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The effects of thrombopoietic stimulation on megakaryocytopoiesis, platelet production, and platelet viability and function were examined in normal volunteers randomized to receive single bolus subcutaneous injections of 3 μg/kg pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo in a 3:1 ratio. PEG-rHuMGDF transiently doubled circulating platelet counts, from 237 ± 41 × 103/μL to 522 ± 90 × 103/μL (P< .0001), peaking on day 12. Baseline and day-12 samples showed no differences in responsiveness of platelets to adenosine diphosphate or thrombin receptor agonist peptide (P > .4 in all cases); expression of platelet ligand-induced binding sites or annexin V binding sites (P > .6 in both cases); or density of platelet TPO-receptors (P > .5). Platelet counts normalized by day 28. The life span of autologous 111In-labeled platelets increased from 205 ± 18 hours (baseline) to 226 ± 22 hours (P < .01) on day 8. Platelet life span decreased from 226 ± 22 hours (day 8) to 178 ± 53 hours (P < .05) on day 18. The theoretical basis for senescent changes in mean platelet life span was illustrated by biomathematical modeling. Platelet turnover increased from 43.9 ± 11.9 × 103 platelets/μL/d (baseline) to 101 ± 27.6 × 103 platelets/μL/d (P = .0009), and marrow megakaryocyte mass expanded from 37.4 ± 18.5 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015). Although PEG-rHuMGDF initially increased megakaryocyte volume and ploidy, subsequently ploidy showed a transient reciprocal decrease when the platelet counts exceeded placebo values. In healthy human volunteers PEG-rHuMGDF transiently increases megakaryocytopoiesis 2-fold. Additionally, peripheral platelets expand correspondingly and exhibit normal function and viability during the ensuing 10 days. The induced perturbation in steady state thrombopoiesis resolves by 4 weeks.
Endogenous thrombopoietin (TPO) initiates receptor-dependent signaling in TPO-receptor–bearing hematopoietic precursors and megakaryocyte lineage cells that induces differentiation, proliferation and endoreduplication.1-9TPO is produced constitutively by the liver and kidneys, and circulates in blood as unbound thrombopoietically active TPO and TPO bound to receptors on peripheral platelets and developing marrow megakaryocytes, in competitive equilibrium.7,10,11 Two recombinant thrombopoietic growth factors have been developed and evaluated in patients: a polyethylene glycol-conjugated, recombinant truncated polypeptide, PEG-rHuMGDF12-14; and the full-length, glycosylated recombinant protein, rHuTPO.2,15 In nonhuman primates, the subcutaneous administration of TPO or PEG-rHuMGDF produce molar-equivalent, log-linear increases in the concentrations of peripheral platelets. The purpose of this study is to examine the effects of single-dose thrombopoietic stimulation on megakaryocytopoiesis, platelet production, platelet life span, and platelet function in healthy human volunteers. Patients and methods
Subjects and study design
This placebo-controlled, blinded study, approved by the Institutional Review Board, involved 16 healthy human volunteers recruited from the Emory campus community. Informed consent was obtained from each participant on admission to the General Clinical Research Center (GCRC) at Emory University, Atlanta, GA, and detailed clinical history, physical examination, complete profile of blood counts, routine serum chemistries, and urinalysis were obtained. The subjects were between the ages of 19 and 50 years, asymptomatic without known disease, and not taking any medications. The blood cell counts and routine blood chemistries were within the normal range for these laboratories.
Volunteers in each of the two 8-subject cohorts were randomized to receive either a single subcutaneous injection of 3 μg/kg PEG-rHuMGDF, or placebo in a 3:1 ratio on day 1. In the first group of 8 subjects (6 receiving active drug and 2 placebo), baseline measurements of platelet life span, platelet function, and megakaryocyte measurements were compared with the results obtained on day 12, the time when the concentration of peripheral platelets was maximum. In the second cohort of 8 subjects (6 receiving active drug and 2 placebo), platelet life span, platelet function, and megakaryocyte measurements were performed on days 8 and 18, times when the rates at which new platelets would enter and leave the circulation were calculated to be maximal, respectively. Thus, baseline (day 1) results with active drug were compared with cohorts of 6 subjects on day 8, day 12, day 18, and 4 placebo subjects (2 from each cohort).
Megakaryocyte growth factor
rHu-MGDF is a nonglycosylated polypeptide produced inEscherichia coli transfected with cDNA encoding the 1 to 163 aminoterminal residues of human thrombopoietin.12 After extraction, refolding, and purification, this truncated protein was derivitized with poly-(ethylene glycol). The resulting pegylated monomeric molecular species was prepared in aqueous buffer, sterilized by filtration, and provided as a gift from Amgen Inc (Thousand Oaks, CA). Endotoxin in the final product was less than 2.5 EU/mL.
Serum levels of pegylated recombinant megakaryocyte growth and development factor (PEG-rHuMGDF) and endogenous thrombopoietin
Daily serum levels of megakaryocyte growth factor were measured immunometrically for 4 weeks using antibody-sandwich enzyme-linked immunosorbant assays (ELISAs). The assay system used a polyclonal antibody raised in rabbits against recombinant human MGDF as the capture antibody. The same antibody conjugated to horseradish peroxidase (HRP) was used as the signal antibody. The sensitivity of the assay was 30 pg/mL.16,17
Determination of thrombopoietin receptors on platelets
Mean TPO receptor numbers per platelet were estimated from platelet-binding isotherms of 125I-labeled recombinant human (rHu) TPO according to Scatchard analysis.18,19 In brief, TPO receptors on platelets were determined using purified rHu-TPO radiolabeled by Iodo-beads iodination reagent (Pierce, Rockford, IL), and incubating rHu-TPO with 50 mmol/L sodium phosphate buffer, pH 7.2, and 125I with Iodo-beads for 15 minutes. Platelets were obtained from blood drawn in acid-citrate-dextrose (ACD) anticoagulant (1:7 v/v), pelleting platelets from platelet-rich plasma by centrifuging at 500 × g for 15 minutes, and resuspending in Tyrode's buffer containing ACD (1:7 v/v), pH 6.2, 1% bovine serum albumin (BSA), and 0.01% Tween. Binding isotherms were obtained by incubating platelets in plasma-free Tyrode's buffer, ACD (1:7 v/v), 1% BSA, 0.01% Tween, pH 6.2, and various amounts of125I-rHuTPO (40 to 640 ng/mL final concentrations) for 1 hour at room temperature. Nonspecific binding was assessed by comparing the effects of 100-fold excess unlabeled rHu-TPO 30 minutes before the addition of 125I-rHuTPO. Nonspecific binding ranged from 10% to 20%. Binding isotherms were analyzed using the Biosoft Ligand Program (Cambridge, UK) to determine the number of binding classes, the number of molecules bound/platelet, and the dissociation constant, Kd. TPO receptors on peripheral platelets were measured before, and 8, 12, and 18 days after initiating rHuPEG-MGDF therapy. rHuTPO, a glycosylated protein produced in Chinese hamster ovary cells transfected with the cDNA encoding for human TPO, was provided as a gift from Kirin Pharmaceutical Laboratories, Takasaka, Japan. The purified molecular species was formulated in an aqueous buffer, and sterilized by filtration. Endotoxin levels in the final product were less than 2.5 EU/mL.
Measurements of platelet function
Platelet aggregation was determined within 1 hour of drawing blood in 1/10 volume 3.2% citrate anticoagulant using a Chrono-Log aggregometer (Havertown, PA) by recording the increase in light transmission through a stirred suspension of platelet-rich plasma (PRP) maintained at 37°C. PRPs and platelet-poor plasmas (PPPs) were prepared by differential centrifugation, as previously described.20,21 The platelet count in the PRP was adjusted to 300 × 103/μL. ADP (Sigma, St Louis, MO), Horm collagen (Nycomed Arzenmittel, Munich, Germany), and TRAP1-6 (Peninsula Labs, Belmont, CA) were added at doses spanning the range of responsiveness. The results were plotted and expressed as the agonist concentration inducing half-maximal aggregation (AC50).22,23
The appearance of activated platelets in the peripheral blood was evaluated by flow cytometry using fluoresceinated monoclonal antibodies (MoAbs) against neoantigens expressed on membrane surfaces of activated platelets, including conformationally altered integrin IIb3ligand-induced binding sites (LIBS), a gift from Dr E. Plow (Cleveland, OH),21,24 and the secretory granule membrane, P-selectin, a gift from Biogen Inc, Cambridge, MA.21,25 In addition, enhanced binding to platelets by fluoresceinated annexin V, a gift from Dr T. Yokoyama, Tokyo, Japan, was also examined using flow cytometry.26-28 Annexin V, a member of the multigene family of calcium-dependent phospholipid binding proteins, exhibits high affinity for phosphatidylserine-rich, negatively charged, phospholipid platelet membrane binding sites promoting assembly of the macromolecular coagulation enzyme complexes.29-31 Flow cytometric platelet studies were performed on blood collected in 1/10 volume 3.8% sodium citrate. PRP was obtained by differential centrifugation; 5 μL PRP was diluted to 50 μL in phosphate buffered saline (PBS) pH 7.4 containing saturating concentrations of fluoresceinated marker protein and 1% bovine serum albumin (BSA), and incubated at 22°C. Annexin V was diluted in HEPES buffer (0.01 mol/L) in 0.15 mol/L sodium chloride and 2.5 mmol/L calcium chloride and 1% BSA pH 7.4. After 30 minutes incubation with the appropriate antibody or binding protein and buffer, the platelets were diluted 10-fold with the incubation buffer without albumin and placed on ice until analyzed by flow cytometry. The findings were standardized each day against calibrated fluorescent beads (Flow Cytometry Standards Corp, San Juan, PR).
Measurements of platelet production
Megakaryocyte number, size, and ploidy were measured by flow cytometry using a previously reported method for multiparameter correlative marrow analysis with a single-argon-ion-laser FACScan analyzer (Becton Dickinson, San Jose, CA).32-34 Cell DNA in aspirated marrow was stained with propidium iodide, and surface membrane receptors were analyzed with antibodies labeled with fluorescein. Megakaryocytes expressing IIb3 integrin (CD41/61) were enumerated in relation to the nucleated erythroid precursors expressing glycophorin A.34,35 Measurements of megakaryocyte diameters were based on the "time-of-flight" principle, ie, time required for a cell in suspension to pass through a focused light beam.36 Aspirated bone marrow (3 mL) obtained from the pelvic bones was collected into 10-mL plastic syringes containing equal volume acid-citrate-dextrose (ACD formula A), 2.5 mmol/L EDTA, and 2.2 μmol/L prostaglandin E1(PGE1; Sigma, St Louis, MO) final concentrations. The marrow was gently pipetted, passed through a 120-mm monofilament nylon filter, and diluted with cold Ca++- and Mg++-free PBS containing 13.6 mmol/L sodium citrate, 2.2 μmol/L PGE1, 1 mmol/L theophylline (Sigma), 3% BSA fraction V (Calbiochem, La Jolla, CA), 11 mmol/L glucose, and adjusted to a pH of 7.3 and an osmolarity of 290 mOsm/L. Megakaryocytes were analyzed in marrow aspirates fractionated with 1.06 density Percoll (Pharmacia Biotech Inc, Piscataway, NJ). The nucleated erythroid marrow cells were analyzed from marrow separated over 1.08 density Percoll (Pharmacia Biotech Inc). Megakaryocytes were selected on the basis of their distinct immunofluorescence at levels above that of control cells labeled with an unrelated MoAb. In each sample, 2000 to 3000 megakaryocytes were analyzed. Each subject had 2 bone marrow studies, ie, either baseline and at day 12, or day 8 and day 18.
Estimates of marrow megakaryocyte mass were used to represent the marrow substrate giving rise to circulating platelets, and were calculated as the product of megakaryocyte numbers and mean megakaryocyte volumes.37
Steady-state platelet mass turnover (platelet concentration multiplied by mean platelet volume and divided by platelet life span and by recovery of injected labeled platelets) was used to estimate the rate at which viable platelet mass was delivered to the peripheral blood.34,37 To measure platelet survival time, autologous platelets were labeled with 111In-oxine using the method described previously.38 Labeling efficiencies averaged 90%, and the labeled platelets functioned normally.20After reinjection, daily blood samples were collected and analyzed for111In-labeled platelet activity to determine the rate at which 111In-labeled platelets were cleared from the circulation. Platelet survival time, ie, the average time in circulation, was then calculated using computer least-squares fitting of the raw data to a gamma-function modeling program.38 The time course of 111In-labeled platelet activity in blood was modeled as:
where Ls(t) is the fraction of remaining111In-platelet activity at time, t, after injection of labeled platelets, and λ is the mean platelet life span.41 The parameter, na, is the number of exponentials in the gamma function, which modulates the variance of λ. The recovery of labeled platelets in the circulation at equilibrium was estimated by extrapolating the survival curve to time zero and estimating the blood volume (70 mL/kg) using the formula: recovery in circulation = total circulating platelet radioactivity/total platelet radioactivity injected.
Nonstationary thrombokinetic model
Peripheral platelet counts and 111In-labeled platelet activity curves were modeled simultaneously using a nonstationary gamma model.39 Modeling was conducted using SAAM II v. 1.1 (SAAM Institute, Seattle, WA). Megakaryocyte proliferation and maturation after stimulation by Mpl ligand were described by a system of nm differential equations as follows:
where nm is the number of catenary-linked megakaryocyte compartments (nm = 5), Mi is the mass in the ith megakaryocyte compartment, S0 is the Mpl ligand-independent platelet production rate, C(t) is the serum concentration of Mpl ligand at time t, kmpl is a proportionality constant relating mpl ligand serum concentration to Mpl ligand-dependent rate of thrombopoiesis, γ modulates steepness of the concentration-response curve, and τm is the mean transit time of cells within the megakaryocyte maturation compartments. C(t) was calculated by linear interpolation of the measured serum Mpl ligand concentrations. The initial condition for the ithmegakaryocyte compartment, Mi(0), was calculated as:
where C0 is the serum concentration of Mpl ligand at baseline.
Megakaryocytopoiesis was described in the model by a black-box process. Without modeling of the time course of megakaryocyte mass, the model parameters estimate the Mpl ligand-dependent and Mpl ligand-independent platelet production rate,
The model assumes that the rate of Mpl ligand-mediated platelet production is proportional to the serum concentration of Mpl ligand, C(t), raised to the power λ. It is recognized that the relationship between serum Mpl ligand concentration and platelet production rate described in Equation 6 is applicable only over the range of Mpl ligand concentrations achieved in this study. At higher Mpl ligand concentrations, the relationship between platelet production rate and serum Mpl ligand may be appropriately described by a sigmoidal concentration-response relationship, such as a logistic function that could describe full receptor occupancy. Subsequent to stimulation of megakaryocytopoiesis, changes in platelet production rate are reflected by changes in platelet count after a transit time delay, τm. Biologic parameters such as the rate of megakaryocytopoiesis, differential release rates of platelets from megakaryocytes of different ploidy, and megakaryocyte death rate, are not directly identifiable from observations of platelet kinetics in blood, per se. However, these biologic events affect the transit time, τm, and the variance of the transit time; hence, the thrombokinetic effects of these events are indirectly represented by the model.
Destruction of peripheral platelets was modeled by random and senescent processes. The kinetics of peripheral platelets were described by the following system of na differential equations:
where na is the number of catenary-linked platelet "age" compartments (na = 20), P is the total number of peripheral platelets, VP is the apparent dilution volume of peripheral platelets (including splenic pooling), Pj is the number of platelets in the jth platelet age compartment, kρ is a constant rate of random platelet utilization, and Λ is intrinsic platelet longevity. The initial condition for the jth platelet age compartment, Pj(0), was calculated as:
where P0 is the baseline peripheral platelet count. The peripheral platelet concentration at time t was calculated as the sum of the platelet numbers within na age compartments divided by the peripheral platelet dilution volume, Vp.
The 111In-labeled platelet activity curves were modeled simultaneously with peripheral platelet counts to account for the effects of platelet count and mean platelet age on the kinetics of111In-labeled platelet decay:
where P*j is the platelet activity (cpm) associated with the jth age compartment. The initial amount of radiolabel, P*j(t*), within the jth platelet age cohort upon injection of the tracer bolus at time t* was assumed to be proportional to the fractional number of platelets within the jth age compartment, ie, incorporation of label was assumed to be independent of platelet age:
where Pj(t*) is the modeled number of platelets within the jth age compartment at time t* and P(t*) is the modeled number of peripheral platelets at time t*, and D* is the dose (cpm) of111In-labeled platelets.
The mean platelet age at time t, A(t), was calculated from the weighted average of na platelet age compartments:
where jΛ/na is the average platelet age in the jthage compartment.
Numerical integration was conducted in SAAM II using the Rosenbrock method.56 Integration was conducted using an adjustable step size. A relative integration error of 0.001 (0.1%) was used.
Blood cell counts
Peripheral platelet counts, mean platelet volumes, red cell counts, and total white cell counts were determined in whole blood collected every day in K3EDTA (2 mg/mL) using Serono/Baker model 9000 whole blood analyzer (Allentown, PA).38 The absolute neutrophil counts (ANCs) were ascertained manually from white cell differential counts on Wright-Giemsa–stained peripheral blood films.
Statistical analysis
The data were analyzed using SIGMA STAT (Jandel Scientific Software, San Rafael, CA). Comparisons between 2 groups were performed using the 2-tailed Student t test, unless the data were not distributed randomly, in which case nonparametric analysis was performed. Analysis of variance (ANOVA) was used to compare value for a particular group at various time points.40 Unless otherwise stated, variance about the mean is given as ± 1 SD.
Serum levels of megakaryocyte growth factor
Serum levels of megakaryocyte growth factor were measured immunometrically each day for 4 weeks (Figure1, Table 1). In subjects receiving active agent, peak concentrations of Mpl ligand occurred 2 days after administering bolus subcutaneous injections of PEG-rHuMGDF, and averaged 500 ± 90 ng/mL (P < .001 compared with placebo). Compared with placebo subjects, significantly elevated concentrations were present in PEG-rHuMGDF–treated subjects for more than 5 days. The average time after injecting PEG-rHuMGDF to achieve half-maximal concentrations was 53 hours. The subjects receiving placebo exhibited serum encogenous TPO levels of 74 ± 20 ng/mL, which remained unchanged throughout the study period (Figure 1; Table 1;P > .6 in all cases).
. | Day 1 . | Day 8 . | Day 12 . | Day 18 . |
Platelet concentration (×103/μL) | ||||
Treated | 230 ± 43 | 354 ± 51 | 522 ± 90 | 420 ± 75 |
Pvs day 1 | .0001 | .0001 | .0001 | |
Placebo | 210 ± 47 | 228 ± 23 | 241 ± 28 | 230 ± 17 |
MPV (fL) | ||||
Treated | 8.0 ± 0.6 | 8.1 ± 0.6 | 7.7 ± 0.8 | 7.7 ± 0.6 |
Pvs day 1 | .2 | .17 | ||
Placebo | 8.1 ± 0.7 | 7.9 ± 0.3 | 8.0 ± 0.7 | 7.9 ± 0.4 |
Platelet life span (hs) | ||||
Treated | 205 ± 18 | 226 ± 22 | 212 ± 23 | 178 ± 53 |
Pvs day 1 | .03 | .45 | .48 | |
Pvs day 8 | .05 | |||
Placebo | 193 ± 12 | 212 ± 9 | 200 ± 10 | 214 ± 30 |
Platelet mass turnover (×105fL/μL/d) | ||||
Treated | 3.6 ± 1.1 | 4.6 ± 0.8 | 6.3 ± 2.1 | 7.5 ± 2.1 |
Pvs day 1 | .0008 | .0009 | ||
Placebo | 3.95 ± 0.9 | 3.5 ± 0.1 | 4.4 ± 0.1 | 3.4 ± 0 |
Mpl ligand level (pg/mL) | ||||
Treated | 119 ± 63 | 150 ± 93 | 102 ± 59 | 98 ± 66 |
Placebo | 74 ± 20 | 71 ± 23 | 76 ± 12 | 75 ± 13 |
. | Day 1 . | Day 8 . | Day 12 . | Day 18 . |
Platelet concentration (×103/μL) | ||||
Treated | 230 ± 43 | 354 ± 51 | 522 ± 90 | 420 ± 75 |
Pvs day 1 | .0001 | .0001 | .0001 | |
Placebo | 210 ± 47 | 228 ± 23 | 241 ± 28 | 230 ± 17 |
MPV (fL) | ||||
Treated | 8.0 ± 0.6 | 8.1 ± 0.6 | 7.7 ± 0.8 | 7.7 ± 0.6 |
Pvs day 1 | .2 | .17 | ||
Placebo | 8.1 ± 0.7 | 7.9 ± 0.3 | 8.0 ± 0.7 | 7.9 ± 0.4 |
Platelet life span (hs) | ||||
Treated | 205 ± 18 | 226 ± 22 | 212 ± 23 | 178 ± 53 |
Pvs day 1 | .03 | .45 | .48 | |
Pvs day 8 | .05 | |||
Placebo | 193 ± 12 | 212 ± 9 | 200 ± 10 | 214 ± 30 |
Platelet mass turnover (×105fL/μL/d) | ||||
Treated | 3.6 ± 1.1 | 4.6 ± 0.8 | 6.3 ± 2.1 | 7.5 ± 2.1 |
Pvs day 1 | .0008 | .0009 | ||
Placebo | 3.95 ± 0.9 | 3.5 ± 0.1 | 4.4 ± 0.1 | 3.4 ± 0 |
Mpl ligand level (pg/mL) | ||||
Treated | 119 ± 63 | 150 ± 93 | 102 ± 59 | 98 ± 66 |
Placebo | 74 ± 20 | 71 ± 23 | 76 ± 12 | 75 ± 13 |
Peripheral platelet concentrations and volumes
The concentration of peripheral platelets after the single bolus subcutaneous injection of PEG-rHuMGDF was detectably increased on day 6 (from baseline 230 ± 43 × 103/μL to 271 ± 35 × 103/μL; P = .01; Figure2), and doubled by day 12 (Figure 2; Table1; from baseline 230 ± 43 × 103/μL; P < .0001). The increase in peripheral platelet counts was associated with a small reduction in mean platelet volume (MPV) from 8.1 ± 0.6 fL on day 8 to 7.7 ± 0.8 fL; P < .05) on day 12. The peripheral platelet concentration and volume gradually normalized by day 28, ie, 243 ± 43 × 103/μL compared with placebo of 230 ± 43 × 103/μL (P > .6 compared with baseline), and 8.0 ± 0.6 fL to 8.0 ± 1.0 fL (P > .8 compared with baseline), respectively.
Platelet responsiveness to physiologic agonists, and expression of platelet receptors
The concentrations of ADP or TRAP inducing half-maximal platelet aggregation were not changed from baseline by administering PEG-rHuMGDF (Table 2; P > .4 in all cases). Similarly, administering PEG-rHuMGDF did not alter the expression of TPO receptors, LIBS, or annexin V binding sites during the study period (data not shown).
Platelet life span
Life span measurements of autologous111In-labeled platelets were computed using gamma function routines, modified for nonsteady- state analysis.38,41,42The disappearance pattern of 111In-labeled platelets obtained at baseline from 8 normal subjects is nearly linear, but exhibits slight curvilinearity (Figure 3). The computed average time platelets remain in the circulation, ie, platelet life span (λ), was 205 ± 18 hours (Table 1). These results agreed closely with the estimates of platelet life span obtained in placebo subjects at 8, 12, and 18 days (P > .5 for each of the 4 estimates). In PEG-rHuMGDF–treated subjects, platelet life span was prolonged to 226 ± 22 hours on day 8 (Figure 3; P = .04), the time when the cohort of platelets produced in response to PEG-rHuMGDF was youngest.
Peripheral platelet counts and 111In-labeled platelet kinetics were simultaneously modeled using a nonstationary thrombokinetic model (Figure 4). The model incorporated the effects of endogenous and exogenous Mpl ligand on megakaryopoiesis, elimination of platelets by senescence and endothelial use, and the effects of nonsteady-state alterations in mean platelet age on 111In-labeled platelet decay curves. Modeling of peripheral platelet counts and platelet tracer kinetics for a subject receiving autologous 111In-labeled platelets at baseline and on day 12 are illustrated in Figure5 (upper and middle plots). Data fitting yielded an intrinsic platelet longevity (Λ) estimate (platelet life span in absence of external hazard) of 9.7 days and a constant rate of platelet utilization (kp) of 8000 platelets per microliter per day. The apparent dilution volume (Vp) of platelets (including the splenic pool) was 7700 mL. The model predicted a linear platelet decay at baseline, due to a relatively uniform distribution of peripheral platelet ages. On day 12, the modeled tracer decay curve was slightly sigmoidal because of an increase in the proportion of younger platelets in circulation just before the platelet zenith. Modeling of peripheral platelet counts and platelet tracer kinetics for a subject receiving autologous 111In-labeled platelets on days 8 and 18 are illustrated in Figure 6 (upper and middle plots). In this subject, the intrinsic platelet longevity (Λ) was 10.3 days, platelet utilization (kp) was 7700 platelets per microliter per day, and the platelet dilution volume (Vp) was 6200 mL. A pronounced sigmoidicity in the platelet tracer decay curve on day 8 was noted, and was attributed to the high influx rate of new platelets into circulation. By contrast on day 18, the platelet tracer dose decayed rapidly and exponentially; the platelet tracer kinetics on day 18 were attributed to an increased proportion of old platelets in circulation with an associated increased destruction rate of the senescent cells.
The theoretical time course of mean platelet age, A(t), after transient stimulation of megakaryocytopoiesis by a bolus dose of PEG-rHuMGDF was calculated (Equation 15) and is illustrated in Figures 5 and 6 (lower plot). The theoretical nadir in mean platelet age was predicted to be around day 9, and the theoretical zenith in mean platelet age was predicted to be around day 19. Modeling of the data calculated a difference in mean platelet age between the theoretical age zenith and nadir of approximately 2.0 to 2.5 days. The modeling projection corresponded closely to the observed platelet life span difference of 48 hours between days 8 and 18 (Table 1). After model correction for nonstationary alterations in the age distribution of peripheral platelets, the model estimates of intrinsic platelet longevity and endothelial use were similar to published steady state determinations (intrinsic platelet longevity, 10.5 days; endothelial utilization rate 7100 platelets per microliter per day) in absence of exogenous cytokine stimulation.52,53
Platelet production
Platelet production was assessed in 2 ways: the rate at which viable platelets appeared in the circulation, calculated as platelet turnover (the peripheral concentration of platelets divided by the mean platelet life span, and corrected for splenic pooling); and megakaryocyte cytoplasmic substrate destined to give rise to platelets, calculated as megakaryocyte mass (the product of megakaryocyte number and megakaryocyte volume). The administration of PEG-rHuMGDF doubled platelet turnover, platelet mass turnover, and marrow megkaryocyte mass (Table 2), ie, platelet turnover increased from baseline of 44 ± 12 × 103 platelets/μL to 101 ± 28 × 103 platelets/μL/d (P = .0009), platelet mass turnover (Table 1) increased from 3.6 ± 1.1 fL/μL/d to 7.5 ± 2.1 fL/μL/d (P = .0009), and marrow megakaryocyte mass (Table 3) expanded from 37 ± 19 × 1010 fL/kg to 62 ± 17 × 1010 fL/kg (P = .015).
. | Baseline . | Day 8 . | Day 12 . | Day 18 . |
Aggregation* | ||||
ADP (μmol/L) | 2.7 ± 1.5 | 2.1 ± 1.4 | 2.6 ± 1.9 | 2.5 ± 2.0 |
TRAP (μmol/mL) | 2.2 ± 1.9 | 1.5 ± 1.2 | 1.6 ± 1.4 | 1.9 ± 1.8 |
Activation epitopes† | ||||
. | Baseline . | Day 8 . | Day 12 . | Day 18 . |
Aggregation* | ||||
ADP (μmol/L) | 2.7 ± 1.5 | 2.1 ± 1.4 | 2.6 ± 1.9 | 2.5 ± 2.0 |
TRAP (μmol/mL) | 2.2 ± 1.9 | 1.5 ± 1.2 | 1.6 ± 1.4 | 1.9 ± 1.8 |
Activation epitopes† | ||||
Agonist concentration producing half-maximal aggregation.
Binding sites × 103/platelet.
Megakaryocyte volume and ploidy
Megakaryocyte volume (Table 3) was significantly decreased on day 12 (from 30 ± 1.8 × 103fL to 24 ± 3.7 × 103 fL; P = .0003). This decline was reciprocally related to the increase in peripheral platelet counts beyond the placebo values. Corresponding changes were noted in megakaryocyte ploidy (Figure 7). The proportion of megakaryocytes with ploidy of 16N and 32N was decreased on days 8 and 12 (P < .05 in both cases) when peripheral platelet counts in treated volunteers significantly exceeded placebo controls (Figure 2; P < .05 in both cases).
. | Day 1 . | Day 8 . | Day 12 . | Day 18 . |
Platelet turnover | ||||
(×103plt/μL/d) | 44 ± 12 | 58.3 ± 9.8 | 78.8 ± 26.0 | 101 ± 28 |
Pvs day 1 | .0008 | .0009 | ||
Megakaryocyte number | ||||
(×106/kg) | 13 ± 6.4 | 17 ± 7.5 | 27 ± 9.4 | 11 ± 7.2 |
Pvs day 1 | .69 | .03 | .69 | |
Megakaryocyte volume | ||||
(×103fL) | 30 ± 1.8 | 28 ± 3.0 | 24 ± 3.7 | 26 ± 3.2 |
Pvs day 1 | .0003 | |||
Megakaryocyte mass | ||||
(×1010fL/kg) | 37 ± 19 | 46 ± 19 | 62 ± 17 | 30 ± 18 |
Pvs day 1 | .42 | .015 |
. | Day 1 . | Day 8 . | Day 12 . | Day 18 . |
Platelet turnover | ||||
(×103plt/μL/d) | 44 ± 12 | 58.3 ± 9.8 | 78.8 ± 26.0 | 101 ± 28 |
Pvs day 1 | .0008 | .0009 | ||
Megakaryocyte number | ||||
(×106/kg) | 13 ± 6.4 | 17 ± 7.5 | 27 ± 9.4 | 11 ± 7.2 |
Pvs day 1 | .69 | .03 | .69 | |
Megakaryocyte volume | ||||
(×103fL) | 30 ± 1.8 | 28 ± 3.0 | 24 ± 3.7 | 26 ± 3.2 |
Pvs day 1 | .0003 | |||
Megakaryocyte mass | ||||
(×1010fL/kg) | 37 ± 19 | 46 ± 19 | 62 ± 17 | 30 ± 18 |
Pvs day 1 | .42 | .015 |
This study demonstrates that administering single bolus subcutaneous PEG-rHuMGDF (3 μg/kg) to healthy human volunteers doubles the peripheral platelet concentration by stimulating megakaryocyte proliferation and endoreduplication, thereby doubling the mass of megakaryocyte cytoplasm destined for release as circulating platelets, without modifying platelet viability, platelet responsiveness to physiologic agonists, or platelet expression of activation epitopes.
Circulating concentrations of unbound endogenous TPO regulate physiologic platelet production to maintain constant peripheral platelet counts by compensating for changes in the peripheral demand for platelets.4,6,9,43 rHuTPO and PEG-rHuMGDF are 2 thrombopoietic growth factors that have been developed and evaluated in patients.8,9 These 2 agents produce molar-equivalent effects on megakaryocytopoiesis. In healthy human volunteers, exogenous stimulation of thrombocytopoiesis by PEG-rHuMGDF produces measurable increases in marrow megakaryocyte endoreduplication and cytoplasmic volume within 24 hours, and in marrow megakaryocyte number several days later (Table 3).44 Corresponding increases in circulating platelets are detectable after 5 to 6 days and peak by 10 to 14 days.45,46 Under steady-state conditions PEG-rHuMGDF produces comparable log-linear dose responses in megakaryocyte mass and platelet mass turnover.47,48 This study in volunteers underscores the reliability of megakaryocyte ploidy as a morphologic indicator of thrombopoietic stimulus, as shown by the transient decreases in megakaryocyte ploidy during induced thrombocytosis.
In healthy human volunteers, minimal reduction in MPV develops as the peripheral platelet counts are elevated (Table 1), consistent with previous reports in nonhuman primates regarding the reciprocal relationship between MPV and the peripheral concentration of platelets.48 Presumably, the blunted decrease in MPV observed in normal subjects (Table 1), compared with the greater reduction in nonhuman primates, is due to the striking difference in the relative total dose and duration of PEG-rHuMGDF administration.
Modulation of peripheral platelet counts in healthy human volunteers provides an opportunity to evaluate the effects of platelet concentration and age on the measurements of platelet life span under physiologic conditions. Current understanding of platelet survival time has evolved from the Mills-Dornhorst equation,41,49 which postulates an intrinsic 10-day time-dependent viability for circulating platelets in normal individuals (analogous to the 120-day life span for normal erythrocytes). However, superimposed on this 10-day life span is ongoing fixed random utilization that is required to maintain physiologic vascular integrity, a process requiring approximately 10% of the circulating platelet mass.38,50 Thus, in normal individuals the disappearance pattern of mixed population111In-labeled platelets is predominately linear, due to senescent removal, with slight curvilinearity resulting from fixed random vascular utilization, so-called "external hazard function" (Figure 3).50,51 Gamma function analysis of these disappearance curves provides good estimates of the average time that platelets survive in the circulation.38,51 As peripheral platelet counts decrease below 50 000/μL the relative importance of vascular utilization progressively increases, resulting in shortened platelet survival times secondary to thrombocytopenia, per se, apart from any other mechanism(s) of platelet destruction.
In this study, platelet life span was measured for platelet populations that were (1) enriched with young, recently formed platelets (day 8); (2) enriched with platelets approaching senescence (day 18); and (3) platelets at twice the baseline concentration (day 12). Gamma function analysis revealed prolonged platelet life span for platelets enriched with newly formed platelets (day 8; Figure 3; Table 1), shortened platelet life span for platelets approaching senescence (day 18; Figure3; Table 1), and minimally prolonged platelet life span at peak-induced thrombocytosis, compared with baseline (day 12; Figure 3; Table 1). These findings are concordant with the concepts that platelets are removed from the circulation predominantly by time-related senescent mechanism(s), with superimposed fixed vascular utilization that becomes proportionately less important when the peripheral concentration of platelets is increased. Clearly, the observed effects reported here would be accentuated by enhancing the degree of thrombocytosis induced by PEG-rHuMGDF or rHuTPO.
To provide a theoretical basis for the effects of changing platelet count and nonsteady-state alterations in mean peripheral platelet age, peripheral platelet counts, and 111In-labeled platelet decay curves were simultaneously modeled using a nonstationary model of platelet destruction and PEG-rHuMGDF-mediated megakaryocytopoiesis (Figure 4). The gamma function used for calculating platelet life span from platelet tracer curves (Table 1) was derived using steady state assumptions. Although the gamma function can approximate mean platelet life span under nonsteady-state conditions, the gamma function, per se, does not correct for nonstationary alterations in platelet age distributions or the effects of changing platelet count on endothelial platelet clearance during the time course of tracer decay. Therefore, to estimate intrinsic platelet longevity and endothelial platelet utilization rate after transient stimulation of megakaryocytopoiesis, a nonstationary thrombokinetic model was implemented. Using this model, excellent fits to peripheral platelet counts and tracer decay curves were attained (Figures 5 and 6). Model estimates of intrinsic platelet longevity and endothelial utilization rate were consistent with published values determined under steady-state conditions,52,53 thereby supporting the notion that the observed shifts in mean platelet life span on days 8, 12, and 18 relative to baseline were attributable to anticipated effects of changing platelet count and mean peripheral platelet age.
Because the Mpl ligands, PEG-rHuMGDF and rTPO, initiate receptor-dependent platelet signaling in vitro,52,53 and enhance platelet aggregative responsiveness to physiologic agonists in vitro and ex vivo,48 it has been postulated that their therapeutic use would increase the risk of developing vascular thrombosis.8,9 However, the negative results reported in Table 3 for normal subjects, and by others for cancer patients,15,54 support the conclusions resulting from studies in nonhuman primates,48 that therapy with Mpl ligands does not increase the risk of developing vascular thrombotic events, at least for the dosing and indications evaluated to date. However, in patients with underlying vascular disease, the degree of thrombocytosis complicating Mpl ligand administration may be of importance in the genesis of thrombotic events.28
The development of PEG-rHuMGDF has been suspended because of the development of neutralizing antibodies that inactivate endogenous TPO in some individuals after repeated injections.55 This observation represents a significant impediment to development of Mpl ligands. Recent descriptions of short peptide Mpl agonists with amino acid sequences unrelated to TPO indicate a possible means of circumventing this problem.55 The findings in this study support the rationale for using modest doses of nonimmunogenic Mpl ligands in volunteer donors undergoing platelet apheresis, to improve yields and efficiency.
In conclusion, 1 week after bolus thrombopoietic stimulation with 3 μg/kg PEG-rHuMGDF, megakaryocytopoiesis doubles, leading to corresponding doubling on day 12 of platelets that exhibit normal function and viability during the ensuing 10 days. All values return to baseline 4 weeks after bolus thrombopoietic stimulation.
Supported in part by Amgen Inc, and in part by PHS Grant MO1-RR00039 from the General Clinical Research Centers Program, National Institutes of Health, National Center for Research Resources.
Reprints:Laurence A. Harker, Blomeyer Professor and Director, Division of Hematology and Oncology, Emory University School of Medicine, 1639 Pierce Dr, Room 1003 WMB, Atlanta, GA 30322.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.


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One is in industrial lending, which includes working with business purchasers to investigate their monetary statements and assist them secure loans to broaden their companies. Another chance is in bank branch management where you would work with particular Business & Finance person prospects to supply a wide range of banking services. Still another option is in trust administration, the place you would assist clients with property issues and the administration of their wealth.
Gain centered monetary expertise, monetary analytics proficiency, and hands-on experiences like consulting for shoppers and managing real-world funds. Complex federal and worldwide accounting rules can throw off your balance sheet. Rely on us to delve into the major points and the way they'll affect your corporation, making recommendations together with your future well-being in thoughts. To graduate with this major, students should complete all university, school, and major necessities. UCR's doctoral program in finance supplies college students with a solid foundation in the theoretical and empirical instruments of finance.
Diploma Areas
This tells you the way a lot income you count on to generate per employee and offers a solid foundation for comparison with opponents and business leaders. An example of what a personnel forecast may look like, for example for personnel working on gross sales and advertising, could be found below Business. An example of what an working bills forecast may appear to be as an example for spending on gross sales and advertising, could be discovered below. Cost of goods bought are these costs that undoubtedly must be made in order for an organization to ship a service or produce a good.
Various colleges and colleges may have necessities along with the requirements listed under. For further data, see the college Undergraduate General Education Requirements section of the Guide.
Monetary Analysis, Planning & Forecasting
From small family companies to multi-million dollar firms, we now have the tools and knowledge to assist virtually any financing need. Extension Credits.Credits earned in credit outreach programs supplied by UW-Eau Claire are treated as resident credit. Credits earned in extension programs offered by different models of the University of Wisconsin System are handled as transfer credits. All other (non-UW) extension and correspondence credit are normally restricted to one-fourth of the entire required for commencement from any curriculum.
Pot Users Welcome: Amazon Won't Test Jobseekers for Cannabis – U.S. News & World Report
Pot Users Welcome: Amazon Won't Test Jobseekers for Cannabis.
Posted: Wed, 02 Jun 2021 00:15:43 GMT [source]
It is tough to create a forecast with a steep development curve if every sale needs to be rationalized and if its level of departure is the maximal capability of your organization . With the bottom up method it is onerous to bear in mind components corresponding to virality or word of mouth.
Compliance Office
It is a term that encompasses a variety of activities and disciplines revolving across the management of money and different valuable assets. Business finance programs Finance in universities familiarize students with accounting methodologies, investing methods and effective debt administration.
- As the $25 million gross sales income mark is high for many small companies, most will solely choose to use the accrual accounting method if their bank requires it.
- Review what inner controls, monitoring and expertise utilities need to appropriately manage grants.
Students on restricted standing must improve their Texas State GPA to at least 2.zero in the subsequent semester or their admission to McCoy College shall be voided. Students are required to fulfill with a representative of the McCoy College Academic Advising Center to remove probation holds; otherwise, the hold will prevent registration or schedule modifications. A scholar whose admission is voided could regain admission to McCoy College by going through the appliance process and competing with other candidates for openings. Students with a Texas State GPA beneath a 2.0 are additionally subject to the University tutorial probation and suspension policies. Students with fewer than 30 semester credit score hours will be automatically admitted to the McCoy College in the event that they obtain assured admission to Texas State and choose a first-choice main within the McCoy College of Business Administration. Students who are admitted to the University but denied admission to McCoy College shall be thought-about for admission to their second choice major or as an exploratory professional major.

subset: commoncrawl


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Bus Timişoara - Bucharest prices from $217.14
Timişoara to Bucharest by bus
The journey from Timişoara to Bucharest by bus is 255.2 mi and takes 6 hr 37 min. There are 1 connections per day, with the first departure at 12:00 AM and the last at 9:20 PM. It is possible to travel from Timişoara to Bucharest by bus for as little as $217.14 or as much as $437.99. The best price for this journey is $217.14.
Lowest Price | $217.14 |
Journey Duration | 6 hr 37 min |
Connection per Day | 1 |
Lowest Price | $217.14 |
Highest Price | $437.99 |
First Departure | 12:00 AM |
Last Departure | 9:20 PM |
Distance | 255.2 mi |
Departure | Timişoara |
Arrival | Bucharest |
Carriers | TAROM, DaytripMore |
Timişoara to Bucharest Bus Times
Get from Timişoara to Bucharest with Virail
Planning to travel from Timişoara to Bucharest? Sometimes it can be hard to know where to start - but that's where Virail comes in. Our powerful search engine will explore all the options available for your trip, combing through more than 2000 transport companies all over the globe. All you have to do is enter your travel dates. In an instant, we'll show you a thorough list of possible routes, with all the information you need to make your decision. Choose from buses, trains, and more, connecting or direct, one-way or round trip - whatever you prefer. When you see your results, select your chosen option and Virail will redirect you to the provider's website, where you can complete your booking. Let us do the legwork for you, and you can just sit back and enjoy your trip.
How can I get the cheapest bus tickets to travel from Timişoara to Bucharest?
The cheapest bus tickets from Timişoara to Bucharest cost $217.14. On average, you can expect to spend around $406.39 for your bus ticket. Virail will help you find a journey that fits your budget. You can expect prices to vary between different travel providers and methods of transport; often, faster journeys will cost more than slower routes. If your travel dates are flexible, you might be able to find cheaper bus tickets, as prices may fluctuate a little from day to day. You may also find lower prices by booking at certain times of day, avoiding rush hour traffic. Another tip for travelers on a tight budget is to look for connecting journeys. Sometimes it can be cheaper to make a change or two along the way, rather than traveling on a direct route from Timişoara to Bucharest.
Unfortunately, no price was found for your trip from Timişoara to Bucharest. Selecting a new departure or arrival city, without dramatically changing your itinerary could help you find price results.
When you're traveling from Timişoara to Bucharest, you can expect to pay an average of approximately $406.39. Some forms of transport are cheaper than others, and prices will vary according to time of day, day of the week, season, and other factors. For the cheapest bus tickets, make your reservation well in advance. If you can, travel at an off-peak time, as bus ticket prices will often be reduced. You might also be able to save money by booking a connecting journey, rather than a direct route. Be sure to check the conditions of the ticket before booking, as sometimes cheaper bus tickets cannot be changed or refunded. Use Virail to check your options before booking, to ensure that you find the lowest price for your journey.
How long will it take me to travel from Timişoara to Bucharest by bus?
The average journey time is 6 hr 37 min. This is how long it takes to cross the 255.2 mi from Timişoara to Bucharest. When planning your journey, remember that delays are a possibility. Some unplanned events could cause your journey from Timişoara to Bucharest to take longer than 6 hr 37 min. The distance between Timişoara and Bucharest is around 255.2 mi. Depending on the exact route and provider you travel with, your journey time can vary. On average, this journey will take approximately 6 hr 37 min. However, the fastest routes between Timişoara and Bucharest take 0 hr 50 min. If a fast journey is a priority for you when traveling, look out for express services that may get you there faster. Some flexibility may be necessary when booking. Often, these services only leave at particular times of day - or even on certain days of the week. You may also find a faster journey by taking an indirect route and connecting in another station along the way.
How many times a day do journeys run from Timişoara to Bucharest?
If you want to get from Timişoara to Bucharest, you can travel with providers such as . You will find a maximum number of departures per day. On average, there are 1 departures per day. Book in advance to make sure that your journey will run smoothly and you depart at a time that suits you best. When you travel, you can look for direct journeys or other routes which offer connections and changes. On average, there are 1 direct journeys between Timişoara and Bucharest. If you don't mind changing along the way, you may find more options available to you. There are usually 0 journeys with at least one change. These numbers may vary depending on the time of year or day of the week. Some travelers prefer direct routes, particularly if they are carrying heavy suitcases or concerned about missing a connection. Others enjoy the flexibility offered by connecting journeys. Whatever you prefer, Virail can help you find the right options to suit you. Unfortunately, no connection was found for your trip from Timişoara to Bucharest. Selecting a new departure or arrival city, without dramatically changing your itinerary could help you find connections.
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Book in advance and save
If you're looking for the best deal for your trip from Timişoara to Bucharest, booking train tickets in advance is a great way to save money, but keep in mind that advance tickets are usually not available until 3 months before your travel date.
Stay flexible with your travel time and explore off-peak journeys
Planning your trips around off-peak travel times not only means that you'll be able to avoid the crowds, but can also end up saving you money. Being flexible with your schedule and considering alternative routes or times will significantly impact the amount of money you spend on getting from Timişoara to Bucharest.
Always check special offers
Checking on the latest deals can help save a lot of money, making it worth taking the time to browse and compare prices. So make sure you get the best deal on your ticket and take advantage of special fares for children, youth and seniors as well as discounts for groups.
Unlock the potential of slower trains or connecting trains
If you're planning a trip with some flexible time, why not opt for the scenic route? Taking slower trains or connecting trains that make more stops may save you money on your ticket – definitely worth considering if it fits in your schedule.
Virail Pro Tip ✌: Booking 10 days in advance save 10% on average
Price as departure date approaches
Bus companies may charge higher ticket prices as the date of departure approaches. Prices in the next days can range from around $70.04 to $557.58 but may be subject to change. Our advice is to book as early as possible to potentially save up to 87% on your trip!
Find the best day to travel to Bucharest by bus
When travelling to Bucharest by bus, if you want to avoid crowds you can check how frequently our customers are travelling in the next 30-days using the graph below. On average, the peak hours to travel are between 6:30am and 9am in the morning, or between 4pm and 7pm in the evening. Please keep this in mind when travelling to your point of departure as you may need some extra time to arrive, particularly in big cities!
Timişoara to Bucharest CO2 Emissions by Bus
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Chicago police officers exchanged gunfire with someone Monday morning in the Loop.
Someone fired at police and officers returned fire near Michigan Avenue and Lake Street, according to a tweet from Chicago police spokesman Tom Ahern. No officers were injured.
The incident happened shortly before 4 a.m. and no one was hit by gunfire, according to Civilian Office of Police Accountability spokesman Ephraim Eaddy. COPA will investigate the shooting.
Police involved shooting Michigan and Lake. Shots fired at police who returned fire. No officers injured. Unknown at this time if offenders were shot. #ChicagoPolice pic.twitter.com/scVQVmT7YG
— Tom Ahern (@TomAhernCPD) August 10, 2020
The incident happened as police responded to widespread looting and property damage downtown.
A day earlier, a person was shot by police and taken into custody after allegedly firing at officers in Englewood.


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Review: Raising Kane by Lorelei James
I received this book for free from Purchased in exchange for an honest review. This does not affect my opinion of the book or the content of my review.
This post contains affiliate links you can use to purchase the book. If you buy the book using that link, I will receive a small commission from the sale.
Book Info
Released: July 2010
Series: Rough Riders #9
Pages: 316
She surrendered the reins. Now he's raising the stakes.
When a patch of ice sends attorney Ginger Paulson head-over-high-heels down a flight of stairs, she has no one to care for her young son and her invalid father – until lethally sexy Kane McKay shows up at the hospital, determined to prove his cowboy chivalry. Past experience has inoculated her against take-charge men, but even Ginger isn't immune to Kane's invasive charm and Built Ford Tough body.
For two years rancher Kane McKay has followed the Little Buddies mentoring program's cardinal rule – hands off his Little Buddy Hayden's mama. But one look at Ginger's bruised body and Kane is through watching the stubborn woman take care of everyone but herself. The feisty, curvy redhead needs his help, and he'll give her the hands-on type whether she likes it or not.
After Kane throws out doctor's orders and issues his own demands – her full sexual submission – Ginger realizes Kane's caring nature extends beyond just fulfilling her physical needs.
Can the former hell-raiser convince the gun-shy single mom to look beyond his past – toward a shared future? From Goodreads
We have heard so much about Kane's hell raising ways in the other books in the series and even had hi featured in a rather tawdry threesome/foursome in "Cowgirl Up and Ride" and now finally Kane (also known as Buck!) has his own book. A book I really enjoyed, I don't know what it is about these twins, but Kane and Kade's book have got to be my top two in this series.
Kane has changed, he is now a responsible guy, despite how he is still seen by some people. However, this just adds to his appeal, he was such a great guy in this book. Loving, kind, hardworking, and ofcourse abso-fricking-lotely gorgeous. And the sex scenes in this were really good, hot and intense, Ginger is one lucky lady!
This series just keeps getting better and better and I really can not get enought of LJ's hot cowboys!
- The Narrator by K.L. Slater Book Review - April 1, 2023
- A Nefarious Engagement by Lynn Messina: A Historical Mystery Review - October 14, 2022
- An Infamous Betrayal by Lynn Messina: Historical Mystery Review - October 6, 2022


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Ultra-rich eye cream targets dehydration lines. This hydrating cream helps increase skin resilience around the delicate eye area with Vitamin A, Wild Yam Extract and Pro-Vitamin B5. Help smooth skin texture as vitamins C and E plus Grape Seed Extract shield the delicate eye area against further environmental damage.
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Perfect for dry skin.
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How do I use it?
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Vitamin A, Wild Yam Extract and Pro-Vitamin B5 help combat signs of premature ageing. Skin-replenishing phytonutrients help increase elasticity and smooth skin texture. Vitamins C, E and Grape Seed Extract shield the delicate eye area against further environmental damage. Formulated without artificial fragrances and colours. Certified cruelty free by Leaping Bunny and PETA.
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'Forty-Year-Old Version' Filmmaker Radha Blank to Be Honored by Sundance Institute
- November 19, 2020
- Dave McNary
- No comments
The Sundance Institute is honoring "The Forty-Year-Old Version" filmmaker Radha Blank with the 2020 vanguard award. The award honors the artistic achievement of her feature film directorial debut, "The Forty-Year-Old Version," which premiered at the 2020 Sundance Film Festival — where it earned her a directing award — and was developed at the Institute's Labs. […]


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2. Sugary cereals
How can you tell the difference between a bad sugary cereal and a good one for your health? Some cereals are full of carbs and sugar. When you eat them, your blood sugar rapidly spikes and then bottoms out. Since you probably do not want your energy to crash at the beginning of the day, it is best to avoid those cereals. Instead, choose cereals with higher fiber and protein. Do you seek for another option? Add ground flaxseed or walnuts for more protein and fiber.
Navigate using pages.
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For various reasons, an user might want editing httpd.conf on OpenShift PaaS. Here is how you can edit and making it working on restart. We have quite good number of articles on OpenShift PaaS, one can use the search feature on this website to get the search result like a list of article by using OpenShift as keyword to search. First you need to know about the Cloud Computing Service Models. Second thing is that, PaaS is not exactly like a cPanel webhosting.
OpenShift has a free tier of usage simply because, when launched, it was intended for the developers. PaaS came with offers but initially had no standards " the big providers presented strategies for Platform as a Service. There are reasons to use PaaS as well as there are shortcomings.
Editing httpd.conf on OpenShift : First Things First
You will need Shell Access on OpenShift, which is possible through the command line tool. Basic UNIX commands and their own commands can be executed on OpenShift PaaS Cloud. MySQL supports interactive mode as well. We have a basic guide on using OpenShift Command Line Tool. We have a small video guide on OpenShift SSH too, please click watch it on YouTube and click the gear icon on YouTube to select the highest resolution (1080p HD).
Editing httpd.conf on OpenShift
May be, you need to make an apache directory alias and define a directory with some permissions. If you add some herdoc statements to deployment script, commit the change, and pushed to rhc, it will not work. Ensure that httpd is installed :
1 | sudo yum install httpd |
We basically need to create a small environment for Apache to run with your username. Our user is abhishekghosh so, should create a directory on this path => /home/abhishekghosh/this-example/
. Commands are easy :
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 | mkdir -p ~/this-example/ # this-example is just an example cd ~/this-example/ # let us create directory for logs, html for static pages, a directory (etc) for configuration, # conf.d for any more changes to Apache mkdir logs run html etc conf.d # copy from original location cp /etc/httpd/conf/httpd.conf etc/ # edit httpd config file looking like this Listen 127.1.2.3:8080 # default is # Listen 80 # 127.1.2.3 is as per documentation # Edit ServerRoot directory ServerRoot œ/home/abhishekghosh/this-example/ # default is /etc/httpd # change abhishekghosh/this-example/ DocumentRoot œhtml/ # it was different by default # symlink the modules ln -s /usr/lib64/httpd/modules/ modules # or ln -s /usr/lib/httpd/modules/ modules # check version of httpd, i.e. 64bit httpd, 32bit httpd etc |
The following command should also start Apache httpd:
1 | httpd -f ~/this-example/etc/httpd.conf |
What we wrote up to this is :
1 2 3 4 | https://github.com/openshift/origin-server/tree/master/cartridges/openshift-origin-cartridge-mock # docs http://openshift.github.io/documentation/oo_cartridge_developers_guide.html http://openshift.github.io/documentation/oo_cartridge_guide.html |
You can clone or download the repo zip from github:
1 | https://github.com/openshift/origin-server/archive/master.zip |
Up to this is a way.
But in case you want an alias pointing to the files outside of the ApacheDocument root, assuming FollowSymLinks is enabled in the cartridge, so all you need is to create a symlink. Here is the Apache conf file :
1 2 3 4 5 6 7 | Alias /your-public-alias "/path/to/outside/of/document/root" <Directory "/path/to/outside/of/document/root"> Options Indexes FollowSymLinks AllowOverride All Order allow,deny Allow from all </Directory> |
1 | ln -s /path/to/outside/of/document/root ${OPENSHIFT_REPO_DIR}/sub-directory/your-public-alias |
The .htaccess file inside of /path/to/outside/of/document/root will override all. Just restart :
1 | rhc app restart |


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If you're looking to buy a business, carrying out a robust analysis of its financial statements will help you understand the financial health of the company.
Amortisation and depreciation are two concepts among many that feature on the balance sheet and income statement, but what exactly are they?
And, more importantly, how can they help you figure out if this business is a viable acquisition?
By calculating its expenses, a business can use them as a tax deduction. In turn, this reduces the business's tax liability.
Depreciation is one method of calculating these expenses, focusing on fixed assets. Fixed assets are purchased with long-term use in mind, such as machinery and office equipment. These items are not bought to be quickly converted into money, as parts would be, but used within the business operationally.
Business owners calculating the depreciation of their fixed assets can do so in many ways, such as:
Straight Line
An asset depreciating in a straight line is when it depreciates the same amount every year, usually spanning a 10-year period.
Declining Balance
A declining balance can be described as an accelerated depreciation method. This way, an asset depreciates more in the early years of life, reducing to smaller amounts towards the end of its useful life.
Units Of Production
Under this method, the amount of depreciation charged is in direct proportion to the total hours the asset has been used for. If machinery is the asset in question, this can be translated to how many units it has produced during its useful life.
An asset depreciating through the sum of years method shows higher levels of depreciation in the first few years which slows down in the later years, similar to declining balance. The difference here is that the remaining useful life of the asset is divided by the sum of its years.
Whilst depreciation is the process by which a fixed asset is charged to expense, amortisation pays attention to intangible assets – assets which have no physical form.
Intangible assets expensed using amortisation can rarely exist outside of the realms of the business they are held in. They can be split into two groups:
- Intellectual property
This category includes things like patents, copyrights, trademarks and computer software. - Goodwill
Goodwill is a miscellaneous category for intangible assets that are hard to quantify but are still identifiable, like customer loyalty and brand reputation. In the sale of a business, goodwill is the extra value paid beyond the fair value of assets.
Imagine that you have acquired a new business. A very popular restaurant. Having added tangible assets to the balance sheet, like kitchen equipment, you'll also need to add the value of the intangible assets, such as the brand. But, if the total intangible assets were added all at once, in this financial year, it would screw up your real numbers and misrepresent the financial health of your business.
Amortisation combats this issue. And, similarly to depreciation, there are various types. It's important to note that the term amortisation is used within both accounting and lending.
Let's have a look at a few of the methods.
Straight Line
Straight line amortisation is the most common method and sees the book value of an intangible asset equally distributed across its useful life. In the context of a loan, the payable interest is spread over the life of a loan.
Declining Balance
As with declining balance depreciation, this technique sees the intangible asset amortised more in the first few years of its useful life, reducing to smaller amounts later on. Within lending, it is similar to paying off a mortgage, in that the interest charged decreases over time.
Amortising a loan through annuity looks like a set of equal payments made between equal time intervals. Examples of annuities could include regular deposits into a savings account and monthly insurance payments.
There are two types of annuity:
- Ordinary annuity
With this method, payments are made at the end of each interval period. - Annuity Due
Under these rules, payments are made at the beginning of each interval period.
Bullet And Balloon
Bullet and balloon style repayments aren't usually amortised over the life of the loan. Instead, borrowers make little or no payments for much of the loan's life, paying most or all of the loan off at the end of the term.
Because they are the most straightforward, straight line amortisation is nearly always the method you will see used in business, closely followed by declining balance.
Depreciation And Amortisation Help To Spread The Cost Of Assets Over Time
The difference is that depreciation expenses fixed assets and amortisation expenses intangible assets.
If you're analysing the financial documents of a business you want to buy, you need to know about these two methods to really understand how profitable it is. But it can be difficult to get your head around.
Feeling stuck? Not everyone has a head for accounting. Book a call with The Business Consultant and take the weight off.


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Diversity, Equity and Inclusion – A Core Value and a Strategic Goal
The Stevens Strategic plan details the core values embedded throughout all that we do and emphasizes that we are one Stevens community.
Overall, we strive to ensure that all members of our community feel welcome, are included and have opportunities to succeed. We work individually and collectively to advance our university and members of our community while engendering pride in our affiliation with Stevens.
As a global community, we are committed to recruiting a diverse population of students, staff, and faculty, while cultivating inclusive learning and working environments where people of all backgrounds can thrive. To maximize efforts to achieve these shared goals, many campus initiatives are the result of collaborations across Stevens' units.
Strategic efforts toward these goals have existed at Stevens for decades. Some recent collaborations include:
Inclusive Workplace Workshops - designed and facilitated by Stevens faculty to help employees learn and practice strategies that foster inclusion through the use of critical incidents and collaborative decision-making. School of Business, Office of the President
ONGIG – A text analyzing tool that provides suggestions on how to remove biased language and gendered words from your writing. Job postings at Stevens are reviewed through ONGIG to reach the most potential candidates. Divisions of Human Resources, Office of the President
NameCoach – A university-wide initiative to foster belonging and inclusion by providing the technology to share and search audio clip name pronunciations of faculty, students, and staff. Office of the President, Division of Information Technology
Faculty Tips for Inclusive Engagement – To build strong faculty-student interactions and create supportive and inclusive learning environments, research-based tips with additional resources are available to all faculty. Office of the President, Office of Faculty Development


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Product Overview
Brand New/Never Used/Original Packaging
30921 Carrera Digital 132 Porsche 935 GT2, #96/69
Porsche celebrated itself for the 70th anniversary and made the hearts of motorsport fans beat faster with the new edition of the Porsche 935 "Moby Dick". With Carrera, two variants jet over the rails. The Porsche 935 GT2 "No.96 / 69" is a very special variant of the anniversary racer at the start line, because the paintwork was designed by Carrera and was a real eye-catcher at the 24-hour race at Spa-Francorchamps 2019. The 1:32 scale digital slot car is powered by an E200 motor. Each car is fitted with replaceable double contact brushes for maximum contact with the slot car track while downforce magnets make for quick cornering and great handling. Carrera digital cars are real head-turners with high-quality tampo-printing over detailed molds designed to faithfully replicate the original car. Working front headlights and rear brake lights add to the stunning look and even the interior and the driver inside looks just as good as the exterior! This Carrera Digital 132 Car in 1:32 scale will work on all Carrera Digital slot car tracks. The switch located on the bottom of the car also allows you to switch to analog mode for racing on an Evolution track.
- Presented in clear display case
- Red and blue in color, #96/69.
- Operating headlights and taillights.
- Powered by an E200 motor and fitted with replaceable double contact brushes for maximum contact with the slot car track. Downforce magnets underneath make for quick cornering and great handling.
- Working headlights and taillights combined with a detailed interior and high-quality tampo-printing over a detailed mold designed to faithfully replicate the original car
- Compatible with Carrera Digital 132 and 124 slot car tracks. Can also be switched to analog mode to race on Carrera Evolution tracks by simply sliding the switch located on the bottom of each car.
- Not Recommended to use on the 1/30 High Bank Curve (2/30 High Bank and wider is okay)


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Everyone today includes a blog site, it is just one of those activities that may be catching on and what folks are using to have their sound observed. Should you wish to see how you can create and keep an internet based existence this report and each of the skills within must assist you to do exactly that.
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Building an internet reputation by way of running a blog is a thing which takes time, take this into account. Remember that this quicker you start out the sooner your possible ways to develop an audience gets to be. So do your best to make use of all the expertise you learned as well as the viewers you've been trying to find should follow.
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Nottingham Forest made it three wins in a row as they came from behind to topple Hull City 2-1 at the City Ground.
A Lewis Grabban penalty and a lovely finish from Brennan Johnson made it nine games unbeaten for Steve Cooper's men.
The hosts dominated the first half, right up until the point they were forced into a formation change when they lost Jordi Osei-Tutu to injury.
But they were punished for not making the most of all the possession they had. Despite playing some lovely football, they didn't create too many clearcut chances - and were unable to finish off the ones they did have.
Grabban had a couple of sights of goal in the opening 20 minutes.
On the first occasion, he couldn't quite catch a first-time effort right when he tried to divert a brilliant Jack Colback ball goalwards. Then, 10 minutes later, he flicked wide at the near post from a James Garner free-kick.
Johnson came closest to a breakthrough, however, following a delightful link-up with Djed Spence. The latter expertly back-heeled the ball into his teammate's path, with Johnson then rolling a cross-shot across goal and inches wide of the far post.
Joe Worrall tried his luck from range soon after, running on to the ball before blasting it - just with rather more power than precision.
Osei-Tutu had required treatment after going down with a knock in the opening stages. He tried to continue, but was forced off with 40 minutes gone.
And that left Forest all out of sorts as they switched from three at the back to four, with Jack Colback slotting in at left-back and Ryan Yates going into the middle.
They were caught napping two minutes later, when a Keane Lewis-Potter beat Spence to a lofted pass, showed a turn of pace and then belted it past Brice Samba to give the visitors the lead.
The Tigers could have increased that advantage when Lewis-Potter was left with a free header at a corner, but the Reds went in at the break 1-0 down.
They responded quickly in the second half as Philip Zinckernagel had Baxter at full stretch with a header from Johnson's cross, before Alex Mighten was brought down to earn a penalty.
The youngster was injured in the process, but Grabban was clinical from the spot to make it 1-1.
And the front man then turned provider in the 72nd minute, setting up Johnson to provide a fine finish for 2-1.
Here's how Reds reporter Sarah Clapson rated the Forest players at the City Ground.
Brice Samba: Some of his distribution wasn't quite up to his usual standard, and was a little careless with the ball at his feet at times. Not tested too often, but made a good save right at the end. Rating: 7/10
Joe Worrall: Growing in confidence whenever he has the ball. Developing into a very good footballer, as well as a solid defender. Not much got past him. Rating: 8/10
Ryan Yates: Started off in defence, where he looked really comfortable - composed on the ball and playing some neat passes. Moved into the centre when Forest had to reshuffle. Rating: 8/10
Scott McKenna: At ease in either a three or four-man defence, just getting on with the job. Made some important clearances when Hull had their moments. Rating: 8/10
Djed Spence: Switched off for the visitors' goal as he was caught out. Going forward, however, he was excellent. Could watch his first-half back-flick to Johnson on repeat all day. Rating: 7.5/10
Jack Colback: Forced into the left-back role again once Osei-Tutu departed. Did a job, but not where he's at his best. Had been pulling the strings in the centre of the pitch. Rating: 8/10
James Garner: Really hitting his stride now, back to the kind of form he showed last season - even if his set-pieces could perhaps do with a bit of work at times. Rating: 7.5/10
Jordi Osei-Tutu: A real shame to see him forced off. Had been really bright when he was on the pitch, starting to show what he can do. Just desperately unlucky with injuries. Rating: 7/10
Brennan Johnson: That touch! Sublime bit of control, and a delightful finish to get on the scoresheet. Complete and utter class. Thoroughly deserved, too - and a joy to watch. Rating: 9/10
Garibaldi Red podcast - listen and subscribe
The Garibaldi Red podcast is our weekly Nottingham Forest podcast featuring fans, ex-players and managers or Forest supporting journalists, such as Darren Fletcher, Daniel Storey and Gregor Robertson.
Interviewees have included Frank Clark, Paul Hart, Sean O'Driscoll, Ian Storey-Moore, Paul Anderson and Nathan Tyson, while Reds legend Garry Birtles is a regular guest.
When and how does it go out?
The show goes out at least once a week, every week, either on Mondays, Wednesdays or both. Episodes tend to be 45 minutes to an hour long.
How to subscribe
It can be found on all major podcast platforms and we have a YouTube channel. The show is often streamed live on our Facebook page, which means we take comments and questions from listeners in real time.
Listen and subscribe on Apple Podcasts here or you can watch episodes and subscribe to our YouTube channel. Most episodes are also streamed on Facebook so like our page for alerts when videos will begin.
You can also subscribe to our weekly newsletter here. Just select latest Nottingham Forest news.
Philip Zinckernagel: Not too far away from getting on the scoresheet, with a header which drew a very good save. Worked incredibly hard, even if he didn't always get the rewards to show for it. Rating: 7/10
Lewis Grabban: Cool as you like from the spot, despite having to wait a while to take the penalty - and having missed a couple of decent chances in the first half. Then teed up the winner with a great bit of play - and turn of pace - down the flank. Rating: 8.5/10
Alex Mighten (for Osei-Tutu, 40mins): Had made an impact when he came on, forging an opening before the break and then winning the penalty. Looked in some discomfort when he went down, though. Rating: 7/10
Joao Carvalho (for Mighten, 57mins): A big chance to impress as he made his first appearance since August. Didn't have too many touches of the ball, though. Rating: 6/10
Tobias Figueiredo (for Zinckernagel, 79mins): Helped his team to see out the win as they kept the Tigers at bay in the closing minutes. Rating: 6/10
Subs not used: Horvath, Bong, Cafu, Taylor.
We've teamed up with LiveScore to invite fans to nominate their favourite results - the games which were much more than a score. Nominate the match that mattered to you here


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Last month NOAA scientists Drs. Andrea Kroetz and John Carlson returned to Everglades National Park and Ten Thousand Islands National Wildlife Refuge. They resumed sampling for endangered smalltooth sawfish in their critical habitat area. NOAA scientists have been monitoring the relative abundance, distribution, and habitat use of juvenile smalltooth sawfish there since 2009. Critical habitat is a habitat area essential to the conservation of a listed species.
Part of our monitoring program includes acoustically tracking this species with long-term acoustic tags (5 and 10 year tags). These help us monitor migration patterns and identify specific habitats used by juveniles and adult sawfish. Learning more about the specific habitats that various life stages of sawfish use can help scientists refine critical habitat or even potentially designate new areas. This is particularly important for large juvenile and adult sawfish for which critical habitat has not yet been designated.
NOAA Fisheries' biologists have deployed 26 acoustic receivers throughout the area to monitor tagged sawfish. During the May research expedition, they retrieved 20 receivers and downloaded thousands of detection data. Many sawfish that NOAA scientists and their partners have tagged since 2016 (more than 70 sawfish) were detected on our receivers.
Additionally, they also detected several species of shark, ray, and teleosts tagged by various state, federal, and non-governmental institutions. Our acoustic array is important for the long-term monitoring of endangered smalltooth sawfish. It has also proven to be important for monitoring the movements of many other species that call Ten Thousand Islands and Everglades National Park home.
Our continued sampling provided seven new sawfish that we captured, sampled, tagged, and released, two of which swam away with acoustic tags. The first sawfish implanted with a 10-year acoustic tag was a 7-foot female and a nearly 5-foot female was tagged with a 5-year tag. Overall, a successful research expedition that adds valuable data contributing to the recovery monitoring of smalltooth sawfish.


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Mumbai is the only major city where these double-deckers are still in service. But are you aware- "Mumbai's Double decker buses are back with a new look & in a new form!" Read to know more.
What "BEST" officials states about double decker buses?
The recognizable double-decker buses of Mumbai are about to return to service in an electric form. Brihanmumbai Electric Supply and Transport Undertaking (BEST) officials predicted that by the end of the year, their numbers will match the peaks of the 1990s.
According to officials, these double-deckers were formerly present in Kolkata, Bengaluru, Delhi, Thiruvananthapuram, and Chennai. However, they state that the financial capital is the sole major location where they are still in operation. Before the numbers began to decline after 1995, BEST had the most double-decker buses. As losses grew and operational expenses increased, the city's top road transportation service had 225 double-deckers by 2006.
Also Read: Neeraj Chopra wins heart again! Becomes first Indian to ace Diamond League.
There are still about 50 of them in Mumbai, with the majority of them operating in the southern part of the city. Other existing buses serve as guides for visitors on historical excursions.
The Very First Launch in India
India's first electric air-conditioned double-decker bus was shown last week. " BEST" general manager Lokesh Chandra promised that it would start travelling the country's highways in September.
What Lokesh Chandra has to say about?
By the end of 2023, two bus vendors will provide BEST with 900 electric double-deckers. Chandra(GM of BEST) said, " We had more than 900 double-decker buses in the 1990s. By the end of next year, we'll be back there".
Double-deckers with a length of 10.5 meters can transport 100 passengers. This is in contrast to the load that regular buses can carry. The BEST GM gave a statement: When smaller buses were introduced, we lost lakhs of passengers. Furthermore, he stated, "These double-deckers would regain the daily carrying capacity of 1.5 lakh seats."
Features of new "double decker Buses"
The new electric AC double-decker bus has a plethora of features. This includes a dual door-stairway configuration, larger windows, bucket seats, CCTVs, charging ports for computers and smartphones, and public address systems.
Changed Outlook?
Commencing with those that resembled London's Routemaster in appearance, they had a single passenger cabin and a sloping engine roof. Over the years, double-deckers have seen some design alterations.
Sunil Ganacharya, a former member of the BEST Committee, the Undertaking's policy-making body, commented on the fall in the number of these vehicles. He states, "Operational expenses were a big concern because it required the deployment of two conductors as well as increased diesel
Read More: "BEST" easiest approach to go between Thane, Kharghar, and BKC!
Moreover, the fall in numbers was also brought on by manufacturers' ceasing to produce these double-decker buses.
During the unveiling of the new double-decker last week, BEST GM Chandra said—"Contractors are responsible for the expense of personnel and maintenance." In support of this, he said, "This happened because the new electric buses are arriving as part of a wet lease arrangement."
He said that although the double-decker and single-decker e-buses on wet lease models cost 56 and 46 dollars per kilometre, respectively, the operational cost of their conventional buses is 150 dollars per kilometre.
Capacity and Charges
According to the authorities, "the capacity of double-decker buses is 78 people, while that of single-deck electric buses is 54 passengers. A double decker bus generates Rs. 79 of income every kilometre, compared to Rs. 43 for a single decker bus.