Multilevel superposition for deciphering the conformational variability of protein ensembles.

The dynamics and variability of protein conformations are directly linked to their functions. Many comparative studies of X-ray protein structures have been conducted to elucidate the relevant conformational changes, dynamics and heterogeneity. The rapid increase in the number of experimentally determined structures has made comparison an effective tool for investigating protein structures. For example, it is now possible to compare structural ensembles formed by enzyme species, variants or the type of ligands bound to them. In this study, the author developed a multilevel model for estimating two covariance matrices that represent inter- and intra-ensemble variability in the Cartesian coordinate space. Principal component analysis using the two estimated covariance matrices identified the inter-/intra-enzyme variabilities, which seemed to be important for the enzyme functions, with the illustrative examples of cytochrome P450 family 2 enzymes and class A $\beta$-lactamases. In P450, in which each enzyme has its own active site of a distinct size, an active-site motion shared universally between the enzymes was captured as the first principal mode of the intra-enzyme covariance matrix. In this case, the method was useful for understanding the conformational variability after adjusting for the differences between enzyme sizes. The developed method is advantageous in small ensemble-size problems and hence promising for use in comparative studies on experimentally determined structures where ensemble sizes are smaller than those generated, for example, by molecular dynamics simulations.

Identification of histidine residues that affect the T/R-state conformations of human hemoglobin using constant pH molecular dynamics simulations.

Human hemoglobin (Hb) is a tetrameric protein consisting of two α and two ß subunits that can adopt a low-affinity T- and high-affinity R-state conformations. Under physiological pH conditions, histidine (His) residues are the main sites for proton binding or release, and their protonation states can affect the T/R-state conformation of Hb. However, it remains unclear which His residues can effectively affect the Hb conformation. Herein, the impact of the 38 His residues of Hb on its T/R-state conformations was evaluated using constant-pH molecular dynamics (CpHMD) simulations at physiological pH while focusing on the His protonation states. Overall, the protonation states of some His residues were found to be correlated with the Hb conformation state. These residues were mainly located in the proximity of the heme (α87 and ß92), and at the α1ß2 and α2ß1 interfaces (α89 and ß97). This correlation may be partly explained by how easily hydrogen bonds can be formed, which depends on the protonation states of the His residues. Taken together, these CpHMD-based findings provide new insights into the identification of titratable His residues α87, α89, ß92, and ß97 that can affect Hb conformational switching under physiological pH conditions.

Sample Entropy in Electrocardiogram During Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is an arrhythmia commonly encountered in clinical practice. There is a high risk of thromboembolism in patients with AF. Nonlinear analyses such as electroencephalogram (EEG), electrocardiogram (ECG), and respiratory movement have been used to quantify biological signals, and sample entropy (SampEn) has been employed as a statistical measure to evaluate complex systems. In this study, we examined the values of SampEn in ECG signals for patients with and without AF to measure the regularity and complexity. METHODS: ECG signals of lead II were recorded from 34 subjects without arrhythmia and 15 patients with chronic AF in a supine position. The ECG signals were converted into time-series data and SampEn was calculated. RESULTS: The SampEn values for the group without arrhythmia were 0.252 ± 0.114 [time lag (τ) = 1] and 0.533 ± 0.163 (τ = 5), and those for the chronic AF group were 0.392 ± 0.158 (τ = 1) and 0.759 ± 0.246 (τ = 5). The values of SampEn were significantly higher in the group with chronic AF than in the group without arrhythmia (P < 0.01 for τ = 1, P < 0.004 for τ = 5). The constructed three-dimensional vectors were plotted in time-delayed three-dimensional space. We used time lags of τ = 5 and τ = 1. The shape of the loops of the three-dimensional space was better for τ = 5. CONCLUSION: The values of SampEn from ECG for chronic AF patients were higher than for subjects without arrhythmia, suggesting greater complexity for the time-series from chronic AF patients. SampEn is considered a new index for evaluating complex systems in ECG.

A Modified Method for Examining the Walking Pattern and Pace of COPD Patients in a 6-min Walk Test Before and After the Inhalation of Procaterol.

Objective The 6-min walk test (6MWT) is a simple test that is used to examine the exercise tolerance and outcomes in patients with chronic obstructive pulmonary disease (COPD). Although the 6MWT is useful for assessing exercise tolerance, it is difficult to evaluate time-dependent parameters such as the walking pattern. A modified 6MWT has been devised to assess the walking pattern by calculating the number of steps per second (NSPS). This study was performed to investigate walking pattern of COPD patients in the modified 6MWT before and after a single inhalation of the short-acting ß2-agonist procaterol. Methods Nine male COPD patients participated in this study. The 6MWT was performed before and after the inhalation of procaterol hydrochloride. A digital video recording of the 6MWT was made. After the 6MWT, the number of steps walked by the subject in each 5-s period was counted manually with a hand counter while viewing the walking test on the video monitor. Results After the inhalation of procaterol, the 6-min walking distance increased significantly in comparison to baseline (p<0.01). The mean NSPS was also significantly increased after the inhalation of procaterol in comparison to baseline (p<0.01). The walking pattern was displayed on a graph of time versus NSPS, and the walking pace was shown by a graph of time versus cumulative steps. Conclusion The analysis of the COPD patients' walking test performance and their walking pattern and pace in the 6MWT may help to evaluate the effects of drug treatment.

Usefulness of Pulse Oximeter That Can Measure SpO2 to One Digit After Decimal Point.

Pulse oximeters are used to noninvasively measure oxygen saturation in arterial blood (SaO2). Although arterial oxygen saturation measured by pulse oximeter (SpO2) is usually indicated in 1% increments, the value of SaO2 from arterial blood gas analysis is not an integer. We have developed a new pulse oximeter that can measure SpO2 to one digit after the decimal point. The values of SpO2 from the newly developed pulse oximeter are highly correlated with the values of SaO2 from arterial blood gas analysis (SpO2 = 0.899 × SaO2 + 9.944, r = 0.887, P < 0.0001). This device may help improve the evaluation of pathological conditions in patients.

Approximate Entropy of Respiratory Movements in Human Newborns during Different Sleep States.

Previous studies have reported that the respiratory cycle of healthy newborns is more irregular during active sleep. This study aimed to apply non-linear analysis to examine the irregularity of respiratory movement in newborns at different sleep states. The respiratory movement signals from an abdominal band during quiet and active sleep were analyzed using approximate entropy (ApEn). The breathing interval of active sleep was significantly shorter than that of quiet sleep [1.30 (0.17) s vs. 1.58 (0.11) s; (P < 0.03)]. The ApEn of respiratory movements during active sleep were significantly larger than that during quiet sleep [0.785 (0.135) s vs. 0.678 (0.083) s; (P < 0.05)]. We found that the ApEn of respiratory movement in healthy newborns could detect irregularities in respiration during sleep.

Modified six-minute walk test: number of steps per second.

The 6-min walk test (6MWT) has been used to examine subjective dyspnea, predict mortality and measure clinical outcomes in studies of patients with chronic pulmonary or heart disease. Although the 6MWT is useful to assess the general ability to perform daily physical activity, it is difficult to evaluate time-dependent responses. To improve the 6MWT, we devised a new index, which is the number of steps walked per second (NSPS). We performed the 6MWT in 11 healthy subjects and 7 patients with chronic obstructive pulmonary disease (COPD) and calculated the NSPS. The mean NSPS was significantly higher in the healthy subjects than in the COPD patients, while the coefficient of variation of the NSPS was significantly smaller in healthy subjects compared with COPD patients. Calculation of the NSPS was useful to evaluate the walking pattern. This modified 6MWT may be helpful for assessing the efficacy of rehabilitation and drug therapy for COPD.

Molecular dynamics study on conformational differences between dGMP and 8-oxo-dGMP: Effects of metal ions.

The modified nucleotide base 7,8-dihydro-8-oxo-guanine (8-oxo-G) is one of the major sources of spontaneous mutagenesis. Nucleotide-sanitizing enzymes, such as the MutT homolog-1 (MTH1) and nudix-type motif 5 (NUDT5), selectively remove 8-oxo-G from the cellular pool of nucleotides. Previous studies showed that, although the syn conformation generally predominates in purine nucleotides with a bulky substituent at the 8-position, 8-oxo-dGMP binds to both MTH1 and NUDT5 in the anti conformation. This study was initiated to investigate the possibility that 8-oxo-dGMP itself may adopt the anti conformation. Molecular dynamics simulations of mononucleotides (dGMP, 8-oxo-dGMP) in aqueous solution were performed. 8-oxo-dGMP adopted the anti conformation as well as the syn conformation, and the proportion of adopting the anti conformation increased in the presence of metal ions. When 8-oxo-dGMP was in the anti conformation, a metal ion was located between the oxygen atom of phosphate and the oxygen atom at the 8-position of 8-oxo-G. The types of stable anti conformations of 8-oxo-dGMP differed, depending on the ionic radii and charges of coexisting ions. These data suggested a role for metal ions, other than as cofactors for the hydrolysis of the di- and tri-phosphate forms of mononucleotides; that the metal ions help retain the anti conformation of the N-glycosidic torsion angle of 8-oxo-dGMP to promote the binding between the 8-oxo-G deoxynucleotide and the nucleotide-sanitizing enzymes.

Prognostic impact of preoperative hematological disorders and a risk stratification model in bladder cancer patients treated with radical cystectomy.

OBJECTIVES: The present study investigated prognostic indicators, including clinicopathological and preoperative hematological factors, and developed a prognostic factor-based risk stratification model in bladder cancer patients treated with radical cystectomy. METHODS: Data were collected from 249 consecutive bladder cancer patients treated with radical cystectomy without neoadjuvant therapy. Prognostic values of the preoperative hematological parameters, along with the patients' clinicopathological parameters were evaluated. A risk stratification model was developed to predict disease-specific survival after radical cystectomy using the regression coefficients of multivariate analysis. RESULTS: In the multivariate analysis, preoperative hemoglobin and C-reactive protein levels, as well as the pathological factors of T stage, positive surgical margin and lymph node metastasis, were independently predictive of disease-specific survival. Low hemoglobin (<10.5 g/dL), a high C-reactive protein (>0.5 mg/dL), extravesical T stage (≥pT3a) and positive surgical margin were independent predictors of poor disease-specific survival. The risk stratification model showed significant differences in disease-specific survival between the three subgroups. CONCLUSIONS: This is the first report to show the significance of combining preoperative hemoglobin with the pathology of radical cystectomy specimens as an independent predictor for disease-specific survival, and it also represents the largest contemporary series to date demonstrating that two types of preoperative hematological disorders, assessed by hemoglobin and C-reactive protein, are independent predictors in bladder cancer patients treated with radical cystectomy. Our risk stratification model could provide physicians with useful prognostic information for identifying patients who might be candidates for multimodal treatments.

Fatty acid binding to serum albumin: molecular simulation approaches.

BACKGROUND: Binding affinity for human serum albumin (HSA) is one of the most important factors affecting the distribution and free blood concentration of many ligands. The effect of fatty acids (FAs) on HSA-ligand binding has long been studied. Since the elucidation of the 3-dimensional structure of HSA, molecular simulation approaches have been applied to studies of the structure-function relationship of HSA-FA binding. SCOPE OF REVIEW: We review current insights into the effects of FA binding on HSA, focusing on the biophysical insights obtained using molecular simulation approaches such as docking, molecular dynamics (MD), and binding free energy calculations. MAJOR CONCLUSIONS: Possible conformational changes on binding of FA molecules to HSA have been observed through MD simulations. High- and low-affinity FA-binding sites on HSA have been identified based on binding free energy calculations. The relationship between the warfarin binding affinity of HSA and FA molecules has been clarified based on the results of simulations of multi-site FA binding that cannot be experimentally observed. GENERAL SIGNIFICANCE: Molecular simulation approaches have great potentials to provide detailed biophysical insights into HSA as well as the effects of the binding of FAs or other ligands to HSA. This article is part of a Special Issue entitled Serum Albumin.

Fas expression in nephrectomized, non-cancerous specimens predicts post-nephrectomy chronic kidney disease progression in patients with renal and upper urinary tract malignancies.

OBJECTIVES: Despite the surgical curability of renal cell carcinoma (RCC) and upper urinary tract urothelial carcinoma (UUT-UC), post-nephrectomy chronic kidney disease (CKD) continues to be a cause of concern. We investigated the correlation between the expression of apoptotic regulatory molecules in the nephrectomized, noncancerous cortex, as well as CKD progression and CKD-related mortality. MATERIALS AND METHODS: Fas and Bcl-2 mRNA and protein expression in surgically resected specimens from 100 patients with RCC and UUT-UC were determined. The estimated glomerular filtration rates (eGFR) were determined sequentially before surgery and up to 5 years after surgery. The relationships between CKD progression, the expression of these molecules in the renal cortex, and the clinical characteristics were analyzed. RESULTS: The mean 1-year postoperative percent eGFR decrease was 30.2 (Standard deviation [SD]: 15.2). The 1-year postoperative percent eGFR decrease greater than the approximate value of mean ± SD (45) was categorized as severe renal functional deterioration (SRFD). Glomerular Fas protein expression and a Fas/ß-actin mRNA ratio >0.3 were independent predictors for SRFD. Significantly increased mortality rates due to cardiovascular events were indicated by glomerular Fas protein expression, Fas mRNA levels >0.3, and SRFD. No significant change in Bcl-2 levels was observed. CONCLUSIONS: This study is the first report to demonstrate the significance of Fas expression in the nephrectomized normal cortex as a predictor of post-nephrectomy CKD progression. The results from nephrectomized kidney showed that the natural course of renal function in the remaining kidney may be affected not only by Fas-induced glomerular cell apoptosis but also by the total amount of Fas mRNA in cortical cells.

Steric and allosteric effects of fatty acids on the binding of warfarin to human serum albumin revealed by molecular dynamics and free energy calculations.

Human serum albumin (HSA) binds with drugs and fatty acids (FAs). This study was initiated to elucidate the relationship between the warfarin binding affinity of HSA and the positions of bound FA molecules. Molecular dynamics simulations of 11 HSA-warfarin-myristate complexes were performed. HSA-warfarin binding free energy was then calculated for each of the complexes by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. The results indicated that the magnitude of the binding free energy was smaller in HSA-warfarin complexes that had 4 or more myristate molecules than in complexes with no myristate molecules. The unfavorable effect on the HSA-warfarin binding affinity was caused sterically by the binding of a myristate molecule to the FA binding site closest to the warfarin binding site. On the other hand, the magnitude of HSA-warfarin binding free energy was largest when 3 myristate molecules were bound to the high-affinity sites. The strongest HSA-warfarin binding was attributable to favorable entropic contribution related to larger atomic fluctuations of the amino acid residues at the warfarin binding site. In the binding of 2 myristate molecules to the sites with the highest and second-highest affinities, allosteric modulation that enhanced electrostatic interactions between warfarin and some of the amino acid residues around the warfarin binding site was observed. This study clarified the structural and energetic properties of steric/allosteric effects of FAs on the HSA-warfarin binding affinity and illustrated the approach to analyze protein-ligand interactions in situations such that multiple ligands bind to the other sites of the protein.

Identification of high affinity fatty acid binding sites on human serum albumin by MM-PBSA method.

Human serum albumin (HSA) has seven common fatty acid (FA) binding sites. In this study, we used the molecular mechanics Poisson-Boltzmann surface area method to identify high affinity FA binding sites on HSA in terms of binding free energy. Using multiple HSA-FA (myristate, palmitate) complex models constructed by molecular dynamics simulations, two methods were performed in molecular mechanics Poisson-Boltzmann surface area, the "three-trajectory method" and the "single-trajectory method". The former, which is less precise than the latter but may be more accurate as it includes the effects of conformational change upon binding, was used to classify high and low affinity sites. As a result, Sites 2, 4, and 5 were identified as high affinity sites for both FAs. The latter method, which is precise because energies are calculated from snapshots of the same trajectory for HSA-FA complex, was performed to compare the magnitude of binding free energy for these sites. The order of magnitude was 5 > 4 > 2, identical to that of a previous publication by others. In this way, a combination of the two methods was effectively used to identify high affinity sites. This study therefore provides an insight into the quantitative identification of high affinity FA binding sites on HSA.

Molecular dynamics study of conformational changes in human serum albumin by binding of fatty acids.

Human serum albumin (HSA) binds with fatty acids under normal physiologic conditions. To date, there is little published information on the tertiary structure of HSA-fatty acid complex in aqueous solution. In the present study, we used molecular dynamics (MD) simulations to elucidate possible structural changes of HSA brought about by the binding of fatty acids. Both unliganded HSA and HSA-fatty acid complex models for MD calculations were constructed based on the X-ray crystal structures. Five myristates (MYRs) were bound in the HSA-fatty acid complex model. In the present MD study, the motion of domains I and III caused by the binding of MYR molecules increased the radius of gyration of HSA. Root-mean-square fluctuations from the MD simulations revealed that the atomic fluctuations of the specific amino acids at drug-binding site I that can regulate the drug-binding affinity were increased by the binding of MYR molecules. Primary internal motions, characterized by the first three principal components, were observed mainly at domains I and III in the principal component analysis for trajectory data. The directional motion projected on the first principal component of unliganded HSA was conserved in HSA-MYR complex as the third principal directional motion with higher frequency. However, the third principal directional motion in unliganded HSA turned into the first principal directional motion with lower frequency in the HSA-MYR complex. Thus, the present MD study provides insights into the possible conformational changes of HSA caused by the binding of fatty acids.

Topographic MN-SSEPs (N18, N20 and N30) might characterize underlying CNS involvements in representative types of cerebral palsy.

This study is aimed at constructing the neurophysiological basis for determining the characteristic features of cerebral motor disturbance in representative cerebral palsy (CP) types using topographical S-SEPs technology. Median-nerve stimulated S-SEPs (MN-SSEPs) were examined for 23 patients with four representative types of cerebral palsy: 6 athetotic (including 3 patients due to hypoxic-ischemic encephalopathy (HIE) and 3 to kernicterus), 7 hemiplegic, 5 diplegic and 5 tetraplegic types, and 13 normal controls. In HIE group of athetotic CP, frontal N30 specifically showed severe amplitude reduction or abolishment. In hemiplegic CP, both N20 and N30 on the affected cerebral side tended either to disappear or to be normally evoked at the same time, and their mean amplitudes declined severely. In diplegic CP, the amplitudes of subcortical N18 and parietal N20 were not small but significantly enlarged. N30 amplitude stayed within normal. The reason for this unexpected enlargement of N18 and N20 is unclear, but may be partly due to premature birth which caused abnormally abundant dendritic spine due to absence from perinatal normal spine elimination in the brainstem. In several quadriplegic patients, both N20 and N30 disappeared. The mean amplitude of N30 severely decreased. In conclusion, topographical results of N18, N20 and N30 may basically suggest the underlying involvement of nervous structures in CP according to their representative type.

Development of hardware accelerator for molecular dynamics simulations: a computation board that calculates nonbonded interactions in cooperation with fast multipole method.

Evaluation of long-range Coulombic interactions still represents a bottleneck in the molecular dynamics (MD) simulations of biological macromolecules. Despite the advent of sophisticated fast algorithms, such as the fast multipole method (FMM), accurate simulations still demand a great amount of computation time due to the accuracy/speed trade-off inherently involved in these algorithms. Unless higher order multipole expansions, which are extremely expensive to evaluate, are employed, a large amount of the execution time is still spent in directly calculating particle-particle interactions within the nearby region of each particle. To reduce this execution time for pair interactions, we developed a computation unit (board), called MD-Engine II, that calculates nonbonded pairwise interactions using a specially designed hardware. Four custom arithmetic-processors and a processor for memory manipulation ("particle processor") are mounted on the computation board. The arithmetic processors are responsible for calculation of the pair interactions. The particle processor plays a central role in realizing efficient cooperation with the FMM. The results of a series of 50-ps MD simulations of a protein-water system (50,764 atoms) indicated that a more stringent setting of accuracy in FMM computation, compared with those previously reported, was required for accurate simulations over long time periods. Such a level of accuracy was efficiently achieved using the cooperative calculations of the FMM and MD-Engine II. On an Alpha 21264 PC, the FMM computation at a moderate but tolerable level of accuracy was accelerated by a factor of 16.0 using three boards. At a high level of accuracy, the cooperative calculation achieved a 22.7-fold acceleration over the corresponding conventional FMM calculation. In the cooperative calculations of the FMM and MD-Engine II, it was possible to achieve more accurate computation at a comparable execution time by incorporating larger nearby regions.

Congenital mirror movement: a study of functional MRI and transcranial magnetic stimulation.

Two male patients (a child and an adult) with congenital mirror movement were studied using functional MRI (fMRI) and transcranial magnetic stimulation (TMS). Bilateral primary sensorimotor cortices were activated during unilateral hand gripping on fMRI when the child patient was 8 years old andthe adult was 37 years old. Bilateral motor evoked potentials were induced from the hand and forearm muscles after TMS of each hemisphere. Bilateral motor responses were also induced from the arm muscles in the adult patient. Bilateral motor responses had short and similar latencies. Contralateral motor responses to TMS were smaller than ipsilateral ones in the hand muscles, while contralateral responses were larger than ipsilateral ones in the arm muscles. Contralateral hand motor responses reduced in amplitude or disappeared with increasing age while in the child patient, mirror movements decreased gradually. Our results suggest that bilateral activation of the primary sensorimotor cortices during intended unilateral hand movement and bilateral motor responses to TMS account, at least in part, for the pathophysiology of congenital mirror movement. Reduction of contralateral hand motor responses may be related to the decrease in mirror movements during development.