Low-Level Expression of a Folding-Incompetent Protein in Escherichia coli: Search for the Molecular Determinants of Protein Aggregation In Vivo

Aggregation of peptides and proteins into insoluble amyloid fibrils or related intracellular inclusions is the hallmark of many degenerative diseases, including Alzheimer's disease, Parkinson's disease, and various forms of amyloidosis. In spite of the considerable progress carried out in vitro in elucidating the molecular determinants of the conversion of purified and isolated proteins into amyloid fibrils, very little is known on factors governing this process in the complex environment of living organisms. Taking advantage of increasing evidence that bacterial inclusion bodies consist of amyloid-like aggregates, we have expressed in Escherichia coli both wild type and 21 single-point mutants of the N-terminal domain of the E. coli protein HypF. All variants were expressed as folding-incompetent units in a controlled manner, at low and comparable levels. Their solubilities were measured by quantifying the protein amount contained in the soluble and insoluble fractions by Western blot analysis. A significant negative correlation was found between the solubility of the variants in E. coli and their intrinsic propensity to form amyloid fibrils, predicted using an algorithm previously validated experimentally in vitro on a number of unfolded peptides and proteins, and considering hydrophobicity, β-sheet propensity, and charge as major sequence determinants of the aggregation process. These findings show that the physicochemical parameters previously recognized to govern amyloid formation by fully or partially unfolded proteins are largely applicable in vivo and pave the way for the molecular exploration of a process as complex as protein aggregation in living organisms.     

Addition to the revision of the Pristaulacus comptipennis species-group: Description of two new species from Laos and Thailand (Hymenoptera: Aulacidae)

Two species belonging to the recently revised Pristaulacus comptipennis species-group, P. manuelae Turrisi and Madl, sp. n. from Laos and P. iosephi Turrisi and Madl, sp. n. from Thailand, are described, illustrated and compared with most related species. The former species most resembles P. watanabei Turrisi and Smith, 2011, from Thailand, but is readily recognizable, mainly by the shape of the head, which is elongated, with a well-developed temple. The latter species resembles two other species, P. nobilei Turrisi and Smith, 2011, from south China, and P. emarginaticeps Turner, 1922, from Vietnam, but it is easily recognizable mainly by the shape of the medial occipital groove, which is shallow and V-shaped, as well as the shape of the mesosoma, with anterior lobe of mesoscutum, anterior to notauli, strongly elevated dorsally and anteriorly and distinctly overhanging pronotum.     

Dysregulation of the Autonomous Nervous System in Patients with Temporomandibular Disorder: A Pupillometric Study

The role of the autonomic nervous system (ANS) was recently investigated in Temporomandibular disorders (TMD). Several authors argue that in subjects with TMD there is a dysregulation of ANS. Recent literature support that Pupillometry is a simple non-invasive tool to study ANS. The aim of this study was to investigate the relationship between TMD and ANS activity using pupillometry recording in Infrared light at rest Mandible Position (RP); Infrared light at Forced Habitual Occlusion (FHO); Yellow-green light at RP; Yellow-green light at FHO. Forty female subjects were enrolled: 20 case patients showed TMD based on the Research Diagnostic Criteria for TMD, and 20 control patients, aged matched, had no signs or symptoms of TMD. Statistical analysis was performed on average pupil size. Ratio between pupil size in FHO and RP (FHO/RP ratio) and yellow-green and infrared (light/darkness ratio) lighting were carried out. Within group differences of pupil size and of “ratio” were analyzed using a paired t test, while differences of pupil size between groups were tested using an unpaired t test. Statistical comparisons between groups showed no significant differences of absolute values of pupil dimension in RP and FHO, both in yellow-green and in infrared lighting. In addition, there were no significant differences within groups comparing RP and FHO in yellow-green light. In within group comparison of pupil size, differences between RP and FHO were significant in infrared conditions. Control subjects increased, whereas TMD patients decreased pupil size at FHO in infrared lightening. FHO/RP ratio in darkness and light/darkness ratio in RP were significantly different between groups. Taken together, these data suggest that TMD subjects have an impairment of the sympathetic-adrenergic component of the ANS to be activated under stress. The present study provides preliminary pupillometric data confirming that adrenergic function is dysregulated in patients with TMD.     

Inactivation of Human Salivary Glutathione Transferase P1-1 by Hypothiocyanite: A Post-Translational Control System in Search of a Role

Glutathione transferases (GSTs) are a superfamily of detoxifying enzymes over-expressed in tumor tissues and tentatively proposed as biomarkers for localizing and monitoring injury of specific tissues. Only scarce and contradictory reports exist about the presence and the level of these enzymes in human saliva. This study shows that GSTP1-1 is the most abundant salivary GST isoenzyme, mainly coming from salivary glands. Surprisingly, its activity is completely obscured by the presence of a strong oxidizing agent in saliva that causes a fast and complete, but reversible, inactivation. Although salivary α-defensins are also able to inhibit the enzyme causing a peculiar half-site inactivation, a number of approaches (mass spectrometry, site directed mutagenesis, chromatographic and spectrophotometric data) indicated that hypothiocyanite is the main salivary inhibitor of GSTP1-1. Cys47 and Cys101, the most reactive sulfhydryls of GSTP1-1, are mainly involved in a redox interaction which leads to the formation of an intra-chain disulfide bridge. A reactivation procedure has been optimized and used to quantify GSTP1-1 in saliva of 30 healthy subjects with results of 42±4 mU/mg-protein. The present study represents a first indication that salivary GSTP1-1 may have a different and hitherto unknown function. In addition it fulfills the basis for future investigations finalized to check the salivary GSTP1-1 as a diagnostic biomarker for diseases.     

Spatially Distributed Effects of Mental Exhaustion on Resting-State FMRI Networks

Brain activity during rest is spatially coherent over functional connectivity networks called resting-state networks. In resting-state functional magnetic resonance imaging, independent component analysis yields spatially distributed network representations reflecting distinct mental processes, such as intrinsic (default) or extrinsic (executive) attention, and sensory inhibition or excitation. These aspects can be related to different treatments or subjective experiences. Among these, exhaustion is a common psychological state induced by prolonged mental performance. Using repeated functional magnetic resonance imaging sessions and spatial independent component analysis, we explored the effect of several hours of sustained cognitive performances on the resting human brain. Resting-state functional magnetic resonance imaging was performed on the same healthy volunteers in two days, with and without, and before, during and after, an intensive psychological treatment (skill training and sustained practice with a flight simulator). After each scan, subjects rated their level of exhaustion and performed an N-back task to evaluate eventual decrease in cognitive performance. Spatial maps of selected resting-state network components were statistically evaluated across time points to detect possible changes induced by the sustained mental performance. The intensive treatment had a significant effect on exhaustion and effort ratings, but no effects on N-back performances. Significant changes in the most exhausted state were observed in the early visual processing and the anterior default mode networks (enhancement) and in the fronto-parietal executive networks (suppression), suggesting that mental exhaustion is associated with a more idling brain state and that internal attention processes are facilitated to the detriment of more extrinsic processes. The described application may inspire future indicators of the level of fatigue in the neural attention system.     

Multi-Scale Analysis of the European Airspace Using Network Community Detection

We show that the European airspace can be represented as a multi-scale traffic network whose nodes are airports, sectors, or navigation points and links are defined and weighted according to the traffic of flights between the nodes. By using a unique database of the air traffic in the European airspace, we investigate the architecture of these networks with a special emphasis on their community structure. We propose that unsupervised network community detection algorithms can be used to monitor the current use of the airspace and improve it by guiding the design of new ones. Specifically, we compare the performance of several community detection algorithms, both with fixed and variable resolution, and also by using a null model which takes into account the spatial distance between nodes, and we discuss their ability to find communities that could be used to define new control units of the airspace.