An original clinical methodology for non-invasive assessment of pivot-shift test

Even if pivot-shift (PS) test has been clinically used to specifically detect anterior cruciate ligament (ACL) injury, the main problem in using this combined test has been yet associated with the difficulty of clearly quantifying its outcome. The goal of this study was to describe an original non-invasive methodology used to quantify PS test, highlighting its possible clinical reliability. The method was validated on 66 consecutive unilateral ACL-injured patients. A commercial triaxial accelerometer was non-invasively mounted on patient's tibia, the corresponding 3D acceleration was acquired during PS test execution and a set of specific parameters were automatically identified on the signal to quantify the test. PS test was repeated three times on both injured and controlateral limbs. Reliability of the method was found to be good (mean intra-rater intraclass correlation coefficient was 0.79); moreover, we found that ACL-deficient knees presented statistically higher values for the identified parameters - than the controlateral healthy limbs, averagely reporting also large effect size.     

Comparison between different markers for sperm quality in the cat: Diff-Quik as a simple optical technique to assess changes in the DNA of feline epididymal sperm

The majority of wild felids, as well as some domestic cats, have low sperm concentration in their ejaculates, and a high proportion of abnormal spermatozoa. We have employed several possible semen quality markers to further characterize cat epididymal sperm. Methods included possible apoptotic reporters, such as the annexin V assay to monitor exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane, as well as cell integrity; and the TUNEL assay to quantify DNA breaks. Sperm surface ubiquitination, another putative marker of sperm quality, was also monitored. The annexin V assay revealed a high percentage of sperm with PS exposure, and the TUNEL assay pointed to high levels (13+/-12%) of sperm with DNA breaks. Correlations were found between apoptotic markers (but not ubiquitination) and semen parameters. In parallel to this analysis, cat sperm morphology was evaluated using the Diff-Quik optical stain, which has been used in human reproduction laboratories. Several types of abnormalities could be characterized with this method. Remarkably, head staining abnormalities detected using the Diff-Quik staining method were strongly correlated with, and could accurately predict, sperm DNA defects detected in the same sample using the TUNEL assay. We therefore suggest that sperm morphology analysis using Diff-Quik could be used in field conditions to assess sperm status, due to the simplicity of the procedure and the equipment involved.     

Simultaneous positive and negative external mechanical work in human walking.

In human walking, the center of mass motion is similar to an inverted pendulum. Viewing double support as a transition from one inverted pendulum to the next, we hypothesized that the leading leg performs negative work to redirect the center of mass velocity, while simultaneously, the trailing leg performs positive work to replace the lost energy. To test this hypothesis, we developed a method to quantify the external mechanical work performed by each limb (individual limbs method). Traditional measures of external mechanical work use the sum of the ground reaction forces acting on the limbs (combined limbs method) allowing for the mathematical cancellation of simultaneous positive and negative work during multiple support periods. We expected to find that the traditional combined limbs method underestimates external mechanical work by a substantial amount. We used both methods to measure the external mechanical work performed by humans walking over a range of speeds. We found that during double support, the legs perform a substantial amount of positive and negative external work simultaneously. The combined limbs measures of positive and negative external work were approximately 33% less than those calculated using the individual limbs method. At all speeds, the trailing leg performs greater than 97% of the double support positive work while the leading leg performs greater than 94% of the double support negative work.     

Performance index as an indicator of the physiological state of trees in urban forest ecosystems

Based on the measurements of fluorescence of bark chloroplasts by means of PAM and PEA fluorometers, the information capacity of the methods for assessing the physiological state of Tilia cordata L. from the maximum quantum efficiency of PS II photochemistry (Fv/Fm) and the performance index (PI) has been compared. The measurements were performed on annual shoots of linden trees growing in different environment. It was shown that the chlorophyll content in the bark of shoots growing near the busy urban street was twice less compared with trees growing out of the city. On the trees from the unsafe environment, a small decrease in the relative fluorescence variable (Fv/Fm) was registered, and there was a significant statistical deviation of this value compared to control trees. It was found that the PI and its constituent parameters calculated on the basis of light fluorescence induction curve (PEA-method) are more informative and allow one to recognize changes in the primary energy transformation processes in PS II when they are comparatively small. The results of our work show that PI can be used as a sensitive and a rapid test to evaluate the physiological state of trees and other plant objects even under minor environmental changes.     

An interactive modeling program for DNA

An interactive program for modeling A-, B- and Z-DNA in a schematic and nonatomic representation has been developed for the Evans and Sutherland PS300. The program lets users display several molecules for which parameters determining the three-dimensional structure can be calculated either on the basis of theoretical models or inferred from experimental data. The calculation of the curvature and torsion of the helical axis, by a method based on Frenet's equations, makes it possible to quantify the effects of the parameters on the helical axis.     

A unified approach for quantifying, testing and correcting population stratification in case-control association studies

The HapMap project has given case-control association studies a unique opportunity to uncover the genetic basis of complex diseases. However, persistent issues in such studies remain the proper quantification of, testing for, and correction for population stratification (PS). In this paper, we present the first unified paradigm that addresses all three fundamental issues within one statistical framework. Our unified approach makes use of an omnibus quantity (delta), which can be estimated in a case-control study from suitable null loci. We show how this estimated value can be used to quantify PS, to statistically test for PS, and to correct for PS, all in the context of case-control studies. Moreover, we provide guidelines for interpreting values of delta in association studies (e.g., at alpha = 0.05, a delta of size 0.416 is small, a delta of size 0.653 is medium, and a delta of size 1.115 is large). A novel feature of our testing procedure is its ability to test for either strictly any PS or only 'practically important' PS. We also performed simulations to compare our correction procedure with Genomic Control (GC). Our results show that, unlike GC, it maintains good Type I error rates and power across all levels of PS.     

Increased muscle regeneration after repair of divided motor nerve with neuronotrophic factors containing glue

Neuronotrophic factors (NTFs) directed to spinal cord motor neurons were collected in rats within silicone nerve regeneration chambers according to LONGO et al. (1983b). Unilateral addition of NTFs to the fibrin glue used for the repair of divided sciatic nerves improved locally nerve regeneration without affecting the controlateral side. Nerve regeneration was assessed by weight gain of the reinnervated muscles and by radioactive labelling of the acid-soluble phosphate fractions of both nerve Schwann cells and reinnervated muscle cells. Fast gastrocnemius and slow soleus muscles, the motor nerve of which had been repaired with added NTFs, were significantly heavier (21 and 28%) than their controlateral controls, and the metabolic dedifferentiation attendant on post-division nerve repair was less marked. It is suggested that this experimental nerve regeneration model is suitable to test potential nerve-active agents in vivo, under conditions close to the usual clinical setting, with, as ultimate goal, the improvement of the end-results of microsurgical repair of peripheral nerve in man.     

Biofilm image reconstruction for assessing structural parameters

The structure of biofilms can be numerically quantified from microscopy images using structural parameters. These parameters are used in biofilm image analysis to compare biofilms, to monitor temporal variation in biofilm structure, to quantify the effects of antibiotics on biofilm structure and to determine the effects of environmental conditions on biofilm structure. It is often hypothesized that biofilms with similar structural parameter values will have similar structures; however, this hypothesis has never been tested. The main goal was to test the hypothesis that the commonly used structural parameters can characterize the differences or similarities between biofilm structures. To achieve this goal (1) biofilm image reconstruction was developed as a new tool for assessing structural parameters, (2) independent reconstructions using the same starting structural parameters were tested to see how they differed from each other, (3) the effect of the original image parameter values on reconstruction success was evaluated, and (4) the effect of the number and type of the parameters on reconstruction success was evaluated. It was found that two biofilms characterized by identical commonly used structural parameter values may look different, that the number and size of clusters in the original biofilm image affect image reconstruction success and that, in general, a small set of arbitrarily selected parameters may not reveal relevant differences between biofilm structures.     

Quantitative measures for fracture healing: an in-vitro biomechanical study

A method is presented to assess the functional capabilities of plated osteotomized canine femora as load bearing structures and to quantify their healing status. In this method the osteotomized bones and their intact contralateral controls are tested in nondestructive bending, in twenty-four planes of loading at 15 degree angular increments. Flexural rigidity (EI), in each of the planes, is determined by using classical beam theory. It has been found that the EI values have the expected elliptical distribution. The 24 EI values, obtained for each bone, are curve fitted by regression analysis and the characteristics of each bone are described by the three parameters defining its ellipse. The parameters of the ellipses of the osteotomized bone and of its contralateral control are used to define four new parameters that may serve as measures for the healing efficiency. One of these serves as an indicator for the fragility of the healed bone and the other three add quantitative information on its mechanical state.     

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

ATP11C mutation is responsible for the defect in phosphatidylserine uptake in UPS-1 cells

Type IV P-type ATPases (P4-ATPases) translocate phospholipids from the exoplasmic to the cytoplasmic leaflets of cellular membranes. We and others previously showed that ATP11C, a member of the P4-ATPases, translocates phosphatidylserine (PS) at the plasma membrane. Twenty years ago, the UPS-1 (uptake of fluorescent PS analogs) cell line was isolated from mutagenized Chinese hamster ovary (CHO)-K1 cells with a defect in nonendocytic uptake of nitrobenzoxadiazole PS. Due to its defect in PS uptake, the UPS-1 cell line has been used in an assay for PS-flipping activity; however, the gene(s) responsible for the defect have not been identified to date. Here, we found that the mRNA level of ATP11C was dramatically reduced in UPS-1 cells relative to parental CHO-K1 cells. By contrast, the level of ATP11A, another PS-flipping P4-ATPase at the plasma membrane, or CDC50A, which is essential for delivery of most P4-ATPases to the plasma membrane, was not affected in UPS-1 cells. Importantly, we identified a nonsense mutation in the ATP11C gene in UPS-1 cells, indicating that the intact ATP11C protein is not expressed. Moreover, exogenous expression of ATP11C can restore PS uptake in UPS-1 cells. These results indicate that lack of the functional ATP11C protein is responsible for the defect in PS uptake in UPS-1 cells and ATP11C is crucial for PS flipping in CHO-K1 cells.     

Three-dimensional model of the SHBG-like region of anticoagulant protein S: new structure-function insights

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.     

Evaluation of a candidate International Standard for Meningococcal Group C polysaccharide

Meningococcal group C (MenC) plain polysaccharide (PS) and conjugate vaccines are primarily evaluated by physicochemical methods to ensure that batches are consistently manufactured. As different assays are employed to quantify the MenC PS content of final formulations and bulk intermediaries, there is a need for an International MenC PS Standard to calibrate internal references used in the different laboratories. Twelve laboratories from nine different countries participated in a collaborative study to determine the MenC PS content of a candidate International Standard MenC PS preparation (08/214) and to assess its suitability. On the basis of the results from this study the candidate standard 08/214 was established as an International Standard for the quantification of MenC PS content in vaccines and components. It has a content of 1.192 ± 0.192 mg MenC PS/ampoule (expanded uncertainty with coverage factor of k = 2.365 corresponding to a 95% level of confidence), as determined by the resorcinol assays carried out by eight of the participating laboratories. The standard is available from The National Institute of Biological Standards and Control who act as guardians and distributors of the material under the auspices of WHO.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20

Polysorbate 20 (PS‐20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS‐20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS‐20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS‐20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS‐20 was measured by a mixed‐mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS‐20 was characterized by ¹H‐NMR and a reverse‐phase chromatography method. Non‐specific adsorption of PS‐20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS‐20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS‐20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di‐esters and tri‐esters. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1503–1511, 2013     

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms.     

Detecting and quantifying global instability during a dynamic task using kinetic and kinematic gait parameters

ObjectivesInstability during gait can be identified in many different ways. Recent studies have suggested utilizing spatiotemporal parameters to detect instability during gait. Detecting instability using kinetic and kinematic gait parameters has not yet been examined fully. In addition, these studies have not yet identified measures that are capable of assessing the magnitude of instability. The objective of the present study was to identify kinetic and kinematic gait parameters that can best identify instability and quantify its magnitude.MethodsTen healthy men underwent successive gait analysis testing under three controlled settings: (1) Stage 0 instability (control setting), (2) Stage 1 instability and (3) Stage 2 instability. The levels of instability were precisely applied with the use of a controlled perturbation device (AposTherapy System). Differences between all stages and between stages were identified using Friedman and Wilcoxon tests.ResultsStride-to-stride variability (STSV) in kinetic and kinematic measures increased significantly between stages 0 and 1 or between stages 0 and 2 for almost all parameters (all P<0.05). A significant increase between stage 0 and both stages 1 and 2 was found for knee flexion moment, knee varus moment, knee flexion angle and hip adduction angle. The increase between stages 1 and 2 was variable. Only the knee varus moment parameter showed a significant increase in STSV between stages 1 and 2 (P=0.026).ConclusionsAlmost all kinetic and kinematic gait parameters are sensitive to changes in global instability in a dynamic task. The most sensitive are parameters measured at the knee. Of these, STSV in knee varus moment can be used to quantify the magnitude of dynamic instability.     

Mass, center of mass, and moment of inertia estimates for infant limb segments.

To quantify limb dynamics, accurate estimates are needed of anthropometric inertia parameters (mass, center-of-mass location, and moments of inertia). These estimates, however, are not available for human infants; therefore, the movement dynamics of infants have not been studied extensively. Here, regression equations for the masses, center-of-mass locations, and transverse moments of inertia of upper and lower limb segments (upper arm, forearm, and hand; thigh, leg, and foot) of 0.04 to 1.50 yr old infants are provided. A mathematical model of the human body was used to determine the anthropometric inertia parameters for upper limbs in 44 infants and for lower limbs in 70 infants. Stepwise linear regressions were used to fit the distributions of the anthropometric inertia parameters. The regression equations accounted for significant amounts of the variance (64-98%), and the R2-values compared favorably when our equations were cross-validated. Consequently, these regression equations can provide, for infants of similar ages, reasonable estimates of upper and lower limb anthropometric inertia parameters, suitable for equations of motion in the analysis of limb dynamics in human infants.     

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.     

Venous volume displacement plethysmography: Its diagnostic value in deep venous thrombosis as determined by receiver operator characteristic curves

The pitfall of several reviews of noninvasive venous assessment has been the expression of the test data solely in terms of diagnostic accuracy (the number of correct tests in ratio to all tests performed), where results of a test will vary according to disease prevalence. The advantages of receiver operator characteristic curve analysis are twofold: (1) it describes the dynamic relationship between sensitivity (the ratio of the number of true positive tests to the patients with deep venous thrombosis) and specificity (the ratio of true negative tests to the number of patients with no deep venous thrombosis) independent of disease prevalence; and (2) the threshold criteria that defines a positive test can be set by the best balance between sensitivity and specificity and then applied to a given patient population for its diagnostic accuracy. Venous volume plethysmography is a widely used, simple and rapid method. It was compared to the "gold standard" of phlebography in a prospective blind study of 70 limbs that were clinically suspect of having deep venous thrombosis (DVT). Venous volume displacement plethysmography was defined objectively by three quantitative parameters: (1) maximum venous outflow, (2) integer ratio, and (3) segmental venous capacitance ratio. The DVT (22 to 70 positive phlebograms) was divided by anatomic location into either calf vein DVT or proximal DVT (popliteal vein or above). By combining these three parameters, a balance between sensitivity and specificity was obtained to provide a rapid, objective method for screening patients with suspected DVT.