A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

New insights into pathophysiology of vestibular migraine.

Vestibular migraine (VM) is a common disorder in which genetic, epigenetic, and environmental factors probably contribute to its development. The pathophysiology of VM is unknown; nevertheless in the last few years, several studies are contributing to understand the neurophysiological pathways involved in VM. The current hypotheses are mostly based on the knowledge of migraine itself. The evidence of trigeminal innervation of the labyrinth vessels and the localization of vasoactive neuropeptides in the perivascular afferent terminals of these trigeminal fibers support the involvement of the trigemino-vascular system. The neurogenic inflammation triggered by activation of the trigeminal-vestibulocochlear reflex, with the subsequent inner ear plasma protein extravasation and the release of inflammatory mediators, can contribute to a sustained activation and sensitization of the trigeminal primary afferent neurons explaining VM symptoms. The reciprocal connections between brainstem vestibular nuclei and the structures that modulate trigeminal nociceptive inputs (rostral ventromedial medulla, ventrolateral periaqueductal gray, locus coeruleus, and nucleus raphe magnus) are critical to understand the pathophysiology of VM. Although cortical spreading depression can affect cortical areas involved in processing vestibular information, functional neuroimaging techniques suggest a dysmodulation in the multimodal sensory integration and processing of vestibular and nociceptive information, resulting from a vestibulo-thalamo-cortical dysfunction, as the pathogenic mechanism underlying VM. The elevated prevalence of VM suggests that multiple functional variants may confer a genetic susceptibility leading to a dysregulation of excitatory-inhibitory balance in brain structures involved in the processing of sensory information, vestibular inputs, and pain. The interactions among several functional and structural neural networks could explain the pathogenic mechanisms of VM.

Functional multidisciplinary rehabilitation versus outpatient physiotherapy for non specific low back pain: randomized controlled trial.

INTRODUCTION: In recent decades the treatment of non-specific low back pain has turned to active modalities, some of which were based on cognitive-behavioural principles. Non-randomised studies clearly favour functional multidisciplinary rehabilitation over outpatient physiotherapy. However, systematic reviews and meta-analysis provide contradictory evidence regarding the effects on return to work and functional status. The aim of the present randomised study was to compare long-term functional and work status after 3-week functional multidisciplinary rehabilitation or 18 supervised outpatient physiotherapy sessions. METHODS: 109 patients with non-specific low back pain were randomised to either a 3-week functional multidisciplinary rehabilitation programme, including physical and ergonomic training, psychological pain management, back school and information, or 18 sessions of active outpatient physiotherapy over 9 weeks. Primary outcomes were functional disability (Oswestry) and work status. Secondary outcomes were lifting capacity (Spinal Function Sort and PILE test), lumbar range-of-motion (modified-modified Schöber and fingertip-to-floor tests), trunk muscle endurance (Shirado and Biering-Sörensen tests) and aerobic capacity (modified Bruce test). RESULTS: Oswestry disability index was improved to a significantly greater extent after functional multidisciplinary rehabilitation compared to outpatient physiotherapy at follow-up of 9 weeks (P = 0.012), 9 months (P = 0.023) and 12 months (P = 0.011). Work status was significantly improved after functional multidisciplinary rehabilitation only (P = 0.012), resulting in a significant difference compared to outpatient physiotherapy at 12 months' follow-up (P = 0.012). Secondary outcome results were more contrasted. CONCLUSIONS: Functional multidisciplinary rehabilitation was better than outpatient physiotherapy in improving functional and work status. From an economic point of view, these results should be backed up by a cost-effectiveness study.

Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease.

Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.

In vitro functional correction of Hermansky-Pudlak Syndrome type-1 by lentiviral-mediated gene transfer.

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by oculocutaneous albinism, bleeding tendency and susceptibility to pulmonary fibrosis. No curative therapy is available. Genetic correction directed to the lungs, bone marrow and/or gastro-intestinal tract might provide alternative forms of treatment for the diseases multi-systemic complications. We demonstrate that lentiviral-mediated gene transfer corrects the expression and function of the HPS1 gene in patient dermal melanocytes, which opens the way to development of gene therapy for HPS.

Personnes âgées aux urgences: défis actuels et futurs [Older patients at the emergency department: current and future challenges].

Emergency departments are and will be at the front line to face the forthcoming increased use of the health care system by the aging baby boomers cohort. Emergency department services will need to adjust on a quantitative as well as on a qualitative basis to manage the impact of these demographic changes. Various models of care have been developed to improve the care of older geriatric patients in the Emergency department that resulted in favorable results on functional, health, as well as health services utilization outcomes. Key components of these successful models have been identified that require a high level of integration between geriatric and emergency teams.

Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

Functional calibration procedure for 3D knee joint angle description using inertial sensors.

Measurement of three-dimensional (3D) knee joint angle outside a laboratory is of benefit in clinical examination and therapeutic treatment comparison. Although several motion capture devices exist, there is a need for an ambulatory system that could be used in routine practice. Up-to-date, inertial measurement units (IMUs) have proven to be suitable for unconstrained measurement of knee joint differential orientation. Nevertheless, this differential orientation should be converted into three reliable and clinically interpretable angles. Thus, the aim of this study was to propose a new calibration procedure adapted for the joint coordinate system (JCS), which required only IMUs data. The repeatability of the calibration procedure, as well as the errors in the measurement of 3D knee angle during gait in comparison to a reference system were assessed on eight healthy subjects. The new procedure relying on active and passive movements reported a high repeatability of the mean values (offset<1 degrees) and angular patterns (SD<0.3 degrees and CMC>0.9). In comparison to the reference system, this functional procedure showed high precision (SD<2 degrees and CC>0.75) and moderate accuracy (between 4.0 degrees and 8.1 degrees) for the three knee angle. The combination of the inertial-based system with the functional calibration procedure proposed here resulted in a promising tool for the measurement of 3D knee joint angle. Moreover, this method could be adapted to measure other complex joint, such as ankle or elbow.

Functional diversification of duplicate genes through subcellular adaptation of encoded proteins.

BACKGROUND: Gene duplication is the primary source of new genes with novel or altered functions. It is known that duplicates may obtain these new functional roles by evolving divergent expression patterns and/or protein functions after the duplication event. Here, using yeast (Saccharomyces cerevisiae) as a model organism, we investigate a previously little considered mode for the functional diversification of duplicate genes: subcellular adaptation of encoded proteins. RESULTS: We show that for 24-37% of duplicate gene pairs derived from the S. cerevisiae whole-genome duplication event, the two members of the pair encode proteins that localize to distinct subcellular compartments. The propensity of yeast duplicate genes to evolve new localization patterns depends to a large extent on the biological function of their progenitor genes. Proteins involved in processes with a wider subcellular distribution (for example, catabolism) frequently evolved new protein localization patterns after duplication, whereas duplicate proteins limited to a smaller number of organelles (for example, highly expressed biosynthesis/housekeeping proteins with a slow rate of evolution) rarely relocate within the cell. Paralogous proteins evolved divergent localization patterns by partitioning of ancestral localizations ('sublocalization'), but probably more frequently by relocalization to new compartments ('neolocalization'). We show that such subcellular reprogramming may occur through selectively driven substitutions in protein targeting sequences. Notably, our data also reveal that relocated proteins functionally adapted to their new subcellular environments and evolved new functional roles through changes of their physico-chemical properties, expression levels, and interaction partners. CONCLUSION: We conclude that protein subcellular adaptation represents a common mechanism for the functional diversification of duplicate genes.

Functional analysis of Ectodysplasin-A mutations causing selective tooth agenesis.

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome hypohidrotic ectodermal dysplasia (MIM 305100), but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis-causing EDA mutations (one novel and five known) that are located in the C-terminal tumor necrosis factor homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth, requires the highest level of EDA-receptor signaling, whereas other ectodermal appendages, including posterior teeth, have less stringent requirements and form normally in response to EDA mutations with reduced activity.

Activities of daily living with reverse prostheses: importance of scapular compensation for functional mobility of the shoulder.

HYPOTHESIS: The nonanatomical design of reverse shoulder prostheses induce medial displacement of the center of rotation, impingements and may reduce the mobility of the shoulder. The aim of this study is to test the hypothesis that during activities of daily living functional mobility of the shoulder can be restored by scapular compensation. MATERIAL AND METHODS: A numerical 3-dimensional model was developed to reproduce the movement of the scapula and humerus, during 4 activities of daily living measured experimentally. This hypothesis was tested in 4 configurations of the aequalis reverse prosthesis (standard 36-mm glenosphere, 42-mm glenosphere, lateralized 36-mm glenosphere, lateralized Bony Increased-Offset Reverse Shoulder Arthroplasty [BIO-RSA]), which were implanted in the virtual model. All impingement positions were evaluated, as the required scapular compensation to avoid impingements. RESULTS: With the 36-mm glenosphere, impingements occurred only for rest of hand to back-pocket positions. The 42-mm partly improved the mobility. The 2 lateralized glenospheres were free of impingement. When impingements occurred, the scapular compensation was less than 10°. CONCLUSION: Most reverse prostheses impingements reported in clinical and biomechanical studies can be avoided, either by scapular compensation or by a glenosphere lateralization. After reverse shoulder arthroplasty, a fraction of the mobility of the gleno-humeral is transferred to the scapulo-thoracic joint.

Evolutionary and Functional Analyses of the Interaction between the Myeloid Restriction Factor SAMHD1 and the Lentiviral Vpx Protein.

SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.