Temporal differences and commonalities between hand and tool neural processing.

Object recognition is a complex cognitive process that relies on how the brain organizes object-related information. While spatial principles have been extensively studied, less studied temporal dynamics may also offer valuable insights into this process, particularly when neural processing overlaps for different categories, as it is the case of the categories of hands and tools. Here we focus on the differences and/or similarities between the time-courses of hand and tool processing under electroencephalography (EEG). Using multivariate pattern analysis, we compared, for different time points, classification accuracy for images of hands or tools when compared to images of animals. We show that for particular time intervals (~ 136-156 ms and ~ 252-328 ms), classification accuracy for hands and for tools differs. Furthermore, we show that classifiers trained to differentiate between tools and animals generalize their learning to classification of hand stimuli between ~ 260-320 ms and ~ 376-500 ms after stimulus onset. Classifiers trained to distinguish between hands and animals, on the other hand, were able to extend their learning to the classification of tools at ~ 150 ms. These findings suggest variations in semantic features and domain-specific differences between the two categories, with later-stage similarities potentially related to shared action processing for hands and tools.

Rapid changes in profiles from stored materials used in scent training of explosive detection dogs.

Canines trained on scents from materials emitting vapours of explosives and related compounds are widely used to detect explosives in civilian, military and forensic applications. Despite the importance of these training materials, there is limited knowledge on how long these subsamples can be stored and whether vapour profiles change over time. We developed a sampling methodology that makes use of a secondary chamber for stabilisation of headspace concentration to allow reliable and reproducible determination of scent profiles. The effect of aging was investigated by following the response of volatile markers emitted from eight common explosives in open and closed containers over two months or two years. The initial headspace air volume consisted of a wide variety of chemical substances related to explosives, with levels varying in magnitude from low ppb to ppm. All included subsamples were affected by aging by demonstrating exponentially lower levels, and five subsamples showed a significant change in their scent profile. The dominant components decreased on a short time scale for plastic explosives based on RDX, PETN and dynamite as well as for granules of octol and ammonium nitrate mixed with fuel. For flakes of TNT, granules of Comp B and nitrocellulose powder, headspace air concentrations declined, but the overall character of their profiles were in general more stable. The overall changes, i.e., lower levels and/or changed profiles, justifies regular checks of the scent status of training materials. Considering these results together with data displaying marginal changes in energetic performance, it is advisable to complement scent training with training materials subjected to different durations of aging.

A reduced level of consciousness affects non-conscious processes.

Being conscious is a profound aspect of human existence, and understanding its function and its inception is considered one of the truly grand scientific challenges. However, the nature of consciousness remains enigmatic, to a large part because "being conscious" can refer to both the content (phenomenology) and the level (arousal) of consciousness, and how these different aspects are related remains unclear. To empirically assess the relation between level and content of consciousness, we manipulated these two aspects by presenting stimuli consciously or non-consciously and by using Propofol sedation, while brain activity was measured using fMRI. We observed that sedation affected both conscious and non-conscious processes but at different hierarchical levels; while conscious processing was altered in higher-order regions (the intraparietal sulcus) and spared sensory areas, the opposite effect was observed for non-conscious processing. The observation that Propofol affected non-conscious processing calls for a reconsideration of what kind of information one can gain on "consciousness" from recording neural responses to sedation without considering both (content) conscious and (content) non-conscious processing.

Burden of spinal cord injury-related neuropathic pain in the United States: retrospective chart review and cross-sectional survey.

STUDY DESIGN: Cross-sectional, observational study. OBJECTIVES: Characterize demographic and clinical characteristics, health status, pain, function, productivity and economic burden in spinal cord injury-related neuropathic pain (SCI-NeP) subjects, by pain severity. SETTING: United States. One hundred and three subjects diagnosed with SCI-NeP recruited during routine primary care or specialty physician office visits completed a questionnaire to assess patient-reported outcomes. Physicians completed a case report form on inclusion/exclusion criteria, subject clinical characteristics and health-care resource use (HRU) based on 6-month retrospective chart review. RESULTS: Subjects' mean age was 48.7, 69.9% were male and 48.5% were unable to walk. The most frequently reported comorbidities were sleep disturbance/insomnia (28.2%), depressive symptoms (25.2%) and anxiety (23.3%). Subjects' mean pain severity score was 5.3 (0-10 scale), and 77.7% reported moderate or severe pain. On a 0-10 scale, subjects' reported moderate pain interference with function: mean 5.4. Subjects' health status, as measured by the EuroQol 5-dimensions health-state utility, was 0.49 (-0.11 to 1.00 scale). Pain interference with function and health status were significantly worse among subjects with more severe pain (P<0.0005). Among employed subjects (13.6%), overall work impairment was 38.0%. The proportion of subjects who were prescribed ≥1 medication was 94.2%, and the mean number of physician office visits in past 6 months due to SCI-NeP was 2.2. Total annualized cost per subject was $26 270 (direct: $8636, indirect: $17 634). CONCLUSION: SCI-NeP subjects exhibited high pain levels, despite active management. Pain levels were associated with poor function, low health status and lost productivity. HRU was prevalent, and costs, particularly indirect, were substantial, highlighting unmet need. SPONSORSHIP: This study was supported by Pfizer, Inc.

The impact of the economic downturn and health care reform on treatment decisions for haemophilia A: patient, caregiver and health care provider perspectives.

Little is known about the impact of the recent US economic downturn and health care reform on patient, caregiver and health care provider (HCP) decision-making for haemophilia A. To explore the impact of the recent economic downturn and perceived impact of health care reform on haemophilia A treatment decisions from patient, caregiver and HCP perspectives. Patients/caregivers and HCPs completed a self-administered survey in 2011. Survey participants were asked about demographics, the impact of the recent economic downturn and health care reform provisions on their treatment decisions. Seventy three of the 134 (54%) patients/caregivers and 39 of 48 (81%) HCPs indicated that the economic downturn negatively impacted haemophilia care. Seventy of the 73 negatively impacted patients made financially related treatment modifications, including delaying/cancelling routine health care visit, skipping doses and/or skipping filling prescription. Treatment modifications made by HCPs included delaying elective surgery, switching from higher to lower priced product, switching from recombinant to plasma-derived products and delaying prophylaxis. Health care reform was generally perceived as positive. Due to the elimination of lifetime caps, 30 of 134 patients (22%) and 28 of 48 HCPs (58%) indicated that they will make treatment modifications by initiating prophylaxis or scheduling routine appointment/surgery sooner. Both patients/caregivers and HCPs reported that the economic downturn had a negative impact on haemophilia A treatment. Suboptimal treatment modifications were made due to the economic downturn. Health care reform, especially the elimination of lifetime caps, was perceived as positive for haemophilia A treatment and as a potential avenue for contributing to more optimal treatment behaviours.

Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites.

BACKGROUND: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin-protease complex have been proposed. In the absence of a spatial structure of a serpin-protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial. RESULTS: We used a recently developed method for making precise distance measurements, based on donor-donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1') were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into beta sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease-inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker. CONCLUSIONS: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.

The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.

The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.