Radial decline of the extracellular action potential

A modified line source model presented earlier has been used to study the decline of the extracellular single muscle fibre action potential. The muscle tissue is modelled as a low-pass filter. The transfer function of the filter declines more slowly than a first order low-pass filter at low frequencies, but much faster at high frequencies. The cutoff frequency of the filter increases when the anisotropy of the muscle decreases. It also increases proportionally with the propagation velocity of the action potential. The decline of different frequency components obtained from the modified line source volume conductor and a filter model derived from experimental measurements are compared and their differences explained. The modified line source model was found to be identical to the volume conductor model in terms of results and at the same time conceptually simple for applications.     

Colistin pharmacokinetics: the fog is lifting

Colistin is a re-emerging old antibiotic that is used to treat multidrug-resistant infections in critically ill patients. It corresponds to a mixture of at least 30 different compounds administered as inactive derivatives. Therefore, colistin pharmacokinetics are quite difficult to investigate and complex to predict. However specific chromatographic methods have been made available in recent years, leading to a series of modern pharmacokinetic studies after intravenous administration of the prodrug to critical-care patients; these have been conducted by a few groups and have only been recently published. The objective of this article was to conduct a critical review of these very informative modern pharmacokinetic studies and to provide prospective thoughts.     

Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel from influenza A virus

The M2 protein of the influenza A virus is activated by low endosomal pH and performs the essential function of proton transfer into the viral interior. The resulting decrease in pH within the virion is essential for the uncoating and further replication of the viral genetic material. The x-ray crystal [Stouffer AL, et al. (2008) Nature 451:596–599] and solution NMR [Schnell JR, Chou JJ (2008) Nature 451:591–595] structures of the transmembrane region of the M2 homo-tetrameric bundle both revealed pores with narrow constrictions at one end, leaving a question as to how protons enter the channel. His-37, which is essential for proton-gating and selective conduction of protons, lies in the pore of the crystallographic and NMR structures. Here, we explore the different protonation states of the His-37 residues of the M2 bundle in a bilayer using molecular dynamics (MD) simulations. When the His-37 residues are neutral, the protein prefers an Open_out-Closed_in conformation in which the channel is open to the environment on the outside of the virus but closed to the interior environment of the virus. Diffusion of protons into the channel from the outside of the virus and protonation of His-37 residues in the tetramer stabilizes an oppositely gated Closed_out-Open_in conformation. Thus, protons might be conducted through a transporter-like mechanism, in which the protein alternates between Open_out-Closed_in and Closed_out-Open_in conformations, and His-37 is protonated/deprotonated during each turnover. The transporter-like mechanism is consistent with the known properties of the M2 bundle, including its relatively low rate of proton flux and its strong rectifying behavior.     

Probing the α-Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis

Reactivation of the p53 cell apoptosis pathway through inhibition of the p53-hDM2 interaction is a viable approach to suppress tumor growth in many human cancers and stabilization of the helical structure of synthetic p53 analogs via a hydrocarbon cross-link (staple) has been found to lead to increased potency and inhibition of protein–protein binding (J. Am. Chem. Soc. 129: 5298). However, details of the structure and dynamic stability of the stapled peptides are not well understood. Here, we use extensive all-atom molecular dynamics simulations to study a series of stapled α-helical peptides over a range of temperatures in solution. The peptides are found to exhibit substantial variations in predicted α-helical propensities that are in good agreement with the experimental observations. In addition, we find significant variation in local structural flexibility of the peptides with the position of the linker, which appears to be more closely related to the observed differences in activity than the absolute α-helical stability. These simulations provide new insights into the design of α-helical stapled peptides and the development of potent inhibitors of α-helical protein–protein interfaces.     

Identification of cytochrome P450 enzymes involved in the metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a designer drug,in human liver microsomes

1. The metabolism of 4′-methoxy-α-pyrrolidinopropiophenone (MOPPP), a novel designer drug, to its demethylated major metabolite 4′-hydroxy-pyrrolidinopropio-phenone (HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes.

2. CYP2C19 catalysed the demethylation with apparent K_m and V_max values of 373.4 ± 45.1?μM and 6.0 ± 0.3?pmol?min^?1?pmol^?1 CYP, respectively (mean ± SD). Both CYP2D6 and HLM exhibited clear biphasic profiles with apparent K_m,1 values of 1.3 ± 0.4 and 22.0 ± 6.5?μM, respectively, and V_max,1 values of 1.1 ± 0.1 pmol?min^?1?pmol^?1 CYP and 169.1 ± 20.5?pmol?min^?1?mg^?1 protein, respectively.

3. Percentages of intrinsic clearances of MOPPP by particular CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4′-hydroxylation or bufuralol-1′-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively.

4. MOPPP, HO-PPP and the standard 3′,4′-methylenedioxy-pyrrolidinopropio-phenone (MDPPP) were separated and analysed by liquid chromatography–mass spectrometry in the selected-ion monitoring (SIM) mode.

5. The CYP2D6 specific chemical inhibitor quinidine (3?μM) significantly (?p<0.0001) inhibited HO-PPP formation by 91.8 ± 0.5% (mean ± SEM) in incubation mixtures with HLM and 2?μM MOPPP.

6. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 is the enzyme mainly responsible for MOPPP demethylation.     

Semi-physiological pharmacokinetic–pharmacodynamic modeling and simulation of 5-fluorouracil for the whole time course of alterations in leukocyte,neutrophil and lymphocyte counts in rats

Abstract

1. We aimed to develop a simple pharmacokinetic–pharmacodynamic (PK–PD) model to predict the onset and degree of severe toxic side effects that severely limit the use of many anticancer agents, such as myelosuppression, in rats.

2. Our PK–PD model consisted of a two-compartment PK model, with one compartment representing proliferative cells and some transit compartments consisting of maturing cells, while the other compartment represented circulating blood cells for the PD model.

3. The semi-physiological PK–PD model effectively captured the features of myelosuppression and the degree of the off-target toxicities observed after 5-fluorouracil (5-FU) chemotherapy, and helped simultaneously simulate the whole time course for alterations in leukocyte, neutrophil and lymphocyte counts after 5-FU treatment in rats. Interestingly, by plotting the nadir period of leukocyte, neutrophil and lymphocyte counts as determined by PK–PD analytical simulation curves against the area under the plasma 5-FU concentration–time curve (AUC_0–∞) after intravenous administration of 5-FU, a linear relationship was inferred, with r^2?=?0.989, 0.877 and 0.956, respectively.

4. The semi-physiological PK–PD model is a valuable tool for evaluating a variety of novel cancer chemopreventive agents or emerging therapeutic strategies that are difficult to address in humans.     

My Career as a Pharmacometrician and Commentary on the Overlap Between Statistics and Pharmacometrics in Drug Development

As part of a series of articles celebrating the American Statistical Association's 175th anniversary in 2014, this article provides a historical perspective of key statistical contributions to pharmacometrics (the design, modeling, and analysis of experiments involving complex dynamic systems) as well as a commentary on the author's career as a pharmaceutical industry statistician and pharmacometrician. Individuals with training in various academic disciplines including pharmacokinetics, pharmacology, engineering, and statistics, to name a few, have pursued careers as pharmacometricians. While pharmacometrics has benefitted greatly from advances in statistical methodology, there continues to be tension and skepticism between biostatisticians and pharmacometricians as they apply their expertise to drug development problems. This article explores some of the root causes for this tension and provides some suggestions for improving collaborations between statisticians and pharmacometricians. The article concludes with a plea for more statisticians to consider careers as pharmacometrics practitioners.     

Deactivation blocks proton pathways in the mitochondrial complex I

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the “active”-to-“deactive” transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

In mitochondrial cellular respiration, the membrane-bound enzyme complexes I, III, and IV convert chemical energy into a flux of electrons toward dioxygen (1–6). The free energy of the process is transduced by pumping protons across the inner mitochondrial membrane (IMM), powering oxidative phosphorylation and active transport (7, 8). The electron transport process is initiated by the respiratory complex I (NADH:ubiquinone oxidoreductase), a 45-subunit modular enzyme machinery that shuttles electrons from nicotinamide adenine dinucleotide (NADH) to ubiquinone (Q₁₀) and transduces the free energy by pumping protons across the IMM, generating a proton motive force (pmf) (1, 4–6) (Fig. 1). This proton-coupled electron transfer reaction is fully reversible, and complex I can also operate in reverse electron transfer (RET) mode, powering ubiquinol oxidization by consumption of the pmf. Such RET modes become prevalent under hypoxic or anoxic conditions that may result, e.g., from stroke or tissue damage (9), during which the electrons leak from complex I to molecular oxygen and result in the formation of reactive oxygen species (ROS) with physiologically harmful consequences (9–11). To regulate this potentially dangerous operation mode, the mammalian complex I can transition into a “deactive” (D) state with low Q₁₀-turnover activity (12, 13). Although some structural changes involved in the “active”-to-“deactive” (A/D) transition were recently resolved (14–16), the molecular details of how this transition regulates enzyme turnover and its relevance during in vivo conditions still remain puzzling. Moreover, it is also debated whether conformational changes linked to this transition are involved in the native catalytic cycle of all members of the complex I superfamily or whether this transition is specific for the mitochondrial enzyme (12, 13, 17).Open in a separate windowFig. 1.Structure and function of the mammalian complex I. (A) Electron transfer from NADH reduces quinone (Q) to quinol (QH₂) and triggers proton pumping across the membrane domain. (Inset) Closeup of the ND1/ND3/ND4L/ND6 interface involved in the "active" to "deactive" transition. TM3^ND6, which has been linked to conformational changes in the A/D transitions, is marked. (B) An intact atomic model of the deactive state was constructed using MDFF based on the cryoEM structure of the “active” state and the density map of the “deactive” state. (C and D) Conformational changes during the A (in pink)/D (in brown) transition during the MDFF simulations at the TM3^ND6 region, with the D state density map shown. Refer to SI Appendix, Figs. S2, S3, and S12 for other conformational changes. (E) The dihedral angle, ϕ, for ND6 residues Leu51(Cβ)-Leu51(Cα)-Phe67(Cα)-Phe67(Cβ) during MD simulations in the “active” and “deactive” states in comparison to refined cryoEM models.The recently resolved cryo-electron microscopy (cryoEM) structures of the “deactive” mammalian complex I at around 4-Å resolution highlighted conformational changes around several subunits close to the interface between the hydrophilic and membrane domains of complex I. Particularly interesting are the conformational changes around the membrane domain subunits ND4L, ND3, and ND6 (ND for NADH Dehydrogenase) that form a bundle of 11 transmembrane (TM) helices, connected by long loop regions (14–16). Notably, it was observed that TM3 of ND6 transitions from a fully α-helical form in the “active” state to a π-bulge around residues 60 to 65 during the deactivation process (14–16). Although the exact relevance of these conformational transitions remains debated, it is notable that several point mutations in the vicinity of these regions have been linked to mitochondrial disease (11), supporting their possible functional relevance. Structural changes during complex I deactivation, inferred from the lack of density in the cryoEM maps (14–16), were also suggested to take place in several loop regions of the membrane domain, near the Q₁₀ binding site tunnel, and around the supernumerary subunits NDUFA5/NDUFA10 (16, 18). However, the functional consequences of these structural changes and their coupling to the biological activity still remain unclear.To probe how structural changes linked to deactivation could affect the protonation and quinone dynamics of the mammalian complex I, we combine here atomistic molecular dynamics (MD) simulations and hybrid quantum/classical (QM/MM) free energy calculations with cryoEM data (16, 19). Our combined findings suggest that conformational changes around the ND1/ND3/ND4L/ND6 interface and conserved loop regions could block the coupling between proton pumping- and redox-modules upon complex I deactivation. The explored molecular principles are of general importance for elucidating energy transduction mechanisms in the mammalian respiratory complex I and possibly other bioenergetic enzyme complexes, but also for understanding the development of mitochondrial diseases.     

An Improved Method of Minimizing Tool Vibration during Boring Holes in Large-Size Structures

The paper presents a thoroughly modified method of solving the problem of vibration suppression when boring large-diameter holes in large-size workpieces. A new approach of adjusting the rotational speed of a boring tool is proposed which concerns the selection of the spindle speed in accordance with the results of the simulation of the cutting process. This streamlined method focuses on phenomenological aspects and involves the identification of a Finite Element Model (FEM) of a rotating boring tool only and validating it with a real object, while dispensing with discrete modelling of a completely rigid workpiece. In addition, vibrations in the boring process in all directions were observed, which implies a geometric nonlinearity of the process model. During the simulation, the values of the Root Mean Square (RMS) of the time plots and the dominant values of the “peaks” in the displacement amplitude spectra were obtained. The effectiveness of the method was demonstrated using a selected mechatronic design technique called Experiment-Aided Virtual Prototyping (E-AVP). It was successfully verified by measuring the roughness of the indicated zone of the workpiece surface. The economic profitability of implementing the method in the production practice of enterprises dealing with mechanical processing is also demonstrated.