Long-term platinum retention after treatment with cisplatin and oxaliplatin

Background  

The aim of this study was to evaluate long-term platinum retention in patients treated with cisplatin and oxaliplatin.     

Pentacycloundecane lactam vs lactone norstatine type protease HIV inhibitors: binding energy calculations and DFT study

Background

Novel pentacycloundecane (PCU)-lactone-CO-EAIS peptide inhibitors were designed, synthesized, and evaluated against wild-type C-South African (C-SA) HIV-1 protease. Three compounds are reported herein, two of which displayed IC₅₀ values of less than 1.00 μM. A comparative MM-PB(GB)SA binding free energy of solvation values of PCU-lactam and lactone models and their enantiomers as well as the PCU-lactam-NH-EAIS and lactone-CO-EAIS peptide inhibitors and their corresponding diastereomers complexed with South African HIV protease (C-SA) was performed. This will enable us to rationalize the considerable difference between inhibitory concentration (IC₅₀) of PCU-lactam-NH-EAIS and PCU-lactone-CO-EAIS peptides.

Results

The PCU-lactam model exhibited more negative calculated binding free energies of solvation than the PCU-lactone model. The same trend was observed for the PCU-peptide inhibitors, which correspond to the experimental activities for the PCU-lactam-NH-EAIS peptide (IC₅₀ = 0.076 μM) and the PCU-lactone-CO-EAIS peptide inhibitors (IC₅₀ = 0.850 μM). Furthermore, a density functional theory (DFT) study on the natural atomic charges of the nitrogen and oxygen atoms of the three PCU-lactam, PCU-lactim and PCU-lactone models were performed using natural bond orbital (NBO) analysis. Electrostatic potential maps were also used to visualize the electron density around electron-rich regions. The asymmetry parameter (η) and quadrupole coupling constant (χ) values of the nitrogen and oxygen nuclei of the model compounds were calculated at the same level of theory. Electronic molecular properties including polarizability and electric dipole moments were also calculated and compared. The Gibbs theoretical free solvation energies of solvation (∆G_solv) were also considered.

Conclusions

A general trend is observed that the lactam species appears to have a larger negative charge distribution around the heteroatoms, larger quadrupole constant, dipole moment and better solvation energy, in comparison to the PCU-lactone model. It can be argued that these characteristics will ensure better eletronic interaction between the lactam and the receptor, corresponding to the observed HIV protease activities in terms of experimental IC₅₀ data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0115-5) contains supplementary material, which is available to authorized users.     

Resolution of three structural states of spin-labeled myosin in contracting muscle.

We have used electron paramagnetic resonance (EPR) spectroscopy to detect ATP- and calcium-induced changes in the structure of spin-labeled myosin heads in glycerinated rabbit psoas muscle fibers in key physiological states. The probe was a nitroxide iodoacetamide derivative attached selectively to myosin SH1 (Cys 707), the conventional EPR spectra of which have been shown to resolve several conformational states of the myosin ATPase cycle, on the basis of nanosecond rotational motion within the protein. Spectra were acquired in rigor and during the steady-state phases of relaxation and isometric contraction. Spectral components corresponding to specific conformational states and biochemical intermediates were detected and assigned by reference to EPR spectra of trapped kinetic intermediates. In the absence of ATP, all of the myosin heads were rigidly attached to the thin filament, and only a single conformation was detected, in which there was no sub-microsecond probe motion. In relaxation, the EPR spectrum resolved two conformations of the myosin head that are distinct from rigor. These structural states were virtually identical to those observed previously for isolated myosin and were assigned to the populations of the M*.ATP and M**.ADP.Pi states. During isometric contraction, the EPR spectrum resolves the same two conformations observed in relaxation, plus a small fraction (20-30%) of heads in the oriented actin-bound conformation that is observed in rigor. This rigor-like component is a calcium-dependent, actin-bound state that may represent force-generating cross-bridges. As the spin label is located near the nucleotide-binding pocket in a region proposed to be pivotal for large-scale force-generating structural changes in myosin, we propose that the observed spectroscopic changes indicate directly the key steps in energy transduction in the molecular motor of contracting muscle.     

Organization and ligand binding properties of the tail of Acanthamoeba myosin-IA. Identification of an actin-binding site in the basic (tail homology-1) domain

The Acanthamoeba myosin-IA heavy chain gene encodes a 134-kDa protein with a catalytic domain, three potential light chain binding sites, and a tail with separately folded tail homology (TH) -1, -2, and -3 domains. TH-1 is highly resistant to trypsin digestion despite consisting of 15% lysine and arginine. TH-2/3 is resistant to alpha-chymotrypsin digestion. The peptide link between TH-1 and TH-2/3 is cleaved by trypsin, alpha-chymotrypsin, and endo-AspN but not V8 protease. The CD spectra of TH-2/3 indicate predominantly random structure, turns, and beta-strands but no alpha-helix. The hydrodynamic properties of TH-2/3 (Stokes' radius of 3.0 nm, sedimentation coefficient of 1.8 S, and molecular mass of 21.6 kDa) indicate that these domains are as long as the whole myosin-I tail in reconstructions of electron micrographs. Furthermore, separately expressed and purified TH-1 binds with high affinity to TH-2/3. Thus we propose that TH-1 and TH-2/3 are arranged side by side in the myosin-IA tail. Separate TH-1, TH-2, and TH-2/3 each binds muscle actin filaments with high affinity. Salt inhibits TH-2/3 binding to muscle actin but not amoeba actin filaments. TH-1 enhances binding of TH-2/3 to muscle actin filaments at physiological salt concentration, indicating that TH-1 and TH-2/3 cooperate in actin binding. An intrinsic fluorescence assay shows that TH-2/3 also binds with high affinity to the protein Acan125 similar to the SH3 domain of myosin-IC. Phylogenetic analysis of SH3 sequences suggests that myosin-I acquired SH3 domain after the divergence of the genes for myosin-I isoforms.     

Relating biochemistry and function in the myosin superfamily

All characterized myosins share a common ATPase mechanism. However, detailed kinetic analyses suggest that modulation of the rate and equilibrium constants that define the ATPase cycle confers specific properties to these motor proteins, suiting them to specific physiological tasks. Understanding the kinetic mechanisms allows potential cellular functions of the different myosin classes and isoforms to be better defined.     

Type-3 BRET,an Improved Competition-Based Bioluminescence Resonance Energy Transfer Assay

The heart adjusts its power output to meet specific physiological needs through the coordination of several mechanisms, including force-induced changes in contractility of the molecular motor, the β-cardiac myosin (βCM). Despite its importance in driving and regulating cardiac power output, the effect of force on the contractility of a single βCM has not been measured. Using single molecule optical-trapping techniques, we found that βCM has a two-step working stroke. Forces that resist the power stroke slow the myosin-driven contraction by slowing the rate of ADP release, which is the kinetic step that limits fiber shortening. The kinetic properties of βCM are affected by load, suggesting that the properties of myosin contribute to the force-velocity relationship in intact muscle and play an important role in the regulation of cardiac power output.The cardiac cycle is a tightly regulated process in which the heart generates power during systole and relaxes during diastole. Appropriate power must be generated to effectively pump blood against cardiac afterload. Dysfunction of this cycle has devastating consequences for affected individuals.Cardiac power output is regulated by several feedback mechanisms (e.g., neuronal, hormonal, mechanical) that ultimately lead to changes in the force and power output of the molecular motor, β-cardiac myosin (βCM). In isolated cardiac fibers and cardiomyocytes, loading the muscle during systole slows contraction and alters power output. It is widely believed that this slowing is partially due to force-induced inhibition of myosin ATPase kinetics, similar to the Fenn Effect in skeletal muscle. However, this hypothesis has not been directly tested at the molecular level. Much of our contemporary view of how power is generated in cardiac muscle is due to in vivo and isolated muscle-fiber studies (1). Substantial progress has been made in understanding the actomyosin interactions required for power generation, but resolving the molecular effects of mechanical load on the ATPase properties of βCM in intact muscle has been challenging. Nevertheless, determining the biophysical parameters that define βCM contractility is key to understanding cardiac regulation and the etiology of several muscle diseases (1).In vitro assays using isolated contractile proteins have been central to advancing our understanding of contractility, although most experiments have been conducted at low resisting loads that do not mimic working conditions. Elegant optical trapping experiments have imposed loads on small ensembles of murine α-cardiac myosin at subsaturating [ATP] (2), and these experiments suggest that force slows α-cardiac myosin kinetics. The kinetic properties of α-cardiac myosin are substantially different from βCM, the primary isoform in the adult human myocardium (3). Thus, experiments using βCM must be performed to determine the unitary force-dependent kinetic parameters of this key molecular motor. We used optical trapping to measure the working-stroke displacement and force dependence of actin-detachment kinetics of single porcine βCM molecules at saturating ATP concentrations. These experiments allow direct measurement of the force-velocity relationship for single βCM molecules and reveal the mechanism of how loads regulate βCM-driven power output.Using the three-bead geometry (4) in which an actin filament is strung between two optically-trapped beads and then lowered over a bead that is sparsely coated with purified full-length porcine ventricular βCM, interactions between single βCM molecules and actin were recorded at 10 μM ATP (Fig. 1 A) (5, 6). Ensemble averages of these interactions were constructed to determine the size and kinetics of the working stroke (7, 8). βCM has an average displacement (6.8 ± 0.04 nm) that is similar to previously characterized muscle myosins (9, 10). Similar to skeletal muscle myosin (10), ensemble averages clearly show that the βCM working stroke is composed of two substeps with average displacements of 4.7 ± 0.05 nm and 1.9 ± 0.05 nm (Fig. 1 B). A single exponential function was fit to the rising-phase of the time-forward ensemble averages, yielding a rate (74 ± 2 s⁻¹) for the transition from state 1 to state 2 (Fig. 1 C). This rate is similar to the biochemical rate of ADP release measured for βCM (64 s⁻¹) (3), indicating that this structural transition is associated with the release of ADP. The rate of the rising phase of the time-reversed ensemble averages (22 ± 0.7 s⁻¹) reports the rate of exit from state 2 and is consistent with the biochemical rate of ATP binding and actomyosin detachment at 10 μM ATP (16 s⁻¹) (3) (Fig. 1 C).Open in a separate windowFigure 1(A) Representative data trace showing actomyosin displacements generated by βCM at 10 μM ATP. (Blue lines) Individual binding events. (B) Ensemble averages of the βCM working stroke generated from averaging 1295 binding interactions collected at 10 μM ATP. Single exponential functions were fit to the data (red lines) and the reported errors are the standard errors from the fit. (C) Cartoon showing an idealized actomyosin interaction with the corresponding mechanical and biochemical states. To see this figure in color, go online.To examine actomyosin detachment kinetics under working conditions, a positional feedback optical clamp was used to apply a dynamic load to the myosin, keeping the myosin at an isometric position during its working stroke (11). We measured the effect of force on the actin-attachment duration at 4 mM Mg.ATP to ensure that the rate of ATP binding is not rate-limiting for detachment. Increases in attachment durations are observed as the force on the myosin is increased (Fig. 2 A, inset). Assuming a two-state model (12), we expect the attachment durations to be exponentially distributed at each force with the force-dependent actin detachment rate, k(F), given by (13)k(F)=k0∗e−F·ddetkB∗T,(1)where k₀ is the rate of the primary force-sensitive transition in the absence of force, F is the force on the myosin, d_det is the distance to the transition state (a measurement of force sensitivity), k_B is Boltzmann’s constant, and T is the temperature. Maximum likelihood estimation (MLE) fitting Eq. 1 to the data yields a detachment rate (k₀ = 71 (−1.0/+0.8 s⁻¹)) that is similar to the rate of ADP release measured for βCM (64 s⁻¹) (3) and the rate of the time-forward ensemble averages (74 ± 2 s⁻¹). Thus, the ADP release step (and the accompanying state-1 to state-2 mechanical transition) is force-sensitive (d_det = 0.97 (−0.014/+0.011) nm). The value of d_det indicates that the ADP release step slows with increasing force, but less than some other characterized myosins (14). Using the values determined from the MLE fitting and the measured size of the working stroke, it is possible to calculate a force-velocity relationship for βCM, assuming the rate of ADP release limits actin motility (Fig. 2 A).Open in a separate windowFigure 2(A, Inset) Single molecule actomyosin interactions were collected in the presence of the isometric optical clamp. The scatter plot shows 262 binding events. Attachment durations are exponentially distributed at each force. (A) The detachment rate as a function of force as determined by MLE fitting. (Black line) Best fit; (small gray shaded area) 95% confidence interval. (Right axis) Velocity, calculated by multiplying the displacement of the working stroke by the detachment rate. (B) The calculated mean detachment rate as a function of force. Attachment durations were binned according to the average force experienced by the myosin during the binding event. Error bars were calculated via bootstrapping simulations of each force bin. (Blue line) Expected mean detachment rate based on the MLE fitting and the limited temporal resolution of our experiment (see the Supporting Material for details). (C) Proposed model for how force slows shortening velocity. Force inhibits the mechanical transition associated with ADP release, slowing the rate of actomyosin detachment. To see this figure in color, go online.The MLE fitting of Eq. 1 assumes an exponential distribution of attachment durations at every force. As such, the MLE fitting of the raw data should yield correct values of the parameters k₀ and d_det, despite limitations of the temporal resolution of our experiment (see Supporting Material for detailed discussion of MLE fitting). Frequently, groups report the mean attachment duration as a function of force. However, the mean attachment duration at each force will be overestimated because some shorter binding events cannot be resolved. We provide a method for calculating the expected mean detachment rate based on the parameters determined from the MLE fitting, given the limited temporal resolution of the experiment, and verify the robustness of the MLE fitting (see the Supporting Material). For demonstration purposes only, Fig. 2 B shows that the measured mean detachment rate agrees well with the expected mean detachment rate based on the MLE fitting and the temporal resolution of the experiment. It should be emphasized that the relevant dissociation values are obtained from the MLE fitting in Fig. 2 A (see also Figs. S1–S3).Our data demonstrate that at saturating [ATP], the detachment rate is limited by the ADP release step, which is the same transition that limits fiber shortening velocity (15). We propose that resisting loads slow ADP release and actin detachment by slowing the mechanical transition that accompanies ADP release (Fig. 2 C), thereby reducing the shortening velocity of muscle fibers. Thus, our data demonstrate that the intrinsic force-dependent properties of βCM contribute to the force-velocity relationship in the heart. It is important to note that our proposed mechanism does not rule out additional mechanisms by which force could directly modulate the activity of actomyosin such as force-induced reversal of the power stroke (11) or population of branched pathways (16, 17).Are the loads in our experiments physiologically relevant to contracting muscle? Modeling of the force per cross-bridge generated in isometric soleus muscle, which contains the βCM isoform, suggests a load of 2–4 pN per myosin (18). At these loads, we expect actin-detachment to slow up to threefold. Interestingly, βCM is substantially less force-sensitive than smooth muscle myosin (d_det = 2.7), suggesting that βCM can generate more power (the product of force and velocity) under load.In conclusion, our data show that cardiac power output can be directly modulated by force at the level of single myosin molecules. These data will enable the comparison of how molecular changes, such as light-chain phosphorylation, pharmacological treatments, or mutations associated with cardiomyopathies, affect the ability of the myosin to generate power against the afterload.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Development of enzyme-immunoassays based on egg yolk antibodies for the detection of mycotoxins

Enzyme-linked immunosorbent assays (ELISA) proved to be a fast and simple method for the detection of mycotoxins and other undesired contaminants in food and feed. The present study is focused on the optimisation and exploitation of the egg yolk antibody technology in order to develop competitive ELISAs for the detection of mycotoxins in cereals. Due to its importance as one of the most relevant Fusarium mycotoxins, the trichothecene deoxynivalenol (DON) was selected as representative. Chickens were immunised with different protein conjugates performing varying booster intervals. The antibodies were isolated by the poly(ethylene glycol) precipitation method according to Polson. By use of these antibodies an indirect competitive ELISA was developed for the detection of DON. First investigations of naturally contaminated wheat samples showed a good correspondence with results obtained by GC-ECD when calibration in blank wheat extracts was performed.