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Loss of the High-Affinity Pentamidine Transporter Is Responsible for High Levels of Cross-Resistance between Arsenical and Diamidine Drugs in African Trypanosomes
Daniel J. Bridges, Matthew K. Gould, Barbara Nerima, Pascal Mäser, Richard J. S. Burchmore and Harry P. de Koning
Molecular Pharmacology April 2007, 71 (4) 1098-1108; DOI: https://doi.org/10.1124/mol.106.031351
Daniel J. Bridges
Matthew K. Gould
Barbara Nerima
Pascal Mäser
Richard J. S. Burchmore
Harry P. de Koning
PDF + SI
Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [3H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1–/– line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4′,6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 μM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.
Trypanosoma brucei subspp. are protozoan parasites that cause human African trypanosomiasis (i.e., sleeping sickness) and the corresponding veterinary condition in livestock. Treatment of both the human and livestock diseases depends on a very small set of mostly very old drugs. The first-line treatment for the late stage of both West African and East African human African trypanosomiasis is melarsoprol, an organoarsenic compound of the melaminophenyl arsenical class, introduced in 1949 (Jannin and Cattand, 2004). A similar but water-soluble melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), is increasingly used for animal trypanosomiasis. Early-stage West African sleeping sickness is routinely treated with the diamidine drug pentamidine, introduced in 1937 (Jannin and Cattand, 2004). The corresponding widely used veterinary diamidine is diminazene aceturate (Berenil). The only new trypanocide to be developed in recent decades, DB75, is also a diamidine and currently in clinical trials as an orally available prodrug.
It has been known for decades that cross-resistance between melaminophenyl arsenicals and diamidine drugs sometimes occurs (Fulton and Grant, 1955; Williamson and Rollo, 1959), but cross-resistance patterns can be unpredictable (Williamson, 1970). Kaminsky and Mäser (2000) distinguished six distinct resistance profiles in Trypanosoma brucei brucei laboratory strains and field isolates, presumably an indication that multiple factors determine (the level of) resistance to the various drugs. They also noted that some resistance profiles seem to be associated with either field or laboratory-induced resistance.
Some progress has been made in understanding drug resistance mechanisms in trypanosomes. The important role of the T. brucei AT1/P2 purine transporter in the accumulation of melaminophenyl arsenical compounds and diamidines is now well understood (Carter and Fairlamb, 1993; Mäser et al., 1999; De Koning, 2001a; Delespaux and De Koning, 2007). The loss of TbAT1/P2 alone was shown to be sufficient for high levels of diminazene aceturate resistance in T. brucei brucei and Trypanosoma evansi (De Koning et al., 2004; Witola et al., 2004). However, the deletion of the TbaT1 gene produced only minor loss of sensitivity to pentamidine and melaminophenyl arsenicals (Matovu et al., 2003). Thus, the action of some diamidines relies (almost) exclusively on TbAT1/P2 for access to intracellular targets, whereas other diamidines, as well as melaminophenyl arsenicals, have at least one additional route of entry.
We have shown previously that adenosine-insensitive uptake of [3H]pentamidine in T. brucei brucei is mediated by two discrete transport activities: a low-affinity pentamidine transporter [LAPT1 (De Koning and Jarvis, 2001)] and a high-affinity pentamidine transporter [HAPT1 (De Koning, 2001b)]. Although this seems to explain why clinical pentamidine resistance has not established itself, despite the many decades of intensive use (for review, see Bray et al., 2003), it was not clear whether high levels of pentamidine resistance might ensue with the consecutive loss of TbAT1/P2 and either HAPT1 or LAPT1. Nor is it yet clear why the tbat1–/– line did not display an appreciable level of resistance to melaminophenyl arsenical drugs (Matovu et al., 2003). It has been argued that higher levels of arsenical resistance in trypanosomes could be the result of the (over)expression of an ABC-type transporter of the multidrug resistance type, TbMRPA. Overexpression of this transporter in T. brucei brucei does induce 10-fold resistance to melarsoprol in vitro (Shahi et al., 2002). Arsenical resistance attributed to loss of TbAT1/P2 and overexpression of TbMRPA were shown to be additive (Lüscher et al., 2006). However, TbMRPA overexpression did not lead to arsenical resistance in vivo, and TbMRPA overexpression could not be demonstrated in melarsoprol-resistant trypanosome isolates from patients with sleeping sickness (Alibu et al., 2006).
In the current article, we address the outstanding questions concerning diamidine and arsenical resistance using Trypanosoma brucei lines adapted to high levels of pentamidine and cross-resistant to melaminophenyl arsenicals. We now provide a model for the biochemical basis of cross-resistance between melaminophenyl arsenicals and the diamidines. The model predicts that HAPT1, together with TbAT1, may be a key determinant for arsenical resistance in African trypanosomes, providing a hypothesis that should now be validated on clinical isolates from patients refractory to melarsoprol.
Trypanosomes and Cultures. Growth of bloodstream trypanosomes in culture was performed using HMI-9 medium (BioSera Ltd., East Sussex, UK) (Hirumi and Hirumi, 1989) supplemented with 2 mM β-mercaptoethanol and 10% fetal bovine serum at 37°C, in a 5% CO2 atmosphere. For transport assays, trypanosomes were grown in adult female Wistar rats and isolated from infected blood as described previously (De Koning and Jarvis, 1997). The cloned Trypanosoma brucei gambiense type 2 stock STIB 386 and the derived melarsamine hydrochloride-resistant clone 386MelCyR (hereafter called 386Ms and 386Mr) were a kind gift of Professor C.M.R. Turner (University of Glasgow). The adaptation of 386Ms to high levels of melarsamine hydrochloride has been described elsewhere (Scott et al., 1996). The T. brucei brucei clone s427 (MiTat 1.2/221) was used to derive the tbat1–/– line (Matovu et al., 2003) and the pentamidine-adapted clonal line WT-D24. tbat1–/– was also adapted to higher levels of pentamidine resistance, generating the clonal lines KO-B6, KO-D6, KO-B48, and KO-D48. Clonal populations were generated by limiting dilution. Cell densities were assessed using a hemocytometer and phase-contrast microscope. The New York line of T. brucei brucei (Wirtz et al., 1999) and the derived MRPA overexpressing line NY-mrpa have been described previously (Lüscher et al., 2006). Overexpression was induced (NY-mrpa+) or repressed (NY-mrpa–) by the inclusion or omission, respectively, of 1 μg/ml tetracycline in the culture medium.
Transport Assays. Transport assays were performed exactly as described previously (Wallace et al., 2002). In brief, trypanosomes were isolated from blood taken from infected Wistar rats by DEAE-52 (Whatman, Maidstone, UK) anion exchange chromatography, washed into assay buffer, and resuspended at ∼2 × 108 cells/ml at room temperature. In a minority of cases, trypanosomes for transport assays were cultured in complete HMI-9 medium. Aliquots (100 μl) of bloodstream trypanosomes in assay buffer were mixed with an equal volume of buffer containing radiolabel and sometimes inhibitor, at twice the final concentration, initiating transport. Transport was terminated by the addition of 1 ml of ice-cold buffer containing unlabeled permeant at a saturating concentration (2 mM for pentamidine) followed by rapid centrifugation through an oil layer, separating cells from radiolabel not internalized. Transport values are expressed as picomoles per 107 cells per second.
Drug Sensitivity Assays. Drug sensitivity was determined using the dye resazurin (Alamar blue) exactly as described previously (Wallace et al., 2002) in a protocol adapted from Räz et al. (1997). In brief, drugs were serially diluted in 100 μl of complete HMI-9 media across a 96-well microtiter plate. Unless limited by solubility, the top drug concentration used was 1 mM. Cultures of bloodstream-form trypanosomes were grown to a maximum density of 2 × 106 cells/ml and diluted to 2 × 105 cells/ml with complete HMI-9 medium, of which 100 μl was added to all wells. Plates were then incubated for 48 h at 37°C in a 5% CO2 atmosphere, before the addition of 20 μl of 5 mM resazurin (Sigma, St. Louis, MO) solution in PBS, pH 7.4. Plates were incubated for an additional 24 h at 37°C, before fluorescence was measured using a LS 55 luminescence spectrometer (λex = 530 nm, λem = 590 nm; PerkinElmer Life and Analytical Sciences, Boston, MA).
Fluorescence Microscopy.T. brucei were pelleted (600g, 10 min at room temperature) before being resuspended in fresh HMI-9 media containing 10 μM concentrations of stilbamidine, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), DB75, or Hoechst 33342 solution at room temperature and assessed by fluorescence microscopy. Glass slides of the culture were prepared and examined using differential interference contrast (DIC) and fluorescence microscopy (λex = 365 nm; λem = 445 nm) on an Axioplan 2 imaging microscope (Carl Zeiss GmbH, Jena, Germany) using Volocity v. 3.7 software (Improvision, Coventry, UK). Where necessary, parasites were fixed by incubating in PBS containing 2.5% glutaraldehyde for 20 min. Cells were then washed in 0.05 M glycine in PBS before being mounted on slides.
Lysis Assays. The lysis assays were performed essentially as described by Fairlamb et al. (1992), using bloodstream trypanosomes of tbat1–/– and KO-B48 grown in vitro. They were washed and resuspended in fresh HMI-9 medium with 10% fetal calf serum at ∼2 × 107 cells/ml, which was transferred to 1-ml cuvettes. Absorption was monitored, at 750 nm, at 30-s intervals. Melarsen oxide or phenylarsine oxide were added after 15 min to allow the recording of a short baseline. Up to 16 cultures were monitored simultaneously, using two HP8453 spectrophotometers (Hewlett-Packard), in the presence or absence of low concentrations of pentamidine, which by themselves did not affect cell viability or motility over the course of the experiment.
Preparation of Plasma Membrane Protein-Enriched Fractions.tbat1–/– and KO-B48 plasma membrane-enriched samples were generated by a modification of the procedure described by Voorheis et al. (1979). In brief, bloodstream-form trypanosomes were cultured in vivo in adult female Wistar rats infected by i.p. injection. Blood was collected at peak parasitemia under terminal anesthesia, and parasites were isolated as for transport assays. Cells were then osmotically stressed by addition of water (at 4°C), and swelling was monitored by phase-contrast microscopy until a spherical morphology had been achieved. Cells were then homogenized in an AO Cell Disruptor (Stansted Fluid Power, Stansted, UK), in the presence of protease inhibitors. The homogenate was then returned to a normal osmotic potential through the addition of 3 M KCl to a final concentration of 140 μM, before pelleting the cells. Cells were then treated with 240 units of DNase in TES buffer (20 mM TES, 150 mM KCl, 5 mM MgCl2, and 1 mM 2-mercaptoethanol, pH 7.4), for 5 min at 20°C. The reaction was terminated by the addition of 5 volumes of TES buffer (20 mM TES, 150 mM KCl, 1 mM EDTA, and 1 mM 2-mercaptoethanol, pH 7.4). Cells were pelleted and then re-suspended in 40% sucrose before being layered on a linear 40 to 60% sucrose gradient (in TES buffer). Samples were then centrifuged for 3 h at 70,000g on a SW28 rotor (Beckman Coulter, Fullerton, CA). The most prominent dense white band, corresponding to the plasma membrane fraction, was isolated and washed twice in TES buffer to remove sucrose, before being aliquoted and stored in TES buffer at –80°C. All procedures were performed at 4°C unless otherwise stated.
Western Blotting. Samples for Western blotting were plasma membrane preparations (described under Preparation of Plasma Membrane Protein-Enriched Fractions) or whole-cell protein extracts prepared from 2 × 107 cells, washed in PBS, and flash-frozen in dry ice/ethanol. Samples solubilized in standard Laemmli buffer (60°C for 10 min) were loaded onto a 10% SDS polyacrylamide gel and run until the bromphenol blue dye front reached the end of the gel. Proteins were then transferred onto a nitrocellulose membrane (Hybond ECL; GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 4°C in MeOH Tris-glycine buffer. Blots were incubated in blocking solution (TBS-Tween 20 containing 5% milk powder) for 1 h at room temperature (RT). Primary antibodies raised to MRPA, kindly provided by Christine Clayton, University of Heidelberg, were diluted 1:500 in blocking solution (TBS-Tween 20 containing 5% milk powder) and incubated with the blot for 1.5 h at RT. Unbound/excess antibodies were removed by washing in TBS-Tween 20 (2 × 10 min) and then TBS (10 min). Secondary peroxidase-conjugated anti-mouse immunoglobulin antibodies (Dako Denmark A/S, Glostrup, Denmark) were then incubated with the blot (diluted 1:1000) in blocking solution at RT for 1 h. Unbound secondary antibodies were removed by washing (as with primary antibodies). Bound antibodies were detected using the ECL Plus western blotting detection System (GE Healthcare) according to the manufacturer's instructions. For normalization, the blot was stripped and reprobed using a polyclonal antiserum against paraflagellar rod protein (PFR; diluted 1:1000) as described previously (Schlaeppi et al., 1989) and a secondary peroxidase-conjugated anti-rat immunoglobulin antibody (diluted 1:10,000) (Dako Denmark A/S).
Induction of pentamidine resistance in T. brucei brucei. A, development of resistance over time in three independent clones. B and C, growth of KO-B48 line in the presence (○) or absence (▪) of 80 nM pentamidine in the growth medium. Frame B, shortly after passage to 80 nM pentamidine; frame C, after 2 month in 80 nM pentamidine.
Infectivity of Pentamidine-Adapted T. brucei brucei Lines in Mice. Groups of three to five ICR mice (Harlan UK Ltd., Bicester, Oxon, UK) were infected with 104 trypanosomes in 200 μl of full HMI-9/fetal calf serum culture medium by i.p. injection. In some cases, the mice had been immunosuppressed by treatment with cyclophosphamide (200 mg/kg cyclophosphamide in water, injected i.p.) 24 h before infection. Parasitemia was estimated daily by the rapid "matching" method (Herbert and Lumsden, 1976) from examinations of wet blood films on microscope slides, collected by tail prick, using a phase-contrast microscope. Mice reaching a parasitemia of antilog 8.4 (2.5 × 108 cells/ml blood) or over were euthanized by inhalation of a rising concentration of CO2.
Induction of High-Level Pentamidine Resistance in T. brucei brucei. To maximize the likelihood of the successful generation of drug-resistant lines, we applied selection pressure to four separate cultures from each of the two parental strains, s427 (WT) and the isogenic clone tbat1–/–, the latter of which has been derived from the former by deletion of both copies of the TbAT1 gene (Matovu et al., 2003). From these 8 cultures initially established, three (WT-D, KO-B, and KO-D) went on to develop a stable drug resistance phenotype after several months of selection in increasing pentamidine concentrations. Two clones were derived from each of these lines: one that tolerated intermediate levels of pentamidine (clones WT-D3, KO-B6, and KO-D6) and one that tolerated high levels of pentamidine (clones WT-D24, KO-B48, and KO-D48), compared with the parental strains (Fig. 1A). The strains displaying the highest levels of resistance were able to survive in pentamidine concentrations 80-fold higher than those tolerated by their parental strain. It was also observed that resistance acquisition was nonlinear over time and seemed to correlate best to an exponential model, with correlation coefficients between 0.87 and 0.93 (plots not shown). The wild-type strain, in particular, took many passages to acquire any pentamidine resistance but subsequently adapted to very significant levels of resistance in two discrete steps.
During each round of selection, it was observed that the initial acquisition of resistance in the presence of drug was associated with a reduced growth phenotype (Fig. 1B), in terms of both doubling time and maximum cell density. For all the lines eventually selected, this was reversed to a parental growth phenotype by maintaining the same selection pressure for an additional period of time (Fig. 1C). Once the resistance phenotype was well established, removal of pentamidine from the media had no effect on growth, and the resistance phenotype was stable for at least 3 months in the absence of drug pressure.
Cross-Resistance Profile of KO-B48 and KO-D48. We have previously reported the cross-resistance phenotype of the tbat1–/– strain (Matovu et al., 2003), which displayed high levels of resistance to diminazene but only minor loss of sensitivity to pentamidine and the melaminophenyl arsenical drugs melarsoprol, melarsen oxide, and melarsamine hydrochloride. Using the same protocol based on the reduction of the dye Alamar blue (resazurin), we have repeated these experiments with WT, tbat1–/–, KO-B48, and KO-D48 in parallel. The results are presented as a series of bar graphs in Fig. 2. The phenotypes of KO-B48 and KO-D48 were very similar. Compared with the WT strain, a large increase in pentamidine resistance was observed, reaching an average 130- and 82-fold resistance in KO-B48 and KO-D48, respectively (n = 8; Fig. 3). Resistance to melarsen oxide was also markedly increased in KO-B48 and KO-D48 (15.4- and 10.7-fold, respectively, relative to WT; Fig. 2B). Sensitivity to propamidine and stilbamidine, already reduced in the KO strain, was modestly decreased further in KO-B48 and KO-D48 (Fig. 2, C and D), whereas resistance to diminazene aceturate was not further increased in the pentamidine resistant lines (Fig. 2E). Sensitivity to phenylarsine oxide, which freely diffuses across membranes, was identical in all the lines (Fig. 2E), showing that the resistance phenotype was most likely associated with changes in drug uptake. A table with exact sensitivities of all four trypanosome lines to all six drugs is included as on-line only Supplementary Data.
Drug sensitivity profile of four T. brucei brucei clones. A, pentamidine. B, melarsen oxide. C, propamidine. D, stilbamidine. E, diminazene aceturate. F, phenylarsine oxide (PAO). Error bars indicate S.E. All values are the average of between 3 and 11 determinations. A table with the exact values and number of replicates is available as on-line only supplementary material.
Pentamidine Transport in Pentamidine-Resistant Trypanosome Lines. Transport of 0.5 μM[3H]pentamidine was drastically reduced in both KO-B48 and KO-D48, compared with the parental tbat1–/– line (Fig. 4A, B), by 74 and 68%, respectively. To exclude the possibility that culture conditions had somehow played a role in the reduced uptake rates, the experiment was repeated with tbat1–/– and KO-B48 cells grown in Wistar rats (Fig. 4C). The result clearly demonstrates that pentamidine transport in the adapted lines is severely impaired yet remains saturable (Figs. 4C and 5). We also confirmed that transport in KO-B48 was not generally impaired, using [3H]2-deoxyglucose. Uptake of [3H]2-deoxyglucose by KO and KO-B48 cells was determined in three independent experiments, each performed in triplicate, and found to be linear over 30 min, completely saturated by 10 mM 2-deoxyglucose, and not significantly different between the two trypanosome lines (Supplementary Data Fig. 1).
Pentamidine transport in the tbat1–/– strain is mediated by two transporters, HAPT1 and LAPT1 (Matovu et al., 2003). To determine whether one or both of these transporters were affected in the highly resistant line KO-B48, a series of transport assays was performed at 15 nM [3H]pentamidine. This very low concentration of radiolabel is necessary to avoid saturating HAPT1 and to obtain a biphasic inhibition curve showing both the high-affinity (but low-capacity) HAPT1 activity and the low-affinity (but high-capacity) LAPT1 activity in the WT s427 and tbat1–/– strains (De Koning, 2001b; Matovu et al., 2003). The presence of a high-affinity pentamidine transport activity could not be confirmed in KO-B48: it was either absent altogether or its transport capacity was very significantly reduced (Fig. 5A), although pentamidine transport was clearly biphasic in the control WT strain (Fig. 5B). In contrast, the low-affinity transporter LAPT1 seemed completely unchanged compared with parental strains (Fig. 5, A and C): Km and Vmax values were identical to those previously reported for the parental line s427 (Table 1).
Kinetic parameters of pentamidine transporters in various cloned trypanosome lines
Arsenical-Induced Lysis in tbat1–/– Is Mediated by HAPT1 and Absent in KO-B48. Lysis of trypanosomes, induced by arsenical drugs, can be monitored spectroscopically at 750 nm (Fairlamb et al., 1992) and was used to demonstrate that melaminophenyl arsenicals enter T. brucei brucei principally through P2 (Carter and Fairlamb, 1993). We have previously used this technique to show that in tbat1–/– cells, a secondary, slower phase of melarsen oxide-induced lysis can be discerned and displays the pharmacological profile of HAPT1 (Matovu et al., 2003). The results depicted in Fig. 6A confirm this observation. Although melaminophenyl arsenicals act more slowly on tbat1–/– than on WT cells (Matovu et al., 2003), 10 μM melarsen oxide induced cell lysis in tbat1–/– cells in approximately 50 min (trace a), but coadministration of as little as 1 μM pentamidine with 10 μM melarsen oxide substantially delayed cell lysis (trace b). In contrast, the same concentration of pentamidine had no influence on lysis induced by phenylarsine oxide (traces c and d), which rapidly diffuses across membranes (Carter and Fairlamb, 1993).
Sensitivity of four T. brucei brucei clones to pentamidine (A) and melarsen oxide (B). Lines used were WT (□), KO, (▪), KO-B48 (▴), and KO-D48 (○). The data shown is a representative experiment using the Alamar blue method as described under Materials and Methods section. IC50 values were calculated by nonlinear regression. Fluorescence is given in arbitrary units on a scale of 0 to 1000. The lowest concentration data point is the "no drug" control.
Transport of 0.5 μM[3H]pentamidine in resistant trypanosome lines. A, uptake of pentamidine by KO-B48 (○) or the parental tbat1–/– strain (▪) grown in culture in the absence of pentamidine pressure and the two strains were assayed simultaneously. Lines were calculated by linear regression. B48, r2 = 0.89, slope = 0.027 ± 0.005; tbat1–/–, r2 = 0.99, slope = 0.10 ± 0.006. B, uptake of pentamidine by KO-D48 (○) or the parental tbat1–/– strain (▪) grown in culture in the absence of pentamidine pressure and the two strains were assayed simultaneously. Lines were calculated by linear regression. D48, r2 = 0.97, slope = 0.038 ± 0.005; tbat1–/–, r2 = 0.99, slope = 0.17 ± 0.01. C, uptake of [3H]pentamidine by KO-B48 (○, •) or the parental tbat1–/– strain (▪, □) grown in Wistar rats, in the presence (open symbols) or absence (filled symbols) of 1 mM nonlabeled pentamidine. Lines were calculated by linear regression. Values in the absence of pentamidine were as follows: B48, r2 = 0.97, slope = 0.0032 ± 0.0001; tbat–/–, r2 = 0.94, slope = 0.021 ± 0.002. Pentamidine uptake is expressed as picomoles per 107 cells. Symbols represent average of triplicate determinations; error bars are S.E., when not shown fall within the symbol.
In KO-B48 cells, melarsen oxide-induced lysis is already much delayed compared with tbat1–/– cells (trace a), and 1 μM pentamidine has no further effect (trace b), demonstrating the absence of HAPT1 activity. Again, coincubation with 1 μM pentamidine had no effect on lysis induced by phenylarsine oxide (traces c and d), and the timing of phenylarsine-induced lysis was identical in both strains.
Assessment of Pentamidine Transporters Using Fluorescent Microscopy. The rate of entry of fluorescent diamidines, monitored by fluorescence-activated cell sorting or fluorescence microscopy, can be used as a probe to investigate changes in membrane permeability and cross-resistance patterns (Frommel and Balber, 1987; Stewart et al., 2005). Like Frommel and Balber (1987), we used 4′,6-diamidino-2-phenylindole dihydrochloride as a fluorescent substitute for pentamidine. This was possible given the fact that DAPI displays considerable affinity for all three pentamidine transporters. Ki values are 0.67 ± 0.12 μM (TbAT1/P2, n = 3), 26 ± 6 μM (HAPT1, n = 3), and 20 ± 6 μM (LAPT1, n = 5) (data not shown). The highest affinity was for TbAT1/P2; nonetheless, this transporter did not primarily mediate DAPI transport. Figure 7 shows that the rate of development of fluorescence was not clearly different in live long-slender WT s427 or tbat1–/– cells. By 5 min, fluorescence was clearly observed, and by 10 min, staining of both nucleus and kinetoplast was very intensive. In contrast, KO-B48 cells showed only faint fluorescence in both organelles after 20 min. Even after 60 min, fluorescence was much less than that observed in the other cell lines at 10 min (Fig. 7). Care was taken to exclude any dead cells from the observations, as they accumulated DAPI very rapidly, presumably as a result of loss of membrane integrity.
It thus seems that the rate of DAPI uptake is much reduced in KO-B48 cells, relative to the parental line tbat1–/–. To assess whether this was caused by the loss of HAPT1 or LAPT1, the development of fluorescence was again monitored in live KO-B48 cells incubated with DAPI alone or in the presence of either 10 μM pentamidine (saturates HAPT1) or 1 mM pentamidine (saturates both transporters). The results in Fig. 8A show accumulation in live cells at 25, 45, and 60 min of incubation with the fluorophore. It is clear that the lower concentration of pentamidine does not delay the rate of DAPI accumulation compared with the no-drug control, whereas 1 mM pentamidine practically abolishes DAPI fluorescence over 60 min. The P-glycoprotein inhibitor verapamil (50 μM) did not affect the accumulation rate of DAPI (Fig. 8A). Using live, motile trypanosomes does not allow the overlay of DIC and fluorescent images and causes imperfect focus on the fluorescence micrographs. At the termination of the experiment, at 60 min, cells were therefore fixed with glutaraldehyde. The higher quality DIC, fluorescence, and overlay images shown in Fig. 8B confirm the conclusions of the timed/real time experiment depicted in Fig. 8A.
Pentamidine uptake in Trypanosoma brucei brucei clone KO-B48 is mediated only by a low-affinity transporter. A, transport of 0.015 μM [3H]pentamidine in KO-B48 over 2.5 min was assayed in the presence or absence of increasing concentrations of unlabeled pentamidine. The data were fitted to a sigmoid curve using nonlinear regression. B, uptake of 0.04 μM[3H]pentamidine over 60 s in LS bloodstream forms of s427 WT was assessed in the presence of a constant concentration of 1 mM adenosine and a variable concentration of unlabeled pentamidine. The data were fitted to a "two-site competition" equation in the GraphPad Prism 4.0 software package. IC50 values were 0.13 and 85 μM for the high- and low-affinity components, respectively. C, conversion of the inhibition data in frame A to a Michaelis-Menten plot. All data shown are the average of triplicate determinations; error bars are S.E. Pentamidine uptake is expressed as picomoles per 107 cells per second.
The Drug Efflux Transporter TbMRPA Is Not Overexpressed in KO B-48. To verify whether the cross-resistance phenotype of KO B-48 can be partly explained through the elevated expression of TbMRPA, an ABC transporter of the multidrug resistance (mdr) family, plasma membrane-enriched extracts of tbat1–/– and KO B-48 were run on one-dimensional SDS polyacrylamide gels and transferred to nitrocellulose for Western blotting with anti-TbMRPA antiserum. Protein preparations from tetracycline-induced and noninduced NY-mrpa cells and parental NY trypanosomes were analyzed in parallel as control cells. MRPA was clearly present in NY-mrpa+ cells but could not be detected in any of the other lines (Fig. 9A). In contrast, the presence of PFR could easily be demonstrated in each of the preparations (Fig. 9B).
Infectivity of the KO-B48 and KO-D48 Lines. During in vivo culturing it was observed that infectivity of both KO-B48 and KO-D48 lines was much reduced compared with WT and tbat1–/– lines. To assess this systematically, groups of five mice were infected with 104T. brucei brucei organisms from tbat1–/– and each of the derived pentamidine resistant lines. In both KO-B6 and KO-B48 groups, all mice developed detectable parasitemia within 3 days, which, however, rapidly disappeared. Two mice of the KO-B48 group suffered 1 or 3 subsequent relapses, otherwise, no parasitaemia was detectable in thick blood smears taken daily. No parasitaemia was observed at all in the KO-D6 group, and only one mouse in the KO-D48 group developed a brief, low parasitaemia on day 5. In contrast, the tbat1–/– line appeared far more virulent. One group of mice was infected with tbat1–/– cells from a long-term culture. All mice rapidly developed substantial parasitaemia, and two mice had to be euthanized. In the last group, infected with the same number of tbat1–/– cells from stabilates kept in liquid nitrogen and only briefly brought back into culture, all mice developed massive parasitaemia and were euthanized (four on day 4, one on day 13). To investigate whether the lack of infectivity was innate to the adapted trypanosome lines or dependent on the immune system, groups of immunocompromised mice were infected. As shown in Fig. 10A, all mice infected with tbat1–/– cells developed similar parasitaemia and were euthanized on day 7. In the KO-B48 and KO-D48 groups (Fig. 10, B and C), all mice similarly established substantial parasitaemia; in all but one mouse in the KO-B48 group, however, these were transient and the mice apparently self-cured.
An Arsenical-Resistant T. brucei gambiense Clone Has Lost TbAT1 and HAPT1. To assess whether loss of HAPT1 activity is peculiar to selection with pentamidine, we also assessed [3H]pentamidine transport in a T. brucei gambiense type 2 clone, 386Mr, adapted for high levels of melarsamine hydrochloride resistance in vivo (Scott et al., 1996). The [3H]pentamidine transport profile of the parental line STIB 386Ms, conformed to the same three-transporter model as the earlier characterized T. brucei brucei s427 (De Koning, 2001b; Bray et al., 2003). Using fluorescence microscopy similar to the experiments shown here, it has previously been shown that 386Mr accumulates the diamidine DB99 much more slowly than the parental line (Stewart et al., 2005).
Figure 11A shows that uptake of 0.015 μM[3H]pentamidine was inhibited partly by adenosine, with a Ki value of 1.8 μM, consistent with inhibition of TbAT1/P2. A second component, assessed in the presence of 1 mM adenosine, was revealed by a dose-dependent inhibition with propamidine, which inhibits HAPT1 but not LAPT1 (De Koning, 2001b), with a Ki value of 9.0 ± 3.0 μM(n = 3). The third transport activity was evident as inhibited by high concentrations of unlabeled pentamidine but not by propamidine or adenosine, consistent with LAPT1 activity. In contrast to 386Ms, transport of [3H]pentamidine in 386Mr was not sensitive to either adenosine or propamidine (Fig. 11B), showing the absence of TbAT1 and HAPT1 activity in these cells.
The Km and Vmax values of HAPT1 and LAPT1 activities in the T. brucei gambiense lines are listed in Table 1 and are very similar to those determined in WT T. brucei brucei. Table 1 also shows that the Vmax of LAPT is not changed in the resistant T. brucei gambiense line, relative to its parental line.
Lysis of trypanosomes induced by 10 μM melarsen oxide. A, strain tbat1–/–. B, strain KO-B48. Melarsen oxide (10 μM) was added after 15 min of recording, as 50 μl [200 μM] in full HMI-9 medium added to 1 × 107 (A) or 1.5 × 107T. brucei brucei in 1 ml of full HMI-9 medium being monitored at 750 nm. Conditions from t = 0 min: a, 10 μM melarsen oxide; b, 10 μM melarsen oxide plus 1 μM pentamidine; c, 0.5 μM phenylarsine oxide; d, 0.5 μM phenylarsine oxide plus 1 μM pentamidine; e, control, no drug. Dotted lines indicate the incubations in the presence of 1 μM pentamidine.
In principle, pentamidine resistance in African trypanosomes could be associated with changes in the intracellular drug target, with reduced uptake of the drug or with active extrusion from the cell by an ABC-type efflux pump.
It has been shown that, in wild-type T. brucei brucei, pentamidine uptake is very rapid and efficient, amounting to a cytosolic concentration corresponding to at least 1 mM, if the drug were to remain unbound (Damper and Patton, 1976). Such an intracellular accumulation of the drug is liable to interfere with multiple vital processes and it has long been hypothesized that resistance would most likely be associated with a reduction in cellular levels (Wang, 1995). Indeed, different authors (Wang, 1995) have demonstrated various effects of pentamidine on cellular processes. Pentamidine resistance in T. brucei brucei based on acquired mutations in any one target, while impossible to rule out, would thus seem unlikely.
Another potential route to drug resistance is the expression of ABC transporters capable of actively removing the toxic compound from the cell. Strong evidence couples this mechanism to drug resistance in some protozoa, including Plasmodium and Leishmania spp. and Entamoeba histolytica (Légaré et al., 2001; Lopez-Camarillo et al., 2003). In T. brucei, three ABC transporter genes have been identified (Mäser and Kaminsky, 1998). However, in studies with the P-glycoprotein inhibitor verapamil, no evidence could be found for the involvement of such transporters in multidrug resistance in T. brucei brucei, either in vitro or in vivo (Kaminsky and Zweygarth, 1991). Experimental overexpression of the T. brucei brucei ABC transporter TbMRPA led to substantial melarsoprol resistance in vitro (Shahi et al., 2002) but not in vivo, and TbMRPA overexpression was not observed in melarsoprol-resistant clinical isolates from a number of patients with sleeping sickness (Alibu et al., 2006).
Thus, although resistance based on target modifications or active efflux cannot be excluded, the available information is consistent with changes to drug entry being the cause of arsenical and diamidine resistance in African trypanosomes. Our findings, using pentamidine-adapted laboratory strains, are strongly in agreement with this hypothesis. We identify HAPT1 as potentially a key drug resistance determinant and for the first time provide a biochemical model for high-level arsenical-diamidine cross-resistance in African trypanosomes.
Assessment of DAPI uptake using fluorescent microscopy over time. LS bloodstream forms of WT s427, tbat1–/– and KO-B48 were incubated with 10 μM DAPI and fluorescence was observed under an Axioplan 2 imaging microscope (λex = 365 nm, λem = 445 nm) at 1000× magnification.
DAPI fluorescence acquisition in live KO-B48 cells incubated in the absence or presence of a low (10 μM) or high (1 mM) concentration of pentamidine, or 50 μm verapamil over time (A). Separate images (DIC, DAPI, and combined overlay) from glutaraldehyde-fixed parasites after 60-min incubation with DAPI in the presence or absence of pentamidine (B). DAPI concentration was 10 μM. Magnification was 1000×. Comb, combined overlay.
High-Level Pentamidine Resistance Is Associated with Loss of HAPT1 but Not of LAPT1 Activity. Several independent lines of evidence suggested that the pentamidine-adapted lines KO-B48 and KO-D48 had lost HAPT1 rather than LAPT1 activity. Because the adaptation started with the tbat1–/– clonal line, P2 transport activity was also not present. We have shown previously that propamidine is an inhibitor of high-affinity pentamidine transport in T. brucei but not of the low-affinity transport component (De Koning, 2001b), and the increase in propamidine resistance is certainly consistent with loss of HAPT1 activity. Furthermore, there was no increased resistance to diminazene aceturate, which is transported only by TbAT1/P2 (De Koning et al., 2004), or to phenylarsine oxide, which diffuses across membranes.
The resistance profiles and [3H]pentamidine transport rates of the two adapted lines were highly similar. These experiments measured initial rates of pentamidine uptake rather than accumulation, and the results can only be explained in terms of deficient uptake rather than active extrusion or intracellular sequestration. Unlike T. brucei brucei s427 and tbat1–/– (De Koning, 2001b; Matovu et al., 2003), transport of 15 nM [3H]pentamidine in KO-B48 was not inhibited by submicromolar concentrations of unlabeled pentamidine, saturated only by very high levels of the substrate, and Km and Vmax values were identical to those reported earlier for LAPT1 (De Koning, 2001b).
In tbat1–/–, lysis induced by melarsen oxide was very considerably delayed, relative to T. brucei brucei s427, by coadministration of as little as 1 μM pentamidine—sufficient to saturate HAPT1 but not LAPT1. Pentamidine was apparently competing with the arsenical drug for transporter-mediated import, because 1 μM of pentamidine had no effect on the rapid lysis induced by phenylarsine oxide. These experiments clearly demonstrate the uptake of melarsen oxide through HAPT1. The same process was assessed by monitoring DAPI fluorescence as a marker for diamidine accumulation. Scant difference was observed between the WT and tbat1–/– lines, but the development of fluorescence was substantially delayed in KO-B48 cells (Fig. 7), indicating a loss of diamidine transport unrelated to TbAT1/P2. The remaining DAPI transport activity was saturated by 1 mM but not by 10 μM pentamidine, consistent with uptake through LAPT1 rather than HAPT1.
TbMRPA expression in various Trypanosoma brucei brucei lines. Western blots with extracts of tbat1–/– (lane 1), KO-B48 (lane 2), NY-mrpa+ (lane 3), NY-mrpa(lane 4), and NY (lane 5), using anti-TbMRPA (A) or PFR antisera (B).
The Role of HAPT1 in Accumulation of Melaminophenyl Arsenicals. Since the seminal observations of Carter and Fairlamb (1993) that a T. brucei brucei line adapted to high levels of resistance to melaminophenyl arsenicals had lost activity of a transporter they named P2, much evidence has accumulated linking TbAT1/P2 to arsenical resistance (Carter et al., 1999; Delespaux and De Koning, 2006). Loss of P2 activity alone was not sufficient to establish substantial arsenical resistance as the tbat1–/– line was less than 3-fold resistant to melarsen oxide and melarsoprol (Matovu et al., 2003). We show here that lysis of trypanosomes induced by melaminophenyl arsenicals, but not by phenylarsine oxide, was 1) sensitive to inhibition by 1 μM pentamidine and 2) much delayed in KO-B48 cells. The level of resistance to melarsen oxide reached 15-fold in KO-B48. We conclude that high levels of arsenical resistance can be induced by selection with pentamidine and can be achieved by the sequential loss of TbAT1 and HAPT1. The example with T. brucei gambiense 386Mr shows that selection with melaminophenyl arsenicals can likewise lead to loss of the same two transporters, thus explaining reported incidences of pentamidine/arsenical cross-resistance (Frommel and Balber, 1987; Kaminsky and Mäser, 2000). High levels of resistance could be far more easily induced when starting from a TbAT1-null background. This is potentially significant given the increasing levels of resistance to melarsoprol and diminazene aceturate in the field, believed to be at least partially linked to loss of TbAT1/P2 activity in the resistant strains (Kaminsky and Mäser, 2000; De Koning et al., 2004; Delespaux and De Koning, 2006).
Parasitaemia in T. brucei brucei infected mice. Progression of T. brucei brucei infections in immunocompromised mice after intraperitoneal inoculation with equal numbers of tbat1–/– (A), KO-B48 (B), and KO-D48 (C). Animals reaching a parasite burden of 108 or more were euthanized, indicated with a double bar. Each group consisted of three mice, represented by symbols ▪, ▴, and ○. PI, postinfection.
Transport of 0.015 μM[3H]pentamidine in T. brucei gambiense 386Ms (A) and 386Mr. Uptake was measured over 60 s in the presence or absence of adenosine (▾), propamidine (○), or unlabeled pentamidine (▪). Incubations with propamidine or pentamidine were performed in the presence of 1 mM adenosine to saturate the TbAT1/P2 transporter. Data shown are the average of triplicate determinations; error bars are S.E. Pentamidine uptake is expressed as picomoles per 107 cells per second.
It must be acknowledged that our findings have been generated under laboratory conditions and that any clinical link between loss of HAPT and diamidine or melarsoprol treatment failure remains theoretical. However, the previous explanation for the observed treatment failures (loss of TbAT1/P2 only) proved unsatisfactory (Matovu et al., 2003), and we here propose an improved model that can now be tested against the situation in the field. Efforts to identify the genes encoding HAPT and LAPT are ongoing and would greatly assist the evaluation of this model against clinical isolates.
It is possible that additional mechanisms of resistance to melaminophenyl arsenicals may exist, because cross-resistance is not always observed (e.g., Fairlamb et al., 1992). However, conclusions based on a review of the literature are complicated by differences in technique (e.g., in vitro or in vivo assessment of resistance), variations in drugs tested, and other confounding factors.
The Role of Drug Efflux: ABC Transporters. It has been demonstrated that overexpression of the T. brucei brucei ABC transporter MRPA resulted in a 10-fold resistance to melarsoprol in vitro (Shahi et al., 2002), an effect that was additive with the effect of TbaT1 deletion (Lüscher et al., 2006). However, we found no detectable levels of TbMRPA in the two pentamidine-adapted lines. In addition, we found that the P-glycoprotein inhibitor verapamil had no effect on DAPI accumulation, and thus we have been unable to find evidence that the increased resistance phenotype is the result of increased activity of P-glycoprotein or mdr-type ABC-transporters.
Is Pentamidine Resistance Associated with Reduced Virulence? Pentamidine is still the first-line drug for early-stage gambiense sleeping sickness. Treatment failures are rare but stable over at least three decades (Pépin and Milord, 1994). In contrast, pentamidine resistance seems to emerge easily in Leishmania species (Papadopoulou et al., 1998), though pentamidine usage against leishmaniasis has been very much less widespread than against African trypanosomiasis. The difference can be explained in part by the involvement of an mdr-type ABC transporter in pentamidine efflux from Leishmania (Coelho et al., 2003) and the fact that pentamidine uptake in Leishmania is apparently mediated by a single transporter (Basselin et al., 2002; Bray et al., 2003). The presence of three pentamidine transporters in T. brucei brucei has been argued to be responsible for the lack of clinical resistance to pentamidine (Bray et al., 2003). However, it has been demonstrated that trypanosomes with defective tbat1 genes can be isolated from sleeping sickness patients (Matovu et al., 2001), and we show here that the further loss of just HAPT1 leads to very significant levels of pentamidine resistance. The presence of multiple accumulation routes alone may thus not be sufficient to prevent the occurrence of resistance. One explanation would be loss of viability or virulence in the resistant parasites. Berger et al. (1995) reported that pentamidine adapted T. brucei brucei clone PR32.6 was far less virulent than the parental strain and excluded increased susceptibility to the host's immune system or changes attributable to prolonged culturing as factors. The phenotype of the KO-B48 and KO-D48 lines seems identical to that of PR32.6. These lines were less infective in mice and rats than the parental line, which had been cultured at least equally long, and immunosuppression of the animals did not lead to substantially higher parasitaemia. There is thus a distinct possibility that high-level pentamidine resistance is not viable in the field, and self-limiting.
We are grateful to Professor Mike Turner for the use of T. brucei gambiense strains 386Ms and 386Mr and to Professor Christine Clayton for the gift of anti-TbMRPA antiserum. We are particularly grateful to Professor Paul Voorheis (University of Dublin) for expert advice and assistance on the preparation of plasma membrane protein-enriched fractions for Western blotting.
This work was funded by the Wellcome Trust.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.106.031351.
ABBREVIATIONS: LAPT1, low-affinity pentamidine transporter; HAPT1, high-affinity pentamidine transporter; ABC, ATP-binding cassette transporter; TbAT1, Trypanosoma brucei adenosine transporter 1; MRPA, multidrug resistance protein A; TbMRPA, Trypanosoma brucei multidrug resistance protein A; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; DIC, differential interference contrast; TES, N-tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; RT, room temperature; PFR, paraflagellar rod protein; WT, wild-type.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology April 1, 2007, 71 (4) 1098-1108; DOI: https://doi.org/10.1124/mol.106.031351
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Free NBA Picks Today for Friday, February 11th, 2022
Our free NBA picks are out as the NBA season continues to roll on. Take a look at our picks for Friday, February 11th, 2022, as we preview the best five games for today.
Fri 11th February 2022
SportsTips
Our free NBA picks are out for Friday, February 11th, 2022! Make sure you check our NBA picks before the action kicks off.
Look below to find our free NBA picks for the best five games on today's schedule!
Cleveland Cavaliers @ Indiana Pacers – 7pm ET
The Cleveland Cavaliers kick off a four game road trip on Friday night when they travel to Indiana and take on the Pacers. The Cavaliers are currently on a three game winning streak, which does include a win over the Pacers, 98-85. The Pacers have lost their last four in a row, which drops their season record to 19-37.
Jeffery Thomas
The new look Packers are expected to have most of, if not all of their new toys at their disposal in their home game against the Cavs on Friday. These new pieces include Tyrese Haliburton, Buddy Hield, and former Cav Tristan Thompson. All in all they had to give up Justin Holiday, Jeremy Lamb and two-time All-Star Domantas Sabonis to get these deals done. The coaches are excited about the 3 point shooting that Haliburton and Hield will bring to the Pacers, both over 40% from range in their careers. It will be interesting to see how all their pieces mesh together for the rest of the season, and it will be a peek into what could be a bright future for the franchise. The Cavs are in a whole different mindset, as they are going for it all this year, sitting at 4th place in the Eastern Conference.
They are winners of three straight in large part due to the play of 6th year center Jarrett Allen. In those 3 games, he is averaging 19.7 points and 17.6 rebounds and this coincidentally aligns perfectly with him being left off the All-Star roster. The Cavs did have an All-Star this year, their PG Darius Garland, who returned to the lineup in the last game, a 105-92 victory over the Spurs at home, where he dropped a game-high 27 points. The Cavs have won the first two meetings this year, most recently a comeback win last Sunday, where they trailed by as much as 20. A lot has changed since that meeting with all the transactions, and the Pacers have gotten a lot younger and will be working out kinks and chemistry issues. The Cavs now have former Pacer Caris LeVert they're working in but their main core is still intact and they are the better team in this one, and it should be a pretty routine victory for them, even as the road team. Give me the Cavs to cover.
Jeffery's NBA Pick: Cleveland Cavaliers -4.5 (-110)
Michael Lantz
Just days after the Cleveland Cavaliers and Indiana Pacers made a huge trade that sent Caris LeVert to the Cavaliers, the two teams face off against each other on Friday night. In LeVert's debut with the Cavaliers on Wednesday, he only scored 11 points, but that was all the team needed from him as they cruised past the San Antonio Spurs 105-92. They'll look for their fourth straight win against a Pacers team that has its fair share of new faces as well.
𝐓𝐡𝐞 𝐇𝐨𝐦𝐞𝐭𝐨𝐰𝐧 𝐊𝐢𝐝𝐬 🌟@dariusgarland22 will be on #TeamLeBron next week at #NBAAllStar! pic.twitter.com/yKrVC7H27e
— Cleveland Cavaliers (@cavs) February 10, 2022
The Pacers lost their All-Star player Domantas Sabonis in a trade with the Sacramento Kings, but they were able to pick up two massive talents as they brought in Tyrese Haliburton and Buddy Hield. Them getting Haliburton was a bit of a shock; many believed he was the centerpiece for the Kings going forward. He's a natural point guard who in his second season already looks incredibly mature. He was a steal for the Pacers who are currently rebuilding but have some nice offensive pieces. While those will all be on display Friday, they're playing a very good Cleveland Cavaliers team who right now sit in fourth in the Eastern Conference standings. They have one of the league's best defenses, so I expect them to have no problem against this Pacers team that might need some time to mesh its new players.
Michael's NBA Pick: Cleveland Cavaliers -4.5 (-110)
Noah Dunlap
With the NBA trade deadline having passed, these are two teams that have different looks to them now. The Cleveland Cavaliers, who were already playing like one of the best teams in the East, added former Indiana Pacer Caris Levert, while the Pacers went crazy before the deadline, giving away Domantas Sabonis, but managing to pick up Tyrese Haliburton and Buddy Hield from Sacramento. We've already seen LeVert suit up for the Cavs, and he seemed to fit in extremely well with their system, and played well alongside Darius Garland, who has finally returned from a bit of an extended absence. The fact that they kept their core intact, and added another weapon in LeVert, to a team that was already competing in the East, should give Cleveland fans a lot of optimism as we head down the homestretch of the regular season.
As for Indiana, they added a few offensive pieces in Hield and Haliburton, but I'm not sure why the deal was made. The biggest issue for this Pacers squad all year has been their defense, not the offense, so getting rid of Sabonis for two primarily offensive threats just seems bizarre to me. While the notion of just playing to outscore your opponents may work in the regular season, in the postseason, defense tends to stiffen up, which is why I think the old adage of "defense wins championships" still rings true, despite an offensive boom in the league over the past few years. These moves from Indiana do nothing to fix an already atrocious defense, so with the fact that they were already struggling, I think that continues against this hugely surprising Cavaliers squad.
Noah's NBA Pick: Cleveland Cavaliers -4.5 (-110)
Oklahoma City Thunder @ Philadelphia 76ers – 7pm ET
The Oklahoma City Thunder also kick off their road trip on Friday night, and they will travel to Philadelphia to take on the 76ers. The Thunder will be looking to get back on track after losing their last three games, and this has taken their season record to 17-37. The 76ers have lost three of their last four, which includes losses to the Wizards, Mavericks and Suns.
What a day it was for the Philadelphia 76ers. After months and months of the Ben Simmons drama, they were finally able to find a trade partner to take on their disgruntled former #1 pick and it was none other than the Brooklyn Nets. In return for Ben? They get former MVP James Harden to pair up with their MVP candidate, Joel Embiid. In addition, they had to give up Seth Curry, Andre Drummond, and 2 first round picks. It is unlikely Harden will be cleared for this game against the Thunder but with Philly already sitting at 5th in the East before this move, it's going to be an interesting second half of the season as they've catapulted themselves right in the thick of title contention with this move.
The Thunder are a ways away from ever being in those kinds of discussions and are just trying to avoid a fourth consecutive loss in this road game against the Sixers. Their last time out was a 98-117 loss to the Raptors that saw their record fall to 17-37. Still very much short-handed, they are just getting all their young guys more experience and seeing what they can do for the future. Even before the James Harden move, it was going to be a tough go of it for the Thunder. I don't see him playing in this one as I mentioned though, and with the roll Joel Embiid is on, James won't be needed to take down this Thunder team. The loss of Curry and Embiid's backup will hurt their depth, but they still kept key pieces in Maxey, and Tobias Harris, and this team will still be very scary come playoff time. On Friday night, they will be scary enough to cover the spread against the Thunder as well.
Jeffery's NBA Pick: Philadelphia 76er -13 (-110)
The Philadelphia 76ers made plenty of headlines yesterday—and they didn't even step on the court. Seriously: Doc Rivers canceled the team's practice yesterday after trade rumors around their disgruntled All-Star Ben Simmons and various other players. The Sixers were able to trade Simmons, as well as Seth Curry and Andre Drummond, and boy did they get a prize back in return. The team sent those players to Brooklyn in exchange for future Hall-of-Famer James Harden, who was upset with his current situation with the Nets. While Harden will unlikely be available for this one, the 76ers definitely have a boost in confidence that a new era is underway and that they'll be able to make a run for the Eastern Conference crown.
While they wait for their new team to fully assemble, the Sixers get a pretty easy task to tide them over. They face an Oklahoma City Thunder team that's without their leading scorer Shai Gilgeous-Alexander. The Thunder have lost their last three games, all by double digits. While they face an incomplete 76ers team, they still will likely have to take on MVP candidate Joel Embiid, and high-scoring supporting players Tobias Harris and Matisse Thybulle. While the Thunder have gotten some solid production from youngsters Lu Dort and Josh Giddey, they likely won't have enough to face this very good 76ers squad. For this one, I'll go with the Sixers to cover at home.
Michael's NBA Pick: Philadelphia 76ers -13 (-110)
The Philadelphia 76ers are another team that made a move before the trade deadline, as they took part in the biggest trade of the year, a move that sent Ben Simmons, Seth Curry, Andre Drummond, and two future first-round picks, to Brooklyn, to pick up James Harden, giving him the chance to pair with Joel Embiid. While I like the fit with Harden and Embiid, I'm not a fan of what it took to make the move, as I think your depth took a huge hit, especially this year, so in my opinion, this is a Philly team that just took themselves out of the Finals race. Add on the fact that they're already struggling, having dropped three of four, and Harden won't yet be available, and I think this has the chance to be a huge upset.
The Oklahoma City Thunder aren't a good team by any means, coming in at 17-37, and losers of three straight, but they do have a few young pieces that have shown glimpses of potential. The issue for this OKC team right now is that they're still without their best player in Shai Gilgeous-Alexander, which absolutely plays a huge role in terms of their offensive production, and none of the other guys on the roster really have the talent or ability to drop 20+ point per game consistently. However, as for this one, while I do have the Sixers picking up the win at home, I think this one ends up being closer than many expect, so I'm taking OKC to at least cover the number, with a chance to steal a huge outright upset victory.
Noah's NBA Pick: Oklahoma City Thunder +13 (-110)
Denver Nuggets @ Boston Celtics – 7.30pm ET
The Denver Nuggets hit the road for a short two game trip, which includes this game in Boston to take on the Celtics, and another stop in Toronto. The Nuggets have turned around their three game losing streak, and are now on a two game winning streak, after defeating the Nets and Knicks. The Celtics roll continues on, they have won six games in a row, which takes their season record to 31-25.
This young and hungry Celtics team is hitting their stride right before the break and will be looking to keep their momentum going as they welcome in the Denver Nuggets in an NBATV televised game on Friday Night. They made a little bit of noise at the trade deadline, getting guard Derrick White from San Antonio and bringing back Daniel Theis from Houston, losing backup PG Dennis Schroeder, and Josh Richardson among others along the way. Their last time out was a dominant 126-91 win over a short-handed Brooklyn team on Tuesday, and was their fifth win of 29 or more in the last nine games. They are also tied with Milwaukee for the most wire-to-wire (never trailed in the game) wins in the whole league with 8.
This recent run has them sitting at 7th in the East and climbing. The Denver Nuggets are winners of two straight, after a three game losing streak. It was a 132-115 win over the Knicks in their last time out, a game in which they had a season-high 83 points at halftime. MVP candidate Nikola Jokic now has four straight games of at least 20 points and 10 rebounds, as the offense continues to revolve around him. They are sitting comfortably at 6th in the West as the All-Star break approaches. It's hard to go against this Celtics team with how hot they've been. It's got their fans and city buzzing and in this nationally televised game, I expect the crowd to be electric. They've been blowing teams out left and right, led by their stellar defense and I see that trend continuing this in one against a Denver team that has been up and down lately. Give me Boston and the spread.
Jeffery's NBA Pick: Boston Celtics -4 (-110)
The Boston Celtics are one of basketball's hottest teams, winning six straight games, to almost instantly take them from a disappointment to a legitimate playoff threat. Still, some question the strength of their opponents during this run. They've beaten lowly teams like the Pistons and Magic, and when they played the Heat and Nets, both teams were missing star players. So, they'll have a true test on Friday as they face a Nuggets team that's been resilient all season long. The Nuggets have been led by reigning MVP Nikola Jokic, who is playing like he's hungry to repeat that honor this season. Jokic is averaging nearly 26 points and just over 13.5 rebounds on the year, as he's led this Nuggets team that's played without Jamal Murray and Michael Porter Jr. to a six-seed.
Thank you @J_Rich1 and @yeahyeah22 🙏🏾☘ pic.twitter.com/4F3czZqSA2
— Boston Celtics (@celtics) February 11, 2022
The Nuggets are coming off back-to-back victories where they scored 120+ points, including a 132-115 win over the New York Knicks where they scored 83 first-half points. In that game they had four players score 20 or more points and almost had a fifth, as Aaron Gordon scored 19. It showed that as much as the Nuggets depend on Jokic's greatness, they also have an able and willing supporting cast as well. While the Celtics are the hotter team right now, I like the way the Nuggets often step up in these situations. They continue to grow more and more cohesive and I expect them to come together and take a win from this Celtics squad.
Michael's NBA Pick: Denver Nuggets (+168)
The Denver Nuggets come into this one as one of the most inconsistent teams in the NBA so far this season. Before a current two game winning streak, they had lost three straight, and honestly that describes a large chunk of their season. There's a bit of talent on this roster, but with a few injuries that they're facing, it isn't much, which has caused them to be too heavily reliant on Nikola Jokic to lead the way. The problem with that is, as great as he is, there's only so much he can do against some of the league's best teams.
Now they take on a Boston Celtics team that, after a rough, ugly start to the year, as well as for much of the year, has seemed to turn things around. They are now looking like one of the better teams in the East, and a team that if you aren't careful, has the talent to make a deep run in the postseason. They've won six straight, and while one of their issues early in the year was shooting, with the likes of Jayson Tatum and Jaylen Brown, they're shooting the ball a bit better, and playing better defense. As for this matchup, I'm going to take Boston to pick up win number seven in their current streak, as I think they lean heavily on a home crowd to get them over the finish line.
Noah's NBA Pick: Boston Celtics -4 (-110)
Minnesota Timberwolves @ Chicago Bulls – 8pm ET
The Minnesota Timberwolves are in game three of their four game road trip, and on Friday night they will go toe-to-toe with the Chicago Bulls. The Timberwolves had their five game winning streak snapped last time out, thanks to the Kings. The Bulls managed to snap their two game losing streak last time out, with a win against the Hornets.
DeMar DeRozan was brought in to help solidify the Bulls stance as an Eastern Conference contender and he's done that and more. Earning MVP consideration, he's averaging over 27 points along with 5 rebounds and 5 assists a game, and being selected to his fifth All-Star game. His contributions have the Bulls sitting a game back of the 1 seed Miami Heat, entering Thursday. The Bulls are coming off a 121-109 victory on the road in Charlotte, where DDR led the way with 36 points. The Timberwolves are coming off a mini 2 game road series against the Kings that they split, losing the last one 119-132. The loss snapped a five-game winning streak as the Wolves are hunting for a top 6 placement (they are 1.5 games back of the 6 seed Nuggets) to avoid the play-in tournament.
They've made the playoffs just once in the past 17 seasons but are finally putting together a solid season behind all their young talent, of Karl-Anthony Towns, Anthony Edwards, and D'Angelo Russell. The Bulls are one of the better teams in the league and the Timberwolves are up and coming trying to reach that level. These are two great offenses and it makes sense why the total is set at 240.5, and both teams thrive in the shootout environment. However, I like getting the spread here with a Wolves team that has won 9 of their past 12 meetings with the Bulls. They are coming off a bad loss to Sacramento and will be extra motivated for this and I don't see the Bulls having an answer for KAT down low. Give me the Wolves to cover.
Jeffery's NBA Pick: Minnesota Timberwolves +4 (-110)
After two tough losses to the Philadelphia 76ers and Phoenix Suns, the Chicago Bulls were able to cruise to a 121-109 win over the offensive juggernaut Charlotte Hornets. With DeMar DeRozan and Zach LaVine feeling healthy this team should be able to continue to stay atop of the Eastern Conference. On Friday they'll face the up-and-coming Minnesota Timberwolves, which also happen to be LaVine's former team. The Minnesota Timberwolves have shown they're ready to make the jump to the next level, as this team has gone from lottery team to playoff contender in two seasons. They're currently the seventh seed and are thrilled to have some outstanding young talent in Karl Anthony Towns, Anthony Edwards, and D'Angelo Russell.
But, after a five-game win streak, the team suffered a surprise defeat to the Sacramento Kings, losing 119-132, despite being at full strength. That's one the Timberwolves will want to forget, and in order to do so, they would love to have a win over one of the league's best teams in the Chicago Bulls. These two teams are neck in neck when it comes to points per game, as both sit in the league's top ten. The Bulls are the most efficient shooting team in the league, but the Timberwolves will in no way be shy of pouring it on. Especially because Chicago's defense has taken a hit since Alex Caruso, Derrick Jones Jr and Patrick Williams went down with injuries. Ultimately, I'll take the Bulls to win this one, as they're fighting to stay atop the Eastern Conference and should win a shootout against Minnesota.
Michael's NBA Pick: Chicago Bulls (-172)
The Minnesota Timberwolves are another team that is somewhat difficult to try to predict. They have one of the best cores in basketball, with the trio of D'Angelo Russell, Anthony Edwards, and Karl-Anthony Towns, but they also struggle from lack of depth. They have multiple decent win streaks on the year so far, including a five game run that was just snapped in their last game, but the problem is that they seem to struggle between these runs. If they were more consistent I think they have the chance to be a dark house out West, but with the inconsistencies, I do have my doubts.
Now they travel southeast to take on the Chicago Bulls, who are currently sitting as the two seed in the Eastern Conference, and have looked like one of the better teams all year long. They are dealing with a few injury problems, as they'll be without Alex Caruso and Lonzo Ball, but they do still have DeMar Derozan and Zach LaVine to lead the way. As for the result of this one, I like Chicago in a tight one, as I think the duo of Derozan and LaVine simply manages to barely outscore their opponents.
Noah's NBA Pick: Chicago Bulls (-172)
Orlando Magic @ Utah Jazz – 9pm ET
The final game for your Friday night viewing in the NBA will see the Orlando Magic and the Utah Jazz face off against each other. The Magic have won two of their last four, but they did get the all important win last time out, 113-95, against the Trail Blazers. The Jazz's winning streak has lifted to four games in a row, which includes wins over the Nuggets, Nets, Knicks and Warriors.
The Jazz will enter this home game against the second-worst team in the league record wise, off a statement victory over the Warriors. It was a 111-85 victory that pushed their February record to 4-0 as they try to keep their play at a high level while they await the return of their defensive stopper Rudy Gobert. The Magic began a four-game road trip in Portland, beating the Blazers who had just traded CJ McCollum 113-95.
Cole Anthony led the way with 23 points and 9 dimes, as it was a much needed confidence boost after two ugly home losses prior to that game. The Jazz will be licking their chops getting a cake matchup here against the Magic after beating down on the Warriors. Donovan Mitchell only had 14 points in the Golden State game so look for him to try and get going in this one as I expect him to exceed his points prop. I also expect the Jazz to ride their great home crowd to a blowout victory over this hapless Magic team.
Jeffery's NBA Pick: Utah Jazz -12 (-110)
After a turbulent last month that's been plagued by injuries, the Utah Jazz were able to score a big win on Wednesday night as they ran over the Golden State Warriors 111-85. Sure, the Warriors didn't have Klay Thompson or Draymond Green, but they still are a very good team without those two, so the Jazz will have to feel good about picking up that victory after a couple of weeks of injuries and disappointing losses. They'll look to carry that play over to their Friday night game, against a poor Orlando Magic squad.
For 8 years in a Jazz uniform, for breaking franchise records, for shaping a community and everything else you couldn't possibly fit on a stat sheet: Thank you, @joeingles7.
Always a Jazzman. Always part of the family ❤☕ pic.twitter.com/VS2JV2WlzB
— Utah Jazz (@utahjazz) February 10, 2022
But, you can't count the Magic out: they beat the Jazz back in November when they were in full strength. The Jazz may have Rudy Gobert out on Friday, so the Magic might have a slight chance at stealing another one from the Jazz. Especially because Orlando has looked a lot better on offense in recent weeks. They've got outstanding performances from young players including Cole Anthony and Franz Wagner, and even though they still have a long way to go, they're happy to see some early success from these young prospects. But don't think the Jazz won't remember that November defeat, and with this one in Salt Lake City and the Jazz coming off a big win, I expect them to hand the Magic a hefty loss this time around.
Michael's NBA Pick: Utah Jazz -12 (-110)
This one shouldn't be close. The Orlando Magic are one of the worst teams in the NBA, coming in at 13-43, and the only reason I don't think they're the worst is because of Detroit and Houston. They don't have a real playmaker or legitimate star that can lead the offense and just take over when needed, as I think Cole Anthony is still too young and inexperienced to be that guy, and all the way around there's just a serious problem with the roster, as they also lack significant depth on this roster.
And now they take on a Utah team that should be ready to get back in action, after completely obliterating the Golden State Warriors in their last outing. That game was somewhat surprising, seeing as they were without Rudy Gobert, who will still be sitting in this one next. This win managed to end an embarrassing stretch for Utah, where they simply couldn't get anything going, largely due to being without Gobert, Donovan Mitchell, and Joe Ingles, who has since been traded. Even with Gobert still missing, and them trading Ingles away, despite him being out with a torn ACL, I think this is a Utah team that can still roll right over the Orlando Magic, especially with Donovan Mitchell in the lineup, as he's been one of the leaders of this team for years.
Noah's NBA Pick: Utah Jazz -12 (-110)
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McConnell to meet with 9/11 first responders amid public tensions with Jon Stewart
(WASHINGTON) — Sen. Mitch McConnell is scheduled to meet with 9/11 first responders this week to discuss the renewal of the September 11th Victim Compensation Fund Act, following a public feud with comedian Jon Stewart.
McConnell's office has not confirmed the meeting, but a Ground Zero recovery worker and longtime activist, John Feal, confirmed the meeting to ABC News. Feal added that he requested the meeting, which will be held Tuesday.
"I don't think he would have just openly invited me," he told ABC News.
Earlier this month, Stewart made an emotional appeal to Congress to make the victim compensation fund permanent. With first responders and their advocates behind him, Stewart ripped Congress for failing to fully fund the program.
"They responded in five seconds, they did their jobs. With courage grace, tenacity, humility. Eighteen years later, do yours!" he shouted.
The September 11th Victim Compensation Fund, which Stewart and others have battled to protect for years, is set to run out of money in December 2020.
"These cuts are real," Feal said Monday. "Tomorrow will be my 270th trip to Washington in 15 years. I've got a chip on my shoulder. I'm seeing people get sick, friends die. Their families are going to be left in financial ruin."
Stewart has called out McConnell on several occasions for his inaction in the past regarding the bill. Stewart promised in his testimony before Congress that he and other advocates won't allow a "certain someone" in the Senate to use the program as a "political football" in spending negotiations, referring to McConnell.
McConnell retaliated on "Fox and Friends" saying he didn't know why Stewart was "bent out of shape," and denied that he was moving slowly on the issue. He also said the extension would pass when it came up for renewal.
Stewart fired back on the "The Late Show with Stephen Colbert," arguing that he was "not bent out of shape." He said he was upset that heroes were getting sick or dying and desperately needed Congress' help.
"These are the first heroes and veterans and victims of the great trillions of dollars War on Terror. And they are currently dying, suffering and in terrible need," he said. "You know you would think that would be enough for Congress to pay attention, but apparently, it's not."
Mark Sanford says beating Trump in potential primary run 'not necessarily the goal'


Youth Tour | Houston County Electric Cooperative, Inc.
Complete your sophomore, junior or senior year of high school by June 2019.
Have a parent or legal guardian who is a HCEC member.
Submit an essay and application.
Tour historical sites, including Arlington National Cemetery, Capitol Hill, the Lincoln Memorial, Mount Vernon, the Smithsonian institution, the U.S. Supreme Court, the Washington National Cathedral and much, much more!
Create lifelong friendships with students from all over Texas and the entire country!


This write up provides you some of the guidelines that are provided to be based on faculty experience. However, in the part two you will be given some more ideas.
The overall objective of project work is to experience the way in which organizations function is given environment. The keyword is experience. Experience has two dimensions.
i. Understand the industry in which the organizations operate.
iii. Understand particular functional area such as Marketing, Finance, HR, etc.
Understand how various principles you have learned during one year of course work is applied in the real time business world.
iii. Learn the changes that are taking place in the business world. One question that immediately comes to your mind. Is it possible for me?
Each of the time periods is very important.
It is requesting for a project work you will be raising certain expectations from the organizations.
Personal interview attended by you.
At all the stages, honestly, integrity, good presentation of self is very important.
Find out the name & contact number of the person to whom you have to report.
Inform in advance if possible of your arrival. Dress code of plain shirt with shoe, tie and clan shave has to be adhered.
Your first interface should project the following traits.
Show patience, organizations have to cater to all stakeholders and yours will be last as the purpose of organization is value addition. This does not mean that you are unimportant; your work comes last in the important things to be done.
The project work is an additional responsibility undertaken by organization; therefore don't expect organizations or the person in charge to respond to you immediately. Some time you may have to wait more than 3 hours or even 1 day. This doesn't mean that organization is not need of you. It only indicates that they have different priorities. However you will be observed during entire phase of waiting.
The first title you arrive at need not be the final title. You can change e as you progress during the project.
Please go through Hindu Industrial Survey 2007.Pertaining to the Industry. In which the business organization operates. You have to use your discussion carefully, if you are working in automobiles spare parts organizations do touch up on automobile Industry, However as a rule of thumb 75 to 80 of your industrial profile should be automobile spare parts industry. Visit various websites such as www.about.com. If you want to go through various research reports please go through www.ssrn.com. Be comprehensive while talking about industry and write about industry of understanding all the dimensions about industry.
Please find at lest ten major companies in your industry and in the international market. Write a brief write up on what happened for the past three years and what is in store for three years for the industry. Identify at least three; Indian CEOs and three international CEO' and find their views about industry. You can use your CMIE Data Base in the college. You can have photographs of the CEO's, being copied.
Please go through past balance sheets, organization's website, press reports and all other data pertaining to the organization. This part will help you to understand your organization in the context of present business situation. Keep a log of what ever you are doing everyday. It will help you where you are and what you are likely to achieve.
You have to explain what "Value addition" you will be doing for the organization. You are working 35 clear working days for project work. Your effort, understanding and value addition to the organization by answering specific questions will provide better experience for you.
This part is mandatory. However it will be a good idea includes information in the project work. However don't exceed five pages. If you are doing research on motivation you may include latest research on motivation as literature survey.
You have to be precise while writing your objectives and should reflect the research you are going to undertake. Restrict yourself not more than 3 objectives, however two will be ideal.
In the process of achieving you objectives you will also be undertaking certain other tasks which are important. You may include them under scope of objectives.
Research Instrument (if it is a questionnaire, you have to write what type of questionnaire. However, do not assume always that you need to have questionnaire to start the research. One of the greatest sins you will commit is to think designing questionnaire to start your research will convent your research putting cart before the horse.) Always remember that you need not have questionnaire to do research. You have different methods and use them to enrich your thought process.
Sample Size. Determine using methods thought to you in Research Methodology.
Do not write history of statistical tools what you have to write is what appropriate tools you have used and reason for using the tools please cross tabulate your data in order to further understanding. From the synopsis to your final report constantly fine tune your project report. It will be rich experience for you to cherish.
Trying to miscommunication with organizations by telling that institute wants a review. We are sending your schedule of project report to all the organizations. You do not have any reviews in between.
While taking to the organization personnel do not degrade your institute or teachers by making statements that are reduce your self image.
Talking negative and trying to be certain about everything. No organization or person likes negative talk and people who ask too may question to be certain of everything.
I want marketing project, I am in a finance organization. Do not ask the manager this question. You are going to an organization to know its business and business do not have functional silos. However, ask questions relating to your project, show enthusiasm. Smile, enjoy hard work.
If some urgent work is to be done by the organization, do it willingly. For example the office has no power and your manager finds that the electricity bill is not paid, volunteer to do the task. MBA is not management by arrogance. Never show that this is not my work. You will be surprised to know that there is no work as not your work. The organizations are boundary less organizations.
Do not take campus culture to organization. Leave letters, excuses, concessions etc. Inculcate organizations culture and come to institute with all ideas to change the way in which you work in institute. Share experience with your classmates and teachers. It is an opportunity to professionalize with you.
Wear your tie and go to organization. Some organizations have informal environment. They may not insist on your attire. That does not mean that you go with casuals.


text:
Veröffentlicht am 7. Oktober 2019 13. Oktober 2020 von kallisthos
Date built: 1935
Location: Washington, D.C.
Architect: Cass Gilbert
Built under: Chief Justice Hughes
The United States Supreme Court building also referred to as the 'Marble Palace' by John P. Frank, was designed based on the Roman temple form. The tall Corinthian columns that are reached by a grand flight of stairs gives the building a monumental entrance. The building is designed to fit into the scale of its surrounding structures. Therefore, the wings beside the central temple are lower in height. Compared to its neighbouring building the Library of Congress, The Supreme Court was designed in a subtle, more held back style, which is now termed as neoclassical revival.
At its core, the building is, in fact, a steel frame structure faced with white marble. The entire façade is approximately 300 ft, including an elaborate entablature which is a part of the central temple-like pavilion supported by a series of 16 Corinthian columns. Lower wings balance the bold central section on the side in the Ionic order. The floor plan purposefully separates the justices working areas from the public to ensure privacy and quiet. Inspired by the courtroom in the U.S. Capitol Building, Gilbert preserved much of its intimacy in the courtroom of the United States Supreme Court.
The main entrance of the building is on its west, which is facing the U.S. Capitol Building. Sculptures are an essential part of the overall design. Seated marble figurines are placed on either side of the grand entrance staircase. These statues are the work of James Earle Fraser. The pediment was also an opportunity to display such symbolic statuary.
Tourists usually don't get to see the back of the building where resides the east entrance of the Supreme Court. The words 'Justice the Guardian of Liberty' are carved on the architrave above the columns. The two entries of the building are sometimes referred to as the East and West façade. The east side has fewer columns and was designed as a 'back-door', with a single row of columns.
Harmen A. McNeil carved the sculptures present on the east side. The three great lawmakers from different civilisations, namely Moses, Confucius and Solon, were placed in the centre. Whereas, the surrounding sculptures are figures that symbolise ideas, including Means of Enforcing the Law, Carrying on Civilisation, Tampering Justice with Mercy and Settlement of Dispute Between States.
Dieser Artikel wurde unter Architektur, Marmor veröffentlicht und mit folgenden Schlagworten versehen: architektur, marmor, naturstein, usa
kallisthos
The Colosseum in Rome
Hierapolis


After hearing about this game on podcasts, I finally got it on sale. It's a good strategy game, perfect for 15-20 minute bursts of play (enough time to run a mission), and with just the right level of difficulty. Controls felt intuitive to me, and I found it just challenging enough although I'm not a very good player of strategy games. I would have liked a bit more customization, but it was entertaining enough as is. I cleared the campaign but will probably go back again.
If you're a Mac gamer frustrated that Arkham Knight is not coming to Mac in the near future, then a revisit to the game that launched the Arkham Franchise is not a bad option at all. I replayed this game all the way through earlier this year (2016) and found it still holds up. The voice acting is really top notch, the stealth and the fighting were both improved with Arkham City but still feel pretty good even by today's standards.
I bought this game after hearing about it on a podcast. I've never played Sim City or other building simulation games this was my first. If you like strategy games I think you'll find a lot to like about Cities:Skylines. It's very satisfying to watch things happen, and figure out what is going wrong and how to fix it. I needed to watch a couple of online video gameplays to get a hang of it though, but now find it's a go-to game for quiet reflection and thinking.
This was my first venture into Homeworld, and I was not disappointed. The learning curve was not steep at all, although it helps if you've played a RTS game before. It took awhile to learn how to move ships up and down in space as well as around but it wasn't that hard. There is nothing quite like seeing your fleet line up on the beautiful backdrop of some nebula or other. The campaign was satisfying and not too long. I didn't try the multiplayer.
Best music in a video game I have heard in a long time. Fits the theme perfectly.
Be careful before you buy!
This game is NOT compatible with anything over OS X 10.6. If you updated to Yosemite then you are just out of luck. I bought the game awhile ago, updated my OS and realised to my chagrin that I now can not play it.
Hopefully it will get an update to fix this, otherwise it should soon be dropped from the Macgamestore as more and more people will be using higher (non-compatible) forms of OS X.