Valence Force Fields as a Tool in Vibrational Spectroscopy and Molecular Mechanics

Force field calculations on conjugated molecules are discussed. The discussion is based on the experience of a series of overlay calculations, recently carried out, where the transferability of force constants was thoroughly studied. Successful applications as well as limitations of the constructed force field are described. The effects of nonbonded interactions are recognized as the most serious restriction of the transferability of valence force fields, and it is suggested that the molecular mechanics method, where the nonbonded interactions are taken explicitly into account, would be advantageous. The treatment of potential energy in the molecular mechanics method is briefly described and theconnections between valence force constants and potential energy parameters in this method are discussed

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus-and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs. IMPORTANCE Similar to all positive-strand RNA viruses, the arthropod-borne alpha-viruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA and in vitro replication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.Peer reviewe

Vortex splitting and phase separating instabilities of coreless vortices in F=1 spinor Bose-Einstein condensates

The low lying excitations of coreless vortex states in F = 1 spinor Bose-Einstein condensates (BECs) are theoretically investigated using the Gross-Pitaevskii and Bogoliubov-de Gennes equations. The spectra of the elementary excitations are calculated for different spin-spin interaction parameters and ratios of the number of particles in each sublevel. There exist dynamical instabilities of the vortex state which are suppressed by ferromagnetic interactions, and conversely, enhanced by antiferromagnetic interactions. In both of the spin-spin interaction regimes, we find vortex splitting instabilities in analogy with scalar BECs. In addition, a phase separating instability is found in the antiferromagnetic regime.Comment: 11 pages, 9 figure

Promoting Parents' Use of Non-Pharmacological Methods and Assessment of Children's Postoperative Pain at Home

Background: Parents have reported challenges in assessing their child's postoperative pain at home.Aims: The purpose of this study was to evaluate the usefulness of the parental use of the Parents' Postoperative Pain Measure -tool (PPPM) on 1-3 -year-old children's non-pharmacological pain alleviation at home.Methodology: This was a non-randomized, prospective study with two parallel groups, where the parents in the intervention group were provided with PPPM in addition to a pain diary consisting of a verbal pain scale. The data were collected from 50 parents whose children had undergone day surgery in three Finnish university hospitals between January 2006 and June 2007. Parents completed questionnaires consisting of background information, verbal pain rating scale and a sub-scale measuring parents' use of non-pharmacological methods in children's postoperative pain alleviation.Results: Most children had mild postoperative pain after discharge, but in some children pain was moderate or severe. Non-pharmacological interventions were used commonly for pain alleviation in both groups, including holding the child in lap, comforting the child and spending time with the child more than usual during the recovery period after discharge. However, the use of non-pharmacological pain alleviation methods was 15% more common in the intervention group than in the control group. Parents of the intervention group had carried the child (p=0.04) and used distraction (p=0.05) more commonly than parents in control group. No group differences were found in parental assessments of the helpfulness of non-pharmacological pain alleviation methods.Conclusions: Children's pain remains under-treated and their pain alleviation can be promoted by providing the parents pain assessment tools, such as PPPM, to be used at home. The results can be utilized to further improve children's pain alleviation. More parental education is needed to promote their skills to alleviate the child's pain. Further research of the usefulness of the PPPM using larger samples is needed

Conversion of ab-initio force fields and structures to molecular mechanics energy functions

The rapid development of computers in recent years has brought increasingly complex compounds into the range of high level ab-initio calculations. Such calculations produce valuable results which in many cases would be difficult or even impossible to obtain, with comparable accuracy, in any other way (Fogarasi & Pulay, Annu. Rev. Phys. Chem. 35, 191, 1984). Thus, it is highly desirable to be able to utilize these results in the construction of potential energy functions used in molecular mechanics (MM), molecular dynamics and Monte-Carlo calculations. For instance, the significance of quadratic cross terms in MM energy functions is still insufficiently explored (Lii & Allinger, J. Am. Chem. Soc. 111, 8566, 1989). In order to make possible the complete utilization of ab-initio results in MM calculations, we have developed a method by which scaled ab-initio (or empirical) force fields and structures can be directly converted to MM potential energy parameters, without sacrificing any of the original accuracy with regard to vibrational frequencies or structure. Here we briefly outline the conversion procedure, a more complete analysis being published separately.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29587/1/0000676.pd

Optimization of parameters of nonbonded interactions in a spectroscopically determined force field

A procedure is given by which parameters of nonbonded interactions in a molecular mechanics energy function can be optimized for maximum compatibility with ab initio force fields and structures. The method is based on a previously derived transformation of ab initio valence parameters to the molecular mechanics formalism. Explicit analytical expressions for the derivatives of the molecular mechanics force constants and reference geometry parameters with respect to the parameters of the nonbonded interactions are derived. The form of the goodness-of-fit function is discussed. A first application to a set of alanine dipeptides is described.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30924/1/0000594.pd

Utilization of overlay and AB initio force fields in the construction of empirical potential energy functions for conjugated molecules

The optimization of the parameters in the molecular mechanics method is discussed. The utilization of spectroscopic force fields is described, and the advantages of utilizing molecular symmetry in the optimization of the parameters on the vibrational frequencies are stressed. The development of the potential functions for the benzene ring and for the vinyl group is briefly described, and the the application of these functions to calculate the geometry of the styrene molecule is discussed.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28713/1/0000534.pd

Comparison of peak serum C-reactive protein and hydroxybutyrate dehydrogenase levels in patients with acute myocardial infarction treated with alteplase and streptokinase

Peak serum C-reactive protein concentrations were measured in 146 patients randomized to receive streptokinase, alteplase, or a combination of streptokinase and alteplase in the GUSTO-I trial. Those receiving alteplase treatment had lower values than those receiving streptokinase or the combination treatment. Irrespective of treatment, complete reperfusion of the infarct-related artery (TIMI grade 3 flow) was associated with low peak serum C-reactive protein values

Towards in cellulo virus crystallography

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.Peer reviewe