2012 ). Recently it has been demonstrated that threonine and SAMe metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation (Shyh-Chang et al., 2013 ). Similarly, disruption in mice of GNMT, the enzyme that catalyses the methylation of glycine to synthesize sarcosine, and overexpression in ovary cancer cells of NNMT, the enzyme that synthesizes 1-methylnicotinamide by methylation of nicotinamide, cause aberrant DNA, histone and phospholipid methylation by altering cellular SAMe (Martínez-Chantar et al., 2008 ; Ulanovskaya et al., 2013 ). GNMT knockdown has been shown to attenuate prostate cancer (Sreekumar et al., 2009 ). This has led to an emerging paradigm in enzyme regulation, where MATα2β synthesizes SAMe locally to support specific methylation reactions (Kaelin & McKnight, 2013 ; Gibson & Kraus, 2011 ; Martinez-Una et al., 2013 ).
Schematic representation of the oligomeric states of mammalian MAT enzymes. The mammalian genes mat1A and mat2A produce the catalytic subunits MATα1 and MATα2, respectively, sharing 84% sequence homology. The MATα1 and MATα2 subunits can be found organized as dimers and tetramers. The MATα1 dimer and tetramer are known as MATIII and MATI, respectively. On the other hand, mat2B encodes the regulatory subunit for which there are two major isoforms, MATβV1 and MATβV2. The MAT(α2)4(βV2)2 complex consists of a MATα2 tetramer flanked by two MATβV2 subunits. In this study we were able to assemble in vitro three different MATαβ complexes: MAT(α2)4(βV1)2, MAT(α2)4(βV2)2 and MAT(α1)4(βV1)2 (the last two complexes are described for the first time).
The synthesis of SAMe has been proposed to follow an SN2 catalytic mechanism (Markham et al., 1987 ) that has gained structural support from the crystallographic analyses of MAT catalytic subunits bound to various ligands (Komoto et al., 2004 ; González et al., 2003 ; Shafqat et al., 2013 ). Briefly, the reaction is initiated by the sulfur atom of the methionine which carries a nucleophilic attack against the C5′ atom of ATP followed by the hydrolysis of the tripolyphosphate (PPPi) (Supplementary Fig. S1A ). Despite the central role of MATα2β, the nature of the complex has remained elusive and its structural elucidation a challenge. Here we report the crystallographic and solution X-ray scattering structures as well as biophysical analysis of the MATα2β complexes, providing the basis for fresh insight into this widely utilized system in biology.
2.1. MATβ isoforms interact with MAT(α2)2 through the C-terminal region
Crystallographic structures of the 258 kDa MATα2βV2 complex determined in different forms to a resolution ranging from 2.35 to 3.3 Å reveal that the complex consists of a 185 kDa MATα2 tetramer which is flanked by two MATβV2 subunits of 36.5 kDa each (Fig. 2 a). The complex has four active sites located at the interface of the MATα2 dimers. The oligomeric state of our crystallographic structure is different to the suggested tetrameric form [MAT(α2)2(β)2] (Kotb & Kredich, 1985 ) or the recently proposed model in which MATαβ was assumed to be a trimer [MAT(α2)2(β)1] (González et al., 2012 ).
Structure of the MAT(α2)4(βV2)2 complex. (a) Crystallographic structure of the MAT(α2)4(βV2)2 complex. Two MATα2 monomers with visible gating loops are coloured in slate whereas the other two MATα2 monomers are coloured in red. MATβV2 is coloured in green with the first visible residues at its N-terminal in orange. The reaction product (SAMe) is represented by spheres. (b) SAXS of the MAT(α2)4(βV2)2 complex, the experimental spectrum (blue) is shown with a simulated fit (red) obtained from the crystal structure (qmax = 0.3 Å−1) (χ2 = 3.2). The radius of gyration, Rg = 50.1 ± 0.05 Å, was estimated from the low-angle scattering region by a Guinier plot. The distance distribution function, P(r), with maximum linear dimension Dmax = 187 Å. We interpret the higher χ2 value as arising in part from the flexibility of the interaction and the missing residues at the N-terminus of both MATβ subunits in the crystal structure. This is supported when SAXS data of the MATα2βV1Δ16 mutant (slightly better quality data, data not shown) are used with the model from the crystal structure (χ2 = 1.7). (c) The ab initio shape reconstruction of MAT(α2)4(βV2)2 by DAMMIN using P1 symmetry, showing a good agreement between the predicted molecular shape (light green mesh) and the crystal structure (Rg = 50.7) (cartoon), after alignment by SUPCOMB (Kozin & Svergun, 2001 ) (NSD = 0.93).
In the first study Kotb and Kredich used analytical ultracentrifugation analysis to estimate the molecular weight of the complex whereas in the second study Gonzales et al. used ITC (isothermal titration calorimetry) to investigate its stoichiometry which matches our model in a 2:1 ratio of MATα2:MATβ subunits. In order to confirm that the crystallographic oligomer is representative of the solution state and is thus physiologically relevant, we performed small-angle X-ray scattering (SAXS) experiments using the HPLC-integrated SAXS set-up at the SOLEIL synchrotron. The scattering curve of the complex in solution obtained by SAXS is in agreement with the theoretical curve calculated from our 2.35 Å crystal structure (χ2 = 3.2) (Fig. 2 b), confirming that the MATα2β complex in solution does indeed have the same composition and overall conformation as observed in our crystal structures, i.e. MAT(α2)4(βV2)2 (Fig. 2 c).
The crystallographic structure of the MAT(α2)4(βV2)2 complex reveals that MATβV2 interacts with MATα2 through the insertion of the C-terminal tail of the β subunit into a cavity created at the interface of the MATα2 dimer. Specifically, residues K315 to H323 of MATβV2 establish extensive hydrophobic and polar interactions with side chains of both MATα2 monomers (Fig. 3 a). In the complex, the usually disordered tail of the MATβV2 C-terminal folds into a helical structure (Fig. 3 b), and within the MAT(α2)2 binding cavity the interaction generates a dilation of the cleft without any change in the orientation of the side chains consistent with a `lock-and-key' mechanism. In order to establish that the C-terminal tail is indeed the key region of the interaction, we generated a truncated version lacking the last 15 residues at the C-terminus in both variants of MATβ, MATβV1 and MATβV2. These deletions, though preserving the secondary structure, as confirmed by circular dichroism (CD) spectra (Fig. S1B ), precluded the assembly of the complex in solution (gel filtration, Fig. 3 c; and ITC, Figs. 3 d, 3 e and Table 1 ). Taken together, these data show that MATβV2 and MATβV1 interact with MAT(α2)2 through the insertion of the C-terminal tail of the β subunit.
Thermodynamic parameters of MATαβ complex formation
MATβV2 → MATα2
[] 36.5 µM → 10 µM 51.2 µM → 10 µM 236.8 µM → 45.1 µM
Ka (M−1) (1.14 ± 0.17) × 108 (2.90 ± 0.82) × 107 (3.38 ± 0.24) × 105
Kd (M) (8.77 ± 1.34) × 10−9 (3.44 ± 0.98) × 10−8 (2.96 ± 0.21) × 10−6
ΔH −2.28 × 104 ± 188.4 −2.37 × 104 ± 367.6 −9301± 140.9
N 0.44 ± 0.001 0.44 ± 0.004 0.325 ± 0.004
MATβV2 C-terminal interactions with MATα2. (a) Each MATβV2 subunit interacts with the core by inserting its C-terminus (green) in a tunnel created at the interface between two MATα2 subunits (slate, red). The residues involved in the interaction are in stick representation (hydrogen bonds in black dotted lines). (b) Superposition of MATβ (PDB entry 2ydy ) in blue with MATβV2 from the complex with MATα2 (green). The black arrow indicates the conformational change of the unfolded C-terminal loop. (c) Gel-filtration profiles for MAT(α2)4(βV2)2 (red), MAT(α2)4(βV1)2 (dark green) and MAT(α2)4 (blue). For complex formation MATα2 was incubated with both MATβ variants prior to being loaded onto a Superdex 200 10/300 column. The gel-filtration profiles clearly show the shift of the peak that contains the complex (top) and absence of complex formation when MATβV2ΔW309 (black) is used; MAT(α2)4 (blue) and MATβV2ΔW309 (light green) were loaded as controls (bottom). (d) ITC of MATα2 with MATβV2; the top graphs represent the differential heat released during the titration of MATβV2ΔW309 or MATβV2 with MATα2. The bottom graphs represent the fitted binding isotherms. (e) As in (d) ITC of MATα2 with MATβV1ΔW320 or MATβV1.
Interestingly, the tunnel created at the interface of the MATα2 dimer has a symmetry that allows two possible conformations of the C-terminal MATβV2 (Figs. 4 a and 4 b). The interchangeability of these two orientations could allow certain rotational flexibility of the MATβV2 subunit.
Close view of the tunnel created at the interface of the MATα2 dimer. (a) Stick representation of the MATα2 residues at the dimer interface, side chains involved in the interaction with MATβV2 (chain_E in green) are coloured in cyan, the symmetry residues are shown in magenta. (b) Side chains involved in the interaction with MATβV2 (chain_F in green) are coloured in magenta, the symmetry residues are shown in cyan. Note that between the conformation represented in (a) and (b) there is a twofold symmetry. (c) Cartoon representation of the MATα2 monomer; the loop that connects the active site with the buried tail of MATβV2 is highlighted in yellow.
2.2. NADP-binding site
Previously, it has been shown that NADP binds to a conserved glycine-rich GXXGXXG motif (G24ATG27LLG30) at the N-terminal domain of MATβ (Shafqat et al., 2013 ) (Figs. S1C and 5 a). The mutants lacking residues involved in NADP binding of the β subunit have been shown to be able to form a complex with MATα2 supporting the idea that NADP is not needed to form the complex (González et al., 2012 ). In our case, co-crystallization and soaking experiments with an excess of NADP yielded crystals of the MAT(α2)4(βV2)2 complex without NADP. This observation prompted us to evaluate whether NADP could interact in solution with the preformed complex. ITC experiments confirmed that, though NADP binds to both MATβ isoforms, no interaction could be observed with the MAT(α2)4(βV2)2 complex (Figs. S2A and S2B ; Table 2 ). The presence of NADP had no effect on MAT(α2)4(βV2)2 complex formation as observed by gel filtration and native PAGE experiments (Figs. S2C and S2D ). The overall structure of MATβV2 within the MAT(α2)4(βV2)2 complex has no significant structural changes as compared with the NADP-bound MATβ structure (r.m.s.d. = 0.42 Å) (Figs. 5 a and 5 b). These data taken together suggest that after complex formation the NADP is displaced from its binding pocket. It is possible that NADP binding to MATβV2 is relevant for other functions, such as the interaction with HuR, GIT1, MEK, ERK or MafK (Xia et al., 2010 ; Peng et al., 2013 ; Katoh et al., 2011 ). Furthermore, the observation that MAT(α2)4 does not block the binding pocket (Fig. 5 c) suggests that the NADP could bind to the MAT(α2)4(βV2)2 complex in the context of the interaction with other proteins. In fact, there is evidence suggesting that MAT enzymes are involved in the regulation of many pathways, some of which are chromatin based and some may be independent of SAMe.
Thermodynamic parameters of NADP binding to MATβ isoforms
NADP → MATβV2
NADP → MAT(α2)4(βV2)2
[] 500 µM → 30 µM 500 µM → 29.8 µM 155 µM→ 10.22 µM
Ka (M−1) (1.12 ± 0.13) × 105 (1.17 ± 0.22) × 105
Kd (M) (8.93 ± 1.07) × 10−6 (8.55 ± 1.6) × 10−6
ΔH 2784 ±117.4 1866 ± 127.8
N 0.85 ± 0.026 1.23 ± 0.060
NADP-binding site. (a) Superposition of the NADP-bound MATβ structure (PDB entry 2ydx , Shafqat et al., 2013 , in blue) over MATβV2 as it is in the MAT(α2)4(βV2)2 complex (in green). Residues directly involved in the interaction with NADP are labelled and shown in stick representation. (b) Surface representation of the model of an NADP molecule within the binding pocket of MATβV2 from the MAT(α2)4(βV2)2 complex. (c) MAT(α2)4 (in red and slate) does not block the access of NADP to MATβV2 (in green).
2.3. Interaction of MATβ isoforms with MATα1
Looking at the structure of the MAT(α2)4(βV2)2 complex it is difficult to understand why, in vivo, MATβ has so far been found to only interact with MATα2, even though MATα1 and MATα2 have a homology of 84% and share the same folding (Fig. S3 ). A plausible explanation may be that in vivo when the expression of mat1A is switched on, mat2A and mat2B expression is switched off. Thus, whereas in adult hepatocytes mat1A is highly expressed and the expression of mat2A and mat2B is low, in HCC where the expression of mat2A and mat2B is turned on, as well as the expression of other proteins that interact with MATβ such as HuR, GIT1, MEK, ERK or MafK (Xia et al., 2010 ; Peng et al., 2013 ; Katoh et al., 2011 ), mat1A expression is low or absent (Lu & Mato, 2012 ). Accordingly, we observed by gel filtration and ITC the formation of the MAT(α1)4(βV1)2 complex upon incubation of MATβV1 with MATα1 (Figs. S4A and S4B ; Table 1 ). Remarkably, we did not observe a strong interaction by gel filtration between MATβV2 with MATα1 (Fig. S4C ), indicating a role of the N-terminus of MATβ in providing the stability of the MATα1β complex. To confirm this hypothesis, we generated a truncated version of MATβV1 lacking the first 16 amino acids (MATβV1Δ16). This mutant produces a much smaller amount of complex with MATα1, supporting the hypothesis that the MATβ N-terminus is indeed important for the formation of stable MATα1β complexes (Fig. S4D ).
2.4. Active site and enzymatic activity
After incubation of the MAT(α2)4(βV2)2 complex with its product SAMe, its substrate MET, ATP or AMPPNP (non-hydrolysing ATP analogue), the presence of SAMe, adenosine (ADO) or PPNP [(β-γ-imido)triphosphate] was clearly observed in different crystals at the active site (Figs. 6 a, 6 b and 6 c) as supported by the corresponding omit maps (Figs. 6 d, 6 e and 6 f). In all of the structures the adenine group makes a π–π stacking interaction with F250 of MATα2, supporting the hypothesis that the substrate (ATP) and product (SAMe) can occupy the active site in similar orientations. The structures show that two of the four active sites are occupied by SAMe or adenosine whereas the other two are empty, thus providing details of structural differences that accompany SAMe formation by comparison of empty and occupied sites.
Active site. MATα2 monomer with visible gating loop is coloured in slate whereas MATα2 monomer with disordered loops is coloured in red. Important hydrogen bonds are shown as dotted lines. (a) Stick representation of the bound product, SAMe, at the active site. (b) Crystal co-crystallized and soaked with only ATP shows adenosine molecule (ADO) and PO4 at the active site, as a result of the ATPases and tripolyphosphatase activity of MATα2. (c) Crystal co-crystallized and soaked with AMPPNP and methionine shows SAMe and PPNP. (d) Omit (Fo − Fc) electron-density map and stick representation of bound molecule, the map is contoured at the 2.5σ level around the SAMe molecule, (e) around the adenosine (ADO) molecule and (f) around SAMe and PPNP.
A comparison of the active site in two different states reveals that loops flanking the empty catalytic pockets are disordered (Fig. 2 a), in particular the `gating loop' (residues 113–131) that has been proposed to act as a dynamic lid controlling access to the active site (Komoto et al., 2004 ). The flexibility of this loop induces two different conformations of the catalytic subunit. The loop is disordered in the open conformation causing the entrance to the active site to be opened. The entrance to the active site is blocked in the closed conformation and the gating loop becomes well ordered (Fig. 7 a). In the case of the catalytic subunit MATα1, S-nitrosylation of residue C121 in the `gating loop' promotes the inactivation of the enzyme (Pérez-Mato et al., 1999 ). A comparison of the SAMe-bound MAT(α2)4(βV2)2 complex with SAMe-bound MAT(α2)4 (PDB entry 2p02 ; Shafqat et al., 2013 ) shows that in the absence of MAT(βV2)2 the four active sites of MAT(α2)4 are in a closed conformation. The complex formation with MAT(βV2)2 causes an asymmetry in MAT(α2)4, in which two sites are found in an open state while the other two are in a closed conformation (Fig. 1 a). In the absence of SAMe, the apo-structures exhibit no density for the gating loop, indicating its flexible nature (Fig. 7 b). However in MAT(α2)4(βV2)2 the open active sites show two additional flexible loops, near the inserted MATβV2 C-terminus (Fig. 7 c). In addition, the N-terminal loop (residues 1–13) of both MATβV2 in the complex, which is orientated to the same side of the open active sites, is disordered. This observation raises the question of whether the N-terminus of MATβ could regulate the gating loop of MATα2. If this was the case, the differences between the N-terminus of MATα2βV1 and MATα2βV2 should affect their enzymatic activities. Thus, we compared the activity of the MAT(α2)4(βV1)2, MAT(α2)4(βV2)2 and MAT(α2)4(βV1Δ16)2 complexes. Notably, the presence of either MATβV1 or MATβV2 increased the Vmax of MAT(α2)4 without altering the Km for methionine. Additionally, the Vmax of MATα2 was 34% higher in the presence of MATβV1 than with MATβV2, thus emphasising that differences at the N-terminus affect the activity of MAT(α2)4. Furthermore, the MAT(α2)4(βV1Δ16)2 complex, in which MATβ has the shortest N-terminus, also has the lowest Vmax which is still higher than the catalytic subunit alone (Fig. 7 d and Table 3 ). Therefore, the increasing N-terminal length correlates with an increase in Vmax of the complex activity, confirming the role of the N-terminal in the regulation of the activity.
Kinetic properties of MATα2β complexes
MAT(α2)4
MAT(α2)4(βV1Δ16)2
MAT(α2)4(βV2)2
kcat 295.08 ± 23.7 688.79 ± 38.39 859.21 ± 57.14 1298.16 ± 47.78
km 53.84 ± 10.5 84.79 ± 9.93 42.45 ± 7.38 53.48 ± 4.79
Comparison of MAT(α2)4(βV2)2 complex with MAT(α2)4. (a) Superposition of SAMe-bound MAT(α2)4 (PDB entry 2p02 in blue) and SAMe-bound MAT(α2)4 (slate, red) from the complex MAT(α2)4(βV2)2; in the closed state both structures are similar. (b) Superposition of apo-MAT(α2)2 from Burkholderia pseudomallei (PDB entry 3iml , Baugh et al., 2013 , in pink) with the SAMe-bound MAT(α2)2 (PDB entry 2p02 in blue); in the absence of SAMe only the gating loop is disordered. (c) Superposition of SAMe-bound MAT(α2)2 (PDB entry 2p02 in blue) with MAT(α2)2 after MATβV2 binding; the open state in this case shows two additional flexible loops, near the inserted MATβV2 C-terminus. (d) ATP was added to MAT(α2)4, MAT(α2)4(βV1Δ16)2, MAT(α2)4(βV2)2 and MAT(α2)4(βV1)2 pre-incubated with 5–200 µM of methionine then SAMe formation was quantified using UPLC–MS/MS. It shows that the Vmax of MATα2β complexes are higher depending on the variant of MATβ.
A more extensive mutational analysis shows by gel filtration that the minimum motif required for the formation of the MAT(α2)4(βV2)2 complex comprises three residues at the end of the C-terminal of MATβV2 (Val321Phe322His323). This motif interacts with the loop that recognizes the tripolyphosphate of the ATP at the active site, suggesting a possible allosteric mechanism that could be responsible for the observed increase of the Vmax of MAT(α2)4(βV1Δ16)2 in comparison with the catalytic subunit alone (Fig. 4 c).
In summary, we propose that the N-terminal loop of MATβ acts in a concerted manner with the C-terminus motif, helping the release of the product by making the active site solvent-accessible for the next substrate to be processed. MATα2β activity and SAMe levels appear to be tightly linked with cell proliferation, e.g. upregulation of MATα2 and/or MATβ provides a growth advantage in hepatoma and colon cancer cells (Attia et al., 2008 ; Yang et al., 2008 ; Lu & Mato, 2012 ; Xia et al., 2010 ; Chen et al., 2007 ). Similarly, T-leukaemic cells exhibit higher MATα2β activity but, remarkably, when MATα2 expression is inhibited SAMe levels decrease and there is more apoptosis (Attia et al., 2008 ; Jani et al., 2009 ). In this regard, regulating SAMe production might be an option for potential anticancer therapies. The structure of the MAT(α2)4(βV2)2 complex presented here has direct implications for a broad range of SAMe-based biochemistry (Landgraf & Booker, 2013 ; Kim et al., 2013 ). Our results show that the complex MAT(α1)4(βV1)2 is stable in vitro, raising the possibility that this complex may exist during the transition of the expression between different isoforms. It also provides a gateway for structure-based drug design with the aim of searching lead compounds that regulate the levels of SAMe without interfering with the catalytic reaction for its synthesis.
3.1. Protein expression and purification
MATα1, MATα2 constructs were kindly provided by SGC Oxford and MATβV1, MATβV2 constructs were from the laboratory of SCL. MATβV1 and MATβV1Δ16 isoforms were cloned in the HIS-parallel vector via NcoI and XhoI sites, and MATβV2 was cloned in the pET-28a(+) vector via Ndel and Xhol sites. The expression of MATβV1 was carried out in Escherichia coli BL21(HC41) and the expression of the other three proteins in E. coli BL21(DE3) strain. Cells were grown in LB medium at 37°C to an A600 = 0.6–0.8 at which protein expression was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (GoldBio), at 20°C overnight.
Cell pellets were lysed at 4°C using high-pressure homogenization at 27 Kpsi (1 Kpsi = 69 × 103 MPa) (Constant System Ltd, UK) in lysis buffer (500 mM NaCl, 5% glycerol, 5 mM imidazole, 10 mM BME (β-mercaptoethanol) and the cell homogenate was clarified by centrifugation at 20 000 rev min−1 for 40 min. The clarified supernatant was loaded onto nickel resin equilibrated with lysis buffer. The column was washed with lysis buffer and then with wash buffer (500 mM NaCl, 5% glycerol, 30 mM imidazole, 10 mM BME). Proteins were eluted with elution buffer (500 mM NaCl, 250 mM imidazole, 10 mM BME) and the His tag was then cleaved from MATα2, MATα1 and MATβV1 by incubation overnight with Tobacco etch virus (TEV) protease and with thrombin in the case of MATβV2. MATα2, MATα1 and MATβV2 were then loaded onto an ion-exchange chromatography column (HiTrap Q HP column, GE Healthcare), and MATβV1 on a HiTrap S HP, that were pre-equilibrated with buffer A (50 mM NaCl, 5 mM BME); purification was then performed using an isocratic gradient from 0.05 to 1 M NaCl. Selected fractions of MATα2 and MATα1 were then concentrated and loaded onto a HiLoad 16/60 Superdex 200 gel-filtration column (GE Healthcare) and fractions of MATβV2 and MATβV1 onto a HiLoad 16/60 Superdex 75. Finally, fractions containing pure protein were pooled and stored at −80°C. The buffer used in each purification step was 25 mM HEPES pH 7.5.
3.2. Complex formation, crystallization and data collection
In order to assemble the complexes, MATα and MATβ were incubated together for 1 h at 4°C in 50 mM HEPES buffer pH 7.5 containing 10 mM MgCl2, 50 mM KCl, 300–500 µM NADP. The complex was then loaded onto a Superdex 200 10/300 column and eluted with buffer consisting of 200 mM NaCl, 25 mM HEPES pH 7.5, 1 mM MgCl2, 5 mM KCl, 1 mM TCEP [tris(2-carboxyethyl)phosphine]. Crystals appeared at 25°C within 1–2 d in drops containing 2 µl MAT(α2)4(βV2)2 complex at 5.8 mg ml−1 mixed with 1 µl precipitant solution of 100 mM MES/imidazole buffer pH 6.5, 10% ethylene glycol, 20% PEG 8K. Before crystallization the MAT(α2)4(βV2)2 complex was incubated with its product SAMe (1 mM), its substrate ATP (1 mM) or AMPPNP (250 µM) and MET (1 mM). These compounds were added to the precipitant and cryosolution. Different data sets were collected at the PROXIMA1, XALOC and I04 beamlines at SOLEIL (St Aubin, France), ALBA (Barcelona, Spain) and Diamond (Oxford, England) synchrotron centres, respectively. Data reduction was carried out with the HKL-2000 (Otwinowski & Minor, 1997 ) and XDS programs (Kabsch, 2010 ). The phases were calculated with Phaser (McCoy et al., 2007 ) using MATα2 (PDB entry 2p02 ) and MATβ (PDB entry 2ydy , Shafqat et al., 2013 ) as search models for molecular replacement. Model building and refinement were performed using Coot (Emsley & Cowtan, 2004 ), PHENIX (Adams et al., 2010 ) and REFMAC (Murshudov et al., 2011 ). Data-collection and refinement statistics are summarized in Table 4 .
Data-collection and refinement statistics
Values in parentheses are for the highest resolution shell.
SAMe-bound
ADO-bound
PPNP-bound
Wavelength (Å) 0.98 0.978 0.979
Detector PILATUS 6M PILATUS 6M PILATUS 6M
Space group P212121 P212121 P212121
Unit-cell dimensions (a, b, c) (Å) 72.44, 115.72, 298.45 72.09, 116.57, 299.48 72.14, 122.18, 298.42
Resolution (Å) 50–2.6 (2.69–2.6) 108.63–3.3 (3.48–3.3) 50–2.35 (2.43–2.35)
Rmerge (%) 14.4 (59.4) 13.9 (49.4) 10.1 (70.4)
I/σI 10.12 (1.75) 6.8 (2.7) 17.3 (1.6)
Completeness (%) 98.7 (90.1) 99.9 (99.9) 98.0 (83.2)
Redundancy 5.6 (4.5) 3.3 (3.5) 7.5 (5.2)
Resolution (Å) 2.6 3.3 2.35
No. of reflections 76590 38907 108283
Rwork/Rfree 21.8/27.9 17.6/26.0 21.1/25.1
No. of atoms
Protein 15061 16370 16463
Ligand/ion 54/1 38/1 54/4
Water 108 37† 498
B factors (Å2)
Protein 76.64 71.19 58.4
Ligand/ions SAMe/Mg 68.16/60.62 ADO/Mg 97.95/49.14 SAMe/PNPP/Mg 47.73/63.48/39.57
Waters 56.13 30.67† 48.39
Ramachandran statistics
Residues in preferred regions 1920 (94%) 1863 (90%) 2001 (96%)
Residues in allowed regions 108 (5%) 185 (9%) 76 (4%)
Outliers 10 (0.5%) 20 (1%) 8 (0.38%)
R.m.s. deviations
Bond lengths (Å) 0.006 0.008 0.005
Bond angles (°) 1.091 1.265 1.026
†The waters in this structure (3.3 Å) were assigned using the high-resolution (2.35 Å) model as reference. Compared to other higher resolution structures very few were assigned (37 compared to 498 in 2.35 Å structure). Many of these waters are located/trapped at the interface of MATα2 dimers accounting for the much lower B factors.
SAXS data were collected on the SWING beamline at the SOLEIL synchrotron, using the HPLC-integrated SAXS set-up with a two-dimensional AVIEX CCD detector over an angular range qmin = 0.01 Å−1 to qmax = 0.5 Å−1. 80 µl MAT(α2)4(βV2)2 complex at 5 mg ml−1 was loaded onto a pre-equilibrated Shodex KW-402.5-4F 150 kDa SEC (size-exclusion chromatography) column, 250 frames of SAXS data were taken over the course of protein elution. Data averaging and reduction were carried out with the Foxtrot suite, developed at SOLEIL for the SWING beamline. Further analyses were performed with the ATSAS suite (Petoukhov et al., 2012 ). In order to generate an ab initio model ten runs of DAMMIN (Svergun, 1999 ) were performed, and after averaging and filtering a model with 732 beads was produced.
3.3. Isothermal titration calorimetry (ITC)
In order to address the association constant (Ka) of MATαβ complexes, MATα2, MATα1, MATβV1, MATβV2 and the mutant variants were buffer-exchanged by gel filtration on a Superdex 200 10/300 GL column equilibrated with 200 mM NaCl, 20 mM HEPES pH 7.5 buffer before ITC analysis. Subsequently, MATβ isoforms and mutants were injected into MATα2 or MATα1 solution in aliquots of 10 or 20 µl, respectively (Table 1 ). To verify the interaction of NADP with different MATβ isoforms, each protein was buffer-exchanged by gel filtration on a Superdex 200 10/300 GL column equilibrated in 5 mM MgCl2, 5 mM KCl, 20 mM HEPES pH 7.5, 200 mM NaCl buffer before ITC analysis. NADP was also diluted in the same buffer. NADP was injected into MATβV2 in aliquots of 15 µl and into MATβV1 in aliquots of 10 µl (Table 2 ). All ITC measurements were carried out at 25°C on a VP-ITC Microcalorimeter (MicroCal/GE Healthcare). The ITC data were processed using Origin software (OriginLab Corp., USA).
3.4. Activity assays
MAT activity was addressed by measuring production of SAMe using a fixed concentration of ATP (1 mM) and different concentrations of methionine (5–200 µM). The concentration of MATα2 and MATαβ complexes was optimized to evaluate the activity at the linear region of the Michaelis curve. The final concentrations of proteins in the reaction were 50, 25 and 12.5 nM for MATα2, MAT(α2)4(βV1)2 and MAT(α2)4(βV2)2, respectively.
The reaction was carried out in a final volume of 200 µl. The mixture contained 40 µl of the enzyme [250 nM for MATα2, 125 nM for MAT(α2)4(βV1)2 and 62.5 nM for MAT(α2)4(βV2)2], 40 µl of methionine (25–1000 µM) and 40 µl of ATP (5 mM) in 80 µl of reaction buffer (50 mM HEPES pH 7.5, 25 mM MgCl2, 25 mM KCl). The blank control was prepared in the absence of ATP. All stock solutions for methionine, ATP and each enzyme were prepared in the dilution buffer [10 mM HEPES pH 7.5, 500 mM NaCl, 5%(v/v) glycerol, 0.5 mM TCEP].
For each reaction the protein, methionine and buffer were pre-incubated for 15 min prior to the addition of ATP. The reaction mixtures were thermostated and agitated (37°C, 1400 rev min−1). After 10 min the reactions were terminated by the addition of 800 µl of 75% acetonitrile and 1.2% formic acid. To ensure the reaction stopped after the addition of acetonitrile/formic acid solution all samples were shaken (4°C, 1400 rev min−1) before centrifugation (14 000 rev min−1) and transferred to 96-well plates for UPLC–MS analysis.
Samples were injected in a randomized order for the detection and quantification of SAMe and methionine. Briefly, upon injection, polar metabolites bind to the UPLC column and are then eluted on a polarity gradient. Each fraction was subjected to mass-spectroscopy analysis to determine the production of SAMe and the remaining methionine levels. A ten-point calibration curve with exponentially spaced concentrations of methionine was used for quantization of each sample (van Liempd et al., 2013 ). The rate of SAMe formation was calculated (pmol s−1 per nmol of complex or pmol s−1 per nmol of MAT2α tetramer) for each substrate concentration. R (R Core Team, 2013 ) software was used to fit the enzyme kinetic data with the Michaelis–Menten equation for calculation of Vmax and Km values. Each reaction was performed in triplicate.
Supporting information. DOI: 10.1107/S2052252514012585/lz5002sup1.pdf
This work was supported by National Institutes of Health Grants R01DK51719 (SCL and JMM), the Plan Nacional of I+D SAF 2011–29851 and the Diputación de Vizcaya (METIOsensor project). We would like to thank the staff and management of Diamond, SOLEIL and ALBA for provision of the crystallographic facilities at their synchrotron centres. We also thank the staff of the metabolomics and proteomics platforms at CIC bioGUNE and Dr Aitor Hierro for discussion and comments on the manuscript. Use of SOLEIL was part funded by the European Community Seventh Framework Program (FP7/2007–2013) under BioStruct-X (grant agreement number 283570 and proposals number 2370/2460). We thank Dr Gareth Wright and Dr James Austin for collecting SAXS data. SVA was supported by Wellcome Trust Fellowship 097826/Z/11/Z. BM was supported partly by BBSRC DTP and the Liverpool–bioGUNE partnership.
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This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.


The best gift for the bass player in your life is both fun and practical, like this Vox Headphone Amp.
If you have a bass player in your life you might be thinking a bass-related gift is the perfect idea for a birthday or upcoming holiday. But if you don't know a whole lot about music, or even if you do, it can be tough to search through your options and find a gift that is both thoughtful and useful.
At first it might seem like a good idea to choose some strings or picks, or even a guitar strap. After all, guitar players always need strings, picks and other accessories! But most bass players are very choosy about what type of strings they use, and many don't use picks at all. If you really want to impress your bassist you have to try a little harder.
Good thing I'm here to help! For this article I've hunted down a few gift ideas for bass players that I feel are fun and super useful. As a bassist myself, I wouldn't mind if my wife happened to stumble over this list just before my birthday or Christmas!
If your bass player is like me, and most other musicians, they'll love a gift that shows you know what's important to them (music, of course) and that you really took the time to find something they can use and love.
So check out some of my top gift ideas for bass players!
A handy maintenance kit is a great idea for any musician. Guitarists and bassists are always tinkering with their gear, and keeping a bass in tip-top condition takes a little work. A bass that is set up correctly and maintained well is easier to play, and even sounds better. Bassists need the right tools for the job.
There are many guitar tech kits on the market, but finding one made especially for bassists is a little harder. The CruzTools GrooveTech Bass Kit has all the tools needed to perform standard maintenance and repairs on a bass guitar, and it is small enough to toss in a gig bag or backpack. It's the kind of gift a musician will use for years to come.
A great set of tools to keep a bass in top performance condition.
Gift cards might seem impersonal, but think about it for a minute. Unless you are a bassist yourself, you likely have no idea what you are getting into if you try to pick out gear for the bass player in your life. Sure you mean well, but there is a strong chance whatever you get is going back to the store. Musicians are choosy about their gear, and unless you know right the brand, size, gauge, color and style you are taking a shot in the dark.
On the other hand, when you give a gift card you are actually giving two gifts. Of course one is whatever they decide to purchase with the card. But you're also giving the gift of going to the music store with free money, which is a musician's dream come true.
So, if nothing else on this list makes sense, find out your bassist's favorite music store and grab them a hefty gift card. They'll be thrilled, and you'll be a hero.
A headphone amp is a really great piece of gear for the aforementioned traveler. They aren't going to be able to fit a bass amp in their luggage, so a tiny, compact personal amplification system is perfect. It literally fits in a pocket, and along with a standard pair of headphones it allows a bass player to practice quietly anywhere they want.
Of course a bassist doesn't have to travel to use a headphone amp. They are also extremely useful for bassists who want to play and practice quietly anywhere. If you are the parent of a budding bassist who still seems to be creating more noise than music you might find this to be an especially smart gift idea! However, even veteran players use headphone amps for practice.
A headphone amp allows a bassist to practice quietly. It's a great gift for the bass player in your life, and possibly the whole family!
Is the bassist in your life all talk and no action? Maybe they've always wanted to learn bass but never took the plunge, and now you want to give them a little nudge in the right direction. That a great idea, and it opens up many more options.
Here are some gift ideas for the newbie who still needs a starter bass, amp and all the other gadgets that go along with learning bass guitar.
Check out the top bass guitar starter packs, the best choices for beginners who want to start playing today! Starter packs come with a bass, an amp and all the accessories you need, including learning materials. It's the easiest way to start on bass.
Here are the 5 best bass guitars under $200 for beginners, quality instruments that will get a newbie started off right! These basses are made by some of the top gear brands in the world, the same companies that make the pro-level gear.
Compare 5 of the best bass amps for beginners, top-rated amplifiers from some of the biggest brand names in the bass guitar world. These little rigs aren't expensive, but they make great amps for beginners and will serve as practice amps later on.
While some of the gift ideas in this article are useful and practical, others are just plain cool. So it is with these tiny little replica bass guitars. These are miniature instruments built to scale and made to look just like the real thing, only smaller. You can't play them, and they aren't toys. These are models of real instruments intended to look awesome on a shelf or desktop.
Some of the coolest are licensed by Fender themselves, one of top bass guitar builders in the world. You can find mini replicas of their classic Jazz Bass and Precision Bass designs that look almost as good as the full-sized versions. Any bassist would love to get one of these little guys as a gift!
Do you know someone who plays a little bass? Well, they can't play this one, but it would look amazing on display in a home or office.
Fender bass guitars are legendary in the music world, and these little replicas are pretty awesome in their own right.
You can't fit a bass is a suitcase, but a travel bass is the next best thing. Traveler is a guitar company that specializes in, as the name would suggest, instruments made especially for travel and stowing away in compact areas. With an acoustic-electric design they can be plugged right into any standard bass amp, or played unplugged. The addition of a lap-rest adapter makes them almost as comfortable to play as a full-size bass.
Travel basses are perfect for people who travel a lot for business, or for college kids who don't have a lot of room in their dorms for full-sized instruments. They are also pretty cool for everyday use, and make a nice addition to the instrument collection of any bassist.
Sometimes bass players can get the music they need from guitar sheet music, especially if it is in tab form. But why not give your bassist a songbook made especially for them? The Bass Tab White Pages features 200 popular songs in various genres transcribed just for bass. At over 1,000 pages there is no shortage of music to learn, whether your bassist is a newbie or a seasoned pro.
Don't worry if your bassist can't read standard musical notation, or even if they are a total beginner. Tablature, or tab, is a way of notating music that anyone can pick up within a few minutes and start playing. That makes the Bass Tab White Pages a great way to expand the repertoire of any level of bassist.
The Bass Tab White Pages is a massive collection of music made just for bassists. Tab is an easy way for guitarists and bassists to read music, no matter their level of experience.
Bassists rock. You can't have a rock band without one, and along with the drummer the bass player lays down the foundation of the song. So why is it that, like a certain comedian, bassists don't get any respect?
The truth is that talented bassists get a whole lot of respect from anyone who knows anything about music. But their role as a supporting cast member to the showboating of the lead guitarist and vocalist can result in some ribbing now and then. It also results in some cool t-shirts!
Bassists tend to take themselves a little less seriously than some other musicians, so a t-shirt with a funny or clever saying on it will go a long way as a gift!
We bass players are unique critters, but finding the perfect gift for us really isn't that hard. Bass-related paraphernalia might be a little harder to locate than guitar stuff, but there are still a lot of great ideas out there.
Hopefully you found this article helpful and you are well on your way to finding the right gift for your bass player!
Read this review and learn why the Hartke HA3500 bass amp or its big brother the Hartke HA550o is the perfect choice for gigging bassists.


text:
Kuwait Government Online > Citizens and Residents > Islamic Services > Overview on Kuwait Mosques
The seed of prominence of the masajed in Muslim hearts was first sown in the prophecy era. As soon as the first masjed was established, it was highly ranked and became the origin of illumination, knowledge, discernment, and pride for Islam and the Muslims. The corridors of the masajed mark the composition of Muslim Scholars and Historians for their literary and scientific masterworks. The masajed, also, contributed in the social development of the Islamic righteous society by spreading solidarity and faith among Muslims. As time passed, Muslims reaped the fruits of the masajed effective role. Pride, sublimity and virtue became among Muslim traits. Moreover, masajed helped in spreading Quran and Sunna sciences, as well as all sciences that benefit people. Muslims were keen to send their children to the masajed in order to memorize the Holy Quran, learn about Islam, and draw forth from the infinite sea of knowledge.
From the dawn of Islam, ancient Muslims perceived the grandeur of the masajed and that the way for the highness of Islam and Muslims pride is through the masajed. The most powerful evidence on this magnificent rank is the establishment of the first masjed in Islam by the Prophet Mohamed (peace be upon him) as soon as he reached the land of Hijra. The Prophet (peace be upon him) made the masjed the source of the dawah, call for Islam, and a fountain of power that never runs dry in the hearts of Al Sahaba, the Prophet's (peace be upon him) companions, and believers. The Prophet used to meet with Al Sahaba in the masjed to teach them Islam doctrine. Once the true concepts of religion were engraved in Al Sahaba's hearts; they followed the righteous way in determination and certainty after the death of the Prophet (peace be upon him). They, then, set out to rehabilitate the world and spread Islam everywhere.
Through out history, the masajed were the starting point for beams of knowledge that radiated in the entire world. Within the walls of the masajed, biographies for many notable and honorable scholars and authors of many valued works of religion, science, and ethics were registered. Starting from the masajed and marching to the farthest places on Earth, armies of Islam derived their strength and determination from the strength of bonds with the masajed, the house of the worship, belief, and Call for Islam. The masajed reinforce the relation between Allah and human heart and soul. All Muslims are gathered to establish prayers as Almighty ordered and as inscribed in the Sunna of the Prophet (peace be upon him). They, also, meet in the masajed to learn about Islam and discuss all maters and affairs. Upon meeting, Muslims present purposive opinions, which are the gains of their precognition and correct understanding. Therefore, ties among Muslims are fortified and their union is unbreakable.
Muslims became aware of the dangers of ruination of the masajed and realized Allah's threats to those who endeavor to do so. The dangers of ruinating the masajed are not only limited to their closing, but extend to secluding their leading role, reducing their rank in Muslim hearts, and putting obstacles against performing their roles as required. The masajed play various roles of dawah, as everyone has his own duties that aim at Islam pride. Al-Imam, Al-Muezzin and the prayers, each one is consecrate to play his role and undertake his responsibilities. Each role is just a brick in the road of delivering Islam mission and spreading it throughout Earth.
When Muslims visit the masajed regularly and vitalize their role on Earth, Muslims become one strong unit. This solidarity and strength of Muslims is necessary for Islam dawah and victory.
If you want to search for the available masajed in Kuwait according to the governorate and the region, please click on the following link of the Ministry of Awqaf and Islamic Affairs. This link, also, provides information on each masjed, including the location of the masjed, its Imam, Muezzin and Orator.
- Mosques List
Ask the Mufti -Zakat House
Quick donation - Zakat House
Subscribe to Awqaf Magazine -Online
Mohammed Alkhurafi Center online Hall reservations

redpajama_set_name: RedPajamaCommonCrawl


The Disciplinary Code describes how British Fencing will deal with alleged disciplinary Offences, as defined in the Code.
If you have any questions please contact headoffice@britishfencing.com.
The following are Disciplinary, Grievance or Welfare cases which have resulted in sanctions being brought either through British Fencing or through the courts. The list does not include minors.
Cases resulting in sanctions will be listed here for 12 months or the duration of the sanction, whichever is the greatest.
201 08.07.2015 Mark Nelson-Griffiths Following the decision of a Sports Resolution Appeal Hearing, British Fencing is required to place an imposition of strictures by immediate preclusion from engaging in a variety of activities including, but not limited to, coaching and refereeing whereby contact with children or vulnerable adults is possible. For a full description of the strictures please contact British Fencing Head Office.
237 14.05.2018 Matthew Dickinson 9 month ban of which 4.5 months suspended until 14.5.2019. Membership suspension currently in place until re-instatement conditions satisfied. Until this time the individual must not partake in any fencing related activities including fencing (training or competing), coaching, refereeing and club participation.


Many kinds of applications require fans to heat an object or environment evenly. These may include dryers, ovens, furnaces and others. CFW Fans manufactures robust products designed to perform reliably for an extended period of time.
Fans for process heating applications are chosen on the basis of whether a positive-pressure (forced draft or FD), negative-pressure (induced draft or ID) or balanced-draft system is required. ID fans are required to handle heated air passing through the fan and must therefore be constructed to perform reliably at higher temperatures, particularly with respect to the bearings.
Care in selecting the impeller, materials of construction and additional features is essential to ensure the smooth airflow and reliability that is demanded.
The fan commonly requires additional components, including silencers and filters, safety features such as guarding and isolators, dampers or inlet guide vanes for improved efficiency.


No other Australian cricketer – even the big Don, Allen Border or Shane Warne – has ever been more popular than Adam Gilchrist simply due to the way he played his cricket. While his entertainment on the field attracted fans all around the world, the human being that Gilchrist is, made him even well-liked among cricket lovers, especially in the sub-continent and other developing parts of the world.
Even more exhilarating is the way Gilly played his one-day cricket. A career strike rate of 97 at an average of 35.89 for an opener means that majority of the bowlers suffered (danda!) at his hands in the brutal display of explosive batting. It was comparable or even better than his contemporaries like Sanath Jayasuriya and Virender Sehwag in terms of converting the number of appearances into team's win. And teaming up with an equally – but more technically equipped – explosive Mathew Hayden meant that it was an era that the world cricket will miss for a long long time.
It is sad that Gilly is going out on lower note with himself scoring not many runs in the last few matches. Even more hurting for the fans and Cricket Australia is the fact that Australia lost the last three matches he was part of and also the triangular CB Series. However, the unselfish (how many successful cricketers are not selfish these days?) way in which Gilly played his cricket will be remembered by world cricket for ever. And retiring at the right time instead of stretching his career for an year or two longer, makes him an even more special person.
he was out! And how many cricketers do that consistently through out their careers? Also, Gilchrist is known to be a friendly character even outside the field – not just with his compatriots but also with other gentlemen cricketers around the world. I read somewhere that Gilchrist consistently tried to get Anil Kumble's home telephone number in Bangalore for almost 10 days – before being successful – to congratulate him on completing his 600 wickets in tests.
For what he has done to the world cricket, Gilly deserves a hall-of-fame entry for it is really hard for the cricket fraternity to get to see a similar personality in the near future!
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Some FutureFund sites have bank accounts that do not allow debits which prevent the issuance of credit card refunds. To help in this, FutureFund supports check/cash refunds. However, this was not enforced and the administrator would need to know which funds supported refunds and which ones didn't.
We've added the ability to restrict refunds on a particular fund. Refunds are still trackable in FutureFund by issuing a refund with a check/cash refund method.
We are not able to detect which funds have debit restrictions. If you would like to enable this feature, please contact our support team.


I had the full intention of reviewing the four casters I set as my season 4 active duty roster but after spending six months figuring out the new pace and structure of the game, I figure I should share my thoughts on the state of the game as I see them. I'm a Cygnar player and I play at a semi-competitive level. All my opinions are seen through that lens.
Warmahordes iconic image has always been around big stompy monsters and big stompy robots. I'm glad to see they are front and center now. The addition of more jack/beast points, the Power Up and Spirit Bond rules, and generally making jacks/beasts more efficient has made the game more focused on the iconic aspect of the game. I agree with this change wholeheartedly. The game had become so infantry heavy, especially medium based multi wound infantry, to the point that jacks and beasts were just filler.
There are downsides to the new jack/beast focus. Infantry becomes less common so infantry to counter infantry becomes less useful. As a Cygnar player I find my pow 10 infantry staying in the foam as I build a list. No need to take a counter to a non-existent threat or a threat that is easily countered by the jacks I'm already going to take. Single wound low damage infantry has almost no place competitively.
With the release of Mark III the new Journeyman League rules dropped. These rule immediately confused me. I was fine with no alternate battle boxes. It is a new release so it might be a little while before alternate battle boxes are finalized. The weird point scaling? Strict army lists? Best painted? Best sport? I thought Journeymen Leagues were to get new players into the game and teach them not for experienced players to just crush the new players. If Privateer Press was trying to write rules to introduce new players into the system, they utterly failed.
The army restriction rule is especially egregious for favoring the experienced players. Someone who has been playing Mark II and has an understanding of the synergies can easily plan out a list all the way to 75 points. A new player has no idea if their new unit, solo, or warcaster works with their list or even if it is something they'd like to play. Then telling them they are stuck with their choice for the rest of the league. Restricting the list building puts new players on the back foot and doesn't allow them to adapt.
The rule would work well for a slow growth league aimed at more experienced players. Add a few alternate battle boxes fix the point scaling and you'd have a great start to a league.
I think this is probably the most telling problem from Mark III release. We had a few rules with unclear wording and some models with broken abilities. That is expected from any major release. Some things won't be caught before they hit a wider audience. The fact that an entire faction need a major rework speaks volumes on the rush that Mark III was released. Internally Privateer Press might felt they were ready for the release but externally, form the point of view a customer and a player, it felt rushed, poorly tested, and released too early.
Overwatch by Blizzard Entertainment is different then most FPS games. Unless there is a huge difference is mechanical skill, teamwork and team composition is more important than individual skill. The game also changes drastically based on the mode you play (Quick Play vs Competitive Season) and who queues with you (solo queue vs small group vs full team). These factors can change how you, as a player, can play to be successful.
During Season 1, I played mostly Lucio or Zenyatta in 4 or 5 person queue. I ended the season floating around mid 40s. Now in Season 2, I'm playing Reinhardt and Zarya in a 6 sixth stack. I'm sitting around 2000 so far. Each season I've occasional played solo, duo, and triple queue.
When solo queuing it always felt like you are rolling the dice. You could get a handful of people who were willing and able to work together but the next game… not so much. My experience was mechanical skill became very important and team strategy less so since most games were a disorganized mess of twelve players flailing at each other. Matchmaking when solo queue will try to put with other players solo queuing but you could teamed up with a five stack or anything inbetween.
I can suggest playing solo queue if you have the personal skill and time to play the odds.
Personally my mechanical skill (aim, reaction, and in game awareness) is pretty low. I'm working on it and getting better… slowly.
Queuing as a full six is a ton of fun but is also one of the most frustrating things I've ever done. When queuing this way strategy and teamwork is king. You and your five friends could jump in to competitive randomly, thinking playing together would be fun. Then an organized sixth stack become your opponents. The have practiced comps, good communication, and teamwork. They wipe the floor with you. No contest. This is where Overwatch's team-based shooter really shows off the team-based aspects.
If you queue as a sixth stack, I suggest doing it with people you can practice with on a regular basis. The team takes the time to form strategies, comps, and synergies. Figure out what works for your team and what doesn't.
This is where I am right now. My team plays together twice a week and we have our roles and basic comps figured out. We are trying to get our strategies panned out and get every member's game knowledge up to par.
From personally experience triple queue is the sweet spot. You have enough people to cover the any roles that are needed and to use teamwork effectively. Also the match making isn't going to pair you up with full groups, so if one or two people are running off trying to "carry" you can still win the game.
No matter which way you decide to play a competitive season and climb that ladder. Remember to try and communication with your teammates and be willing to be flexible. Change to a hero to fill a role that needs to be filled. Be willing to change roles if the team asks. If someone asks to play a hero you've locked in, be willing to switch with them.
No matter how mechanically good you are at the game. You will do better if you talk to your team and work together.


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5 of the 10 Wealthiest Americans Are Jewish
Taggedisrael, jewish
Click here to view original web page at therealistreport.com
According to the organized Jewish community and professional Jewish ethnic activists, anyone claiming that Jews are over-represented in key aspects of American political, cultural, and economic life are "irrational, paranoid, hateful, anti-Semitic conspiracy theorists."
Making basic, factual, and easily verifiable statements about the reality of Jewish power and influence in America – pointing out their domination and control of the mainstream mass news media, Hollywood and entertainment industry, for example, or their influence in the Federal Reserve banking system, Wall Street, and corporate America – is considered "anti-Semitic" and "hateful," according to Jews, despite how obvious it all is.
Here we have even more proof of the total Jewish domination of our society: according to a recent report published by The Times of Israel, 5 of the top 10 wealthiest individuals in America are Jewish. That means that 50% of the richest people in the United States are ethnically Jewish, despite Jews representing less than 5% of the total U.S. population.
Think about that for a moment. And think about why we are prevented from openly and honestly discussing these basic facts, despite them being reported in mainstream publications, including explicitly Jewish publications like The Times of Israel.
Forbes published its 2018 roster of America's wealthiest this week, and five members of the tribe made the top 10 list.
Facebook CEO Mark Zuckerberg leads the Jewish pack at number 4, with a net worth of $61 billion. He is followed by software giant Oracle's Larry Ellison at #5 with $58.4b and Google co-founder Larry Page at #6 with $53.8b.
Fellow co-founder Sergey Brin falls a bit behind with $52.4b, leaving him at #9. Finally, former New York mayor Michael Bloomberg closes out the top 10 with a respectable $51.8b. […]
The simple fact of the matter is that Jews do in fact largely run our country.
They own and control the mass media, allowing them to essentially control the narrative, especially on key issues that are central to their insidious international agenda to subvert and destroy traditional Western civilization. They own and control our banking system and much of corporate America. They own and control some of the top internet giants, including top social media companies like Facebook. They play a huge role in American politics, and essentially control the U.S. Congress and executive branch, and thus American foreign and domestic policy.
And it's easy to demonstrate all of this. It's not some crazy "conspiracy theory" – it's openly reported and documented, often by explicitly Jewish media outlets. We're just not allowed to honestly discuss this reality and what it means for our nation and world – that would be "anti-Semitic," after all.
Subscribe to The Realist Report today, and support pro-White independent media!
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Democrats and China End American Democracy As It Was
Democrat Party Leaders Increasingly Old and Out of Touch
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Rightwing (read: Jewish Supremacist) think tank which quietly runs the Knesset (and our Congress)
The Nazi Ghosts of a South American Hideaway


A DVD about my life story, "I Have a Dream", is now available nationwide. This DVD reflects on my experience of hearing loss over 29 years and the miracle of being able to hear again after bilateral cochlear implants in 2011. Click HERE for more info on this DVD.
The DVD can be ordered at R120 per DVD – VAT included/Postage excluded. Click HERE to download order form.
Fanie du Toit is the Patron of the South African Hearing Institute.
He is the Manager of Public Education and Awareness for the National Council for Persons with Physical Disabilities in South Africa. Fanie is also the presenter of the radio program " Leefwêreld van die Gestremde" on Radio Sonder Grense (RSG). Fanie and Francis have come a long way in working together in developing tools and support to the Hearing Impaired community and continue to work together in support of each other.


Last Tuesday was no doubt an exciting day to be in Chicago—the Cubs advanced to the National League Championship Series for the first time since 2003 and boisterous cheers could be heard throughout every bar with a TV in the city. But Anthony Rizzo wasn't the only heavy hitter in Chicago last week. Health insurance MVPs were also out and about attending AHIP's Consumer Experience & Digital Experience conference. The show aimed to bring health plans practical ways that digital engagement can help transform how consumers interact with their health and the health system.
There's no doubt that consumers have high expectations that a brand should "know them" and communicate with them based on how they actually are interacting with that brand e.g. don't send me a promotional email after I just made a purchase from you (cue consumer eyerolls). As consumers yourselves, you know that there's nothing more personal to you than your health needs and motivations—one size definitely does not fit all. Digital healthcare experiences are no exception, and health consumers expect healthcare companies to deliver the same personalized experience they get with any other consumer brand.
Personalization: Not just delivering a personalized message but truly communicating on an individual basis and knowing when, how often, and on which channels they want to be engaged with.
One Transformation Team: Looking inside to see why consumer engagement has been so difficult in healthcare. Transformation comes from breaking down organizational silos to centering all activities around the consumer, not around the function or fear.
New Ways to Engage: From wearables to the formerly feared mobile and social channels, the digital world has blown open a whole new way to reach the digital health consumer.
If you're familiar with the principles of engagement marketing, you're starting to see how the ABCDEs of engagement marketing would really pay off for healthcare marketers. And with the landscape forever shifted by healthcare reform and more Americans now choosing their own health plans, healthcare companies need to find effective and innovative ways to demonstrate their value to this new empowered consumer, but how? That's just the question we wanted to answer during our session on Transformational Consumer Engagement Marketing.
Lesson 1: Collaboration and alignment are essential: define roles and process clearly and bring in executive sponsors early—and keep them informed!
Lesson 2: Think omni-channel throughout the customer journey.
Lesson 3: Get your core technology stack in place as early as you can (MA, CRM, CMS).
Lesson 4: Have courage! It takes bravery to be the change agent but you can do it.
Lesson 5: Test and learn—an agile approach works best.
Lesson 6: Be aspirational but set realistic expectations—especially in the beginning.
To learn more about how healthcare companies can transform their consumer marketing, check out the Marketo and Intelligent Demand presentation from AHIP and the Health Insurance Plans' Guide to Consumer Marketing.
The post Hitting Consumer Healthcare Home Runs with Digital Engagement appeared first on Marketo Marketing Blog – Best Practices and Thought Leadership.


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Presented By Close-Act
i-Puppets
Part of GDIF Opening Night and Greenwich Fair
Type: Spectacle, Street Games
A trio of unusual optical creatures have landed and as they look down from above, they are curious to find out more about Woolwich and Greenwich. An intriguing interactive walkabout show which encourages you to reflect on seeing the world from a different perspective.
i-Puppets will be performed as part of GDIF's spectacular Opening Night, transforming the streets and public spaces of Woolwich with art, food, dance, street theatre and a dazzling finale, and will also be performed as part of Greenwich Fair, GDIF's two-day "festival within a festival" from 21-23 June, bringing together UK and international street arts companies to Greenwich Town Centre in a fun-filled, family-friendly extravaganza.
For both the Opening Night and Greenwich Fair, i-Puppets will be a roaming performance. In Woolwich the performance can be seen all over Powis Street and Greens End, and in Greenwich it will be all over Greenwich Fair.
i-Puppets in Woolwich
Our photo route details the most accessible route between the nearest public transportation and performance locations. Download our photo route for Woolwich.
Download our photo route for Greenwich Fair.
Where to find it in Woolwich
i-Puppets is a roaming show which can be seen all over Powis Street and Greens End.
Where to find it in Greenwich
i-Puppets is a roaming show that will be performed in the Old Royal Naval College and Cutty Sark Gardens.
Close-Act Theatre was founded in 1991 and originates from a collaboration between designers, actors, dancers, choreographers and musicians. The artistic directors of Close-Act are Hesther Melief and Tonny Aaerts.
Greenwich Fair is supported by the Royal Borough of Greenwich, Royal Greenwich Festivals, Arts Council England and Without Walls.


text:
Category : Scholars
Trump's 'Muslim Ban' foolish
Press Shia – Hujjat al-Islam al-Qubanchi said, "Donald Trump's measure to ban citizens of certain countries from entering the United States is foolish, crazy and against the mores of global diplomacy."
Press Shia – In his Friday prayer sermon delivered to a large and fervent crowd of believers in the Iraqi city of Najaf's al-Fatimiyah al-Kubra Husayniyah, Hujjat al-Islam Sayyid Sadruddin al-Qubanchi praised the liberation of several areas in Mosul and the surrounding Nineveh Governorate by the Iraqi forces.
The Iraqi scholar said, "The courage of the warriors in the operation to liberate the western part of Mosul has led to the admiration of the world in such a way that some newspapers, including the Washington Times, has expressed surprise at the rapid advances of the warriors."
He appreciated the Iraqi parliament for passing a law granting the same rights to members of the Popular Mobilization Forces that members of the country's Armed Forces enjoy.
Hujjat al-Islam al-Qubanchi reacted to conference held by a group of Iraqi Sunni leaders in the Swiss city of Geneva who call themselves "The Iraqi National Coalition," and said, "The sense of the destruction of Daesh, the strength of the political process in Iraq and an understanding of the power of the Shi'ahs of Iraq are among the factors for this meeting in Geneva."
He invited those participating in this conference to respect Iraq's constitution and to engage in the country's political process and added, "The participants of this conference must avoid engaging in acts of terrorism, sectarianism and insulting the Popular Mobilization Forces."
Hujjat al-Islam al-Qubanchi referred to the decision made by the new American President, Donald Trump to exclude Iraq from the list of countries whose citizens are banned from entering the United States and said, "Trump's measure to ban citizens of certain countries from entering the US is foolish, crazy and against the mores of global diplomacy."
On Monday, Trump signed the order placing a 90-day ban on people from six mainly Muslim countries.
The new order maintains a 90-day ban on travel to the US by citizens of Iran, Syria, Libya, Yemen, Somalia and Sudan, but excludes Iraq and applies the restriction only to new visa applicants. It also removed an indefinite ban on Syrian refugees.


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Home Community Thinking Outside of the 'Man Box'
Thinking Outside of the 'Man Box'
Sally Harding
Assoc Prof, Dr Michael Flood, Tom Bell, Phil Barker & Cr Kat Bennett, City of Wodonga. Photos: Sally Harding
Positive masculinity is a matter of life and death, according to journalist and former advertising man, Phil Barker.
Barker is the author of the book, "The Revolution of Man – rethinking what it means to be a man", one of three top-notch speakers brought to town by Wodonga City Council as part of a community event titled, "Thinking Outside Of The Man Box."
The 100-plus crowd at The Cube was silent as Barker recalled how he had hit rock bottom a few years ago after his successful business went belly-up, for reasons beyond his control.
Phil Barker
Bogged down by burdens he never expressed at the time, Barker says he came very, very close to becoming one of Australia's shocking suicide statistics – on average, six men a day take their own lives.
Barker takes the subject of masculinity in business up a few notches and refers to former media bosses he's worked under as an editor, big names such as the Packers and the Murdochs. Big names don't hit rock bottom like the rest of us, surely? "Money is such a big part of the performance of being a man," Barker says.
The secret to happiness, he believes, is not from money but rather "deep, rich relationships."
Assoc Prof, Dr Michael Flood
To be honest, as one of three girls raised by a single mother, I'd never thought deeply about the importance of male identity, gender roles and positive male relationships …until I had a son, now 14.
I do remember a bit of sinking feeling of raising a boy when we found out in-utero – I was hoping my partner would take the lead. After all, he had been a boy himself.
Quite by accident our son was given a gender-neutral name but without really thinking about it, his nursery became coloured-in with mainly blue things and cars and trucks for toys. (The line was definitely drawn at toy guns, however at age three he made one out of a strand of spaghetti and attempted to 'pew pew' everyone at the dinner table).
We didn't consciously impose gendered anything on our son – something that is always raised as a social construct rather than a natural choice – but we didn't consciously avoid it either. It's how I was raised myself and I settled into being more of a tom-boy than a pretty-in-pink girl.
Thinking about it now, though, the baby doll I was given was from an Aunty in Germany, a country a little more progressive at the time.
The doll was a boy, something you could tell every time you changed its pretend nappy. So does this explain why I spent more time playing with the neighbourhood boys than girls, or why my workplaces have always tended to be male dominated or why I have as many male friends as female friends?
Not sure, but what I do know is that I hung on every word at the Wodonga Council workshop, admiring the research of Associate Professor, Dr Michael Flood, and the research findings of a recent study on being a young man in Australia, titled "The Man Box".
The 'box' is a metaphor for social expectations about what it is to be a man. So the theory goes, young men inside the Man Box are those who endorse these Man Box rules.
According to the findings, these are also the young men who report "poorer levels of mental health, are more likely to engage in risky drinking, to be involved in car accidents and to report committing acts of violence, online bullying and sexual harassment."
Tom Bell, who has a business called Tomorrow Man, gave a condensed version of the workshops he is commissioned to run for high school students and men in big-league sporting clubs.
Tom Bell
He encourages big and little men to have the confidence to express themselves on their own terms, not according to peer pressure or the expectations of others, or at least that's how I took it.
Therefore, I encouraged the men in my house to attend Tom's evening session, held at Wodonga Bowling Club and both father and son came home with a bit of spring in their step.
I was desperate to find out from my son what he thought but it would seem he was more expressive at the workshop than back at home.
"Do you think it's a good idea for men to share their problems with other men?", I asked.
He shrugged his shoulders, paused, then replied: "I think it's a good idea for everyone to share their problems with anyone."
Not a bad start, hey?
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A Response to be Proud of
#SayThankYouCampaign


View a list of all PSAP System Administrators (updated 01/03/19) by county of service.
Administrative Code (ARS § 38-613 et. seq.) (9-1-1 Title 2, Chapter 1, Article 4, R2-1-401_R2-1-411) Article 4 consisting of Sections R2-1-401 through R2-1-411 adopted effective June 22, 1985. Amended, effective June 14, 1990. Amended by final rulemaking a 6 A.A.R. 1971, effective May 12, 2000.
In the provision of 911 services, a person, a provider as defined in Section 43-5251 or a public entity or any employee of the public entity is not liable for damages in any civil action for injuries, death or loss to a person or property that are incurred by any person with respect to all decisions made and actions or omissions taken that are based on good faith implementation except in the cases of wanton or willful misconduct, regardless of technology platform including a public safety radio communications network, that receives, develops, collects or processes information for the service's location information databases, relays, transfers, transfers, operates, maintains or provides emergency notification services or systems capabilities, or provides emergency communications or services for ambulances, police and fire department or other public safety entities.
Revolving Fund Administration (ARS § 41-704) Emergency Telecommunications Services; Administration; Revolving Fund.
Telecommunication Service Excise Tax - (9-1-1 Title 42, Chapter 5, Article 6, ARS § 42-5252) Levy of tax; applicability.
Middle Class Tax Relief and Job Creation Act of 2012 (Public Law 112-96).
Signed into law Feb. 22, 2012. Title VI, known as the Public Safety and Spectrum Act, reallocated spectrum in the 700MHz block to public safety. Authorized the FirstNet program. Other provisions related to public safety communications and interoperability.
FCC Implementation of the Middle Class Tax Relief and Job Creation Act of 2012 (FCC 12-85).
July 30, 2012. Order adopted by the FCC implementing the Public Safety Broadband Provisions of the Middle Class Tax Relief and Job Creation Act of 2012 (PS Docket No. 12-94); Service Rules for the 698-746,747-762 and 777-792 MHz Bands (Docket No. 06-150); Implementing a Nationwide Broadband Interoperable Public Safety Network in 700 MHz Band (PS Docket No. 06-229).
Signed into law July 23, 2008. The NET 911 Improvement Act is a law to promote and enhance public safety by facilitating the rapid deployment of IP-enabled 911 and E-911 services, encourage the nation's transition to a national IP-enabled emergency network, and improve 911 and E-911 access to those with disabilities.
October 28, 2008. FCC adopted rules implementing certain key provisions of the New and Emerging Technologies 911 Improvement Act of 2008 (NET 911 Act). This included rules that give interconnected VoIP providers rights of access to any and all capabilities necessary to provide 911 and E911 service from entities that own or control those capabilities. The FCC took steps to ensure that the nation's E911 network remains secure as an expanded number of entities are granted rights to access this system.


The Penn Fierce Spinning Reels are saltwater spinning reels designed for many saltwater species and their applicable techniques. The Fierce Spinning Reels, from Penn, lives up to its name with a durable full metal body and sideplate to keep precise gear alignment under heavy loads. The 4+1 stainless steel bearing system provides protection even in the harshest saltwater conditions. The Fierce Spinning Reel possesses a machined and anodized aluminum spool and handle which keeps the weight down and displays excellent durability. Stainless steel ball bearings and main shaft exhibit rust resistance which will keep your reel performing at it's highest level for years to come. Smooth retrieves are derived from Penn's Techno-balanced rotor technology.


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Q: Twitter-Bootstrap Modal not working Properly I had created a modal dialogue
Launch demo modal

        <button type="button" class="close" data-dismiss="modal" aria-hidden="true">×</button>

Modal header

One fine body…

i just called the modal from..
$("a#LaunchDemo").click();

but the modal is just blinking on the screen, i also called the modal through button click .same problem iam getting , where i went wrong?,Please help ..

A: You need to execute your javascript code when the DOM is fully loaded. And use modal instead of click
$(document).ready(function() {
   $('#LaunchDemo').modal(options)
});

Demo: http://jsfiddle.net/MgcDU/1998/

A: It wrong when the with of model was too big.
You can try when set the stylesheet for modal class in bootstrap file {width:900}
It will happen....

A: Try removing the class hide from the modal div:

A: Remove the href="#myModal" in the hyperlink tag because that is trying to link you to a part of the page.
Also, you shouldn't need to specify the element type in your selector. It will be faster to just search for the element by ID.

redpajama_set_name: RedPajamaStackExchange


If you go to work at an office and feel like you want to make an impression, then maybe you should start looking at your body language. Job performance and charisma isn't the only way to communicate to the world that you are ready for that promotion.
Slouching in a way that makes you look way too relaxed.
I mean, who doesn't love to slouch in the privacy of their own home? I know I do! But when you're at work, it's game on and that applies to your posture. Sitting in a relaxed manner is sending off the message that you're lazy at work as if you were completely uninterested in what you're working on. Make sure to sit up straight.
Letting everyone at work know that you're bored out of your mind.
No one's expecting you to act like this is your first day at the job. People know you're only human, but when it comes to slouching, slacking, and dozing off into Lalaland, it's best to put on your "work face," and act like you're happy to be there. Your boss will take it as if you're an active participant who wants to actually be there.
You're constantly rolling your eyes, even if it's just a gesture you're used to making.
You're going to have to work extra hard to avoid doing this at work. It's sending off the message that you have no regard for whatever it is that people are saying. You may have accidentally learned to express your disgust or contempt by rolling your eyes, but that might lead to people thinking you feel like you're above them and their opinions and feelings don't matter to you.
Sitting or talking too close to someone, essentially invading their personal space.
Ugh! We've all had a coworker who's done that. They simply don't get socially accepted etiquette. Why would anyone need to get 1 inch away from someone's face to communicate something? That makes people feel instantly threatened and uncomfortable when someone decides to invade their personal space. Be polite, and stay a healthy distance. Somewhere around 3 to 8 ft is more than okay.
Sneaking into the office, avoiding your coworkers, without anyone noticing you.
That's a bad, bad, office faux pas. Not only is it extremely rude to your colleagues, but it's also a sign that you have poor people skills. Sure, you may just want to avoid people until you get your morning coffee, and you're absolutely entitled to do so, but don't be surprised to find out that coworkers and higher ups see you as antisocial and arrogant. Always greet others with a smile, and make sure to acknowledge the people you run into at the hallways.


This section of our website is dedicated to showing you all of the potential side effects that you may experience when you start taking Adderall.
Blistering, peeling, or loosening of the skin, chest pain or discomfort, confusion, convulsions, dark-coloured urine, diarrhoea, difficulty breathing, difficulty with speaking, difficulty with swallowing, dizziness, double vision, faintness, headache, inability to move the arms, legs, or facial muscles, inability to speak, itching and joint or muscle pain.
You may also experience skin rash, slow speech, sore throat, sores, ulcers, or white spots in the mouth or on the lips, sudden loss of consciousness, sweating, swelling of the feet or lower legs, tightness in the chest, troubled breathing, uncontrolled repeated movements, uncontrolled vocal outbursts, unusual tiredness or weakness and vomiting.