Colistin is increasingly being utilized against Gram-negative pathogens, including Pseudomonas aeruginosa, resistant to all other antibiotics. Since limited data exist regarding killing by colistin at different initial inocula (CFUo), we evaluated killing of Pseudomonas aeruginosa by colistin at several CFUo and developed a mechanism-based mathematical model accommodating a range of CFUo. In vitro time-kill experiments were performed using >8 concentrations up to 64 ؋ the MIC of colistin against P. aeruginosa PAO1 and two clinical P. aeruginosa isolates at CFUo of 10 6 , 10 8 , and 10 9 CFU/ml. Serial samples up to 24 h were simultaneously modeled in the NONMEM VI (results shown) and S-ADAPT software programs. The mathematical model was prospectively "validated" by additional time-kill studies assessing the effect of Ca 2؉ and Mg 2؉ on killing of PAO1 by colistin. Against PAO1, killing of the susceptible population was 23-fold slower at the 10 9 CFUo and 6-fold slower at the 10 8 CFUo than at the 10 6 CFUo. The model comprised three populations with different second-order killing rate constants (5.72, 0.369, and 0.00210 liters/h/mg). Bacteria were assumed to release signal molecules stimulating a phenotypic change that inhibits killing. The proposed mechanism-based model had a good predictive performance, could describe killing by colistin for all three studied strains and for two literature studies, and performed well in a prospective validation with various concentrations of Ca 2؉ and Mg 2؉ . The extent and rate of killing of P. aeruginosa by colistin were markedly decreased at high CFUo compared to those at low CFUo. This was well described by a mechanism-based mathematical model, which should be further validated using dynamic in vitro models.

The mitochondrion of Trypanosoma brucei bloodstream form maintains a membrane potential, although it lacks cytochromes and several Krebs cycle enzymes. At this stage, the ATP synthase is present at reduced, although significant, levels. To test whether the ATP synthase at this stage is important for maintaining the mitochondrial membrane potential, we used RNA interference (RNAi) to knock down the levels of the ATP synthase by targeting the F 1 -ATPase ␣ and ␤ subunits. RNAi-induced cells grew significantly slower than uninduced cells but were not morphologically altered. RNAi of the ␤ subunit decreased the mRNA and protein levels for the ␤ subunit, as well as the mRNA and protein levels of the ␣ subunit. Similarly, RNAi of ␣ subunit decreased the ␣ subunit transcript and protein levels, as well as the ␤-subunit transcript and protein levels. In contrast, ␣ and ␤ RNAi knockdown resulted in a 60% increase in the F 0 complex subunit 9 protein levels without a significant change in the steady-state transcript levels of this subunit. The F 0 -32-kDa subunit protein expression, however, remained stable throughout induction of RNAi for ␣ or ␤ subunits. Oligomycin-sensitive ATP hydrolytic and synthetic activities were decreased by 43 and 44%, respectively. Significantly, the mitochondrial membrane potential of ␣ and ␤ RNAi cells was decreased compared to wild-type cells, as detected by MitoTracker Red CMXRos fluorescence microscopy and flow cytometry. These results support the role of the ATP synthase in the maintenance of the mitochondrial membrane potential in bloodstream form T. brucei.The mitochondrial ATP synthase couples the electrochemical proton gradient to the synthesis or hydrolysis of ATP (5,11,26,39). The ATP synthase is composed of the soluble F 1 moiety, which contains the catalytic sites and the membranebound F 0 moiety, which is involved in proton translocation. The F 1 moiety of the mitochondrial ATP synthase is highly conserved and is composed of five subunits present in a stoichiometry of ␣ 3 ␤ 3 ␥ 1 ␦ 1 ε 1 . The F 0 moiety in Escherichia coli is composed of three subunits, a 1 b 2 c 10-14 , but in eukaryotes its subunit composition increases in complexity to include up to eight additional subunit types (5,22,26). The Trypanosoma brucei mitochondrial ATP synthase has been isolated and characterized. The molecular composition of the enzyme complex is similar to that of other eukaryotic mitochondrial ATP synthases (43). Both functional assays and analysis of protein levels indicate that the complex is developmentally regulated through the life cycle of the organism (7,41,42).A striking feature of T. brucei is its ability to adapt to diverse environments encountered through the stages of its life cycle (21, 34). In the tsetse fly, the mitochondrion of the procyclic trypanosomes is fully developed with many cristae, a complete respiratory chain, Krebs cycle enzymes, and abundant levels of mitochondrial ATP synthase. In contrast, the sparse and tubular mitochondrion of the early (slender) mammalian blood...

Efficacious therapy is of utmost importance to save lives and prevent bacterial resistance in critically ill patients. This review summarizes pharmacokinetic (PK) and pharmacodynamic (PD) modeling methods to optimize clinical care of critically ill patients in empiric and individualized therapy. While these methods apply to all therapeutic areas, we focus on antibiotics to highlight important applications, as emergence of resistance is a significant problem. Nonparametric and parametric population PK modeling, multiple-model dosage design, Monte Carlo simulations, and Bayesian adaptive feedback control are the methods of choice to optimize therapy. Population PK can estimate between patient variability and account for potentially increased clearances and large volumes of distribution in critically ill patients. Once patient- specific PK data become available, target concentration intervention and adaptive feedback control algorithms can most precisely achieve target goals such as clinical cure of an infection or resistance prevention in stable and unstable patients with rapidly changing PK parameters. Many bacterial resistance mechanisms cause PK/PD targets for resistance prevention to be usually several-fold higher than targets for near-maximal killing. In vitro infection models such as the hollow fiber and one-compartment infection models allow one to study antibiotic-induced bacterial killing and emergence of resistance of mono- and combination therapies over clinically relevant treatment durations. Mechanism-based (and empirical) PK/PD modeling can incorporate effects of the immune system and allow one to design innovative dosage regimens and prospective validation studies. Mechanism-based modeling holds great promise to optimize mono- and combination therapy of anti-infectives and drugs from other therapeutic areas for critically ill patients.

We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to bind 5S rRNA in Trypanosoma brucei. These two proteins are nearly identical, with one major difference, an 18-amino-acid insert in the N-terminal region of p37, as well as three minor single-aminoacid differences. Homologues to p34 and p37 have been found only in other trypanosomatids, suggesting that these proteins are unique to this ancient family. We have employed RNA interference (RNAi) studies in order to gain further insight into the interaction between p34 and p37 with 5S rRNA in T. brucei. In our p34/p37 RNAi cells, decreased expression of the p34 and p37 proteins led to morphological alterations, including loss of cell shape and vacuolation, as well as to growth arrest and ultimately to cell death. Disruption of a highermolecular-weight complex containing 5S rRNA occurs as well as a dramatic decrease in 5S rRNA levels, suggesting that p34 and p37 serve to stabilize 5S rRNA. In addition, an accumulation of 60S ribosomal subunits was observed, accompanied by a significant decrease in overall protein synthesis within p34/p37 RNAi cells. Thus, the loss of the trypanosomatid-specific proteins p34 and p37 correlates with a diminution in 5S rRNA levels as well as a decrease in ribosome activity and an alteration in ribosome biogenesis.Ribosomes are essential in all organisms, and their assembly is highly conserved and coordinated. Over 100 accessory proteins are necessary in order for proper processing of ribosomal RNAs and ribosome assembly to occur (8). Ribosomal proteins must be imported from the cytoplasm. The 45S precursor rRNA must be processed to yield 5.8S, small-subunit (SSU) (18S), and large-subunit (LSU) (28S) rRNAs. 5S rRNA, which is independently transcribed within the nucleoplasm, must be imported into the nucleolus by the L5 ribosomal protein for ribosome assembly to occur (16). Ribosomal subunits are subsequently exported to the cytoplasm, where the pre-40S ribosomal subunit undergoes its final processing step (29). In eukaryotes, RNA binding proteins mediate a variety of cellular activities, including mRNA maturation, trafficking, stability, and translational control of mRNA as well as having roles in ribosomal biogenesis (14).The parasite Trypanosoma brucei and its subspecies cause human sleeping sickness (T. brucei gambiense and T. brucei rhodesiense) and nagana in livestock (T. brucei brucei) (31). These organisms continue to pose a serious threat to human health and to cause devastating economic losses (1). Little is currently known about RNA binding proteins and small nucleolar RNAs that are involved in rRNA processing and posttranscriptional modifications in T. brucei. Two proteins with homology to 5S rRNA binding proteins in higher eukaryotes, the La autoantigen and the ribosomal L5 protein, have been identified in T. brucei (19, 34) A family of nucleolar phosphoproteins termed NOPP44/46 proteins have also been identified in this organism and implicated in large ...

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