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In this paper, a new solvation model is proposed for simulations of biomolecules in aqueous solutions that combines the strengths of explicit and implicit solvent representations. Solute molecules are placed in a spherical cavity filled with explicit water, thus providing microscopic detail where it is most needed. Solvent outside of the cavity is modeled as a dielectric continuum whose effect on the solute is treated through the reaction field corrections. With this explicit/implicit model, the electrostatic potential represents a solute molecule in an infinite bath of solvent, thus avoiding unphysical interactions between periodic images of the solute commonly used in the lattice-sum explicit solvent simulations. For improved computational efficiency, our model employs an accurate and efficient multiple-image charge method to compute reaction fields together with the fast multipole method for the direct Coulomb interactions. To minimize the surface effects, periodic boundary conditions are employed for nonelectrostatic interactions. The proposed model is applied to study liquid water. The effect of model parameters, which include the size of the cavity, the number of image charges used to compute reaction field, and the thickness of the buffer layer, is investigated in comparison with the particle-mesh Ewald simulations as a reference. An optimal set of parameters is obtained that allows for a faithful representation of many structural, dielectric, and dynamic properties of the simulated water, while maintaining manageable computational cost. With controlled and adjustable accuracy of the multiple-image charge representation of the reaction field, it is concluded that the employed model achieves convergence with only one image charge in the case of pure water. Future applications to pKa calculations, conformational sampling of solvated biomolecules and electrolyte solutions are briefly discussed.

Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4'-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 +/- 1.3 kcal/mol from 3.5 +/- 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k approximately 30 sec(-1)) with greater solvent accessibility was followed by rearrangement (k approximately 0.01 sec(-1)), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state.

Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn(2+)-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn(2+)-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme.

The stability of biocatalysts is an important criterion for a sustainable industrial operation economically. T1 lipase is a thermoalkalophilic enzyme derived from Geobacillus zalihae strain T1 (T1 lipase) that was isolated from palm oil mill effluent (POME) in Malaysia. We report here the results of high temperatures molecular dynamics (MD) simulations of T1 lipase in explicit solvent. We found that the N-terminal moiety of this enzyme was accompanied by a large flexibility and dynamics during temperature-induced unfolding simulations which preceded and followed by clear structural changes in two specific regions; the small domain (consisting of helices alpha3 and alpha5, strands beta1 and beta2, and connecting loops) and the main catalytic domain or core domain (consisting of helices alpha6- alpha9 and connecting loops which located above the active site) of the enzyme. The results suggest that the small domain of model enzyme is a critical region to the thermostability of this organism.

Understanding multivariate relationships is an important task in multivariate data analysis. Unfortunately, existing multivariate visualization systems lose effectiveness when analyzing relationships among variables that span more than a few dimensions. We present a novel multivariate visual explanation approach that helps users interactively discover multivariate relationships among a large number of dimensions by integrating automatic numerical differentiation techniques and multidimensional visualization techniques. The result is an efficient workflow for multivariate analysis model construction, interactive dimension reduction, and multivariate knowledge discovery leveraging both automatic multivariate analysis and interactive multivariate data visual exploration. Case studies and a formal user study with a real dataset illustrate the effectiveness of this approach.

We present a novel analytical method to calculate conformational entropy of ideal cross-linking polymers from the configuration integral by employing a Mayer series expansion. Mayer-functions describing chemical bonds within the chain and for cross-links are sharply peaked over the temperature range of interest, and, are well approximated as statistically weighted Dirac delta-functions that enforce distance constraints. All geometrical deformations consistent with a set of distance constraints are integrated over. Exact results for a contiguous series of connected loops are employed to substantiate the validity of a previous phenomenological distance constraint model that describes protein thermodynamics successfully based on network rigidity.

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP.P(i)) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer (FRET) to monitor conformational changes in the upper-50-kDa domain of the actin binding cleft in the weak and strong actin binding states. Steady-state and lifetime data monitoring the FRET signal suggest that the cleft is in a more open conformation in the weak actin binding states. Transient kinetic experiments suggest that a rapid conformational change occurs, which is consistent with cleft closure before actin-activated phosphate release. Our results have identified a pre-force-generation actomyosin ADP.P(i) state, and suggest force generation may occur from a state not yet seen by crystallography in which the actin binding cleft and the nucleotide binding pocket are closed. Computational modeling uncovers dramatic changes in the rigidity of the upper-50-kDa domain in different nucleotide states, which suggests that the intrinsic flexibility of this domain allows myosin motors to accomplish simultaneous tight nucleotide binding (closed nucleotide binding pocket) and high-affinity actin binding (closed actin binding cleft).

Additivity principles in chemistry, biochemistry, and biophysics have been used extensively for decades. Nevertheless, it is well known that additivity frequently breaks down in complex biomacromolecules. Nonadditivity within protein double mutant free energy cycles of spatially close residue pairs is a generally well-understood phenomenon, whereas a robust description of nonadditivity extending over large distances remains to be developed. Here, we test the hypothesis that the mutational effects tend to be nonadditive if two structurally well-separated mutated residues belong to the same rigid cluster within the wild type protein, and additive if they are located within different clusters. We find the hypothesis to be statistically significant with P-values that range from 10(-5) to 10(-6). To the best of our knowledge, this result represents the first demonstration of a statistically significant preponderance for nonadditivity over long distances. These findings provide new insight into the origins of long-range nonadditivity in double mutant cycles, which complements the conventional wisdom that nonadditivity arises in double mutations involving contacting residues. Consequently, these results should have far-reaching implications for a proper understanding of protein stability, structure/function analyses, and protein design., ((c) 2007 Wiley-Liss, Inc.)

Two techniques of vena cava filter placement with intravascular ultrasound (IVUS) guidance have been described previously. Placement with real-time IVUS imaging requires two venous access sites, one for the filter delivery system and one for the IVUS catheter, which makes the procedure more invasive. Alternatively, a single-access technique of IVUS imaging of the vena cava requires measuring the distance from the access site to the desired location for filter placement and then delivering the filter to that distance blindly, risking filter misplacement. We describe in this article a single puncture technique that allows for real-time imaging to position the filter delivery sheath using IVUS and reduces the uncertainty of the blind positioning of the filter delivery system.

The time it takes for proteins to fold into their native states varies over several orders of magnitude depending on their native-state topology, size, and amino acid composition. In a number of previous studies, it was found that there is strong correlation between logarithmic folding rates and contact order for proteins that fold with two-state kinetics, while such correlation is absent for three-state proteins. Conversely, strong correlations between folding rates and chain length occur within three-state proteins, but not in two-state proteins. Here, we demonstrate that chain lengths and folding rates of two-state proteins are not correlated with each other only when all-alpha, all-beta, and mixed-class proteins are considered together, which is typically the case. However, when considering all-alpha and all-beta two-state proteins separately, there is significant linear correlation between folding rate and size. Moreover, the sets of data points for the all-alpha and all-beta classes define asymptotes of lower and upper limits on folding rates of mixed-class proteins. By analyzing correlation of other topological parameters with folding rates of two-state proteins, we find that only the long-range order exhibits correlation with folding rates that is uniform over all three classes. It is also the only descriptor to provide statistically significant correlations for each of the three structural classes. We give an interpretation of this observation in terms of Makarov and Plaxco's diffusion-based topomer-search model.

The variability within calculated protein residue pKa values calculated using Poisson-Boltzmann continuum theory with respect to small conformational fluctuations is investigated. As a general rule, sites buried in the protein core have the largest pKa fluctuations but the least amount of conformational variability; conversely, sites on the protein surface generally have large conformational fluctuations but very small pKa fluctuations. These results occur because of the heterogeneous or uniform nature of the electrostatic microenvironments at the protein core or surface, respectively. Atypical surface sites with large pKa fluctuations occur at the interfaces between significant anionic and cationic potentials.

Objective: The acute technical failure of endovascular treatment of chronic total occlusions (CTOs) is most often due to the inability to re-enter the true lumen after occlusion is crossed in a subintimal plane. This study reports our initial experience with true lumen re-entry devices in the treatment of CTOs., Methods: Patients with treatment of CTOs were identified from our vascular registry. All patients in whom the Pioneer catheter or the Outback catheter were used were also identified from a prospectively maintained separate database of cases in which true lumen re-entry devices were used. We used procedural data from the prospective database and reviewed the medical records. Lesion character and location, access type, location of true lumen re-entry, stent usage, procedural times, and complications, were tabulated., Results: From August 2003 to December 2004, endovascular techniques were used to treat 87 CTOs in 58 iliac and 29 superficial femoral arteries. In 24 (26%), the true lumen could not be re-entered by using standard catheter and wire techniques. The true lumen was not initially re-entered in 20 (34%) of 58 of treated iliac CTOs and four (13%) of 29 of treated superficial femoral artery CTOs (73% TASC C and D lesions). Intravascular ultrasound-guided true lumen re-entry using the Pioneer catheter (21 CTOs), or fluoroscopic-guided true lumen re-entry using the Outback catheter (3 CTOs) was successful in achieving true lumen re-entry in all cases at the location desired. Total time of re-entry catheter manipulation required to achieve re-entry was <10 minutes and was routinely accomplished in <3 minutes. All occlusions were stented. No cases were converted to open repair. Bleeding from the recanalization and angioplasty site occurred in four patients (15%). It was controlled with use of covered stents in two cases, and resolved after placement of uncovered stents in the other two. No significant bleeding occurred at the sites of true lumen re-entry needle deployment. All occlusions treated with true lumen re-entry devices remain clinically patent at a mean follow-up of 5.8 months., Conclusions: Endovascular treatment of chronic total occlusions is often limited by the inability to re-enter the true lumen after subintimal crossing of the occluded segment. This occurs more commonly with treatment of iliac occlusions than in superficial femoral artery occlusions. True lumen re-entry catheters are very effective at gaining wire passage back to the true lumen and facilitating successful endovascular treatment of chronic total occlusions that would otherwise require open bypass.

Numerous quantitative stability/flexibility relationships, within Escherichia coli thioredoxin (Trx) and its fragments are determined using a minimal distance constraint model (DCM). A one-dimensional free energy landscape as a function of global flexibility reveals Trx to fold in a low-barrier two-state process, with a voluminous transition state. Near the folding transition temperature, the native free energy basin is markedly skewed to allow partial unfolded forms. Under native conditions the skewed shape is lost, and the protein forms a compact structure with some flexibility. Predictions on ten Trx fragments are generally consistent with experimental observations that they are disordered, and that complementary fragments reconstitute. A hierarchical unfolding pathway is uncovered using an exhaustive computational procedure of breaking interfacial cross-linking hydrogen bonds that span over a series of fragment dissociations. The unfolding pathway leads to a stable core structure (residues 22-90), predicted to act as a kinetic trap. Direct connection between degree of rigidity within molecular structure and non-additivity of free energy is demonstrated using a thermodynamic cycle involving fragments and their hierarchical unfolding pathway. Additionally, the model provides insight about molecular cooperativity within Trx in its native state, and about intermediate states populating the folding/unfolding pathways. Native state cooperativity correlation plots highlight several flexibly correlated regions, giving insight into the catalytic mechanism that facilitates access to the active site disulfide bond. Residual native cooperativity correlations are present in the core substructure, suggesting that Trx can function when it is partly unfolded. This natively disordered kinetic trap, interpreted as a molten globule, has a wide temperature range of metastability, and it is identified as the "slow intermediate state" observed in kinetic experiments. These computational results are found to be in overall agreement with a large array of experimental data.

Hostile neck anatomy remains the predominant reason that patients are denied endovascular aneurysm repair (EVAR). We reviewed our experience of EVAR with use of prophylactic adjunctive proximal balloon-expandable stents in patients with hostile neck anatomy and adjunctive proximal balloon-expandable stents in patients with type I endoleaks. Of 140 patients who underwent EVAR between 2000 and 2004, we reviewed data for 19 patients in whom we used proximal balloon-expandable stents. By high-resolution computed tomography scan or angiography, hostile neck anatomy was classified as length <15 mm, neck diameters > or =26 mm, circumferential thrombus at the proximal neck, angulated neck > or =60 degrees, and neck bulge or reverse taper necks. Patients were considered to have hostile anatomy if they met 1 or more of the above-cited criteria. All patients underwent AAA repair with commercially available endograft systems, Zenith (Cook, Bloomington, IN) and AneuRx (Medtronic/AVE, Minneapolis, MN). Balloon-expandable stents utilized included Cordis-Palmaz stents (17/19) and eV3 Max stents (2/19). Stents were deployed in the proximal graft with transrenal extension. AneuRx (18/19) and Zenith (1/19) endografts were used in all of the patients. Of the 19 patients, 15 had prophylactic stent placement for known hostile neck anatomy and 4 patients had stent placement for type I endoleak. Assisted primary technical success was achieved in all patients. Three patients had maldeployment of the endograft or proximal stent requiring additional endovascular interventions at the time of surgery. No endografts were deployed too low requiring stent placement. Procedure-related complications occurred in 2 of 19 patients. These included 1 operative death secondary to pneumonia and 1 patient who developed progressive renal failure. Short-term clinical success was achieved in 17 of 19 patients. Two patients required secondary interventions, 1 due to device migration with secondary conversion to open repair, and an endoleak, which, on angiogram, was a large type II endoleak successfully treated with coiling of the inferior mesenteric artery. One patient was observed to have a type II endoleak with no associated aneurysm enlargement. Short-term results suggest the use of prophylactic adjunctive balloon-expandable stents may decrease the incidence of secondary interventions related to hostile neck anatomy when used as an adjunctive measure with EVAR. Based on our experience, we feel EVAR may be offered to an expanded patient population with hostile neck anatomy with use of prophylactic balloon-expandable stents.

The protein actin can polymerize from monomeric globular G-actin to polymeric filamentary F-actin, under the regulation of thermodynamic variables such as temperature, pressure, and compositions of G-actin and salts. We present here new measurements of the extent of polymerization (phi) of actin under pressure (P), for rabbit skeletal muscle actin in H2O buffer in the presence of adenosine triposphate and calcium ions and at low (5-15 mM) KCl concentrations. We measured phi using pyrene-labeled actin, as a function of time (t) and temperature (T), for samples of fixed concentrations of initial G-actin and KCl and at fixed pressure. The phi(T,P) measurements at equilibrium have the same form as reported previously at 1 atm: low levels of polymerization at low temperatures, representing dimerization of the actin; an increase in phi at the polymerization temperature (Tp); a maximum in phi(T) above Tp) with a decrease in phi(T) beyond the maximum, indicating a depolymerization at higher T. From phi(T,P) at temperatures below Tp, we estimate the change in volume for the dimerization of actin, DeltaVdim, to be -307+/-10 ml/mol at 279 K. The change of Tp with pressure dTp/dP=(0.3015+/-0.0009) K/MPa=(30.15+/-0.09) mK/atm. The phi(T,P) data at higher T indicate the change in volume on propagation, DeltaVprop, to be +401+/-48 ml/mol at 301 K. The phi(t) measurements yield initial relaxation times rp(T) that reflect the behavior of phi(T) and support the presence of a depolymerization temperature. We also measured the density of polymerizing actin with a vibrating tube density meter, the results of which confirm that the data from this instrument are affected by viscosity changes and can be erroneous.

Given the three-dimensional structure of a protein, its thermodynamic properties are calculated using a recently introduced distance constraint model (DCM) within a mean-field treatment. The DCM is constructed from a free energy decomposition that partitions microscopic interactions into a variety of constraint types, i.e., covalent bonds, salt-bridges, hydrogen-bonds, and torsional-forces, each associated with an enthalpy and entropy contribution. A Gibbs ensemble of accessible microstates is defined by a set of topologically distinct mechanical frameworks generated by perturbing away from the native constraint topology. The total enthalpy of a given framework is calculated as a linear sum of enthalpy components over all constraints present. Total entropy is generally a nonadditive property of free energy decompositions. Here, we calculate total entropy as a linear sum of entropy components over a set of independent constraints determined by a graph algorithm that builds up a mechanical framework one constraint at a time, placing constraints with lower entropy before those with greater entropy. This procedure provides a natural mechanism for enthalpy-entropy compensation. A minimal DCM with five phenomenological parameters is found to capture the essential physics relating thermodynamic response to network rigidity. Moreover, two parameters are fixed by simultaneously fitting to heat capacity curves for histidine binding protein and ubiquitin at five different pH conditions. The three free parameter DCM provides a quantitative characterization of conformational flexibility consistent with thermodynamic stability. It is found that native hydrogen bond topology provides a key signature in governing molecular cooperativity and the folding-unfolding transition.

Thermodynamic stability in polypeptides is described using a novel Distance Constraint Model (DCM). Here, microscopic interactions are represented as constraints. A topological arrangement of constraints define a mechanical framework. Each constraint in the framework is associated with an enthalpic and entropic contribution. All accessible topological arrangements of distance constraints form an ensemble of mechanical frameworks, each representing a microstate of the polypeptide. A partition function is calculated exactly using a transfer matrix approach, where in many respects the DCM is similar to the Lifson-Roig model. The crucial difference is that the effect of network rigidity is explicitly calculated for each mechanical framework in the ensemble. Network rigidity is a mechanical interaction that provides a mechanism for long-range molecular cooperativity and enables a proper treatment of the nonadditivity of a microscopic free energy decomposition. Accounting for (1) helix <--> coil conformation changes along the backbone similar to the Lifson-Roig model, (2) i to i + 4 hydrogen-bond formation <--> breaking similar to the Zimm-Bragg model, and (3) structured <--> unstructured solvent interaction (hydration effects), a six-parameter DCM describes normal and inverted helix-coil transitions in polypeptides. Under suitable mixed solvent conditions heat and cold denaturation is predicted. Model parameters are fitted to experimental data showing different degrees of cold denaturation in monomeric polypeptides in aqueous hexafluoroisopropanol (HFIP) solution at various HFIP concentrations. By assuming a linear HFIP concentration dependence (up to 6% by mole fraction) on model parameters, all essential experimentally observed features are captured.

A statistical mechanical distance constraint model (DCM) is presented that explicitly accounts for network rigidity among constraints present within a system. Constraints are characterized by local microscopic free-energy functions. Topological rearrangements of thermally fluctuating constraints are permitted. The partition function is obtained by combining microscopic free energies of individual constraints using network rigidity as an underlying long-range mechanical interaction, giving a quantitative explanation for the nonadditivity in component entropies exhibited in molecular systems. Two exactly solved two-dimensional toy models representing flexible molecules that can undergo conformational change are presented to elucidate concepts, and to outline a DCM calculation scheme applicable to many types of physical systems. It is proposed that network rigidity plays a central role in balancing the energetic and entropic contributions to the free energy of biopolymers, such as proteins. As a demonstration, the distance constraint model is solved exactly for the alpha-helix to coil transition in homogeneous peptides. Temperature and size independent model parameters are fitted to Monte Carlo simulation data, which includes peptides of length 10 for gas phase, and lengths 10, 15, 20, and 30 in water. The DCM is compared to the Lifson-Roig model. It is found that network rigidity provides a mechanism for cooperativity in molecular structures including their ability to spontaneously self-organize. In particular, the formation of a characteristic topological arrangement of constraints is associated with the most probable microstates changing under different thermodynamic conditions.

Background: The aim of this study was to identify risk factors, clinical characteristics, and outcome of patients with colon ischemia., Methods: A 10-year (1992-2002) retrospective study was undertaken. Patients were identified from computerized hospital discharge information. Patient variables were entered into a computerized database and analyzed., Results: One hundred twenty-nine patients were identified. The mean age was 66 years (range, 29-98 years); 47% were male. Forty-three patients (33%) had chronic renal failure; 73 patients (57%) were receiving vasoactive drugs, and 72 patients (56%) had atherosclerosis. Fifty-four of 129 patients (42%) had ischemic colitis in-hospital. Fifty-six of 129 patients (43%) had melena; 49 of 56 patients (88%) survived. Forty-three of 129 patients (33%) had an acute abdomen; 22 of 43 patients (51%) died. Seventy of 129 patients (54%) were treated nonoperatively initially; the condition of 17 of 70 patients (24%) required surgery. Of 76 patients who were treated operatively, 31 patients (41%) died. Eleven patients at operation had ischemia without colon infarction or perforation; 5 of these patients (45%) died. The overall mortality rate was 29% (37/129 patients)., Conclusion: Ischemic colitis is associated with chronic renal failure and atherosclerosis. Patients commonly have an acute abdomen. The absence of colonic infarction does not ensure a favorable outcome. Patients who are felt to be candidates for nonoperative therapy have significant mortality rates. Mortality rates remain high, despite treatment.