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Fully synthetic endoperoxide antimalarials, namely, OZ277 (RBx11160; also known as arterolane) and OZ439 (artefenomel), have been approved for marketing or are currently in clinical development. We undertook an analysis of the kinetics of the in vitro responses of Plasmodium falciparum to the new ozonide antimalarials. For these studies we used a K13 mutant (artemisinin resistant) isolate from a region in Cambodia and a genetically matched (artemisinin sensitive) K13 revertant. We used a pulsed-exposure assay format to interrogate the time dependence of the response. Because the ozonides have physicochemical properties different from those of the artemisinins, assay optimization was required to ensure that the drugs were completely removed following the pulsed exposure. Like that of artemisinins, ozonide activity requires active hemoglobin degradation. Short pulses of the ozonides were less effective than short pulses of dihydroartemisinin; however, when early-ring-stage parasites were exposed to drugs for periods relevant to their in vivo exposure, the ozonide antimalarials were markedly more effective.
Plasmodium falciparum caused 200 million malaria infections and 438,000 deaths in 2014 (1). While the rate is still very high, malaria deaths have in fact dropped steadily over the last decade, as countries where malaria is endemic have adopted World Health Organization (WHO)-recommended artemisinin-based combination therapies (ACTs) (2). The clinically used derivatives of artemisinin, such as artesunate and dihydroartemisinin (DHA), clear P. falciparum infections rapidly, providing prompt therapy for both uncomplicated and severe infections (3). A disadvantage of the artemisinins is their short in vivo half-lives (∼1 h), with the consequent need for multidose treatment regimens (3) and coadministration with a longer-lived partner drug (4).
Because malaria treatment is so heavily reliant on artemisinin-based therapies, it is extremely concerning that resistance to this drug class is now evident in six Southeast Asian countries (5, 6). Resistance is associated with mutations in the β-propeller domain of a Kelch protein, K13 (PF3D7_1343700) (7). Resistance initially manifested as delayed parasite clearance, but reports of clinical failure (recrudescence of infections) are now increasing in areas with concomitant partner drug resistance (8, 9).
Another issue with the widespread application of the artemisinins is the difficulty of maintaining the supply. The parent compound is prepared by large-scale extraction from plants, and artemisinin derivatives are generated semisynthetically, with growth, harvest, and production processes taking about 18 months (10). Recent advances in production protocols (11) and heterologous production systems (12, 13) are helping to provide the >350 million artemisinin-based treatments supplied annually (14). Nonetheless, there is an urgent need for wholly synthetic endoperoxides that are as effective and as affordable as the currently used artemisinins. Preferably, these synthetic endoperoxides will show efficacy in shorter-course treatment regimens, and ideally, they will maintain activity against artemisinin-resistant strains (10).
Artemisinins have a 1,2,4-trioxane core incorporating an endoperoxide linkage that is essential for activity (15). In the early 1990s, fully synthetic symmetrical dispiro-1,2,4,5-tetraoxane compounds with promising antimalarial activity were generated (16). Further medicinal chemistry efforts revealed that the antimalarial activity is maximized when the steric environment of the peroxide bond is carefully controlled. Asymmetrical 1,2,4-trioxolanes, in which one side of the ozonide heterocycle is sterically hindered and the other is more accessible, exhibited excellent antimalarial activity along with good in vivo exposure, and they were developed for clinical use (17–20).
OZ277 (arterolane maleate; also called RBx11160) was the first synthetic ozonide to undergo clinical trials and is now marketed by Ranbaxy Pty. Ltd. in India (10, 21, 22). It has good activity against all asexual blood stages of P. falciparum; however, the half-life of OZ277 is only 2- to 3-fold longer than that of DHA, and plasma exposure is lower in malaria patients than in volunteers (23, 24). OZ439 (artefenomel) appears to be more promising, exhibiting a much longer in vivo elimination half-life (46 to 62 h) (25, 26), and is currently undergoing phase II clinical trials (10, 25). The extended exposure profile offers the possibility that it might be effective, in combination with a second agent, as a single-dose oral cure for malaria (10, 25).
As for the artemisinins, the peroxide bond is key to the antimalarial activity of the ozonides (27), consistent with the suggestion that they need to be activated by a reduced iron source in order to exert their activity. The involvement of carbon-centered radicals as the toxic species is supported by the observation that nitroxide radical spin trap compounds antagonize the activity of both artemisinins and OZ277 (28). Nonetheless, until now, it has not been demonstrated formally that the ozonides are activated via the same mechanism as that for the artemisinins, nor is it clear whether they offer improved efficacy against artemisinin-resistant parasites. We present here an analysis of the ability of ozonide antimalarials to prevent the multiplication of the laboratory strain 3D7 as well as that of a K13 mutant isolate from Cambodia and its genetically matched K13 revertant.
Culture and tight synchronization of parasites.3D7, Cam3.II, and Cam3.II_rev parasite-infected red blood cells (RBCs) were cultured in complete medium containing RPMI 1640, 25 mM HEPES, pH 7.4, 2 g/liter sodium bicarbonate, 4 mM l-glutamine, 0.2% (wt/wt) d-glucose, 22 μg/ml gentamicin, and 0.5 mM hypoxanthine and supplemented with 5% (vol/vol) human serum and 0.25% AlbuMAX II. Cultures were incubated at 37°C in 1% O2, 5% CO2, and 94% N2 (29).
To generate tightly synchronized parasites, cultures were presynchronized to a 5- to 10-h window with two sorbitol treatments, and schizonts were harvested using a 70% Percoll cushion (30) when the ring:schizont ratio was about 2:1 or when the flux of ring formation was judged to be sufficient for tight synchronization (31). The harvested schizonts were added to precultured RBCs (5% hematocrit) and incubated for 0.5 to 3 h on a shaker to maximize the generation of singly infected rings (32). Excess schizonts were removed by sorbitol treatment, and the tightly synchronized ring culture was adjusted to 1 to 2% parasitemia and incubated on a shaker to achieve the parasite age postinvasion needed for the drug pulse assay. The harvested schizonts were added to precultured RBCs (5% hematocrit) and incubated for 0.5 to 1.5 h on a shaker to maximize the generation of singly infected rings.
Drug pulse assays, interaction assays, and washing protocols.Drugs were serially diluted in complete medium, with or without 0.2% uninfected RBCs, in V-bottomed 96-well microplates. Culture was added to the drugs (0.2% final hematocrit, 1 to 2% final parasitemia) and incubated as required before washing four times with 200 μl of complete medium, with or without transferring the cultures to wells of a new plate (after the first wash). Unwashed samples containing drugs at >10× the 50% lethal concentration (LC50) (standard extended-exposure assay) acted as controls for 100% parasite killing (Ptkill). Parasitemia was also monitored in samples with no drug (Ptcontrol). Serial dilution factors were chosen in an effort to enable measurement of the minimum viability at high drug concentrations while giving good coverage of the concentration range resulting in 50% of the maximum killing effect. All assays were performed as technical duplicates.
Parasite viability was determined by measuring parasitemia in the cycle following drug treatment by flow cytometry using SYTO 61 (33). The culture medium was removed, and the cell pellets were resuspended in 20 μl SYTO 61 (2 μM in phosphate-buffered saline [PBS]), incubated for 15 min at room temperature, diluted to 200 μl, and incubated for 0.5 to 2 h before being measured. Viability was defined as the fraction of the parasite population that survived drug exposure and was able to multiply in the cycle following exposure to the drug with the same efficiency as that of the control. It is possible that some parasites enter a dormant state where they remain "viable" but unable to replicate within the period (>48 h) examined. Therefore, we used the term "viability (replication competence)" (V) to indicate that replication competence in the second cycle was measured. V was calculated using the measured parasitemias in the presence of zero drug (Ptcontrol) and in the presence of >10× the LC50 (Ptkill), as follows: V = (Pt − Ptkill)/(Ptcontrol − Ptkill).
Nonlinear regression models were fitted to the data in Microsoft Excel by using the Solver add-in (34). LC50 values correspond to the drug concentrations producing a 50% loss of parasite viability in a particular assay and were determined by fitting a simple sigmoidal function to the data.
For studies of drug activation, tightly synchronized parasites (1 to 1.5% parasitemia) were preincubated in the absence or presence of sublethal concentrations of BiPy (Sigma-Aldrich) or E64d (Sigma-Aldrich) for 1 h. Serial dilutions of DHA, OZ277, and OZ439 were prepared in separate plates and added to plates containing the preincubated parasites.
Drug stability assays.DHA, OZ277, and OZ439 (1 μM) were incubated for different times under the conditions used for standard assays in our laboratory (wells containing 200 μl RBCs at 0.2% hematocrit in V-bottomed plates) (31). The amount of drug activity remaining in the supernatant was determined by incubating serial dilutions of the supernatant with a parasite culture for >48 h under standard assay conditions before determinations of parasitemia and viability. The relative drug concentration was estimated from the supernatant dilution producing 50% viability, with reference to a parallel assay established using fresh drug.
A modified washing procedure is required to assess ozonide potency in pulsed-exposure assays.We previously examined the responses of laboratory and field strains of P. falciparum to artemisinins by using drug assays designed to mimic in vivo exposure (31, 34). In this assay format, parasites are subjected to short drug pulses, and viability (replication competence) is monitored in the next life cycle. Parasites exhibit complex drug responses that depend on their age postinvasion, their genotype, and the duration of the drug exposure (34, 35).
In this work, we examined the responses of parasites to OZ277 and OZ439 pulses of different durations. Because the physicochemical properties of the ozonides are different from those of DHA, it was important to ensure complete removal of the drugs after the drug pulse to ensure that the activity was not overestimated. That is, the wash procedure needed to be sufficiently stringent to ensure that the drug was diluted to a level well below the inhibitory level.
Our standard protocol for performing pulse assays involves incubating cultures (200 μl; 0.2% hematocrit) with drugs in V-bottomed microplates for a specified period and then removing the drug by washing the cells four times with complete culture medium (containing 5% serum and 0.25% AlbuMax II) (31). In this procedure, the infected RBCs are maintained within the same microplate wells during the course of the assay. This approach is sufficient to dilute the concentration of highly soluble drugs present in the supernatant >10,000-fold. To test the efficiency of this procedure in removing the ozonides, suspensions of uninfected RBCs (0.1% hematocrit) were incubated for 3 h in the presence of drugs at concentrations of up to 2 μM, and the RBCs were washed using the standard protocol. In order to detect any drug activity remaining in the wells, we added aliquots of RBCs infected with the laboratory parasite 3D7 (2 to 4% parasitemia) to the washed uninfected RBCs and measured the parasitemia after culture for >48 h to allow the parasites to enter the next parasite cycle.
We found that DHA at concentrations of up to 2 μM was efficiently removed by the washing procedure, such that the parasitemia was unaffected (Fig. 1A, top panel). In contrast, wells incubated with ≥500 nM ozonides retained significant drug activity after the standard washing protocol, producing a reduction in the parasitemia during the postwash incubation period (Fig. 1A). For OZ439, 50% of parasites were rendered nonviable when the initial added concentration was between 500 and 1,000 nM. Given that the LC50 for exposure times of >48 h (LC50_ >48 h) for OZ439 is ∼9 nM (Table 1), this suggests that ∼1% of the added OZ439 remained associated with the well. In contrast, when we modified the washing procedure so that the uninfected RBCs were transferred to fresh microplate wells after the first wash, there was little or no drug activity remaining (Fig. 1A). These results indicate that transferring the samples to fresh wells substantially improves the efficiency of washing.
Inefficient drug removal compromises the accuracy of determinations of drug potency, particularly under conditions where the concentration producing a 50% loss of viability is high, as in the case of the very early ring stage of artemisinin-resistant parasites from the field. To test this, we examined different washing protocols by using a laboratory-adapted field isolate from Cambodia (Cam3.II; K13 mutant) that was previously shown to exhibit reduced sensitivity to DHA (35, 36). We also employed a reverted transfectant in the same line (Cam3.II_rev), in which the K13 wild-type genotype has been restored (36).
We added drugs at the least sensitive (very early ring) stage of the K13 mutant for a period of 6 h, washed the cultures, and examined viability after >48 h (Fig. 1B, top panels). The dose-response profile obtained with DHA was unaffected by the washing protocol, with both the standard and enhanced washing methods associated with residual viability values of about 30% at physiologically relevant drug doses (∼700 nM) (Fig. 1B, top left panel; see Table S1 in the supplemental material). This failure to render all of the parasites nonviable, even at high drug concentrations, confirms the resistance phenotype, while the overlapping profiles show that DHA is efficiently diluted using the standard washing protocol. In contrast, the loss of viability following pulsed exposure to the ozonides was substantively dependent on the washing regimen. The standard washing regimen resulted in an almost complete loss of parasite viability (>95%) by OZ439 and OZ277 at the highest concentrations examined (Fig. 1B, top panels). In contrast, the residual viability was much higher when the transfer washing regimen was employed (∼40% residual viability) (Fig. 1B, top panels). The problem was less obvious for the sensitive very early ring stage of the K13 revertant (Cam3.11_rev) (Fig. 1B, bottom panels), because the LC50 values were much lower and inefficient washing was thus more difficult to detect.
Because OZ439 is lipophilic, there is the possibility that a substantial fraction of the added drug is associated with cell membranes and the walls of the wells during the initial incubation period. This would mean that the added concentration of ozonides would not reflect the concentration in the medium, which would influence the ability to compare the in vitro efficacies of different drugs and to extrapolate to an in vivo setting. We were therefore interested in determining whether OZ439 activity in the culture supernatant was significantly decreased as a result of binding to surfaces. To estimate the proportion of the drug activity that remains in the supernatant, we mixed different concentrations of OZ439 with uninfected RBCs in wells, recovered the supernatants, and serially diluted them in new wells, with the addition of fresh 3D7-infected RBCs (0.2% hematocrit, 1 to 2% parasitemia). The drug activities remaining in the supernatant, determined with reference to equivalent freshly prepared samples of OZ439, were found to be ∼90% of the added concentration over a broad concentration range (see Fig. S1 in the supplemental material). This demonstrates that under the conditions of our assays, ∼90% of the added drug is retained in the culture supernatant.
Another possible source of error in estimates of drug efficacy may arise due to degradation of the endoperoxide during the incubation pulse. To estimate degradation losses, we incubated drugs (1 μM) with uninfected RBCs under our culture conditions for different periods before serially diluting the supernatants, with the addition of fresh infected RBCs, and examining parasite viability after >48 h. The effective drug activity remaining was determined with reference to freshly prepared drugs by determining the drug dilution required to render 50% of the parasites nonviable. DHA lost activity with a half-life of ∼8 h (Fig. 2). OZ277 lost activity more slowly, with a half-life of ∼17 h. In contrast, OZ439 was stable under culture conditions, with a half-life of ≫48 h and <20% loss after 48 h (Fig. 2). The observed difference in the stabilities of OZ277 and OZ439 is broadly consistent with previously reported differences in stability in blood (19).
Exposure time dependence of the response of parasites to ozonides.We initially compared the activities of the ozonides and DHA against the K13 mutant and revertant parasites and against the laboratory strain 3D7 by using a "standard" assay format where ring-stage parasites (∼20-h synchronization window) are left in contact with drug for more than one parasite life cycle (>48 h) before assessment of parasitemia. With extended exposure, the parasites exhibited very similar LC50 values for all three drugs (1 to 9 nM) (Table 1 and Fig. 3), in agreement with previous work (17, 19). However, we previously showed that drug activity determined using a >48-h exposure is a poor predictor of the in vivo efficacy of artemisinins, as these drugs exhibit different (usually much shorter) exposure times in vivo (34, 35). We therefore used our improved washing protocols to compare the exposure time dependence of the parasite responses, focusing on the stages where parasites exhibit relatively low sensitivity.
We found that the very early ring stage of the Cam3.II strain showed markedly decreased sensitivity to a 3-h pulse of DHA compared with the Cam3.II_rev strain (Fig. 3A, left panels; see Table S1 in the supplemental material), as reported previously for other K13 mutant strains (35). In particular, >50% of Cam3.II parasites survived a 3-h 700 nM DHA pulse, compared to ∼5% of Cam3.II_rev parasites. Similarly, the K13 mutant exhibited significantly lower sensitivities to 3-h and 6-h pulses of OZ277 and OZ439 (Fig. 3, top panels; see Table S1). It is important that the decreased efficacy (in a 6-h exposure) was less evident when the standard (no transfer) washing procedure was employed (Fig. 1). This emphasizes the need for particular care in pulse assays of the efficacy of ozonides.
We also examined the mid-ring stage of 3D7 parasites, which we previously showed to exhibit relatively low sensitivity to artemisinins (34). In agreement with our previous report, the mid-ring stage showed low sensitivity to a 1.5-h pulse exposure to DHA (LC50_3 h, 180 nM) but exhibited a marked increase in sensitivity at longer exposure times (see Fig. S2 in the supplemental material). We found that mid-ring-stage 3D7 parasites exhibited an even lower sensitivity to 1.5-h pulsed exposures to OZ277 and OZ439 (LC50_1.5 h > 1,000 nM). Again, the parasites became sensitive to the ozonides as the exposure time was increased (see Fig. S2). When treatment was initiated at the trophozoite stage, the exposure time dependence of drug action was less dramatic but was still apparent (see Fig. S2).
The complex dependence of drug activity on different exposure factors makes it difficult to directly quantitate or compare the activities of different endoperoxides. We previously developed a cumulative effective dose (CED) model that is able to describe the complex concentration and exposure time dependence of artemisinin-mediated loss of viability and that facilitates comparisons of different drugs and their effects on different strains (34). According to this model, the extent to which a particular endoperoxide renders parasites nonviable (replication incompetent) is dependent on its "effective dose" (likely representing the amount of activated endoperoxide) rather than the administered concentration (37). The effective dose is a saturable function of the administered drug concentration, with the parameter KM representing the drug concentration that produces half the maximum effect (34). The minimum time required to render 50% of the parasites nonviable at saturating drug concentrations (t^50) and the slope of the sigmoidal function that relates the effective dose to the parasite viability (γ) are also determined through application of the CED model to viability data. In performing this analysis, we do not explicitly take into account the changing drug concentrations over time (as revealed in Fig. 2), as the CED model can characterize only the cumulative effect on viability over an entire drug pulse, independently of any detailed changes in kinetics during that drug pulse. In the case of OZ277 and OZ439, this does not have a major influence on the analysis (i.e., parameter estimation), as the experimental observations occur over a period (9 h) much shorter than the in vitro half-lives of the drugs (Fig. 2). In the case of DHA (with a half-life of 8 h), drug instability will lead to an overestimation of KM values and will also influence the underlying slope of the dose-response curve (γ) but will not affect the magnitude of t^50.
The CED model is able to describe the observed dependence of drug activity against early-ring-stage Cam3.II and Cam3.II_rev parasites on drug concentration and exposure time (Fig. 3, fitted solid lines [these provide good fits to the 3-, 6-, and 9-h exposure data]). Analysis of the fit parameters showed that the KM value is essentially independent of the strain but shows a more pronounced dependence on the identity of the drug (Table 2). It is interesting that the average KM value obtained for DHA treatment of the K13 revertant and mutant strains used in this work (mean ± standard error of the mean [SEM], 15 ± 2; n = 4; values were obtained by using the combined data for Cam3.II_rev and Cam3.II from Table 2) is similar to values reported earlier for another pair of wild-type and K13 mutant strains (14 and 19 nM for PL2 and PL7, respectively [35]). In contrast, the combined KM value for OZ439 (60 ± 20; n = 4) is markedly greater than that for DHA, while the value for OZ277 (12 ± 7; n = 4) is similar to that for DHA. Although the KM values determined in this analysis are macroscopic constants and are influenced by the different chemical properties of the drugs (including different levels of binding to proteins), the observed trends are broadly in agreement with their relative abilities to be activated by iron (38).
The CED analysis shows that sensitivity to endoperoxides is largely determined by the t^50 value (34). The use of isogenic lines in the present study revealed that the K13 mutation (R539T) increased the t^50 value for DHA from ∼1.3 h to ∼3.2 h (Table 2), a trend in general agreement with that previously reported for wild-type (PL2) and K13 mutant (PL7) field strains (values of 1.4 and 4.9 h, respectively [35]). Interestingly, early-ring-stage K13 revertant parasites exhibited similar t^50 values when exposed to either OZ277 or DHA (Table 2). K13 mutant parasites exhibited larger t^50 values, but again they were similar for OZ277 and DHA. OZ439 also showed an increased t^50 value for the K13 mutant; however, in this case, the times required to render 50% of parasites nonviable (t^50 values) were 1 to 2 h longer for both strains than the value for DHA.
Simulation of in vivo parasite responses.To consider the possible clinical implications of our findings, we modeled parasite responses to drug exposure at levels that might be expected to be achieved in patients. The different in vivo pharmacokinetic properties of the drugs, as well as their different pharmacodynamic behaviors, preclude a simple comparison using the observed in vitro concentration-response profiles shown in Fig. 3. Moreover, traditional approaches for assessing drug efficacy in vivo, such as measuring peak concentrations and areas under the curve (AUC), are inappropriate for drugs, such as the artemisinins, which exhibit short in vivo half-lives and lag times in their action.
In the present study, we investigated how efficiently a single dose of drug might reduce the parasite burden in an infection comprised of early-ring-stage parasites. We chose early rings because this is the stage at which K13 mutants exhibit the highest ability to survive drug treatment (7, 39). For this simulation, we took advantage of the fact that the reported pharmacokinetics of each drug (summarized in Table S2 in the supplemental material) indicate that saturating concentrations of all the drugs are achieved relatively quickly. That is, the time required (following drug administration) for drug concentrations to reach KM values is typically less than 20 min, and these saturating levels are maintained for many hours. As a result, the CED model predicts that the viability (V) will vary as a function of drug exposure time (te) according to the equation V(te) = [1 + (te/t^50)γ]−1 while these saturating conditions are maintained. That is, early killing events will be independent of the precise pharmacological concentration. In contrast, viability at longer times will depend on both the administered drug dose and the drug pharmacokinetics (i.e., how long it takes for the drug to decay to sublethal levels). In this work, we used the CED model parameters that describe the response of early rings to exposure times of up to 9 h (Table 2) and incorporated knowledge of the pharmacokinetic profile for each drug (see Table S2) (24, 25, 40) in order to generate simulations of parasite viability in vivo over the first 9 h following a single-dose exposure (Fig. 4).
One consequence of the fact that saturating drug concentrations are achieved rapidly and sustained for several hours in vivo is that the times required to achieve a 50% reduction in viability of early rings are well approximated by the t^50 values shown in Table 2. Thus, the CED model analysis indicates that the much slower reduction of parasitemia observed for resistant strains in vitro will be reflected in vivo. The simulations show that a single-dose exposure to DHA would reduce the parasite load to a much smaller degree in the K13 mutant than in the revertant strain, leading to an ∼20-fold greater parasite burden ∼7 h after treatment (Fig. 4, top panel). There is no further substantial parasite killing at times longer than this, as the in vivo DHA concentrations become sublethal. As a result, a substantial fraction of the early ring population of the resistant Cam3.II strain (∼20%) (Fig. 4, top panel) is able to survive a single-dose DHA exposure, which helps to explain the in vivo resistance phenotype. The simulations also show that, as with the trends observed in vitro, the times taken to reduce the parasite load by 50% are similar for OZ277 and DHA treatments but somewhat longer for OZ439 treatment (up to ∼5 h for resistant strains). In the case of a short-lived drug, such as DHA (∼1-h half-life), such an increase in the time taken to kill 50% of parasites would present a major disadvantage, further increasing the risk of treatment failure in a resistant infection. However, the much longer in vivo half-life of OZ439 (see Table S2) means that the in vivo drug concentration is maintained for >72 h at a level much greater than the LC50 value observed in a standard kill assay (Table 1). This means that unlike the situation for DHA, where the drug is essentially depleted after 9 h, OZ439 will continue to efficiently kill both sensitive and resistant parasites well beyond 9 h, with anticipated activity for more than 72 h. (This is evident from Fig. 3, where a comparison is made of the loss of viability for parasites exposed to OZ439 for >48 h [black symbols] and the data for parasites exposed to DHA for 3 h [blue symbols] and 6 h [red symbols].) Thus, our analysis suggests that a single-dose exposure to OZ439 will reduce the parasite load in a resistant infection much more efficiently than a single dose of DHA.
The activities of DHA and the ozonides are antagonized by a hemoglobinase inhibitor.The activation of artemisinins via a reduced iron source is integral to their mode of action (34, 41). Recent work indicates that in the case of DHA, this iron source is heme generated during hemoglobin digestion, even when artemisinins are applied at the very early ring stage (34, 37). Accordingly, the falcipain inhibitor E64d exhibited significant antagonism of the action of a 3-h pulse of DHA against K13 revertant (Cam3.II_rev) parasites (Fig. 5). Like that of DHA, the activity of the ozonides was strongly antagonized by E64d (Fig. 5), indicating that their activity is dependent on hemoglobin degradation. In contrast, the iron chelator BiPy slightly synergized the activities of DHA and the ozonides, indicating that free iron makes little contribution to activation of the endoperoxides in very early rings (Fig. 5). Similar interactions were observed when E64d or BiPy was used in combination with a 6-h pulse of DHA or the ozonides against mid-ring-stage 3D7 parasites (see Fig. S3 in the supplemental material).
In this work, we used a pulsed exposure assay to examine the activity of ozonides at different parasite stages. To achieve reliable estimates of efficacy, it was essential to ensure that the drugs were efficiently removed by the washing procedures employed at the end of the exposure period. We found that significant levels of ozonides remained in the culture wells when a standard washing procedure was employed. This was particularly true for OZ439, the most lipophilic of the three compounds tested. While the adherent fraction is likely quite low (on the order of 1% at micromolar concentrations), killing is caused by the continued exposure of the culture to the remaining drug for the duration of the incubation (>48 h). This can result in overestimation of the efficacy, particularly for resistant strains subjected to short exposures, which need a high level of drug to effect killing. This is important because it could result in the mischaracterization of resistant parasites as sensitive.
Artemisinins are activated via a reduced iron source generated during hemoglobin digestion (34, 37). Recent work (42) confirmed earlier studies (43) suggesting that artemisinins exert their activity by alkylating multiple targets within the parasite. Like the artemisinins, the ozonide antimalarials harbor an endoperoxide bridge; however, it is still unclear whether they are activated in the same way and are subject to the same mechanisms of resistance.
In this work, we showed that, as for DHA, short pulses of ozonides are more active against the trophozoite stage than against the mid-ring stage of 3D7 parasites, consistent with an enhanced hemoglobin degradation flux. Moreover, we found that falcipain inhibitors markedly antagonized the activity of the ozonides, as would be anticipated upon disruption of the supply of heme. In contrast, the iron chelator BiPy had very little effect under the conditions of our experiments, indicating that, like artemisinins (37), the ozonides are dependent on hemoglobin-derived heme rather than free iron for activation.
There is evidence that the parasite mounts a proteostasis response that counteracts the artemisinin-induced insult and that killing is achieved only when this defense system is overwhelmed (35). As a result, the extent of killing is highly dependent on the duration of drug exposure (34, 35, 37). In this work, we found that the killing of parasites by both DHA and the ozonides is highly nonlinear with respect to exposure time. For example, mid-ring-stage 3D7 parasites are insensitive to a short (3 h) exposure to drugs, but exposure to double that time (6 h) results in 100% killing, with an LC50 value that is not much higher than that observed for a 9-h pulse.
We demonstrated previously that in order for artemisinins to render parasites nonviable, the parasites need to be exposed to a sufficient concentration of activated endoperoxide for a sufficient time (34). This manifests as a delay following the application of endoperoxides before 50% of parasites are rendered nonviable. This delay is stage and strain dependent. The CED model predicts that killing will be more efficient if the endoperoxide is more efficiently activated (e.g., due to a higher level of activator or a more readily activated endoperoxide) (37). This is responsible for the particular sensitivity of trophozoite-stage parasites to endoperoxides (34).
The extent of killing will also be determined in part by stage- and strain-dependent differences in the ability of the parasite to deal with damaged proteins (34, 35, 37). This is expected to manifest as a change in the t^50 (time required to render 50% of parasites nonviable at a saturating drug concentration). For example, the very early rings of strains with wild-type K13 are particularly sensitive to DHA, likely because their cellular defense systems are particularly weak (35, 44). Similarly, the early rings of K13 mutant strains are less sensitive to DHA than those of wild-type strains, likely because K13 mutants exhibit an enhanced cellular defense system (35, 44). In this work, we showed that the differential sensitivity of different stages and strains to DHA was matched by similar differences in sensitivity to the ozonides, consistent with the suggestion that the basic mechanism of action of ozonides is similar to that of DHA and that they are prone to the same resistance mechanisms.
We found that the KM and t^50 values varied substantively depending on the chemical features of the endoperoxide, presumably due to differences in the efficiency of activation and/or the toxicity of the activated species. Access to the peroxide bond is more sterically restricted in OZ439 than in DHA (17, 18). We found that OZ439 exhibits moderately larger t^50 values than those of DHA, leading to lower levels of parasite inactivation when the exposure time is short. One possible interpretation is that the higher stability of OZ439 results in slower heme-mediated activation in the parasite, requiring a longer exposure time to render parasites nonviable. It is also possible that differences in the physicochemical properties of OZ439 may slow the rate of accumulation of the drug in the parasite or lead to different protein target interactions. A combination of these factors may be responsible for the requirement for a slightly longer exposure time to render 50% of parasites nonviable.
In an effort to understand the potential in vivo consequence of the in vitro behavior of the ozonides, we used our estimates of drug pharmacodynamics (CED model parameters) to predict parasite behavior in a patient. Parasite viability was simulated for the first 9 h following a single drug dose administered at the very early ring stage to patients harboring wild-type or K13 mutant parasites. We did not attempt to extend the simulation beyond 9 h because stage-dependent differences in sensitivity complicate predictions of the behavior of surviving parasites at longer time points (35). We employed the available pharmacokinetic profiles for the different drugs in patients or volunteers to represent the drug concentrations likely experienced by the parasites. Our simulations indicate that following drug exposure, reduction of parasite numbers to 50% of initial levels may take 1 to 2 h longer when OZ439 is employed than when DHA is used. Indeed, in a retrospective analysis of phase II clinical data, OZ439 was assessed for efficacy in patients with K13 mutant and wild-type parasites (25). In that study, a trend toward an extension of the clearance half-life was observed for patients infected with K13 mutant compared with wild-type parasites (5.5 h versus 4.4 h), although the results were not statistically significant.
Despite the predicted lower initial levels of killing of very early rings, the longer in vivo half-life of OZ439 means that it will perform much more effectively than OZ277 or DHA at reducing parasite burdens in clinical infections, particularly in the case of K13 mutant parasites. Because the in vivo concentrations of OZ439 are maintained well above the LC50_ >48 h value for more than a full life cycle, K13 mutant parasites that survive exposure at the very early ring stage will continue to be exposed to OZ439 in the more sensitive trophozoite stage. Thus, our work supports clinical trials that are under way to assess OZ439 for use (in combination with an effective partner drug) as a single-dose treatment and for multidose treatment of infections in areas with a high penetrance of K13 mutations.
Clearly, there are a number of factors that complicate comparisons of the activities of different drugs in vitro, as well as attempts to extrapolate from in vitro to in vivo behavior. In particular, the differences in physicochemical properties between the three compounds suggest likely differences in protein binding both in the in vitro culture medium and in vivo in blood and plasma, leading to differences in unbound concentrations for the same total drug concentration. Since it is generally assumed that only unbound drug is available for permeating cell membranes and interacting with biological targets (45), these differences would be expected to affect the in vitro and in vivo activities. It also remains to be determined whether the ozonides induce dormancy in a subpopulation of parasites, as has been reported for the artemisinins (46, 47). Given these caveats, it will be critical to validate the predictions from our simulation by using detailed measurements of parasite killing and clearance times in vivo.
In summary, our data provide information about the mode of activation of the ozonides and show that a parasite harboring a K13 mutation exhibits reduced sensitivity to the ozonides. Despite this reduced sensitivity, the data strongly suggest that the longer half-life of OZ439 will result in significantly improved efficacy against K13 mutant parasites and that OZ439 will prove valuable in global efforts to battle artemisinin-resistant malaria. We anticipate that predictions of in vivo efficacy provided by our CED model will help to inform decisions about clinical use of the new endoperoxides.
We thank Shannon Kenny for technical assistance. We thank David Fidock, Columbia University, for providing Cam3.II and Cam3.II_rev parasites. We thank the Medicines for Malaria Venture for providing samples of OZ277 and OZ439.
- Received 13 March 2016.
- Returned for modification 28 March 2016.
- Accepted 3 May 2016.
- Accepted manuscript posted online 9 May 2016.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.00574-16.
- Copyright © 2016 Yang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.


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It would seem T-Mobile's TV-based streaming solution for mobile should now not be expected to arrive until sometime in 2019. This is based on comments provided by T-Mobile's President & Chief Operating Officer, Mike Sievert who specifically stated T-Mobile "next year will be expanding into mobile" when responding to a question about the company's general TV ambitions. The comments along with the updated time-frame were provided during the company's latest quarterly earnings call – following the announcement T-Mobile had another great quarter during Q3, 2018 which saw 1.6 million net customer additions and a record low level of churn.
Although this seemingly confirms 2019 as when mobile TV from T-Mobile will be announced, that's not to say a new TV solution in general won't arrive sooner as Sievert also confirmed the company's TV plans "will start later this year." A point which when taken in context with the next year sentiment does highly indicate the new TV solution will first arrive as a home-based solution. In other words, a fixed wireless solution.
Background: The understanding T-Mobile is working on a new TV service started to gain significant traction when the company announced earlier in 2018 that it had acquired Layer3 TV. Since then, it's been more a matter of when rather than if T-Mobile would announce a new and refreshed TV solution to take on the existing status quo. Although the details on the service have remained somewhat elusive since the first announcement with only snippets of information becoming available infrequently. Previously, it has been thought T-Mobile wanted to offer more of a mobile-focused experience although the comments made this week seem to suggest otherwise and instead highlights how mobile will simply be one of the many arms of a TV service that's offered. Not necessarily the main one.
What has become increasingly clear recently and following the release of new FCC filings which seemed to suggest the new TV service might only become available to T-Mobile customers, is that the service in general will be a wireless-first service. This point was again reiterated multiple times during the earnings call with Sievert describing the upcoming service as one that's "free from having to have wires" as well as being "free from a particular cable box." Also of note, Sievert stated the service will connect to a user's "social, digital and mobile life in a way that TV has never been before." All of these comments when taking together do seem to further suggest that in spite of the mobile implementation arriving at a later date, the service will launch primarily as a web and/or app-based service.
On a more general point, Sievert also made multiple references to 5G making the case this is one of the reasons the service has taken as long as it has in arriving, and also how the service will operate and connect across different platforms and devices. With the suggestion the T-Mobile service most certainly will be a 5G-focused one. On that point, there was also the suggestion made that T-Mobile is more concerned with announcing and/or launching a nationwide 5G (and presumably TV) service than opening up availability in a limited capacity and on a select city-by-city basis – in the way in which AT&T and Verizon have been doing recently with their respective 5G solutions.
Impact: The comments made this week, while acting as some form of an official stance, were not made as a proper announcement and therefore should be taken more in terms of an unofficial update than any clear indication of when or where T-Mobile is rolling out a TV service or its 5G coverage. However, if taken as intended by the company, the comments do paint the picture that T-Mobile's TV solution will be a more robust solution which caters to all types of users and not just those who are mobile. In reality, this point might not be as surprising as it first sounds as the groundwork for the home side of the TV service was largely already put in place by Layer3 TV. What's likely to be the more pertinent question is how much will the new service offer compared to the previous Layer3 solution. Especially considering Layer3 is already being offered by T-Mobile to customers in select regions in the US at a discounted rate. That already available version is largely understood to be the same service in general as what was being offered prior to the acquisition. If nothing does change much on that end, then it could be the case it's only really the mobile side that is being padded out in a new direction. That, or the company will actually launch two different TV-based packages, one which is wireless in nature and spanning home and mobile, as well as more traditional solution akin to what Layer3 has and still is offering.
However and whenever it does arrive, it still remains completely unclear what the price point will be, or how robust the service will be in terms of what additional features will be included or for that matter what channels will lead the package(s). The only comments of note during the earnings call on the content point was when Sievert stated the service will offer "hundreds of high-definition choices." It also remains to be seen how well it will compete in the market in general, as while T-Mobile has done well to climb the cellular rankings in the last couple of years and closing the gap between itself and AT&T and Verizon, the TV market is likely to be a very different hurdle for T-Mobile. Not only is the streaming market still an emerging market but there is already an abundance of choices available from companies and brands that have cemented their position as leaders in the industry and most importantly, the ones who have in effect decided the pricing that newcomers like T-Mobile will have to compete with. This includes AT&T who already has two different streaming options available targeting different price points, and who also happens to be developing yet another streaming solution.


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Literally one of the least interesting places I have ever been. It had little to offer and little of interest. I suppose I had some adventures with my cab driver though. He insisted on taking me out for a quick dinner before dropping me off at my hotel. And when I refused, he simply went to the restaurant anyway and ordered us food. He was about one standard deviation cuter than your average Chinese male, but I was still rather hesitant taking a meal with a strange dude I had just met. Absolutely beguiled by such service, I saved his number and promised to have him take me to the airport on my return flight.
Back to why I was in this frozen, wintry, underdeveloped dump of a Chinese city (I'm spoiled by Beijing, I know). We were doing a training camp for about 30 Chinese girls who were interested in going abroad to the States. They were a cute and friendly bunch, and we managed to get a lot of excitement and participation out of them. And I can't say I did too bad for my first outing; I was subject to torturous hours posing for pictures with each recruit. Their English levels varied dramatically, from bad to worse, from mediocre to ridiculously excellent. I got to hone some of my non-existent acting skills — we did skits demonstrating several problem scenarios — which were very well received.
Would I come here again? Not if I were paying my own way. However, I am quite looking forward to my up-coming business trips. I'll be in Guangzhou in late April doing a longer training camp for a bunch of foreigners, and in Qingdao in May for our quarterly meeting. Nonstop, nonstop!
Location: Changchun, China


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George Washington Research Paper - Term Paper
Apr 25, · Like George Washington, Grant was a President who earned his political capital through his storied military career. And so, in a odd way, the meeting between Ulysses S. Grant and Prince George Washington was an anachronistic encounter between two of America's most celebrated heroes.5/5. Established in at the University of Virginia, The Washington Papers is working to publish comprehensive letterpress and digital editions of the Washington family's correspondence. Search across the papers of the American founders, including George Washington, with this free-to-use database. Rotunda. Creating the American Nation George Washington, the commander in chief of the American Revolutionary Army, was the ever practical military leader, president of the Federal Constitutional Convention, and first president of the United States. The collections of this famous former British colonial reflect his public career and his personal interests.
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When the war was concluded, george washington research paper, he left military service and returned to Virginia to concentrate on domestic responsibilities. He focused on his farm and the tobacco market. His political and military abilities led him to be elected as a representative to the First, and more importantly, the Second Continental Congress.
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The Gear Sling is a monosling bag built specifically to keep your digital devices in check while you're out there enjoying the analog world. It has a dedicated compartment for your iPad, tablet or eReader, and its padded shoulder strap features a built-in pocket for your smartphone. It has a place for everything digital, so everything digital can stay in its place.
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#Indianapolis Colts
# indianapolis-colts - Saturday 21 March, 2020
NFL free agency winners and losers: Improvements in Indy, Bucs wrong to bet on Brady
The Tampa Bay Buccaneers made the splash signing of Tom Brady, but they are not among the winners from free agency.
# indianapolis-colts - Tuesday 17 March, 2020
Colts reportedly sign 8-time Pro Bowl quarterback to $25 million contract
The next domino in free agency has fallen, with the Indianapolis signing Philip Rivers.
# indianapolis-colts - Tuesday 17 December, 2019
Brees makes history as Saints crush Colts
It was the Drew Brees show in the New Orleans Saints' victory over the Indianapolis Colts.
# indianapolis-colts - Sunday 1 December, 2019
Ravens beat 49ers for 8th-straight win, Dolphins kicker scores TD in victory
Elsewhere, the Cincinnati Bengals ensured they will not go 0-16.
# indianapolis-colts - Monday 11 November, 2019
Rudolph's outrageous one-handed catch helps sink Cowboys, Packers and Steelers also win
The highlight of the night came as Minnesota Vikings star Kyle Rudolph scored with a one-handed catch.
# indianapolis-colts - Monday 23 September, 2019
Giants rookie Jones wins first start, Steelers & Broncos sink to 0-3
The post-Eli era started with a win.
# indianapolis-colts - Monday 16 September, 2019
Mahomes erupts for Chiefs, Patriots and 49ers win big
Patrick Mahomes led the Kansas City Chiefs past the Oakland Raiders, while the New England Patriots and San Francisco 49ers won big.
# indianapolis-colts - Sunday 25 August, 2019
'Injuries have taken my joy of NFL away' - Colts star Luck retires
The 29-year-old made the announcement official on Saturday evening.
# indianapolis-colts - Friday 4 January, 2019
Long shots, road warriors, and your NFL wild card weekend preview
Playing this weekend and going on to win the Super Bowl is not easy, writes Steve O'Rourke.
# indianapolis-colts - Monday 12 November, 2018
Titans take down Tom Brady three times in surprise win over Patriots
Marcus Mariota passed for 228 yards as the Pats were well beaten on Sunday night.
# indianapolis-colts - Monday 29 October, 2018
Rams stop Packers to remain unbeaten as Eagles triumph in London's Wembley Stadium
Meanwhile, Adam Vinatieri become the NFL's all-time points leader in the Indianapolis Colt's win over the Oakland Raiders.
# indianapolis-colts - Monday 22 October, 2018
Cowboys, Ravens lose after missed kicks in final seconds
We had some dramatic late finishes in the late games.
# indianapolis-colts - Friday 5 October, 2018
Brady throws 500th touchdown pass as Patriots beat Colts
Tom Brady helped pick apart the Indianapolis Colts' defense, recording 341 yards and three touchdowns.
# indianapolis-colts - Saturday 28 April, 2018
Derrius Guice finally gets the call after dramatic slide down the board
Elsewhere on day two, the Pittsburgh Steelers appeared to find a successor to Ben Roethlisberger.
# indianapolis-colts - Monday 12 February, 2018
The Colts finally have a new head coach, and this one looks like he'll stick around
Indianapolis have appointed former Eagles' offensive coordinator Frank Reich to their top job.
# indianapolis-colts - Thursday 8 February, 2018
'My word is my bond: Agent drops Josh McDaniels after Colts coaching controversy
Bob LaMonte, who also represents Indianapolis Colts general manager Chris Ballard, ended his professional relationship with McDaniels.
# indianapolis-colts - Wednesday 7 February, 2018
5 hours after the Colts announced Josh McDaniels as their new head coach, he says he doesn't want the job
Josh McDaniels was supposed to be moving on from New England two days after they lost in the Super Bowl.
# indianapolis-colts - Sunday 4 February, 2018
NFL player Jackson killed by suspected drunk driver
The 26-year-old Indianapolis Colts man has passed away in tragic circumstances.
# indianapolis-colts - Friday 15 December, 2017
Even with Brock Osweiler, the Broncos cruised past the Colts last night
The Denver Broncos were too good for the Indianapolis Colts as Brock Osweiler impressed in the NFL.
# indianapolis-colts - Monday 11 December, 2017
Blizzard bowl as Bills beat Colts in 'Snowvertime'
The arctic conditions affected most aspects of play, forcing both quarterbacks to keep passing to a minimum.
# indianapolis-colts - Tuesday 17 October, 2017
Marcus Mariota returns to lead Titans past Colts as they leave losing streak behind
Tennessee Titans quarterback Marcus Mariota threw for over 300 yards in Monday's victory.
# indianapolis-colts - Tuesday 6 December, 2016
Colts blow out the Jets to take first place in the AFC South
It was a Monday Night Football to forget for Gang Green.
# indianapolis-colts - Friday 25 November, 2016
Rookie phenom Ezekiel Elliott helps Dallas Cowboys set franchise record
All three of Thursday's NFL winners ended the day in first place in their respective divisions.
# indianapolis-colts - Sunday 2 October, 2016
As It Happened: NFL Week 4 Hangout
It's an early start this week as the first of the London games takes place.
# indianapolis-colts - Monday 19 September, 2016
Jimmy Garoppolo and Adrian Peterson go down hurt as injuries ravage NFL rosters
The Patriots will have to turn to a rookie at quarterback as their win over the Miami Dolphins proved a costly one.
# indianapolis-colts - Thursday 15 September, 2016
Divisional rivalry dominates week 2 of the NFL
The Steelers and Bengals provide the game of the weekend.
# indianapolis-colts - Monday 7 December, 2015
Antonio Brown got creative with his touchdown celebration
Goalpost, meet Antonio.
# indianapolis-colts - Tuesday 3 November, 2015
The Carolina Panthers are 7-0, but they didn't make it easy for themselves last night
Panthers let 17-point lead slip before grinding out win in overtime.
# indianapolis-colts - Monday 19 October, 2015
Colts try trick play straight out of an Adam Sandler film, America shakes its head
We're still trying to work this one out.
# indianapolis-colts - Tuesday 22 September, 2015
Colts out of Luck as Jets continue to soar
Andrew Luck played his second successive terrible game for the pre-season favourites.
# indianapolis-colts - Tuesday 12 May, 2015
Brady suspended for four games over 'Deflategate,' Patriots fined $1 million
The NFL stripped the Super Bowl champions of both their 2016 first round draft pick and the 2017 fourth round draft pick.
# indianapolis-colts - Friday 1 May, 2015
Titans stick with Mariota as Jets and Rams provide the big surprises in round one
Gang Green fans will be particularly happy with how things fell into place last night.
# indianapolis-colts - Wednesday 22 April, 2015
The Patriots will kick off the 2015 NFL season against the Super Bowl's most successful team
The NFL schedule for next season has been released.
# indianapolis-colts - Saturday 24 January, 2015
The NFL acknowledged the 'Deflate-gate' investigation with a statement yesterday
The NFL confirmed they are investigating the New England Patriots use of under-inflated footballs.
# indianapolis-colts - Wednesday 21 January, 2015
11 of 12 footballs found to be 'not properly inflated' in NFL investigation
The New England Patriots remain under investigation for tampering the footballs during the AFC Championship Game.
# indianapolis-colts - Monday 19 January, 2015
The Redzone: Packers made to pay for conservatism while Seahawks pull out all the tricks
It was a long, long night of NFL action but we've a very predictable Super Bowl at the end of it all.
# indianapolis-colts - Sunday 18 January, 2015
As it happened: NFL Championship Games - Colts @ Patriots and Packers @ Seahawks
We brought you the best videos, tweets and more from tonight's two big games.
The final countdown - The42.ie's NFL Championship Games preview
Will there be any surprises this week?
McGregor is just one attraction in what is a seismic sports Sunday in Boston
The New England Patriots will be playing for a place in the Super Bowl tomorrow.
# indianapolis-colts - Friday 16 January, 2015
Remember kids... tattoos are permanent
Belichick yourself before you wreck yourself.


text:
Thank you for being part of the AGE.FYI experience.
Breathing is the bodily process of inhalation and exhalation; the process of taking in oxygen from inhaled air and releasing carbon dioxide by exhalation.
Your lungs have two main functions. Ove it to get oxygen from the air into the body, toh other is to remove carbon dioxide from the body.
Symptoms of lung problems:
Some effective ways to help:
Share an idea, experience or thought with us here.
Some effective ways to help:
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text:
How to understand the rate of x2 (the second team will not lose)
Bet 2 – win of the second team or a draw. Players will be satisfied with any result except home win. This dual outcome is popular in football and hockey. In other team sports where there is a draw, such an event almost never occurs. Rate x2 are principal: they will be able to find at any bookmaker.
2x bet against the victory of the first team – insurance. A player deliberately sacrifices a high coefficient in the name of reliability. When you are satisfied with just 2 of the 3 outcomes of the match, there is a greater sense of confidence. Sometimes double chance is an attempt to take the risk. For example, instead of the handicap (+2) on growth in away match with Zenit, the bettor takes x2. The ratio here is much higher.
Bet on 2 identical форе2 +0.5. Some Western bookmakers prefer to carry double the chance to kinds of odds, but the content remains the same. To distinguish bet on "x2 will not lose" from the handicap 2(0). For a draw at a zero handicap player gets a refund, and the winnings only if you win. But the coefficient for F0 above.
The calculation of rates 2x is no refunds. For example, to meet real Madrid and Catalan Barcelona. We decided to play on the strength of "blue garnet" putting on the x2 If you will win or play a draw, then the bookie will pay winnings. With the victory of real Madrid – calculate the loss. Payment with cafam "1" is impossible.
Betting strategy 2 (draw or away win) in bookmakers
1. Bet against a home win x2 is more often used in multiples. The player chooses 2 or 3 games, seeking at the expense of peremerki to achieve a decent ratio. This format we do not recommend, as home teams often present sensation. The support of the fans, home and transcendent motivation is an essential attribute of the home matches of most European clubs and national teams.
2. As bets a bet of 2 is appropriate to use when the coefficient of 1.50. Your team is a slight underdog at the line, but the chance to take points away from the notebook favorite, according to bookmaker, great. In modern football, and hockey, the opponents equalized in the clubs investors, there is money and status players. So, the old leaders will increasingly give points underdogs.
3. A bet on the "x2 the second team will not lose," it is possible to insure during the match. Almost all football and hockey matches in the office of Leon are live. If your team is ahead, the odds on the home win will rise. On it and you can put a small amount in order to obtain a guaranteed profit. To wilkawatt to match c x2 – we do not recommend. Sooner or later the security service of the bookmaker to block you, and for 1-2% of the profits.
4. Consider the movement of the line. The home team, especially a favorite, believe battery. They are beginning to "ship" to win 1, inadvertently increasing the ratio at 2x. This confidence of colleagues you need to turn in their favor. So better bet shortly before the match, when the line was finally formed. Again, many bookmakers on the match day increase the odds.
Where to bet x2 (aprogress away team)
2x is everywhere, therefore we suggest to choose the office reliable and proven – as Leonbets, where beginners will find exclusive bonus up to 300$ and points for every bet.
Related articles: 1x (1st team doesn't lose), win in 1 goal or draw
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Barristers Bournemouth
Barristers in Bournemouth (Results 1 - 9 of 9)
- 3PB Barristers Bournemouth30 Christchurch RoadBH1 3PD Bournemouth 01202 292102
... Lawyer, barristers
- Paul Gunn & Co1-3 Alum Chine RdBH4 8DT Bournemouth Westbourne 01202 752594
... solicitors, barristers
- Request for QuotationAre you looking for Barristers ?Use our 'Request for Quotation' service and get customized offers from companies in Bournemouth.
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- Genesis : BournemouthBeacon House, 15 Christchurch RoadBH1 3LB Bournemouth 01202 552255Open
At Genesis Solicitors are a team of highly experienced professionals having worked for many years in our respective fields. What defines us is our shared passion to make a difference to our clients...
- S Nightingale3 POOLE ROADBH2 5QJ Bournemouth WESTBOURNE 01202 551991
... legal adviser, advocate, solicitor services, solicitors, attorney, counsel, counsellor, advocacy,...
- Hammond & CoHAMMOND HOUSE, 41 THE GRBH9 2TS Bournemouth 01202 462888
... solicitor, counsellor, solicitor services, barrister, attorney, law society, legal profession, law,...
- Emerson Chase Ltd4 Regal House, Mengham RoadBH8 9RU Bournemouth 02392462255
Whatever stage you are at in your private or business life Ellis Jones can support you through the legal or practical issues you may be facing. We are proud of what we do and the excellent service we...
- Hallmark International Marketing LtdWILSON HOUSE 2 LORNE PARK ROADBH1 1JN Bournemouth 01202 587 787
... a wide choice of professionally designed Ready Made Websites and web design for IFAs, mortgage brokers, estate agents, Solicitors, Barristers, Accountants and websites for Financial Advisers.
- Nick Robinson Barrister30 CHRISTCHURCH ROADBH1 3PD Bournemouth 01202 292102
... law, Criminal Law specialists, employment, firm, Solicitor, counsel, employment law solicitors,...
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- A K HorseyGreyhound House, 2 Market PlaceDT11 7EB Blandford Forum 01258 452555
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text:
Welcome to Musical Mind Music Studios. We are a collaboration of fine musicians from around the Mercer Island and Seattle area.
Our goal is to provide all youth and adults music instruction and therapy that works, and create programs that stimulate the musical mind and bring out everyone's inner musician.


text:
British Rail Pensiion Funds (No. 3)
EDM number 2323 in 1992-93, proposed by Hugh Bayley on 14/07/1993.
That this House notes that 200,000 British Rail pensioners are being warned in a letter from Vernon Hince, the Chairman, and David Adams, the Chief Executive of the BR Pension Fund Trustee Company Ltd., that the Government proposal in the Railway Bill 'places their pensions at risk'; condemns the Government for failing to honour the promise given in the railway privatisation White Paper that 'Privatisation will not affect pensions already being paid from or preserved in British Rail schemes'; recognises that for most retired railway employees their pensions are their life savings; and calls upon the Government to bring forward proposals to revise the Bill so as (a) to retain a single pension scheme for all railway employees, (b) to offer pensions and annual pension increases which are no less favourable than at present and (c) to retain both an annual increase equal to the rate of inflation and the ability to pay a bonus reflecting the investment performance of the scheme as are currently enjoyed.
This motion has been signed by a total of 103 MPs.
Download raw data as csv or xml.


text:
Restoration has stalled and owner now wants to sell. Frame off restoration has frame completed, new just about everything. Body partially complete and primer sprayed. Enough new parts to complete the job. Contact Paul Mitchell @ 508.789.2580. Email firstname.lastname@example.com for photos.
© 2012-2019 Cape Cod British Car Club, Ltd. - PO Box 1661 - Sandwich, MA 02563


text:
Read from the experts about the latest printing news, varying from screen and digital printing, laser cutters, printing inks, packaging and more.
Explore FESPA's global portfolio of events, exhibitions and conferences aimed at the international print trade.
FESPA is a global federation of 37 national associations for the screen printing, digital printing and textile printing community.
Join today and benefit from a wealth of benefits to boost your business.
As more printers move their operations or storefronts online, and more workers base themselves at home, they're more exposed to cyber crime and online fraud than ever before.
Martin Bailey, CTO of Global Graphics Software, shows how printers can make colours uniform and keep buyers happy.
A spectrophotometer and linearization plans are essential to maintain consistency and quality, say industry experts from EFI and Caldera.
A clunky website can turn off customers both new and old. Here are some top tips for sprucing up your user experience.
It's been called the "Sistine Chapel of Rotterdam", but how did printers cope with the challenges of rendering an 11,000 square-metre artwork onto this dizzying 40 metre arch?
With 95 million photos uploaded to Instagram each day, learn to stand out from the crowd.
As the shift to digital payments gathers pace, what are the best options for printers?
Trade shows aren't just an opportunity to meet face to face with fellow printers – here's how visitors can make the most of exhibitions after the event.
What are printing companies doing to help brands protect their products? One unique solution comes from Swiss specialists IQDEMY.
If you plan to sell on or gift your print business to a family member, it pays to know the pros and cons, and how to do it legally.
LinkedIn is an essential online networking tool for thousands of companies and millions of people – but how can PSPs best use it to boost their business?
Networking at print expos and trade shows remains a key skill for all businesses to master. Here's how to do it the right way…
Ensure you're covered with this checklist for PSPs.
New business is your lifeblood, so here are 14 simple tips to keep it flowing in.
The people you hire can be the difference between success and failure. How can smaller PSPs recruit and retain the very best talent?
Plan ahead to alleviate cashflow problems.
eBay has transformed into a shopfront for brand-new products – how can PSPs take advantage of this huge digital marketplace?
Disputes with customers can lose you business and damage your standing. Effective management of complaints ensures you retain your customers.
What the increasing demand for low-impact textiles means for manufacturers.
Build sustainable waste disposal into your workflow and boost your green credentials in the process.
Just choose your country in the list below and we'll find your local association
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text:
Monday wrote:
you can use "do" as a simple include-ish sort of thing> Hello, I am new to Perl. I want to do something simple but seem to be
> unable to do this. I want to call a subroutine and have it access the
> global variables in the caller program, similar to below where
> index.cgi is the calling program and called.cgi is the called program.
> How do I do this since there is no 'include' type of statement in
> Perl? -Monday
> index.cgi
> =========
> #!c:/perl/bin/perl.exe
> our $globalVar = "Chicago";
> callNow();
> return 1;
> called.cgi
> ==========
> callNow() {
> print "global variable is $globalVar";
> }
in index.cgi:
do "called.cgi";
"use" is better on the whole but you need to get into the idea of packages /
modules and name spaces for that. Use "do" if you want dead simple code,
use "use" if you want to import a "package" or "module".
Real address: chris at lowth dot sea oh em.
World's first wrist-watch PDA with Palm OS, available June 30
from Amazon.com. Order now to beat the rush!


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Foundation University Islamabad is organizing 5 Days session of Aspiring TeamsTEPPS starting from October 21, 2019
Thai Government Scholarship Chulalongkorn University Scholarship 2021 in Bangkok, Thailand. Deadline to apply is October 15, 2020
Riphah International University is organizing 2nd Integrative Lifestyle Medicine Conference 2020 on October 24 & 25, 2020
Admission Fall 2020 Open at Mohi-ud-Din Islamic University Nerian Sharif, AJ & K. last date to apply is August 15, 2020
Riphah International University is organizing 2nd International Summit on Ideology of Pakistan on September 24, 2020
Fall Admission 2020 open at Riphah International University across campuses in Islamabad, Rawalpindi, Lahore, and Malakand.
Quaid-i-Azan University Islamabad issued the date sheet for BS Home Economics programme – Terminal Examinations (Spring – 2020) 2nd, 4th, 6th & 8th Semesters
Quaid-i-Azam University Islamabad has issued the schedule for submission of examination forms and fee for MA, MSc, M.Com (part I-II) & BSCS (part I, II, III, IV) annual examination 2020
Pakistan Innovation Foundation – PIF and PIDE are jointly organizing an informal network of Pakistanis with the title re-imagine Pakistan on August 14, 2020
Westminster Academy Islamabad is organizing The Seema Farheen Qureshi Debating Championship 2020 from 8th of August till 10th of August
National Incubation Center and Jazz xlr8 is arranging online session on The Art of Pivoting with Aamir Ibrahim on August 10, 2020
Under the Prime Minister's Electric Wheelchair Scheme, HEC invites applications from the students. submit the online application by September 15, 2020
National Defence University is organizing National Security Workshop 22. Last date of submitting applications is August 25, 2020
Friedrich Naumann Foundation is accepting the applications for DAAD Scholarships application in Germany. Application Deadline April 30, 2021


text:
Despite repeated stern warnings from the Electoral Commission against early campaigns, the election fever is already gathering momentum in Mbarara city ahead of the 2021 Parliamentary election with numerous people expressing their interest so far.
Among them is a cinema manager who has vowed to challenge the incumbent MP Michael Tusiime come 2021.
Others are Western Youth MP, Mwine Mpaka and former Mbarara district chairman, Deus Tumusiime who are vying for a new constituency. Mbarara was named among the four municipalities which with effect from next year will be gazetted as cities by the Ministry of Local Government, and politicians have their eyes on the prize – political offices.
Mbarara city will be divided into two Divisions – South and North, each represented by a Member of Parliament.
Karungi Nassulu Sean, 27, who is currently the manager of Country Cinema Mbarara declared over the weekend that he will contest as an MP for Mbarara North. He will be up against now legislator Tusiime who hails from what will be Mbarara North Division.
The same Division will include what is now Kamukuzi, Kakiika and Biharwe, while Nyakayojo, Nyamitanga and Kakoba will form Mbarara South Divisions.
Western Youth MP, Mwine Mpaka and Imam Shafik Kajiko the area councilor for Katete Ward in Nyamitanga Division are among the individuals that have so far expressed interest in representing Mbarara South Division in Parliament.
The other interested candidate is former Mbarara district chairman, Deus Tumusiime popularly known as Omuteganda.
Karungi told SoftPower News said he took interest in the political seat due to gap that exists between voters and the current area MP. He says that after since the 2016 elections, the MP has ignored the constituency. Tusiime has officiated at numerous occasions in Mbarara municipality, some non-political.
Karungi also claims that Tusiime has failed to fulfill on service delivery and empowering the youths, things he pledged in his 2016 manifesto.
"I have not just joined politics, I have been following politics, and I feel like I am already come 2021. I know people will support me. I will help youths acquire loans to buy motorcycles so they can work," he said.
But MP Tusiime disregarded his political challengers as insignificant.
"Someone tells you 'I am going to stand here' and next day they tell you something different. Some are local leaders who have no idea about how Mbarara was divided. How can you say that I will kill a candidate who steps in my division forgetting that when we have Mbarara South and North, then divisions will no longer be your territories as you are claiming?"
Tusiime instead advised all aspirants to wait for the right time for the voters to decide which candidate they prefer.
On the district woman MP seat, Emma Boona who formerly occupied the seat has revealed her intentions to unseat the incumbent, Rosette Mutambi Kajungu. She claims she was cheated in the 2016 elections.
Boona who accused Kajungu of doing little for the women constituency has been attending a number of functions and making pledges to different churches and women groups of women in the municipality.
The Mbarara city Woman MP seat is yet another position that is likely to be highly contested for. So far, four people have already gone public with their interest.
Jolly Kagira Tumwine, a councilor representing Nyakinengo and Kinshasa Wards in Biharwe Division says she will be contesting for the Woman MP seat in Mbarara city. She doubles as the Secretary for Health in the Municipal council.
Kagira has been championing the campaign to restore River Rwizi whose fast-dropping water levels have raised an outcry among conservationists and residents.
Other intending contestants include Dona Kyomugisha, a lady councilor representing Kakoba Division, Juliet Kamushana, a councilor for Kamukuzi and Jinja's Resident District Commissioner (RDC), Hanifah Murerwa Karadi.
In 2011 and 2016, Karadi stood for the Mbarara district Woman MP seat and lost.
Meanwhile, in Rwampara which has since become a new district, Molly M. Asiimwe, a State House official is eyeing the Woman MP seat against Anna Kansiime, a district councilor representing Rugando sub-county.


text:
<jats:title>ABSTRACT</jats:title><jats:p>The dynamical activity of the human brain describes an extremely complex energy landscape changing over time and its characterisation is central unsolved problem in neuroscience. We propose a novel mathematical formalism for characterizing how the landscape of attractors sustained by a dynamical system evolves in time. This mathematical formalism is used to distinguish quantitatively and rigorously between the different human brain states of wakefulness and deep sleep. In particular, by using a whole-brain dynamical ansatz integrating the underlying anatomical structure with the local node dynamics based on a Lotka-Volterra description, we compute analytically the <jats:italic>global attractors</jats:italic> of this cooperative system and their associated directed graphs, here called the <jats:italic>informational structures</jats:italic>. The informational structure of the global attractor of a dynamical system describes precisely the past and future behaviour in terms of a directed graph composed of invariant sets (nodes) and their corresponding connections (links). We characterize a brain state by the time variability of these informational structures. This theoretical framework is potentially highly relevant for developing reliable biomarkers of patients with e.g. neuropsychiatric disorders or different levels of coma.</jats:p>
Journal article
Cold Spring Harbor Laboratory


text:
Arca Swiss L Brackets for Compact Cameras (Olympus, Sony, Panasonic, Samsung, Canon, Nikon)
I have several comments on this very welcome development (see the press release further below)—
- The press release suggests not just small EVF cameras, but also small DSLRs. It seems curious to offer those brackets, but to say nothing about professional cameras, like the Nikon D3x or Canon 1 series.
- Is the dovetail design actually compatible with the variance in the various clamps on the market, particularly the Really Right Stuff quick release and screw clamps. I have encountered incompatibilities with clamps and dovetails before.
- No doubt the bracket will be very high quality as is typical of Arca-Swiss, but one concern is whether the projecting dovetail makes it uncomfortable and awkward for carrying the camera in one's pocket.
- There seems to be no provision for a grip, and since some of the smaller cameras suffer from very poor grip-ergonomics, the Really Right Stuff bracket with its optional grip might be more practical for varied shooting.
ARCA-SWISS INTERNATIONAL — October 11, 2011
Arca-Swiss presents the 'NEX Kit' for NEX-3 and NEX-5 Sony cameras.
The kit consists of an L-bracket and a viewfinder shoe for an optical finder.
Previously Arca-Swiss has released the Mini L-bracket for compact cameras such as the Olympus E-PL1, E-PL2, Panasonic Lumix GF and LX3, Samsung NX-100, Canon G10, The new smallest DSLR offerings from Canon, Nikon, Olympus, Pentax and Sony.
Today we present a new mini L-bracket. Light and small, it is specifically designed to mate specifically with the Nex 3 and Nex 5 cameras from Sony.
The Nex L-bracket is fully adaptable with our monoball®Fix and monoball®Classic quick release systems. It takes into account the compact design of the Sony Nex cameras allowing a fast change from landscape to portrait format and vise-versa. Secure handling of the cameras when hand holding is improved due to the L-bracket as well.
The design further allows free access to cable ports allowing unhindered connection to the computer without having to remove or shift the bracket.
The kit includes a shoe allowing easy attachment of an optical finder. An optical finder is a great accessory allowing the photographer to see better, yielding faster, more accurate composing and shooting in low light conditions.
ARCA-SWISS is committed to the constant evolution and innovation of its products!
price : 159 € (Taxe free)
Further informations : ARCA-SWISS : 03 81 85 40 60 or : email@example.com