Modeling balance control during sustained waking allows posturographic sleepiness testing

We develop a method to quantify sleepiness. Sleepiness is a major risk factor in traffic and occupational accidents, but lack of convenient tests precludes monitoring impending sleepiness. Posturographic balance testing could address this need because sleepiness increases postural sway. It is, however, unclear how sleepiness influences balance control. Our results, for 12 subjects, show that balance control is more susceptible to increasing time awake (TA) compared to neuromuscular processes. This conclusion is reached since during sustained waking the control process slows down by 3.4% per hour of increased TA. This slowdown accounts for 65% of the variance in diurnal balance. We quantified balance control by modeling the body as an inverted pendulum and by expressing the control as the critical time interval for open-loop control (Deltat(c)) of the center-of-mass movements of this pendulum. To estimate the subjects' TA, we regressed the Deltat(c) scores recorded during sustained waking against increasing TA, and equated separate Deltat(c) test scores with the diurnal Deltat(c) scores. We estimated TA with 68% positive predictive value. The results encourage implementing balance modeling into a device that performs clinical or industrial balance testing because the model-based Deltat(c) score responded to increasing TA.     

Combined rheological and ultrasonic study of alginate and pectin gels near the sol-gel transition

The sol-gel transition of biopolymer mixtures has been investigated by rheological and ultrasonic measurements. A scaling analysis of the data was performed for both types of measurements. A gel time was determined from rheology for the pure pectin samples, and the data could be fitted to a universal scaling form near the transition point. Its critical exponents are in good agreement with the predictions of scalar percolation theory. In addition, the ultrasonic signal of the pectin samples close to the transition was analyzed in terms of a high-frequency scaling approach for the attenuation and the velocity. For the alginate samples and the mixtures, for which the gel point cannot be determined reliably from rheology, the ultrasonic measurements were analyzed using the same scaling form as for the pectin sample, thus providing a method for estimating the gel point, even in the absence of rheological data.     

Microheterogeneity and microrheology of wheat gliadin suspensions studied by multiple-particle tracking

By monitoring the thermally driven displacements of imbedded polystyrene microspheres via video fluorescence microscopy, we quantified the microstructural and micromechanical heterogeneities of wheat gliadin suspensions. We found that the degree of heterogeneity of the suspensions, as measured by the width and skewness of the microspheres' mean squared displacement (MSD) distribution, increased dramatically over a narrow range of gliadin concentrations. The ensemble-averaged MSD of a 250 mg/mL gliadin suspension exhibited a power-law behavior scaling linearly with time, a behavior similar to that observed for a homogeneous aqueous glycerol solution. However, the MSD distribution was wider and more asymmetric than for glycerol. With increasing concentration of gliadin, the ensemble-averaged MSD rapidly displayed a plateau at small time scales, the MSD distribution became wider and more asymmetric, and the local viscoelastic moduli extracted from multiple-particle-tracking measurements showed an increasingly wide range.     

Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes

Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes.     

Role of the NH2-terminal membrane spanning domain of multidrug resistance protein 1/ABCC1 in protein processing and trafficking

Multidrug resistance protein (MRP)1/ABCC1 transports organic anionic conjugates and confers resistance to cytotoxic xenobiotics. In addition to two membrane spanning domains (MSDs) typical of most ATP-binding cassette (ABC) transporters, MRP1 has a third MSD (MSD0) of unknown function. Unlike some topologically similar ABCC proteins, removal of MSD0 has minimal effect on function, nor does it prevent MRP1 from trafficking to basolateral membranes in polarized cells. However, we find that independent of cell type, the truncated protein accumulates in early/recycling endosomes. Using a real-time internalization assay, we demonstrate that MSD0 is important for MRP1 retention in, or recycling to, the plasma membrane. We also show that MSD0 traffics independently to the cell surface and promotes membrane localization of the core-region of MRP1 when the two protein fragments are coexpressed. Finally, we demonstrate that MSD0 becomes essential for trafficking of MRP1 when the COOH-terminal region of the protein is mutated. These studies demonstrate that MSD0 and the COOH-terminal region contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated. These data explain apparent differences in the trafficking requirement for MSD0 and the COOH-terminal region of MRP1 compared with other ABCC proteins.     

Steady-state kinetics of solitary batrachotoxin-treated sodium channels. Kinetics on a bounded continuum of polymer conformations.

The underlying principles of the kinetics and equilibrium of a solitary sodium channel in the steady state are examined. Both the open and closed kinetics are postulated to result from round-trip excursions from a transition region that separates the openable and closed forms. Exponential behavior of the kinetics can have origins different from small-molecule systems. These differences suggest that the probability density functions (PDFs) that describe the time dependences of the open and closed forms arise from a distribution of rate constants. The distribution is likely to arise from a thermal modulation of the channel structure, and this provides a physical basis for the following three-variable equation: [formula; see text] Here, A0 is a scaling term, k is the mean rate constant, and sigma quantifies the Gaussian spread for the contributions of a range of effective rate constants. The maximum contribution is made by k, with rates faster and slower contributing less. (When sigma, the standard deviation of the spread, goes to zero, then p(f) = A0 e-kt.) The equation is applied to the single-channel steady-state probability density functions for batrachotoxin-treated sodium channels (1986. Keller et al. J. Gen. Physiol. 88: 1-23). The following characteristics are found: (a) The data for both open and closed forms of the channel are fit well with the above equation, which represents a Gaussian distribution of first-order rate processes. (b) The simple relationship [formula; see text] holds for the mean effective rat constants. Or, equivalently stated, the values of P open calculated from the k values closely agree with the P open values found directly from the PDF data. (c) In agreement with the known behavior of voltage-dependent rate constants, the voltage dependences of the mean effective rate constants for the opening and closing of the channel are equal and opposite over the voltage range studied. That is, [formula; see text] "Bursts" are related to the well-known cage effect of solution chemistry.     

The use of basis functions in modelling joint articular surfaces: application to the knee joint

This article introduces a new method to represent bone surface geometry for simulations of joint contact. The method uses the inner product of two basis functions to provide a mathematical representation of the joint surfaces. This method guarantees a continuous transition in the direction of the surface normals, an important property for computation of joint contact. Our formulation handles experimental data that are not evenly distributed, a common characteristic of digitized data of musculoskeletal morphologies. The method makes it possible to represent highly curved surfaces, which are encountered in many anatomical structures. The accuracy of this method is demonstrated by modeling the human knee joint. The mean relative percentage error in the representation of the patellar track surface was 0.25% (range 0-1.56%) which corresponded to an absolute error of 0.17mm (range 0-0.16mm).     

Intracellular Transport Dynamics of Endosomes Containing DNA Polyplexes along the Microtubule Network

We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales (t < 7 s) with their mean-square displacement (MSD) 〈Δx(t)²〉 scaling as t^1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t^0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself.     

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-)

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.     

Dynamics of the transition between open and closed conformations in a calmodulin C-terminal domain mutant

BACKGROUND: Calmodulin is a ubiquitous Ca(2+)-activated regulator of cellular processes in eukaryotes. The structures of the Ca(2+)-free (apo) and Ca(2+)-loaded states of calmodulin have revealed that Ca(2+) binding is associated with a transition in each of the two domains from a closed to an open conformation that is central to target recognition. However, little is known about the dynamics of this conformational switch. RESULTS: The dynamics of the transition between closed and open conformations in the Ca(2+)-loaded state of the E140Q mutant of the calmodulin C-terminal domain were characterized under equilibrium conditions. The exchange time constants (tau(ex)) measured for 42 residues range from 13 to 46 micros, with a mean of 21 +/- 3 micros. The results suggest that tau(ex) varies significantly between different groups of residues and that residues with similar values exhibit spatial proximity in the structures of apo and/or Ca(2+)-saturated wild-type calmodulin. Using data for one of these groups, we obtained an open population of p(o) = 0.50 +/- 0.17 and a closed --> open rate constant of k(o) = x 10(4) s(-1). CONCLUSIONS: The conformational exchange dynamics appear to involve locally collective processes that depend on the structural topology. Comparisons with previous results indicate that similar processes occur in the wild-type protein. The measured rates match the estimated Ca(2+) off rate, suggesting that Ca(2+) release may be gated by the conformational dynamics. Structural interpretation of estimated chemical shifts suggests a mechanism for ion release.     

Modelling tendon excursions and moment arms of the finger flexors: anatomic fidelity versus function

A detailed musculoskeletal model of the human hand is needed to investigate the pathomechanics of tendon disorders and carpal tunnel syndrome. The purpose of this study was to develop a biomechanical model with realistic flexor tendon excursions and moment arms. An existing upper extremity model served as a starting point, which included programmed movement of the index finger. Movement capabilities were added for the other fingers. Metacarpophalangeal articulations were modelled as universal joints to simulate flexion/extension and abduction/adduction while interphalangeal articulations used hinges to represent flexion. Flexor tendon paths were modelled using two approaches. The first method constrained tendons with control points, representing annular pulleys. The second technique used wrap objects at the joints as tendon constraints. Both control point and joint wrap models were iteratively adjusted to coincide with tendon excursions and moment arms from a anthropometric regression model using inputs for a 50th percentile male. Tendon excursions from the joint wrap method best matched the regression model even though anatomic features of the tendon paths were not preserved (absolute differences: mean<0.33 mm, peak<0.74 mm). The joint wrap model also produced similar moment arms to the regression (absolute differences: mean<0.63 mm, peak<1.58 mm). When a scaling algorithm was used to test anthropometrics, the scaled joint wrap models better matched the regression than the scaled control point models. Detailed patient-specific anatomical data will improve model outcomes for clinical use; however, population studies may benefit from simplified geometry, especially with anthropometric scaling.     

Time-resolved small-angle X-ray scattering investigation of the folding dynamics of heme oxygenase: implication of the scaling relationship for the submillisecond intermediates of protein folding

Polypeptide collapse is generally observed as the initial folding dynamics of proteins with more than 100 residues, and is suggested to be caused by the coil-globule transition explained by Flory's theory of polymers. To support the suggestion by establishing a scaling behavior between radius of gyration (Rg) and chain length for the initial folding intermediates, the folding dynamics of heme oxygenase (HO) was characterized by time-resolved, small-angle X-ray scattering. HO is a highly helical protein without disulfide bridges, and is the largest protein (263 residues) characterized by the method. The folding process of HO was found to contain a transient oligomerization; however, the conformation within 10 ms was demonstrated to be monomeric and to possess Rg of 26.1(+/-1.1) A. Together with the corresponding data for proteins with different chain lengths, the seven Rg values demonstrated the scaling relationship to chain length with a scaling exponent of 0.35+/-0.11, which is close to the theoretical value of 1/3 predicted for globules in solutions where monomer-monomer interactions are favored over monomer-solvent interactions (poor solvent). The finding indicated that the initial folding dynamics of proteins bears the signature of the coil-globule transition, and offers a clue to explain the folding mechanisms of proteins with different chain lengths.     

Use of the exciton chirality method in the investigation of ligand-gated synthetic ion channels

The objective of this brief highlight is to point out the central role of the exciton chirality method to gain insights on the structural basis of the recently achieved ligand gating of synthetic ion channels. This unprecedented ligand gating was achieved with an equally unprecedented transmembrane rigid-rod pi-stack architecture that is designed to adopt a closed conformation with helically stacked naphthalenediimide (NDI) acceptors. The intercalation of the complementary electron-rich dialkoxynaphthalene ligands then stimulates the untwisting of the closed pi-helices into hollow barrel-stave supramolecules. During this helix-barrel transition, the angle between the transition moments of the exciton-coupled NDI chromophores decreases toward zero. The corresponding disappearance of the split CD provides, according to the exciton chirality method, the otherwise elusive experimental support that ligand-gated ion channel formation really occurs by this rationally designed helix-barrel transition.     

Cation-mediated conformational variants of surfactant protein A

Surfactant protein A (SP-A) is the major protein of pulmonary surfactant. This protein is implicated in regulating surfactant secretion, alveolar processing, recycling, and in non-serum-induced immune response. An increasing body of work indicates the importance of cations, particularly calcium, on SP-A function. However, little information exists on the effects of cations on SP-A quaternary structure. Here, the quaternary organisation of bovine surfactant protein A in the presence of cations has been quantitatively and systematically studied by transmission electron microscopy. The conformation of SP-A is altered by the presence of cations, especially calcium, then sodium, and to a small extent, magnesium. There is a transition concentration, unique for each cation, at which a conformational switch occurs. These transition concentrations are: 5 mM for CaCl₂, 100 mM for NaCl and 1 mM for MgCl₂. Below these concentrations, SP-A exists primarily in an opened form with a large head diameter of 20 nm; above it, SP-A is mostly in a closed form due to a compaction of the headgroups resulting in a head diameter of 11 nm. There is a corresponding increase in particle length from 17 nm for opened SP-A to 20 nm for closed SP-A. The fact that the transition concentrations are within physiological range suggests that cation-mediated conformational changes of SP-A could be operative in vivo.     

The efficacy of non-surgical platelet-rich fibrin application on clinical periodontal parameters and periostin level in periodontitis: Clinical trial

Platelet-rich fibrin (PRF) has been widely used in regenerative dentistry due to many growth factors produced. Periostin, a matricellular protein, is a reliable marker for tissue regeneration. Periostin is part of the cellular matrix and regulates bone homeostasis. This study aims to explore the efficacy of PRF in improvement of the clinical periodontal parameters as an adjunct to the scaling and root planing and to evaluate periostin level in gingival crevicular fluid (GCF) at baseline, 1- and 3-month recall visits. Fourteen periodontitis patients who met the inclusion criteria were recruited in this study. Two contralateral periodontal pockets with 4–6 mm in depth in each patient were selected. The sites in every participant were randomly allocated into control sites or test sites. In control sites, only conventional scaling and root planing was carried out. In test sites, however, scaling and root planing method and PRF were applied. Periostin level in GCF and clinical periodontal parameters were measured. The test sites revealed greater relative attachment gain (2.614 ± 0.606 mm and 3.321 ± 0.668 mm) than control sites (1.285 ± 0.671 mm and 1.839 ± 0.632 mm) and a significant pocket reduction (2.535 ± 0.664 mm and 3.321 ± 0.668 mm) than the control sites (1.21 ± 0.508 mm and 1.892 ± 0.655 mm) at 1- and 3-month recall visits respectively. In the test sites, level of periostin (48.83 ± 9.3 ng/μl and 98.90 ± 24.94 ng/μl) were greater than periostin levels in the control sites (42.65 ± 7.03 ng/μl and 49.29 ± 15.14 ng/μl) at 1- and 3-month recall visits respectively. In conclusion, the non-surgical application of PRF as an adjunct to scaling and root planing significantly improved the clinical periodontal parameters through raising periostin level in GCF.     

Upstream curved sequences influence the initiation of transcription at the Escherichia coli galactose operon.

The two overlapping promoters that control mRNA synthesis at the galactose operon contain three phased stretches of adenine residues, located around positions -84.5, -74 and -63, with respect ot the start of the P1 promoter. As a result, the corresponding DNA sequence is bent, an anomaly that is relieved by the addition of small concentrations of drugs like distamycin A or netropsin. By abortive initiation assays performed on several DNA fragments derived from the wild-type promoter or from various mutants we show that the curved sequence increases the strength of the P1 promoter. In the absence of cyclic AMP (cAMP) and of the corresponding receptor protein (CRP), the upstream curved sequences enhance the rate of isomerization from the closed to the open complex at P1. This effect is abolished when distamycin A is bound in the bent region. In the presence of cAMP-CRP, a more drastic change is observed: activation of the gal P1 promoter takes place at a different formal step, depending whether the upstream curved sequence is present or not (enhancement of the rate of conversion from a closed to an open complex instead of an increase in the affinity of the enzyme during closed complex formation). These data, together with previous results obtained with other mutants of the gal control region, suggest that several closed complexes corresponding to different nucleoprotein arrangements are formed during open complex formation at gal P1, in the presence of CRP.     

A classical and ab initio study of the interaction of the myosin triphosphate binding domain with ATP.

We used classical molecular mechanics (MM) simulations and quantum mechanical (QM) structural relaxations to examine the active site of myosin when bound to ATP. Two conformations of myosin have been determined by x-ray crystallography. In one, there is no direct interaction between switch 2 and the nucleotide (open state). In the other (closed state), the universally conserved switch 2 glycine forms a hydrogen bond with a gamma-phosphate oxygen. MM simulations indicate that the two states are thermodynamically stable and allow us to investigate the extent to which the P-loop, switch 1, and switch 2 are involved in hydrolysis. We find that the open structure has a higher affinity for ATP than the closed structure, and that ATP is distorted toward a transition state by interactions with the protein. We also examine how the structure of the binding site changes with either MgATP or CaATP as the nucleotide in myosin in the open conformer. Our analyses suggest that higher CaATPase rates occur because the leaving phosphate (P(i)) group is more weakly bound and dissociation occurs faster. Finally, we validate the use of a particular formulation of a QM methodology (Car-Parrinello) to further refine the structures of the active site.     

The stability and characteristics of a DNA Holliday junction switch

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.     

S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (<20 cigarettes smoked per day), (3) non-smokers, and (4) vegetarian non-smokers. Samples were evaluated by a gas chromatography/mass selective detector (GC/MSD) method as a reference and using NicCheck I? (DynaGen, Inc.). Colour intensity can range from 0 (no colour) to 14 (deep pink). Qualitative values were assigned as negative (0), low (1-6) and high (7-14). Comparison of the test strip and GC/MSD results showed: (1) 43 (10.75%) false negatives using the criterion of a GC/MSD cotinine level above 200 ng ml^-1 and test strip reading of 0, (2) 31 (7.75%) false positives using the criterion of a GC/MSD cotinine level below 1 ng ml^-1 and a test strip reading of 1 or greater, and (3) no correlation between the test strip and GC/MSD results (r = 0.597, p < 0.05). The fact that the colorimetric reaction is sensitive to many nicotine metabolites and/or heterocyclic amine structures whereas the GC/MSD method measures nicotine and cotinine selectively might explain the false positive results. False negative results were likely to be due to a lack of sensitivity of the test strip.     

Intracellular transport dynamics of endosomes containing DNA polyplexes along the microtubule network

We have explored the transport of DNA polyplexes enclosed in endosomes within the cellular environment by multiple particle tracking (MPT). The polyplex-loaded endosomes demonstrate enhanced diffusion at short timescales (t<7 s) with their mean-square displacement (MSD) Deltax(t)2 scaling as t1.25. For longer time intervals they exhibit subdiffusive transport and have an MSD scaling as t0.7. This crossover from an enhanced diffusion to a subdiffusive regime can be explained by considering the action of motor proteins that actively transport these endosomes along the cellular microtubule network and the thermal bending modes of the microtubule network itself.