Computer simulations of the diffusion of a substrate to an active site of an enzyme.

Computer simulations of the diffusion of a substrate to an enzyme active site were performed. They included the detailed shape of the protein and an accurate description of its electrostatic potential. Application of the method to the diffusion of the superoxide anion to the protein superoxide dismutase revealed that the electric field of the enzyme enhances the association rate of the anion by a factor of 30 or more. Calculated changes in the association rate as a function of ionic strength and amino acid modification paralleled the observed behavior. Design principles of superoxide dismutase are considered with respect to insights provided by the simulations. A possible means of enhancing the enzyme turnover rate through site-directed mutagenesis is proposed.

Reconciling the magnitude of the microscopic and macroscopic hydrophobic effects.

The magnitude of the hydrophobic effect, as measured from the surface area dependence of the solubilities of hydrocarbons in water, is generally thought to be about 25 calories per mole per square angstrom (cal mol-1 A-2). However, the surface tension at a hydrocarbon-water interface, which is a "macroscopic" measure of the hydrophobic effect, is approximately 72 cal mol-1 A-2. In an attempt to reconcile these values, alkane solubility data have been reevaluated to account for solute-solvent size differences, leading to a revised "microscopic" hydrophobic effect of 47 cal mol-1 A-2. This value, when used in a simple geometric model for the curvature dependence of the hydrophobic effect, predicts a macroscopic alkane-water surface tension that is close to the macroscopic value.

How much is a stabilizing bond worth?

It is commonly supposed that the contribution of a bond to protein or nucleic acid stability is equal to the in situ stability of the bond itself. This is not true for the noncovalent bonds that stabilize molecular folding. In general, a bonding interaction contributes a free energy increment to protein or nucleic acid stability that is larger, an enthalpy increment that is smaller, and entropy and heat capacity increments that are more positive than the corresponding bond parameter.

Salt effects on nucleic acids.

Salt-dependent electrostatic effects are a major factor in determining the stability, structure, reactivity, and binding behavior of nucleic acids. Increasingly detailed theoretical methods, especially those based on Monte Carlo and Poisson-Boltzmann methodologies, combined with powerful computational algorithms are being used to examine how the shape, charge distribution and dielectric properties of the molecules affect the ion distribution in the surrounding aqueous solution, and how they play a role in ligand binding, structural transitions and other biologically important reactions. These studies indicate that inclusion of detailed structural information about the nucleic acid and its ligands is crucial for improving models of nucleic acid electrostatics, and that better treatment of the ion atmosphere and dielectric effects is also of major importance.

Entropy in protein folding and in protein-protein interactions.

The reduction of conformational entropy is a major barrier that has to be overcome in protein folding and binding. Changes in solvent entropy are also a major factor. Recent advances include clarification of the fundamental issues concerning the separation of entropy into components, the treatment of association entropy in binding, and the role of size and shape effects in solvation entropy. Advances in the application of entropy calculations include an emerging consensus for estimates of backbone and sidechain entropy loss in protein folding via use of numerically intensive methods for sampling, and use of the expanding protein-structure database.

Heller myotomy vs Heller myotomy plus Dor fundoplication: cost-utility analysis of a randomized trial.

BACKGROUND: The addition of a Dor antireflux procedure reduces the risk of pathologic gastroesophageal reflux (GER) by ninefold following laparoscopic Heller myotomy for achalasia. It is not clear, however, how these benefits compare with the increased cost of the fundoplication. The objective of this study was to estimate the cost-effectiveness of Heller myotomy plus Dor fundoplication compared with Heller alone in patients with achalasia. METHODS: We conducted a cost-utility analysis using the Markov simulation model to examine the two treatment alternatives. The model estimated the total expected costs of each strategy over a 10-year time horizon. Data for the model were derived from our randomized clinical trial. The strategies were compared using the method of incremental cost-effectiveness analysis. RESULTS: The incidence of pathologic GER was 47.6% (10 of 21 patients) in the Heller group and 9.1% (2 of 22 patients) in the Heller plus Dor group using an intention-to-treat analysis (p = 0.005). Heller plus Dor was associated with a significant reduction in the risk of GERD (relative risk 0.11; 95% confidence interval 0.02-0.59; p = 0.01). The cost of surgery was significantly higher for Heller plus Dor than for Heller alone (mean difference $942; p = 0.04), secondary to a longer operating room time (mean difference 40 min; p = 0.01). At a time horizon of 10 years, when proton pump inhibitor (PPI) therapy costs are considered, the cost-utility analysis demonstrates that Heller plus Dor surgery is associated with a total cost of $6,861 per patient and a quality-adjusted life expectancy of 9.9 years, whereas Heller-alone surgery is associated with a cost of $9,541 per patient and a quality-adjusted life expectancy of 9.5 years. CONCLUSIONS: In achalasia patients, Heller myotomy plus Dor fundoplication is preferred to Heller alone because it is both more effective in preventing postoperative GERD and more cost-effective at a time horizon of 10 years.

Management of general surgical problems in patients with left ventricular assist devices.

Congestive heart failure (CHF) is a major health issue resulting in significant patient morbidity and mortality. Left ventricular assist devices (LVADs) are becoming an increasingly popular method of treatment for patients with end-stage CHF. As the use of LVADs increases, there is a greater likelihood that some of these patients will live to develop general surgical problems. It is important for general surgeons to be aware of the often complex evaluation and treatment of patients with these problems. We retrospectively reviewed the charts of three patients with LVADs who underwent nonthoracic general surgical procedures. We reviewed duration of LVAD, time from LVAD implantation to operation, type of anesthesia, and any postoperative complications. Three patients with LVADs underwent five nonthoracic general surgical procedures. Anticoagulation was reversed with heparinization in four cases, the fifth case requiring fresh-frozen plasma. There was no perioperative mortality. Two morbidities occurred in separate patients, a wound infection and driveline site infection. These were managed nonoperatively. These patients raise several important concerns. They are often anticoagulated and require reversal. Staff needs to be familiar with these devices, their operation and physiology. The placement of the LVAD imposes limitations on surgical site location that require the surgeon to be prepared, flexible, and often creative.

Boundary effects on the electrophoretic motion of cylindrical particles: concentrically and eccentrically-positioned particles in a capillary.

The bounded electrophoretic motion of a cylindrical particle in a circular cylindrical microchannel is explored for two cases: (1) the particle is located on the centerline of a channel (concentrically), with a symmetric wall boundary condition since gap width is constant throughout; and (2) the particle is at an eccentric location in the channel, with an asymmetric boundary condition set by the walls. The objective is to determine the effect of different boundary conditions, geometries, and physical properties on the velocity and orientation of the cylinder with respect to the boundary. A theoretical model for the motion of the cylinder is presented and the problem is solved numerically. The steady-state simulations show that the velocity of the cylinder is reduced at small gap widths for the concentric case, but the velocity is increased at small gap widths for the eccentric case. When the cylinder is angled with respect to the horizontal in the symmetric case or is near the boundary in the asymmetric case, vertical and rotational components of velocity are predicted. In such cases, transient simulations are appropriate for most accurately representing particle motion. Two such simulations are included herein and show both horizontal and vertical translation plus rotation of the particle as a function of time.

Evaluation of long-term surgical site occurrences in ventral hernia repair: implications of preoperative site independent MRSA infection.

PURPOSE: Previous work demonstrated that prior MRSA infection [MRSA(+)] is associated with 30-day surgical site infection (SSI) following ventral hernia repair (VHR). We aimed to determine the impact of MRSA(+) on long-term wound outcomes after VHR. PARTICIPANTS: A retrospective cohort study was performed at a tertiary center between July 11, 2005, and May 18, 2012, of patients undergoing elective VHR with class I wounds. Patients with documented preoperative MRSA infection at any site (urinary, bloodstream, SSI, etc.) were considered MRSA(+). Primary outcome was 2-year surgical site occurrence (SSO), defined as SSI, cellulitis, necrosis, nonhealing wound, seroma, hematoma, dehiscence, or fistula. SSOs were subdivided into those that required procedural intervention (SSOPI) and those that did not. RESULTS: Among 632 patients, 46 % were female with average age 53 ± 13 years. There were 368 SSOs in 193 patients (31 %); an SSOPI occurred in 9.8 % (n = 62). The most common SSOs were cellulitis (91/632), seroma (91/632), and serous drainage (58/632). The rate of 2-year SSO was higher with MRSA(+) compared to those without (46 vs. 29 %, p = 0.023), attributed to increased soft tissue necrosis, purulent drainage, serous drainage, cellulitis, and fistula. In multivariable analysis, MRSA(+) was not associated with 2-year SSO (HR 1.5, 95 % CI 0.91-2.55, p = 0.113); factors associated with SSO included obesity, immunosuppression, mesh repair, and operative times. CONCLUSIONS: This study is the first to evaluate long-term SSOs and SSOPIs after VHR, highlighting the importance of long-term follow-up. Though not independently associated with SSOs, MRSA(+) may be a marker of hernia complexity.

Decomposition of interaction free energies in proteins and other complex systems.

A recent analysis of Mark and van Gunsteren has questioned the validity of separating different free energy components in proteins, or indeed in any complex system. The separability of free energy terms is re-examined from both a theoretical and a numerical perspective. Using a power series expansion of the free energy, it is found that the leading terms are free energy components that arise from individual contributions to the Hamiltonian ("in situ" free energies). The energetic part of an in situ free energy component is given by the ensemble average of the corresponding Hamiltonian component, while the leading term in the entropic part, which was missing in the analysis of Mark and van Gunsteren, is given by the mean square fluctuation. In addition there are correlations between fluctuations in each Hamiltonian component, which give rise to a coupling, or correlation entropy. A simple system, whose configurational degrees of freedom can be completely sampled, was examined in order to determine the relative sizes of these different contributions to the free energy. Under certain conditions, the change in system free energy observed when a particular component of the Hamiltonian is removed or altered is well approximated by the change in the in situ free energy of that component. In practical terms, this allows one in these cases to separate out different free energy contributions.

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.

On the decomposition of free energies.

The decomposition of free energies and entropies into components has recently been discussed within the framework of the free energy perturbation (FEP) and thermodynamic integration (TI) methods. In FEP, the cumulant expansion of the excess free energy contains coupling terms in second and higher orders. It is shown here that this expansion can be expressed in terms of temperature derivatives of the mean energy, suggesting a natural decomposition of the free energy into components corresponding to each term in the Hamiltonian. This result is derived in such a way that it establishes the equivalence to a particular form of component analysis based on TI in which all terms in the interaction energy are turned on simultaneously using 1/kT as the coupling parameter.

Salt effects on ligand-DNA binding. Minor groove binding antibiotics.

Salt dependent electrostatic effects play a central role in intermolecular interactions involving nucleic acids. In this paper, the finite-difference solution to the nonlinear Poisson-Boltzmann (NLPB) equation is used to evaluate the salt dependent contribution to the electrostatic binding free energy of the minor groove binding antibiotics DAPI, Hoechst 33258 and netropsin to DNA using detailed molecular structures of the complexes. For each of these systems, a treatment based on the NLPB equation accurately describes the variation of the experimentally observed binding constant with bulk salt concentration. A solvation formalism is developed in which salt effects are described in terms of three free energy contributions: the electrostatic ion-molecule interaction free energy, delta delta G degrees im; the electrostatic ion-ion interaction free energy, delta delta G degrees ii; and the entropic ion organization free energy, delta delta G degrees org. The electrostatic terms, delta delta G degrees im and delta delta G degrees ii, have both enthalpic and entropic components, while the term delta delta G degrees org is purely a cratic entropy. Each of these terms depends significantly on salt dependent changes in the counterion and coion concentrations around the DNA. In each of the systems studied, univalent ions substantially destabilize charged ligand-DNA complexes at physiological salt concentrations. This effect involves a salt dependent redistribution of counterions near the DNA. The free energy associated with the redistribution of counterions upon binding is dominated by the unfavorable change in the electrostatic ion-molecule interactions, delta delta G degrees im, rather than the change in the cratic entropy of ion organization, delta delta G degrees org. In addition, the observed slope of the salt dependence of the free energy is determined by electrostatic ion-molecule and ion-ion interactions as well as the cratic entropy of ion release. These findings are in contrast to models in which the cratic entropy of counterion release drives binding.

Empirical free energy calculations: a blind test and further improvements to the method.

Empirical Gibbs functions estimate free energies of non-covalent reactions (deltaG) from atomic coordinates of reaction products (e.g. antibody-antigen complexes). The function previously developed by us has four terms that quantify the effects of hydrophobic, electrostatic and entropy changes (conformational, association) upon complexation. The function was used to calculate delta deltaG of ten lysozyme mutants affecting the stability of the HyHEL-10 antibody-lysozyme complex. The mutants were computer-modeled from the X-ray structure of the wild-type, and free energy calculations produced a correlation coefficient of 0.5 with the experimental delta deltaG data (average absolute error +/-3 kcal). The following changes were then introduced into the Gibbs function: (1) the hydrophobic force was made proportional to the molecular surface, as calculated by the GEPOL93 algorithm, with the scaling constant of 70 cal/mol/A2; (2) calculation of the electrostatics of binding was carried out by the finite difference Poisson-Boltzmann algorithm, which employed uniform grid charging, dielectric boundary smoothing and charge anti-aliasing; and (3) side-chain conformational entropy was estimated from the CONGEN sampling of torsional degrees of freedom. In the new calculations, correlation with experimental data improved to 0.6 or 0.8 if a single outlying mutant, K96M, was neglected. Analysis of the errors remaining in our calculations indicated that molecular mechanics-based modeling of the mutants, rather than the form of our amended Gibbs function, was the main factor limiting the accuracy of the free energy estimates.

Salt effects on protein-DNA interactions. The lambda cI repressor and EcoRI endonuclease.

In this paper, finite-difference solutions to the nonlinear Poisson-Boltzmann (NLPB) equation are used to calculate the salt dependent contribution to the electrostatic DNA binding free energy for both the lambda cI repressor and the EcoRI endonuclease. For the protein-DNA systems studied, the NLPB method describes nonspecific univalent salt dependent effects on the binding free energy which are in excellent agreement with experimental results. In these systems, the contribution of the ion atmosphere to the binding free energy substantially destabilizes the protein-DNA complexes. The magnitude of this effect involves a macromolecular structure dependent redistribution of both cations and anions around the protein and the DNA which is dominated by long range electrostatic interactions. We find that the free energy associated with global ion redistribution upon binding is more important than changes associated with local protein-DNA interactions (ion-pairs) in determining salt effects. The NLPB model reveals how long range salt effects can play a significant role in the relative stability of protein-DNA complexes with different structures.

The role of protonation and metal chelation preferences in defining the properties of mercury-binding coiled coils.

To define the delicate interplay between metal chelation, protein folding and function in metalloproteins, a family of de novo-designed peptides was synthesized that self-assemble in aqueous solution to form two and three-stranded alpha-helical coiled coils. Each peptide contains a single Cys residue at an a or d position of the heptad repeat. Peptide association thus produces a Cys-rich coordination environment that has been used to bind Hg(II) ions. These peptides display a pH-dependent association, with trimers observed above the pKa of Glu side-chains and dimers below this value. Finite-difference Poisson-Boltzmann calculations suggest that the dimeric state decreases the unfavorable electrostatic interactions between positively charged Lys side-chains (relative to the trimer). The Cys-containing peptides bind Hg(II) in a position-dependent fashion. Cys at a positions form three-coordinate Hg complexes at high pH where the trimeric aggregation state predominates, and two-coordinate complexes at lower pH. A d position Cys, however, is only able to generate the two-coordinate complex, illustrating the difference in coordination geometry between the two positions in the coiled coil. The binding of Hg(II) was also shown to substantially increase the stability of the helical aggregates.

Induction of Larval Settlement in the Reef Coral Porites astreoides by a Cultivated Marine Roseobacter Strain.

Successful larval settlement and recruitment by corals is critical for the survival of coral reef ecosystems. Several closely related strains of γ-proteobacteria have been identified as cues for coral larval settlement, but the inductive properties of other bacterial taxa naturally occurring in reef ecosystems have not yet been explored. In this study, we assayed bacterial strains representing taxonomic groups consistently detected in corals for their ability to influence larval settlement in the coral Porites astreoides. We identified one α-proteobacterial strain, Roseivivax sp. 46E8, which significantly increased larval settlement in P. astreoides. Logarithmic growth phase (log phase) cell cultures of Roseivivax sp. 46E8 and filtrates (0.22µm) from log phase Roseivivax sp. 46E8 cultures significantly increased settlement, suggesting that an extracellular settlement factor is produced during active growth phase. Filtrates from log phase cultures of two other bacterial isolates, Marinobacter sp. 46E3, and Cytophaga sp. 46B6, also significantly increased settlement, but the cell cultures themselves did not. Monospecific biofilms of the three strains did not result in significant increases in larval settlement. Organic and aqueous/methanol extracts of Roseivivax sp. 46E8 cultures did not affect larval settlement. Examination of filtrates from cell cultures showed that Roseivivax sp. 46E8 spontaneously generated virus-like particles in log and stationary phase growth. Though the mechanism of settlement enhancement by Roseivivax sp. 46E8 is not yet elucidated, our findings point to a new aspect of coral-Roseobacter interactions that should be further investigated, especially in naturally occurring, complex microbial biofilms on reef surfaces.

Entropy-enthalpy compensation: fact or artifact?

The phenomenon of entropy-enthalpy (S-H) compensation is widely invoked as an explanatory principle in thermodynamic analyses of proteins, ligands, and nucleic acids. It has been suggested that this compensation is an intrinsic property of either complex, fluctuating, or aqueous systems. The questions examined here are whether the observed compensation is extra-thermodynamic (i.e., reflects anything more than the well-known laws of statistical thermodynamics) and if so, what does it reveal about the system? Compensation is rather variably defined in the literature and different usages are discussed. The most precise and interesting one, which is considered here, is a linear relationship between DeltaH and DeltaS for some series of perturbations or changes in experimental variable. Some recent thermodynamic data on proteins purporting to show compensation is analyzed and shown to be better explained by other causes. A general statistical mechanical model of a complex system is analyzed to explore whether and under what conditions extra-thermodynamic compensation can occur and what it reveals about the system. This model shows that the most likely behavior to be seen is linear S-H compensation over a rather limited range of perturbations with a compensation temperature Tc = dDeltaH/dDeltaS within 20% of the experimental temperature. This behavior is insensitive to the details of the model, thus revealing little extra-thermodynamic or causal information about the system. In addition, it will likely be difficult to distinguish this from more trivial forms of compensation in real experimental systems.

Diagnosis and management of retroperitoneal hematomas after femoral vein cannulation for hemodialysis.

Six patients were identified who suffered retroperitoneal hemorrhage during or after hemodialysis via a femoral vein approach. Hypotension developing during dialysis or lower quadrant abdominal pain was the common presenting symptom. All patients had decreasing hematocrits and five of the six patients required blood transfusions. The method of diagnosis was clinical, with plain abdominal x-ray examination and cystography being the most helpful adjuncts. All patients were successfully treated with volume resuscitation and removal of the femoral vein catheters; none required operative intervention or suffered adverse late effects. Femoral vein dialysis is useful for uremic patients without other peripheral vascular access options, and its associated morbidity may be minimized provided complications are recognized and promptly treated.

Pancreatoduodenectomy with pyloric preservation for carcinoma of the pancreas: a cautionary note.

Radical pancreatoduodenectomy for treatment of pancreatic carcinoma has been the surgical standard of care for the past four decades. The recent popularization of pylorus-sparing pancreatoduodenectomy to treat benign pancreatic disease, because of its decreased morbidity and long-term nutritional consequences, has led to the use of this procedure in cases of pancreatic carcinoma. We report recent experience with three patients with pancreatic carcinoma in whom pyloric preservation would have compromised the potential chance for curative resection or compromised palliation because of occult spread of tumor to a region not resected with this new operative approach. Two patients had proximal, microscopic intramural spread of pancreatic adenocarcinoma within the duodenum or antrum--a mode of spread not previously reported with pancreatic carcinoma. Both patients had no other evidence of metastatic involvement, and both would have had positive surgical margins in a pylorus-sparing pancreatoduodenectomy. A third case demonstrates a true submucosal recurrence of pancreatic carcinoma after a pylorus-sparing pancreatoduodenectomy. It is debatable that any case demonstrating intramural spread within the duodenum could be cured with a standard Whipple resection as this may well represent another sign of incurability, like lymphatic or perineural spread, but it is clearly a major potential obstacle to palliation if submucosal recurrences occur as a result of the use of the pylorus-sparing pancreatoduodenectomy in cases of pancreatic cancer. The use of pylorus-sparing pancreatoduodenectomy in resectable pancreatic cancers must be viewed skeptically at this time.