Serial assessments to determine normalization of gait following anterior cruciate ligament reconstruction.

There is an increased emphasis to identify clinically applicable methods that quantify gait deficits following anterior cruciate ligament reconstruction (ACLR). The purpose of this study was to perform serial gait assessments in a clinical setting to determine whether and when clinical gait parameters normalize in patients following ACLR. The hypothesis was that a clinically available gait treadmill would quantify gait deficits measured at 4 weeks post-reconstruction. The secondary hypothesis was that patients would demonstrate incremental improvements in these gait parameters measured at each interval up to 12 weeks post-reconstruction, and that the objectively measured improvements would correlate to the patient's subjective rating of function. Fifteen subjects, five male and 10 female, who had initial unilateral anterior cruciate ligament injury were selected for this study on the basis of operative data. All subjects were evaluated in a physical therapy clinic within 3 days following ACLR and were enrolled in a standardized rehabilitation program. The dependent gait variables of step length, stance time and gait velocity were measured at 4, 6, 8 and 12 weeks post-ACLR on a commercially available gait treadmill. A 2 x 4 multivariate analysis of variance (2 within factors) with measures for limb involvement (uninvolved and involved) and repeated measures for time (4, 6, 8 and 12 weeks) was used to assess the interactions and the main effects on the gait variables of stance time and step length. The results of this study supported the hypothesis that gait deficits and serial improvements can be objectively quantified in a clinical setting (P<0.001). Specifically, stance time, step length and gait velocity deficits evaluated at 4 weeks showed significant improvements at the measured intervals. Step length normalized at week 8. Stance time and gait velocity reached normal levels at the 12-week time interval. Subjective activity of daily living scores (ADLS) also improved following the 12-week rehabilitation, from 53+/-17% to a mean score of 88+/-11% (P<0.001). ADLS scores significantly correlated to step length (R=0.63) and stance time (R=0.53) in the involved limb. Self-selected gait velocity also correlated to ADLS scores and significantly predicted 49% of the variance in the subjective outcome measure. A clinically available gait treadmill can be used to quantify gait deficits and improvements following ACLR. Serial assessments of walking gait may aid clinicians to identify and target deficits in their patients during rehabilitation.

Characterization of the methylation status of five imprinted genes in sheep gametes.

Genomic imprinting is a mammalian developmental process that uses epigenetic mechanisms to induce monoallelic and parental-specific expression of particular autosomal genes. A crucial epigenetic event consists of DNA methylation of CpG-islands, which become differentially methylated regions (DMRs) on the maternal and paternal alleles during oogenesis or spermatogenesis (germline DMRs). By contrast, somatic DMRs are acquired after fertilization. While there are several studies referring to methylation acquisition within germline DMRs in the mouse and human, a comparable methylation analysis of orthologous sequences is still lacking in sheep. To identify germline DMRs, this study analysed the methylation status of the available CpG-islands of five ovine imprinted genes (H19, IGF2R, DLK1, DIO3 and BEGAIN) in mature spermatozoa and in female gametes at different stages of their follicle growth, including in vitro matured oocytes. The 5'-end CpG-island of H19 showed a full methylation in spermatozoa and an absent methylation in growing and fully grown oocytes. The intron 2 CpG-island of IGF2R was unmethylated in male gametes, while it showed a high level of methylation in early stages of oogenesis. The promoter CpG-islands of DLK1 and DIO3 were found to be unmethylated both in spermatozoa and oocytes. Finally, the exon 9 CpG-island of BEGAIN was hypermethylated in mature male gametes, while it showed an almost complete methylation only in late stages of oocyte development. Our findings suggest that DNA methylation establishment during early stages of sheep oogenesis and subsequent in vitro maturation is gene-specific and that, of the five genes investigated, only the CpG-islands of H19 and IGF2R might represent ovine germline DMRs.

Tracking the time-varying cortical connectivity patterns by adaptive multivariate estimators.

The directed transfer function (DTF) and the partial directed coherence (PDC) are frequency-domain estimators that are able to describe interactions between cortical areas in terms of the concept of Granger causality. However, the classical estimation of these methods is based on the multivariate autoregressive modelling (MVAR) of time series, which requires the stationarity of the signals. In this way, transient pathways of information transfer remains hidden. The objective of this study is to test a time-varying multivariate method for the estimation of rapidly changing connectivity relationships between cortical areas of the human brain, based on DTF/PDC and on the use of adaptive MVAR modelling (AMVAR) and to apply it to a set of real high resolution EEG data. This approach will allow the observation of rapidly changing influences between the cortical areas during the execution of a task. The simulation results indicated that time-varying DTF and PDC are able to estimate correctly the imposed connectivity patterns under reasonable operative conditions of signal-to-noise ratio (SNR) ad number of trials. An SNR of five and a number of trials of at least 20 provide a good accuracy in the estimation. After testing the method by the simulation study, we provide an application to the cortical estimations obtained from high resolution EEG data recorded from a group of healthy subject during a combined foot-lips movement and present the time-varying connectivity patterns resulting from the application of both DTF and PDC. Two different cortical networks were detected with the proposed methods, one constant across the task and the other evolving during the preparation of the joint movement.

Adenovirus E1B 55-kilodalton oncoprotein inhibits p53 acetylation by PCAF.

The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

Variability among polysulphone calibration curves.

Within an epidemiological study regarding the correlation between skin pathologies and personal ultraviolet (UV) exposure due to solar radiation, 14 field campaigns using polysulphone (PS) dosemeters were carried out at three different Italian sites (urban, semi-rural and rural) in every season of the year. A polysulphone calibration curve for each field experiment was obtained by measuring the ambient UV dose under almost clear sky conditions and the corresponding change in the PS film absorbance, prior and post exposure. Ambient UV doses were measured by well-calibrated broad-band radiometers and by electronic dosemeters. The dose-response relation was represented by the typical best fit to a third-degree polynomial and it was parameterized by a coefficient multiplying a cubic polynomial function. It was observed that the fit curves differed from each other in the coefficient only. It was assessed that the multiplying coefficient was affected by the solar UV spectrum at the Earth's surface whilst the polynomial factor depended on the photoinduced reaction of the polysulphone film. The mismatch between the polysulphone spectral curve and the CIE erythemal action spectrum was responsible for the variability among polysulphone calibration curves. The variability of the coefficient was related to the total ozone amount and the solar zenith angle. A mathematical explanation of such a parameterization was also discussed.

A passive brain-computer interface application for the mental workload assessment on professional air traffic controllers during realistic air traffic control tasks.

In the last decades, it has been a fast-growing concept in the neuroscience field. The passive brain-computer interface (p-BCI) systems allow to improve the human-machine interaction (HMI) in operational environments, by using the covert brain activity (eg, mental workload) of the operator. However, p-BCI technology could suffer from some practical issues when used outside the laboratories. In particular, one of the most important limitations is the necessity to recalibrate the p-BCI system each time before its use, to avoid a significant reduction of its reliability in the detection of the considered mental states. The objective of the proposed study was to provide an example of p-BCIs used to evaluate the users' mental workload in a real operational environment. For this purpose, through the facilities provided by the École Nationale de l'Aviation Civile of Toulouse (France), the cerebral activity of 12 professional air traffic control officers (ATCOs) has been recorded while performing high realistic air traffic management scenarios. By the analysis of the ATCOs' brain activity (electroencephalographic signal-EEG) and the subjective workload perception (instantaneous self-assessment) provided by both the examined ATCOs and external air traffic control experts, it has been possible to estimate and evaluate the variation of the mental workload under which the controllers were operating. The results showed (i) a high significant correlation between the neurophysiological and the subjective workload assessment, and (ii) a high reliability over time (up to a month) of the proposed algorithm that was also able to maintain high discrimination accuracies by using a low number of EEG electrodes (~3 EEG channels). In conclusion, the proposed methodology demonstrated the suitability of p-BCI systems in operational environments and the advantages of the neurophysiological measures with respect to the subjective ones.

ZnO Luminescence and scintillation studied via photoexcitation, X-ray excitation, and gamma-induced positron spectroscopy.

The luminescence and scintillation properties of ZnO single crystals were studied by photoluminescence and X-ray-induced luminescence (XRIL) techniques. XRIL allowed a direct comparison to be made between the near-band emission (NBE) and trap emissions providing insight into the carrier recombination efficiency in the ZnO crystals. It also provided bulk luminescence measurements that were not affected by surface states. The origin of a green emission, the dominant trap emission in ZnO, was then investigated by gamma-induced positron spectroscopy (GIPS) - a unique defect spectroscopy method that enables positron lifetime measurements to be made for a sample without contributions from positron annihilation in the source materials. The measurements showed a single positron decay curve with a 175 ps lifetime component that was attributed to Zn vacancies passivated by hydrogen. Both oxygen vacancies and hydrogen-decorated Zn vacancies were suggested to contribute to the green emission. By combining scintillation measurements with XRIL, the fast scintillation in ZnO crystals was found to be strongly correlated with the ratio between the defect luminescence and NBE. This study reports the first application of GIPS to semiconductors, and it reveals the great benefits of the XRIL technique for the study of emission and scintillation properties of materials.

Circular dichroism observations on the lipoxygenase from reticulocytes.

CD spectra of reticulocyte lipoxygenase were recorded at various times during aerobic incubation, which leads to loss of catalytic activity, and after inactivation by the reaction product, 13-LS-hydroperoxylinoleic acid. Neither process showed any correlation with a decrease in the alpha-helix content, which was only observed over much longer times. It is concluded that inactivation of reticulocyte lipoxygenase by the suicidal reaction with 13-LS-hydroperoxylinoleic acid is not accompanied by gross structural alterations.

Gene transfection efficiency of tracheal epithelial cells by DC-chol-DOPE/DNA complexes.

We evaluated the transfection efficiency of five different cationic liposome/plasmid DNA complexes, during the in vitro gene transfer into human epithelial tracheal cell lines. A dramatic correlation between the transfection efficiency and the charge ratio (positive charge of liposome to negative charge of DNA) has been found. DC-Chol-DOPE was found to be the most effective liposome formulation. Therefore, a morphological and structural analysis of DC-Chol-DOPE liposomes and DC-Chol-DOPE/DNA complexes, has been performed by transmission electron microscopy (TEM) and by confocal laser scanning microscopy (CLSM), respectively. The process of interaction between DC-Chol-DOPE/DNA complexes and human epithelial tracheal cells has been studied by CLSM. These results raise some issues for in vivo gene therapy.

Structure and expression of the human ubiquitin fusion-degradation gene (UFD1L).

We report the genomic organization, RNA and protein expression patterns of the gene encoding for the human homolog of the yeast ubiquitin fusion-degradation protein-1 (UFD1L). This enzyme is involved in a ubiquitin-dependent proteolytic pathway (UFD), firstly described in yeast. The human UFD1L gene is organized into 12 exons ranging in size from 33 to 161 bp. Sequence analysis of the 5'-flanking region of the gene revealed a high GC content, multiple CCAAT-binding motifs, CREB, CFT, and AP-2 sites. RNA transcripts were detected in all tissues and cell lines examined, including thymus, thymocytes, T- and B-cells, fibroblasts, chorionic villi, and amniocytes. In Western blot, the UFD1L antibody demonstrated the presence of multiple protein isoforms in all the tested tissues. Expression profile and promoter characteristics suggest UFD1L is a housekeeping gene with implications in the pathogenesis of DiGeorge/velo-cardio-facial syndrome, due to 22q11.2 deletions.

Mapping of a new autosomal dominant non-syndromic hearing loss locus (DFNA43) to chromosome 2p12.

Hearing impairment (HI) is the most frequent sensory defect with wide genetic heterogeneity. Approximately 80% of genetic hearing loss is non-syndromic and 15-25% of exhibit autosomal dominant inheritance. We analysed an Italian three generation family in which non-syndromic hearing impairment is transmitted as an autosomal dominant trait. Onset of HI in all affected subjects occurred in the second decade of life, with subsequent gradual progression from moderate to profound loss. HI was bilateral and symmetrical, involving all frequencies. After exclusion of the known DFNA loci with markers from the Hereditary Hearing Loss Homepage (URL: http://dnalab-www.uia.ac.be/dnalab/hhh), a genome wide scan was carried out using 358 highly informative microsatellite markers. Significant linkage (Zmax=4.21, theta=0) was obtained with chromosome 2p12 markers. The results were confirmed by multipoint analysis (Zmax=4.51), using the location score method. Haplotype analysis defined a 9.6 cM disease gene interval on chromosome 2 without overlap with the other identified loci. Fine mapping and identification of candidate genes are in progress.

Alpha EEG Frontal Asymmetries during Audiovisual Perception in Cochlear Implant Users. A Study with Bilateral and Unilateral Young Users.

OBJECTIVES: The aim of the present study is to investigate the variations of the electroencephalographic (EEG) alpha rhythm in order to measure the appreciation of bilateral and unilateral young cochlear implant users during the observation of a musical cartoon. The cartoon has been modified for the generation of three experimental conditions: one with the original audio, another one with a distorted sound and, finally, a mute version. METHODS: The EEG data have been recorded during the observation of the cartoons in the three experimental conditions. The frontal alpha EEG imbalance has been calculated as a measure of motivation and pleasantness to be compared across experimental populations and conditions. RESULTS: The EEG frontal imbalance of the alpha rhythm showed significant variations during the perception of the different cartoons. In particular, the pattern of activation of normal-hearing children is very similar to the one elicited by the bilateral implanted patients. On the other hand, results related to the unilateral subjects do not present significant variations of the imbalance index across the three cartoons. CONCLUSION: The presented results suggest that the unilateral patients could not appreciate the difference in the audio format as well as bilaterally implanted and normal hearing subjects. The frontal alpha EEG imbalance is a useful tool to detect the differences in the appreciation of audiovisual stimuli in cochlear implant patients.

BRCA1 and BRCA2 mutations in breast/ovarian cancer patients from central Italy.

We report on the screening of the entire BRCA1/BRCA2 coding sequence by SSCP, PTT, and direct sequencing in 68 Italian families with recurrent breast or ovarian cancer. For each investigated proband, the probability of being carrier of a BRCA1/BRCA2 mutation was evaluated using the BRCAPRO software. We detected BRCA1/BRCA2 mutations in 8 patients (11.7%). However, if considering only patients with a carrier probability >10%, the detection rate was 36.8%, confirming the usefulness of the BRCAPRO software. One change (BRCA1 4172insT) was a novel mutation not reported in BIC database.

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Effect of bepridil on the activity of cytochrome c oxidase in solution and in proteoliposomes.

The interaction between bepridil and mitochondrial cytochrome c oxidase has been studied using the purified enzyme either in aqueous suspensions in the presence of detergents, or embedded into phospholipid vesicles. The investigation, systematically extended to nonactin and valinomycin for comparison, showed that: (a) valinomycin and nonactin induce similar changes in the visible absorption spectrum of cytochrome oxidase; these changes are quite different from those induced by bepridil. (b) The three compounds have an effect on the functional properties of purified, solubilized oxidase which may be related to binding. In particular, bepridil displays a complex pH-dependent effect which at concentrations below 50 microM results in a stimulation of the activity of approximately 30% starting with the oxidized resting enzyme. At variance with valinomycin and nonactin, the stimulatory effect is the same, within the errors, for the detergent-suspended, the vesicle-embedded and even the Keilin-Hartree particles. (c) In the case of detergent-suspended oxidase, the stimulatory effect of bepridil is also similar whether the enzyme is in the resting or in the pulsed state. If the oxidase is embedded into vesicles, however, the pulsed state is significantly more sensitive to bepridil than the resting one. These results are discussed in the light of the possible role assigned to pulsed oxidase in the regulation of the electron flux through the respiratory chain.

Revealing deterministic structures in click-evoked otoacoustic emissions.

Click-evoked otoacoustic emissions (CEOAEs) were studied by means of recurrence quantification analysis (RQA) and were found to be endowed with a relevant amount of deterministic structuring. Such a structure showed highly significant correlation with the clinical evaluation of the signal over a data set including 56 signals. Moreover, 1) one of the RQA variables, Trend, was very sensitive to phase transitions in the dynamical regime of CEOAEs, and 2) appropriate use of principal component analysis proved able to isolate the individual character of the studied signals. These results are of general interest for the study of auditory signal transduction and generation mechanisms.

On thermal transitions in biological macromolecules.

The thermal transitions shown by macromolecules are to be understood as an allosteric phenomenon. They can be dealt with in terms of the same linkage principles as those governing the binding of a chemical ligand. This provides a basis for analyzing the observed differences between the reversible heat denaturation of proteins and the melting of nucleic acids. It also adds to our understanding of cooperativity and heterotropic linkage.

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin.

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.

What the intermediate compounds in ligand binding to hemoglobin tell about the mechanism of cooperativity.

The populations of the intermediates in concentrated solutions of hemoglobin A0 equilibrated at various PCO values, pH 7.0, 0.1 M KCl, and 20 degrees C, have been determined using cryogenic methods. Data on CO saturations and distributions of intermediates were analysed in terms of the free energies of dimer-tetramer assembly of the intermediates (G.K. Ackers and F.R. Smith, Annu. Rev. Biophys. Chem. 16 (1987) 583). The cooperative free energy value of the singly ligated species was approximately one-half the total cooperative energy. The cooperative free energy value of the doubly ligated species was not significantly different from that of carboxyhemoglobin. Because of experimental error, the observed difference in concentrations among the populations of the doubly ligated species cannot be taken as indicative of their functional heterogeneity. Additional studies on some NO intermediates have emphasized that (alpha 1 beta 1)(alpha 2 beta 2)X, a key intermediate in the formulation of the 'third-state' hypothesis in the deoxy/cyanomethemoglobin system, has a free energy value for dimer-tetramer assembly which is critically dependent on the nature of the ligand X as suggested by Ackers and Smith (reference as cited above).

Pulsed cytochrome c oxidase.

The identification of two functionally distinct states, called pulsed and resting, has led to a number of investigations on the conformational variants of the enzyme. However, the catalytic properties of cytochrome oxidase may depend on a number of experimental conditions related to the solvent as well as to the protocol followed to determine the turnover number of the enzyme. This paper reports results which illustrate that the steady-state differences between pulsed and resting oxidase may, or may not, be detected depending on experimental conditions.