The Role of Key Amino Acids in the Antimicrobial Mechanism of a Bacteriocin Model Revealed by Molecular Simulations.

The AS-48 bacteriocin is a potent antimicrobial polypeptide with enhanced stability due to its circular sequence of peptidic bonds. The mechanism of biological action is still not well understood in spite of both the elucidation of the molecular structure some years ago and several experiments performed that yielded valuable information about the AS-48 bacterial membrane poration activity. In this work, we present a computational study at an atomistic scale to analyze the membrane disruption mechanism. The process is based on the two-stage model: (1) peptide binding to the bilayer surface and (2) membrane poration due to the surface tension exerted by the peptide. Indeed, the induced membrane tension mechanism is able to explain stable formation of pores leading to membrane disruption. The atomistic detail obtained from the simulations allows one to envisage the contribution of the different amino acids during the poration process. Clustering of cationic residues and hydrophobic interactions between peptide and lipids seem to be essential ingredients in the process. GLU amino acids have shown to enhance the membrane disrupting ability of the bacteriocin. TRP24-TRP24 interactions make also an important contribution in the initial stages of the poration mechanism. The detailed atomistic information obtained from the simulations can serve to better understand bacteriocin structural characteristics to design more potent antimicrobial therapies.

Hydrodynamic and Electrophoretic Properties of Trastuzumab/HER2 Extracellular Domain Complexes as Revealed by Experimental Techniques and Computational Simulations.

The combination of hydrodynamic and electrophoretic experiments and computer simulations is a powerful approach to study the interaction between proteins. In this work, we present hydrodynamic and electrophoretic experiments in an aqueous solution along with molecular dynamics and hydrodynamic modeling to monitor and compute biophysical properties of the interactions between the extracellular domain of the HER2 protein (eHER2) and the monoclonal antibody trastuzumab (TZM). The importance of this system relies on the fact that the overexpression of HER2 protein is related with the poor prognosis breast cancers (HER2++ positives), while the TZM is a monoclonal antibody for the treatment of this cancer. We have found and characterized two different complexes between the TZM and eHER2 proteins (1:1 and 1:2 TZM:eHER2 complexes). The conformational features of these complexes regulate their hydrodynamic and electrostatic properties. Thus, the results indicate a high degree of molecular flexibility in the systems that ultimately leads to higher values of the intrinsic viscosity, as well as lower values of diffusion coefficient than those expected for simple globular proteins. A highly asymmetric charge distribution is detected for the monovalent complex (1:1 complex), which has strong implications in correlations between the experimental electrophoretic mobility and the modeled net charge. In order to understand the dynamics of these systems and the role of the specific domains involved, it is essential to find biophysical correlations between dynamics, macroscopic transport and electrostatic properties. The results should be of general interest for researchers working in this area.

A new insight into the conformation and melt dynamics of hydrogenated polybutadiene as revealed by computer simulations.

Extensive molecular dynamics simulations of the macromolecular conformation and the melt dynamics for model polymers of different molecular weights have been carried out. The selected models are hydrogenated polybutadienes with a 2% content of ethyl branches and linear polyethylene. It will be shown that the density and chain stiffness are clearly affected by both the molecular weight and the presence of ethyl branches. Furthermore, the results obtained from the simulations on the molecular size and, more remarkably, chain dynamics, perfectly match the neutron scattering experiments performed by Zamponi et al. in hydrogenated polybutadienes. We observe a clear chain contraction and a slow dynamics for the hydrogenated polybutadiene with respect to the linear chain of the same molecular length. Using the Likhtman-McLeish definitions, the obtained values of the entanglement relaxation time (τe) and the tube diameter (a) are found to be in agreement with the available experimental data (by rheology and neutron spin echo) as well as with those obtained by the simulations. Finally, a very good agreement of diffusion coefficients as a function of the molecular weight between simulations and experiments is observed. Therefore, there exists a clear difference between the results obtained for branched and linear polyethylene, accounting for a definitive effect of the short chain branching on the conformational properties and the melt dynamics of polyolefins.

Bacteriocin AS-48 binding to model membranes and pore formation as revealed by coarse-grained simulations.

Bacteriocin AS-48 is a membrane-interacting peptide that acts as a broad-spectrum antimicrobial against Gram-positive and Gram-negative bacteria. Prior Nuclear Magnetic Resonance experiments and the high resolution crystal structure of AS-48 have suggested a mechanism for the molecular activity of AS-48 whereby the peptide undergoes transition from a water-soluble to a membrane-bound state upon membrane binding. To help interpret experimental results, we here simulate the molecular dynamics of this binding mechanism at the coarse-grained level. By simulating the self-assembly of the peptide, we predict induction by the bacteriocin of different pore types consistent with a "leaky slit" model.

Exploring the dynamics and interaction of a full ErbB2 receptor and Trastuzumab-Fab antibody in a lipid bilayer model using Martini coarse-grained force field.

Coarse grained (CG) modeling has been applied to study the influence of the Trastuzumab monoclonal antibody on the structure and dynamics of the full ErbB2 receptor dimer, including the lipid bilayer. The usage of CG models to study such complexes is almost mandatory, at present, due to the large size of the whole system. We will show that the Martini model performs satisfactorily well, giving results well-matched with those obtained by atomistic models as well as with the experimental information existing on homolog receptors. For example, the extra and intracellular domains approach the bilayer surface in both the monomer and dimer cases. The Trastuzumab-Fab hinders the interaction of the receptors with the lipid bilayer. Another interesting effect of the antibody is the disruption of the antiparallel arrangement of the juxtamembrane segments in the dimer case. These findings might help to understand the effect of the antibody on the receptor bioactivity.

Size and conformational features of ErbB2 and ErbB3 receptors: a TEM and DLS comparative study.

ErbB2 and ErbB3 receptors belong to the epidermal growth factor receptor family. The members of this family are able to form homo- and heterodimers that trigger diverse downstream signalling concerned to multiple cellular events. In the absence of a ligand, ErbB3 adopts a characteristic tethered conformation, which differs from ErbB2 extended conformation. In this work, transmission electron microscopy (TEM) and dynamic light scattering (DLS) have been used to characterize the conformational features and the size of ErBb2 and ErbB3 receptors. Two main objectives are presented. The first one is to evaluate the use of TEM as a tool for structural studies for this family of receptors. The low molecular weight of these proteins represents a challenging purpose for TEM studies. The other one is to search for a relationship between the results obtained by TEM and those obtained for the hydrodynamic size measured by DLS. This comparison has allowed us to identify the conformational differences of the receptors and to anticipate the use of these experimental techniques for the study of the ligand activated heterodimerization, a process related to a significant number of human malignancies.

Dissimilar interaction of CB1/CB2 with lipid bilayers as revealed by molecular dynamics simulation.

Cannabinoid receptors CB1 and CB2 are a striking class of transmembrane proteins involved in a high number of important biological processes. In spite of the inherent similarity (40% in aminoacid sequence) these receptors are found in different cell environments. In addition to this, CB1 activity has been intimately associated with lipid rafts whereas CB2 has not. In this work we have performed a 50 nanosecond molecular dynamics simulation of the inactive conformations of both receptors inserted in a POPC lipid bilayer. Although in both cases the overall protein structure is maintained along the entire simulation we have found important differences in the protein-lipid interaction. While CB1 tends to distort the lipid bilayer regularity, especially in the extracellular moiety, CB2 has a minor influence on the lipid distribution along the plane of the bilayer. This observation is consistent with some experimental facts observed in these cannabinoid receptors with regard to lipid/protein interaction.

Mapping the Mechanical Properties of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Banded Spherulites by Nanoindentation.

Nanoindentation provides clear evidence that spherulite banding can be associated with a continuous modulation of mechanical properties from the more compliant peaks to the stiffer valleys. The structural arrangement in polymer-banded spherulites has intrigued scientists for many decades, and the debate has been recently intensified with the advent of new experimental evidence. The present paper approaches this issue by exploring the local mechanical properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-ringed spherulites via nanoindentation and discussing the confidence of the results. It was found that storage modulus and hardness across the banding morphology can be described as a sequence of regular oscillations with a periodicity that exactly matches the one observed using optical and atomic force microscopy. Results are consistent with the model of regular twisting of the lamellae, with flat-on arrangement in the low regions and edge-on lamellae in the crests.

Evidences of Changes in Surface Electrostatic Charge Distribution during Stabilization of HPV16 Virus-Like Particles.

The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development.

Transabdominal ultrasound-guided embryo transfer does not increase pregnancy rates in oocyte recipients.

OBJECTIVE: To determine whether transabdominal ultrasound guidance during embryo transfer (ET) is a useful tool for increasing pregnancy rates in patients undergoing oocyte donation. DESIGN: Prospective, randomized, controlled trial. SETTING: In vitro fertilization academic center. PATIENT(S): Three hundred seventy-four infertile patients undergoing oocyte donation. INTERVENTION(S): Transabdominal ultrasound-guided ET. MAIN OUTCOME MEASURE(S): We measured the pregnancy rate and implantation rate after transabdominal ultrasound-guided ET versus the rates in a control group who did not receive transabdominal ultrasound-guided ET. RESULT(S): Clear visualization at ultrasound during ET was achieved in 90.8% of the patients who had ultrasound-guided ET. A similar number of easy transfers were performed in both the ultrasound-guided and the control groups (84.5% vs. 86.6%). The pregnancy rate was comparable between the groups (59.9% ultrasound vs. 55.1% control), as was the implantation rate (30.6% ultrasound vs. 26.3% control). No differences were found in the miscarriage rate (10.7% ultrasound vs. 9.1% control) or in the multiple pregnancy rate (21.4% ultrasound vs. 22.5% control). Although all ectopic pregnancies occurred in the group that did not receive ultrasound guidance, the differences were not statistically significant (0 vs. 2.7%). CONCLUSION(S): We could not show any benefit in terms of pregnancy rate in oocyte recipients for whom ET was performed under direct transabdominal ultrasound visualization of the endometrial cavity. There was a lower ectopic pregnancy rate when ultrasound guidance was used, but this rate was not statistically significant in comparison with the pregnancy rate without ultrasound guidance.

Simulation of homology models for the extracellular domains (ECD) of ErbB3, ErbB4 and the ErbB2-ErbB3 complex in their active conformations.

Epidermal growth factor receptors (EGFR) are associated with a number of biological processes and are becoming increasingly recognized as important therapeutic targets against cancer. In this work, we provide models based on homology for the extracellular domains (ECD) of ErbB3 and ErbB4 in their active conformations, including a Heregulin ligand, followed by further refinement of the models by molecular dynamics simulations at atomistic scale. We compare the results with a model built for ErbB2 based on crystallographic information and analyze the common features observed among members of the family, namely, the periscope movement of the dimerization arm and the hinge displacement of domain IV. Finally, we refine a model for the interaction of the ECDs corresponding to a ErbB2-ErbB3 heterodimer, which is widely recognized to have a high impact in cancer development.

Conformational flexibility of the ErbB2 ectodomain and trastuzumab antibody complex as revealed by molecular dynamics and principal component analysis.

Human epidermal growth factor receptor 2 (ErbB2) is a transmembrane oncoprotein that is over expressed in breast cancer. A successful therapeutic treatment is a monoclonal antibody called trastuzumab which interacts with the ErbB2 extracellular domain (ErbB2-ECD). A better understanding of the detailed structure of the receptor-antibody interaction is indeed of prime interest for the design of more effective anticancer therapies. In order to discuss the flexibility of the complex ErbB2-ECD/trastuzumab, we present, in this study, a multi-nanosecond molecular dynamics simulation (MD) together with an analysis of fluctuations, through a principal component analysis (PCA) of this system. Previous to this step and in order to validate the simulations, we have performed a detailed analysis of the variable antibody domain interactions with the extracellular domain IV of ErbB2. This structure has been statically elucidated by x-ray studies. Indeed, the simulation results are in excellent agreement with the available experimental information during the full trajectory. The PCA shows eigenvector fluctuations resulting in a hinge motion in which domain II and C(H) domains approach each other. This move is likely stabilized by the formation of H-bonds and salt bridge interactions between residues of the dimerization arm in the domain II and trastuzumab residues located in the C(H) domain. Finally, we discuss the flexibility of the MD/PCA model in relation with the static x-ray structure. A movement of the antibody toward the dimerization domain of the ErbB2 receptor is reported for the first time. This finding could have important consequences on the biological action of the monoclonal antibody.

Assessment of the intrinsic conformational preferences of dipeptide amino acids in aqueous solution by combined umbrella sampling/MBAR statistics. A comparison with experimental results.

The propensities of 19 amino acid dipeptides have been calculated by a distributed umbrella sampling molecular dynamics simulation procedure using the OPLS-AA force field. The potential of mean force maps was estimated with the multiple Bennett acceptance ratio statistics. The resulting propensities compare satisfactorily well with very recently published experimental data on equivalent systems. In particular, α conformation-probabilities for all of the dipeptides remain much lower than either ß or P(II) propensities. This result is in agreement with most experimental data for dipeptides. However, it is also in contrast with most simulation studies performed so far with other force fields, where α conformations result even more probable than P(II) or ß ones. We discuss the behavior of the OPLS-AA force field, which can be useful for the improvement of this model in reproducing the recent experimental observations on amino acid dipeptides.

A Curtin-Hammett mechanism for the copolymerization of ethylene and methyl acrylate monomer using a PymNox nickel catalyst as revealed by DFT computational studies.

In this work, the copolymerization of ethylene and methyl acrylate (MA) as catalyzed by a new Ni-based PymNox organometallic compound was studied computationally. We recently tested the behavior of this type of catalyst in ethylene homopolymerization. Experimental results show that the unsubstituted catalyst Ni2 (aldimino PymNox catalyst) is unable to incorporate the MA monomer, whereas methyl-substituted Ni1 (acetaldimino PymNox catalyst) is able to achieve copolymerization. The reactivities of both catalysts were examined using density functional theory (DFT) models. Based on energy profiles calculated at the BP86 level, a Curtin-Hammett mechanism was proposed to explain the different reactivities of the catalysts in ethylene/MA copolymerization. Our results indicate that the methyl substituent Ni1 introduces additional steric hindrance that results in a catalyst conformation that is better suited to polar monomer incorporation. This model provides insights into the design of new catalysts to produce polar functionalized copolymers based on ethylene.

Structural insights on the plant salt-overly-sensitive 1 (SOS1) Na(+)/H(+) antiporter.

The Arabidopsisthaliana Na(+)/H(+) antiporter salt-overly-sensitive 1 (SOS1) is essential to maintain low intracellular levels of toxic Na(+) under salt stress. Available data show that the plant SOS2 protein kinase and its interacting activator, the SOS3 calcium-binding protein, function together in decoding calcium signals elicited by salt stress and regulating the phosphorylation state and the activity of SOS1. Molecular genetic studies have shown that the activation implies a domain reorganization of the antiporter cytosolic moiety, indicating that there is a clear relationship between function and molecular structure of the antiporter. To provide information on this issue, we have carried out in vivo and in vitro studies on the oligomerization state of SOS1. In addition, we have performed electron microscopy and single-particle reconstruction of negatively stained full-length and active SOS1. Our studies show that the protein is a homodimer that contains a membrane domain similar to that found in other antiporters of the family and an elongated, large, and structured cytosolic domain. Both the transmembrane (TM) and cytosolic moieties contribute to the dimerization of the antiporter. The close contacts between the TM and the cytosolic domains provide a link between regulation and transport activity of the antiporter.

Water-mediated conformations of the alanine dipeptide as revealed by distributed umbrella sampling simulations, quantum mechanics based calculations, and experimental data.

An exhaustive umbrella sampling simulation of the alanine dipeptide in solution is reported. The presence of stable Y conformations in solution is assessed by both quantum calculations and experimental data from X-ray and NMR protein-solved structures available in the protein coil library. The agreement between experimental and simulation Ramachandran plots of the dipeptide in solution is excellent. A suitable explanation of the stabilization of the Y conformation mediated by water for the alanine dipeptide is discussed on the basis of Car-Parrinello MD calculations of the dipeptide in water.

Cycle scheduling with oral contraceptive pills in the GnRH antagonist protocol vs the long protocol: a randomized, controlled trial.

OBJECTIVE: To compare cycle outcomes after scheduling with the standard long protocol versus the use of oral contraceptive pills (OCPs) in patients undergoing GnRH antagonist cycles. DESIGN: Prospective, randomized, controlled trial. SETTING: University-affiliated private assisted reproduction center. PATIENT(S): Regularly cycling women aged ≤38 years with fewer than three previous IVF attempts were enrolled. Previous low responses to controlled ovarian hyperstimulation, ovarian surgery, or polycystic ovary were exclusion criteria. INTERVENTION(S): One hundred fifteen patients received OCP (0.030 ethinyl E(2)/0.15 desogestrel) for 12-16 days, and controlled ovarian hyperstimulation was started on day 5 after OCP treatment; similarly, 113 patients received the long protocol from day 20-22 of the previous cycle. MAIN OUTCOME MEASURE(S): The primary outcome was ongoing pregnancy rate; secondary outcome variables were clinical pregnancy rate, live birth rate, implantation rate, and miscarriage rate. RESULT(S): Patients receiving the GnRH antagonist treatment showed a lower peak serum E(2) (1,334 vs. 1,823 pg/mL) but similar peak serum PE (0.58 vs. 0.65 ng/mL), lower duration of the stimulation (10.3 vs. 11.4 days) with similar FSH consumption (1,613 vs. 1,807 IU), and ovarian response (10.2 vs. 11.7 oocytes). No differences were observed in the fertilization rates (68.1% vs. 64.8%), total number of embryos obtained (5.9 vs. 6.2), mean number of embryos transferred (1.8 vs. 1.8), implantation rate (36% vs. 39%), miscarriage rate (8.9% vs. 17%), ongoing pregnancy rate (47.8% vs. 53.9%), or live birth rate (44.3% vs. 47%). CONCLUSION(S): Comparable outcomes can be obtained using OCP containing 0.030 ethinyl E(2)/0.15 desogestrel to schedule patients undergoing the antagonist protocol.

Differential regulation of VEGF after final oocyte maturation with GnRH agonist versus hCG: a rationale for OHSS reduction.

In a prospective cohort study, we compared the effect of hCG and GnRH agonist triggering of final oocyte maturation on vascular endothelial growth factor production. Vascular endothelial growth factor follicular fluid concentration was significantly lower in response to GnRH agonist versus hCG, which may partially explain the absence of OHSS in these of women.

Estradiol supplementation during the luteal phase of IVF-ICSI patients: a randomized, controlled trial.

OBJECTIVE: To evaluate the effectiveness of transdermal E(2) administration in the luteal phase of IVF/ICSI cycles. DESIGN: Prospective, open-label, randomized clinical trial. SETTING: University-affiliated assisted reproduction center. PATIENTS: 1) Pilot trial to test serum E(2) behaviour during the luteal phase in women undergoing agonist as well as antagonist protocol; 2) women undergoing IVF/ICSI with good-quality embryos available. INTERVENTION(S): One hundred seventy-six patients were randomized by random number list on the day of embryo transfer to either: 1) progesterone (P) only as luteal support (200 mg bid starting the following night after oocyte retrieval); or 2) E(2) and P combined, applying E(2) patches (100 microg/day) twice per week beginning on the day of embryo transfer with P, as in the P-only group. MAIN OUTCOME MEASURE(S): The primary outcome was implantation rate per embryo transfer; secondary outcome variables were pregnancy rate per embryo transfer, early pregnancy loss, multiple pregnancy rate, and midluteal P and E(2) levels. RESULT(S): Hormonal levels did not differ between groups. There were no statistically significant differences in terms of implantation rate (34.9% [51 of 146] vs. 28.9% [41 of 142]), ongoing pregnancy rate 42% ([34 of 81] vs. 41.8% [33 of 79]), early pregnancy loss (15% [6 of 40] vs. 13.2% [5 of 38]), or multiple pregnancy rate (28.6% [12 of 42] vs. 24.4% [10/41]) in patients receiving P versus E(2) + P. CONCLUSION(S): The addition of transdermal E(2) to the luteal-phase P support of IVF cycles did not improve cycle outcomes in terms of implantation and pregnancy rates.

Ethylene-styrene copolymerization with constrained geometry catalysts: a density functional study.

A density functional theory (DFT) study of the ethylene-styrene copolymerization process with titanium-based constrained geometry catalyst (CGC) is presented. To establish the difference between simplified CGC or real CGC models, i.e., considering all ligands of the catalyst, we have performed calculations for ethylene and styrene insertions in both models. Thus, we have used two different DFT functional, BP86 and B3L YP along with two basis set, LANL 2DZ (without polarization functions) and DZVP (including polarization functions). We have noted certain differences between theoretical results published by other authors and our theoretical and experimental data.