Comprehensive analysis of proteins secreted by dermatophytes in a protein medium which to some extent mimic "in vivo" growth parameters

RESUME : Les dermatophytes sont les agents infectieux les plus fréquents responsables de la plupart des mycoses superficielles chez les humains et chez les animaux. Ces infections, dermatophytoses, également appelées tineas ou teignes, sont fréquentes et causent des problèmes de santé publique au niveau mondial. La capacité d'envahir et de progresser au sein des structures kératinisées est probablement liée à la sécrétion de différentes enzymes kératinolytiques, qui sont considérées comme la principale caractéristique liée à la pathogénicité de ces champignons. L'objectif de ma thèse a été premièrement de progresser dans l'identification et la caractérisation des nouvelles protéines sécrétées, afin de mieux comprendre a) la capacité globale des dermatophytes à envahir les structures kératinisées, et b) les différences dans la virulence et la spécificité d'hôte que présentent les espèces étudiées .Pour progresser dans l'identification et la caractérisation de ces nouvelles protéines, les secretomes de six espèces de dermatophytes (Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii et Trichophyton tonsurans) ont été étudiés. Bien qu'il y ait un niveau globalement élevé de similitude entre les protéases sécrétées, les différentes espèces de dermatophytes sécrètent des profiles protéiques distincts lorsqu'elles sont cultivées dans les mêmes conditions de culture, et donc une signature spécifique a pu être associé à chaque espèce. Ces profiles ont été un outil avantageux pour identifier et cartographier les protéines orthologues aux six espèces et ont aussi permit la discrimination d'espèces très proches comme T. tonsurans et T. equinum qui ne peuvent pas être différenciées par l'ADN ribosomal. Ce travail également présente ce que l'on croit être la première identification global des protéines sécrétées par les dermatophytes dans des conditions de culture que incitent l'activité protéolytique extracellulaire. Ce catalogue de protéines, comprenant des endo- and exo- proteases, autres hydrolases, oxydoreductases et des protéines avec fonction inconnue, représente probablement le spectre d'enzymes qui permettent la dégradation des tissus kératinisés en composés qui peuvent être assimilés par le champignon. Les résultats suggèrent qu'un changement écologique pourrait être associé à une expression différentielle des gènes codant les protéines sécrétées, en particulier, les protéases, plutôt qu'à des divergences génétiques au niveau des gènes codant les protéines orthologues. Une sécrétion différentielle des protéines par les dermatophytes pourrait également être responsable de la variabilité inflammatoire qui causent ces agents infectieux chez les différents hôtes. Par conséquent, les protéines identifiées ici sont également importantes pour faire la lumière sur la réponse immunitaire de l'hôte au cours du processus infectieux. SUMMARY : Dermatophytes are the most common infectious agents responsible for superficial mycosis in humans and animals. Dermatophytoses, also called tineas or ringworm, are frequent and cause public health problems worldwide. The secretion of different keratinolytic enzymes is believed to be a key pathogenicity-related characteristic of these fungi. The aim of this work was first to progress in the identification and characterization of novel secreted proteins, in order to better understand a) the overall capability of dermatophytes to invade keratinised structures, and b) differences in virulence and host-specificity of the investigated species. To progress in the identification and characterization of novel proteins, the secretomes from Trichophyton rubrum, Trichophyton violaceum, Trichophyton soudanense, Trichophyton equinum, Arthroderma vanbreuseghemii and Trichophyton tonsurans were studied. Although there is a high global level of similarity among the secreted proteases, different dermatophyte species produce distinct patterns of proteins when grown in the same culture medium, and so a specific signature could be associated to each species. These patterns were useful to identify and map orthologous proteins among the six species, as well as to discriminate the closely related species T. tonsurans and T. equinum, which cannot be differentiated by ribosomal DNA. This work also presents the first in-depth identification of the major proteins secreted by dermatophytes growing under conditions promoting extracellular proteolytic activity. This catalogue of proteins, which include several endo- and exo- proteases, other hydrolases, oxydoreductases, and proteins of unknown function, probably represents the spectrum of enzymes that allow the degradation of keratinized tissues into compounds which can be assimilated by the fungus. The results suggest that ecological switching could be related to a differential expression of genes encoding secreted proteins, particularly, proteases, rather than genetic divergences of the genes encoding orthologous proteins. Differential secretion of proteins by Dermatophyte species could also be responsible for the variable inflammation caused by the infectious agent within the host. Therefore, the proteins here identified are also important to shed light into the immune response of the host during the infection process.

Outcome evaluation of ankle osteoarthritis treatments using spatio-temporal gait parameters and plantar pressure during unconstrained long distance walking : P25

Introduction: Ankle arthrodesis (AD) and total ankle replacement (TAR) are typical treatments for ankle osteoarthritis (AO). Despite clinical interest, there is a lack of their outcome evaluation using objective criteria. Gait analysis and plantar pressure assessment are appropriate to detect pathologies in orthopaedics but they are mostly used in lab with few gait cycles. In this study, we propose an ambulatory device based on inertial and plantar pressure sensors to compare the gait during long-distance trials between healthy subjects (H) and patients with AO or treated by AD and TAR. Methods: Our study included four groups: 11 patients with AO, 9 treated by TAR, 7 treated by AD and 6 control subjects. An ambulatory system (Physilog®, CH) was used for gait analysis; plantar pressure measurements were done using a portable insole (Pedar®-X, DE). The subjects were asked to walk 50 meters in two trials. Mean value and coefficient of variation of spatio-temporal gait parameters were calculated for each trial. Pressure distribution was analyzed in ten subregions of foot. All parameters were compared among the four groups using multi-level model-based statistical analysis. Results: Significant difference (p <0.05) with control was noticed for AO patients in maximum force in medial hindfoot and forefoot and in central forefoot. These differences were no longer significant in TAR and AD groups. Cadence and speed of all pathologic groups showed significant difference with control. Both treatments showed a significant improvement in double support and stance. TAR decreased variability in speed, stride length and knee ROM. Conclusions: In spite of a small sample size, this study showed that ankle function after AO treatments can be evaluated objectively based on plantar pressure and spatio-temporal gait parameters measured during unconstrained walking outside the lab. The combination of these two ambulatory techniques provides a promising way to evaluate foot function in clinics.

Identification of Wax Esters in Latent Print Residues by Gas Chromatography-Mass Spectromertry and Their Potential Use as Aging Parameters

Recent studies show that the composition of fingerprint residue varies significantly from the same donor as well as between donors. This variability is a major drawback in latent print dating issues. This study aimed, therefore, at the definition of a parameter that is less variable from print to print, using a ratio of peak area of a target compound degrading over time divided by the summed area of peaks of more stable compounds also found in latent print residues.Gas chromatography-mass spectrometry (GC/MS) analysis of the initial lipid composition of latent prints identifies four main classes of compounds that can be used in the definition of an aging parameter: fatty acids, sterols, sterol precursors, and wax esters (WEs). Although the entities composing the first three groups are quite well known, those composing WEs are poorly reported. Therefore, the first step of the present work was to identify WE compounds present in latent print residues deposited by different donors. Of 29 WEs recorded in the chromatograms, seven were observed in the majority of samples.The identified WE compounds were subsequently used in the definition of ratios in combination with squalene and cholesterol to reduce the variability of the initial composition between latent print residues from different persons and more particularly from the same person. Finally, the influence of a latent print enhancement process on the initial composition was studied by analyzing traces after treatment with magnetic powder, 1,2-indanedione, and cyanoacrylate.

Widespread age-related differences in the human brain microstructure revealed by quantitative magnetic resonance imaging.

A pressing need exists to disentangle age-related changes from pathologic neurodegeneration. This study aims to characterize the spatial pattern and age-related differences of biologically relevant measures in vivo over the course of normal aging. Quantitative multiparameter maps that provide neuroimaging biomarkers for myelination and iron levels, parameters sensitive to aging, were acquired from 138 healthy volunteers (age range: 19-75 years). Whole-brain voxel-wise analysis revealed a global pattern of age-related degeneration. Significant demyelination occurred principally in the white matter. The observed age-related differences in myelination were anatomically specific. In line with invasive histologic reports, higher age-related differences were seen in the genu of the corpus callosum than the splenium. Iron levels were significantly increased in the basal ganglia, red nucleus, and extensive cortical regions but decreased along the superior occipitofrontal fascicle and optic radiation. This whole-brain pattern of age-associated microstructural differences in the asymptomatic population provides insight into the neurobiology of aging. The results help build a quantitative baseline from which to examine and draw a dividing line between healthy aging and pathologic neurodegeneration.

Continuous monitoring and quantification of multiple parameters of daily physical activity in ambulatory Duchenne muscular dystrophy patients.

Multiple motor function and strength assessment tools exist for the evaluation of neuromuscular diseases, but most do not directly assess functional ability in the patients' daily physical activity in their home environment. In this study our aim was to assess: 1) the feasibility and accuracy of physical activity monitoring during two days in a home environment of five DMD patients using a non-commercialized monitor containing a 3D accelerometer and a gyroscope, 2) if a difference in the physical activity parameters could be measured before and one month after starting prednisolone. We reliably quantified the time spend sitting, standing, lying, walking, the number of steps taken, the cadence, the number of walking episodes and their duration as well as how these were distributed over the day. Parameters possibly reflecting endurance, such as the duration of the walking episodes or the succession of two or three walking episodes lasting more than 30 s were the most improved after prednisolone treatment. This degree of detailed determination of physical activity in a home environment has not been previously reported in neuromuscular disorders to our knowledge and some of the reported parameters are potential new outcome measures in clinical trials.

Pharmacokinetic parameters of artesunate and dihydroartemisinin in rats infected with Fasciola hepatica

OBJECTIVES: The pharmacokinetic (PK) parameters of artesunate, recently discovered to possess promising trematocidal activity, and its main metabolite dihydroartemisinin (DHA) were determined in rats infected with hepatic and biliary stages of Fasciola hepatica and compared with uninfected rats after single intragastric and intravenous (iv) doses. METHODS: Rats infected with F. hepatica for 25 and 83 days and uninfected rats were cannulated in the right jugular vein and blood samples were withdrawn at selected timepoints following 10 mg/kg of iv and a single 100 mg/kg oral dose of artesunate. Plasma was analysed for artesunate and DHA by liquid chromatography coupled to tandem mass spectrometry. RESULTS: Rats harbouring juvenile and adult F. hepatica infections revealed considerable changes in PK parameters of artesunate and DHA. Following oral administration, maximum plasma concentrations (C(max)) of artesunate and DHA were 1.8-2.3-fold higher in infected rats [artesunate: 1334 +/- 1404 ng/mL (no infection) versus 2454 +/- 1494 ng/mL (acute infection) and 2768 +/- 538 ng/mL (chronic infection); DHA: 3802 +/- 2149 ng/mL (no infection) versus 6507 +/- 3283 ng/mL (acute infection) and 9093 +/- 884 ng/mL (chronic infection)]. The AUCs of artesunate and DHA were 2.1-4.4-fold greater in infected rats. An opposite trend was observed after iv injection. C(max) and AUC of artesunate and DHA following iv dosing were 5784 +/- 3718 and 140 938 +/- 128 783 ng.min/mL and 3849 +/- 3060 and 86 107 +/- 41 863 ng.min/mL, respectively, in uninfected rats versus 2623 +/- 1554 and 21 617 +/- 12 230 ng.min/mL and 2835 +/- 980 and 64 290 +/- 29 057 ng.min/mL, respectively, in rats harbouring a chronic infection. The elimination half-lives (t(1/2)) of artesunate and DHA were considerably altered in infected rats following oral and iv administration of artesunate. CONCLUSIONS: F. hepatica infections strongly influence the disposition kinetics of artesunate and its metabolite in rats. The clinical implications of this finding need to be carefully studied.

Effects of computing parameters and measurement locations on the estimation of 3D NPS in non-stationary MDCT images.

The goal of this study was to investigate the impact of computing parameters and the location of volumes of interest (VOI) on the calculation of 3D noise power spectrum (NPS) in order to determine an optimal set of computing parameters and propose a robust method for evaluating the noise properties of imaging systems. Noise stationarity in noise volumes acquired with a water phantom on a 128-MDCT and a 320-MDCT scanner were analyzed in the spatial domain in order to define locally stationary VOIs. The influence of the computing parameters in the 3D NPS measurement: the sampling distances bx,y,z and the VOI lengths Lx,y,z, the number of VOIs NVOI and the structured noise were investigated to minimize measurement errors. The effect of the VOI locations on the NPS was also investigated. Results showed that the noise (standard deviation) varies more in the r-direction (phantom radius) than z-direction plane. A 25 × 25 × 40 mm(3) VOI associated with DFOV = 200 mm (Lx,y,z = 64, bx,y = 0.391 mm with 512 × 512 matrix) and a first-order detrending method to reduce structured noise led to an accurate NPS estimation. NPS estimated from off centered small VOIs had a directional dependency contrary to NPS obtained from large VOIs located in the center of the volume or from small VOIs located on a concentric circle. This showed that the VOI size and location play a major role in the determination of NPS when images are not stationary. This study emphasizes the need for consistent measurement methods to assess and compare image quality in CT.

Estimating parameters for generalized mass action models using constraint propagation.

As modern molecular biology moves towards the analysis of biological systems as opposed to their individual components, the need for appropriate mathematical and computational techniques for understanding the dynamics and structure of such systems is becoming more pressing. For example, the modeling of biochemical systems using ordinary differential equations (ODEs) based on high-throughput, time-dense profiles is becoming more common-place, which is necessitating the development of improved techniques to estimate model parameters from such data. Due to the high dimensionality of this estimation problem, straight-forward optimization strategies rarely produce correct parameter values, and hence current methods tend to utilize genetic/evolutionary algorithms to perform non-linear parameter fitting. Here, we describe a completely deterministic approach, which is based on interval analysis. This allows us to examine entire sets of parameters, and thus to exhaust the global search within a finite number of steps. In particular, we show how our method may be applied to a generic class of ODEs used for modeling biochemical systems called Generalized Mass Action Models (GMAs). In addition, we show that for GMAs our method is amenable to the technique in interval arithmetic called constraint propagation, which allows great improvement of its efficiency. To illustrate the applicability of our method we apply it to some networks of biochemical reactions appearing in the literature, showing in particular that, in addition to estimating system parameters in the absence of noise, our method may also be used to recover the topology of these networks.

The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase.

Hsp70-Hsp40-NEF and possibly Hsp100 are the only known molecular chaperones that can use the energy of ATP to convert stably pre-aggregated polypeptides into natively refolded proteins. However, the kinetic parameters and ATP costs have remained elusive because refolding reactions have only been successful with a molar excess of chaperones over their polypeptide substrates. Here we describe a stable, misfolded luciferase species that can be efficiently renatured by substoichiometric amounts of bacterial Hsp70-Hsp40-NEF. The reactivation rates increased with substrate concentration and followed saturation kinetics, thus allowing the determination of apparent V(max)' and K(m)' values for a chaperone-mediated renaturation reaction for the first time. Under the in vitro conditions used, one Hsp70 molecule consumed five ATPs to effectively unfold a single misfolded protein into an intermediate that, upon chaperone dissociation, spontaneously refolded to the native state, a process with an ATP cost a thousand times lower than expected for protein degradation and resynthesis.

Recurrent TET2 mutations in peripheral T-cell lymphomas correlate with TFH-like features and adverse clinical parameters.

Inactivating mutations of the Ten-Eleven Translocation 2 (TET2) gene were first identified in myeloid malignancies and more recently in peripheral T-cell lymphomas (PTCLs). In the present study, we investigated the presence of TET2 coding sequence mutations and their clinical relevance in a large cohort of 190 PTCL patients. TET2 mutations were identified in 40 of 86 (47%) cases of angioimmunoblastic T-cell lymphoma (AITL) and in 22 of 58 (38%) cases of peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), but were absent in all other PTCL entities, with the exception of 2 of 10 cases of enteropathy-associated T-cell lymphoma. Among PTCL-NOS, a heterogeneous group of lymphoma-comprising cases likely to derive from Th follicular (T(FH)) cells similarly to AITL, TET2 mutations were more frequent when PTCL-NOS expressed T(FH) markers and/or had features reminiscent of AITL (58% vs 24%, P = .01). In the AITL and PTCL-NOS subgroups, TET2 mutations were associated with advanced-stage disease, thrombocytopenia, high International Prognostic Index scores, and a shorter progression-free survival.

A comparative study of ballpoint ink ageing parameters using GC/MS.

For more than a decade scientists tried to develop methods capable of dating ink by monitoring the loss of phenoxyethanol (PE) over time. While many methods were proposed in the literature, few were really used to solve practical cases and they still raise much concern within the scientific community. In fact, due to the complexity of ink drying processes it is particularly difficult to find a reliable ageing parameter to reproducibly follow ink ageing. Moreover, systematic experiments are required in order to evaluate how different factors actually influence the results over time. Therefore, this work aimed at evaluating the capacity of four different ageing parameters to reliably follow ink ageing over time: (1) the quantity of solvent PE in an ink line, (2) the relative peak area (RPA) normalising the PE results using stable volatile compounds present in the ink formulation, (3) the solvent loss ratio (R%) calculated from PE results obtained by the analyses of naturally and artificially aged samples, (4) a modified solvent loss ratio version (R%*) calculated from RPA results. After the determination of the limits of reliable measurements of the analytical method, the repeatability of the different ageing parameters was evaluated over time, as well as the influence of ink composition, writing pressure and storage conditions on the results. Surprisingly, our results showed that R% was not the most reliable parameter, as it showed the highest standard deviation. Discussion of the results in an ink dating perspective suggests that other proposed parameters, such as RPA values, may be more adequate to follow ink ageing over time.

A comparative study of ballpoint ink ageing parameters using GC/MS

For more than a decade scientists tried to develop methods capable of dating ink by monitoring the loss of phenoxyethanol (PE) over time. While many methods were proposed in the literature, few were really used to solve practical cases and they still raise much concern within the scientific community. In fact, due to the complexity of ink drying processes it is particularly difficult to find a reliable ageing parameter to reproducibly follow ink ageing. Moreover, systematic experiments are required in order to evaluate how different factors actually influence the results over time. Therefore, this work aimed at evaluating the capacity of four different ageing parameters to reliably follow ink ageing over time: (1) the quantity of solvent PE in an ink line, (2) the relative peak area (RPA) normalising the PE results using stable volatile compounds present in the ink formulation, (3) the solvent loss ratio (R%) calculated from PE results obtained by the analyses of naturally and artificially aged samples, (4) a modified solvent loss ratio version (R%*) calculated from RPA results. After the determination of the limits of reliable measurements of the analytical method, the repeatability of the different ageing parameters was evaluated over time, as well as the influence of ink composition, writing pressure and storage conditions on the results. Surprisingly, our results showed that R% was not the most reliable parameter, as it showed the highest standard deviation. Discussion of the results in an ink dating perspective suggests that other proposed parameters, such as RPA values, may be more adequate to follow ink ageing over time.