Greenstick fractures of the middle third of the forearm. A prospective multi-centre study.

BACKGROUND: Greenstick fractures suffered during growth have a high risk for refracture and posttraumatic deformity, particularly at the forearm diaphysis. The use of a preemptive completion of the fracture by manipulation of the concave cortex is controversial and data supporting this approach are few. AIM: Aim of this study was to determine the factors which predispose to refracture and deformities, and to define therapeutic strategies. METHODS: We prospectively gathered clinical and radiographic data over a period of one year on greenstick fractures of the middle third of the forearm in children as part of a multi-centre study. Endpoint was a follow-up visit at one year. Radiographic deformity, state of consolidation at resumption of physical activities and refracture rate were analysed statistically (ANOVA, Student's t-test and Pearson's chi-square test) with regard to patient age, gender, fracture type, therapy and time in plaster. RESULTS: We collected the data of 103 patients (63 boys, 40 girls), average age 6.6 years (1.3-14.5 years), the vast majority of whom had a combined greenstick fracture of the radius and ulna. 6.7% of the patients sustained a refracture within 49 days (29-76) after plaster removal. They were significantly older (p=0.017) with a significantly higher incidence of manual completion of the fracture with radiographic signs of partial consolidation (p=0.025). Residual deformities were significantly smaller after completion of the fracture compared to reduction without completion (p=0.019) or plaster fixation alone (p<0.005). CONCLUSIONS: Completion of a greenstick fracture does not prevent refracture. Nevertheless, it diminishes the extent of secondary deformities in cases where the primary angulation exceeds the remodelling capacity. Prevention of refracture should include a routine radiographic follow-up 4-6 weeks after injury with continuation of plaster fixation in cases of partial consolidation.

On metapopulation resistance to drift and extinction.

The spatial configuration of metapopulations (numbers, sizes, and localization of patches) affects their ability to resist demographic extinction and genetic drift, but sometimes with opposite effects. Small and isolated patches, for instance, contribute marginally to demography but may play a large role in genetics by maintaining a sizeable amount of genetic variance among demes. In source-sink systems, similarly, connectivity may be beneficial in terms of effective size, but detrimental in terms of survival, by lowering the reproductive value of source populations. How to reconcile these opposite effects? Here we propose an analytical framework that integrates fixation time (ability to resist genetic drift) and extinction time (ability to resist demographic extinction) into a single index of resistance, measuring the ability of a metapopulation to maintain its demo-genetic integrity. We then illustrate with numerical examples how conflicting demands may be resolved.

Sex differences: From mental rotation to object location memory, an ERP study

Sex differences in cognition have been largely investigated. The most consistent sex differences favoring females are observed in object location memory involving the left hemisphere whereas the most consistent sex differences favoring males are observed in tasks that require mental rotation involving the right hemisphere. Here we used a task involving these two abilities to see the impact of mental rotation on object location memory. To that end we used a combination of behavioral and event-related potential (ERP) electroencephalography (EEG) measures.A computer screen displayed a square frame of 4 pairs of images (a "teddy" bear, a shoe, an umbrella and a lamp) randomly arranged around a central fixation cross. After a 10-second interval for memorization, images disappeared and were replaced by a test frame with no image but a random pair of two locations marked in black. In addition, this test frame was randomly displayed either in the original orientation (0° rotation) or in the rotated one (90° clockwise - CW - or 90° counterclockwise - CCW). Preceding the test frame, an arrow indicating the presence or the absence of rotation of the frame was displayed on the screen. The task of the participants (15 females and 15 males) was to determine if two marked locations corresponded or not to a pair of identical images. Each response was followed by feedback.Findings showed no significant sex differences in the performance of the original orientation. In comparison with this position, the rotation of the frame produced an equal decrease of male and female performance. In addition, this decrease was significantly higher when the rotation of the frame was in a CCW direction. We further assessed the ERP when the arrow indicated the direction of rotation as stimulus-onset, during four time windows representing major components C1, P1, N1 and N2. Although no sex differences were observed in performance, brain activities differed according to sex. Enhanced amplitudes were found for the CCW compared to CW rotation over the right posterior areas for the P1, N1 and N2 components for men as well as for women. Major topographical differences related to sex were measured for the CW rotation condition as marked lateralized amplitude: left-hemisphere amplitude larger than right one was measured during P1 time range for men. These similar patterns prolonged from P1 to N1 for women. Early distinctions were found in interaction with sex between CCW and CW waveform amplitudes, expressing over anterior electrode sites during C1 time range (0-50 ms post-stimulus).In conclusion (i) women do not outperform men in object location memory in this study (absence of rotation condition); (ii) mental rotation, in particular the direction of rotation, influences performance on object location memory; (iii) CCW rotation is associated with activity in the right parietal hemisphere whereas the CW rotation involves the left parietal hemisphere; (iv) this last effect is less pronounced in males, which could explain why greater involvement of right parietal areas in men and of bilateral posterior areas in women is generally reported in mental rotation tasks; and (v) the early distinctions between both directions of rotation located over anterior sites could be related to sex differences in their respective involvement of control mechanisms.

Transcription factor PROX1 modifies Wnt signaling in colon cancer

PR0X1 est un facteur de transcription très conservé au cours de l'évolution. PROX1 joue un rôle essentiel dans de nombreuses étapes de l'embryogenèse, telles que le développement du système lymphatique ou la migration des hépatocytes. Récemment, il a été démontré que PROX1 contribue à la progression des tumeurs colorectales, en tant que gène cible de la voie de signalisation Wnt. En utilisant des approches de co- immunoprécipitation et de ligature de proximité, nous avons trouvé que PROX1 fait également partie du complexe transcriptionnel TCF/ß-catenin, à la fois dans les cellules humaines de cancer du colon et dans les cellules murines de l'épithélium de l'intestin, dans lesquelles la voie de signalisation Wnt est activée. Dans le but de comprendre le mécanisme d'action de PROX1, nous avons analysé le génome des cellules cancéreuses de colon à la recherche des sites de fixation de PROX1, TCF4 et ß-catenin. Nous avons ainsi pu montrer que TCF4, ß-catenin et PROX1 se fixent simultanément sur une sous- population d'amplificateurs génomiques, sur lesquels PROX1 agit comme répresseur. Ces résultats suggèrent que, spécifiquement dans le cadre du cancer du colon, PROX1 agit en tant que modificateur de la voie de transduction du signal Wnt/ß-catenin. De plus, nous proposons que ceci constitue un des mécanismes par lesquels la signalisation durable de Wnt, qui est observée dans la majorité des cancers du colon, transforme le programme génétique des progéniteurs intestinaux, initialement normal, en output spécifique de ce type de cancers, ce qui contribue plus tard à la croissance infinie de la tumeur, à son caractère invasif et à sa dissémination.

Traitement des fractures de l'enfant par enclouage centro-médullaire élastique stable (ECMES) [Treatment of pediatric fractures by intramedullary stable elastic pinning]

During the past decade several new techniques for the treatment of children's fractures respecting the specificity of the growing bone have been described. The goal of all these techniques was to mechanically stabilise the fracture however to preserve a certain instability of the fracture gap itself inducing early callus formation and subsequent consolidation. The dynamic external fixation as well as the elastic stable intramedullary pinning have become accepted means in the treatment of long bone fractures in the paediatric age group. We report our experience of the last seven years with the intramedullary pinning of 105 fractures. Eighty-four were fractures of the femur, 9 of the humerus, 8 of the forearm, and a further 4 of the tibial shaft. The intramedullary elastic pinning represents a simple technique which supports or even enhances the natural process of fracture healing of the growing bone. The method is not very invasive, is cost effective, and allows short hospitalisation. Early physical activity is guaranteed due to early consolidation of the fracture. Complications are rare and the final orthopedic and cosmetic outcome is excellent.

Revue du traitement chirurgical de la pseudarthrose de la diaphyse fémorale

Ce travail consiste en l'analyse et la revue des différentes prises en charge de la pseudarthrose de la diaphyse fémorale dans un groupe nommé « Echantillon CHUV » formé par 16 patients. Les patients ont tous été opérés au Centre Hospitalier Universitaire Vaudois, CHUV, entre 2008 et 2011 pour cure de pseudarthrose. Une présentation succincte des fractures de la diaphyse fémorale ainsi que de la pseudarthrose de la diaphyse fémorale consiste en la première partie du rapport. La deuxième partie est une revue des cas des patients de l'échantillon CHUV. Des paramètres tels que le mode initial de fracture, la durée effective de la pseudarthrose, le nombre de révisions avant la consolidation ou la répartition des pseudarthroses selon leur type font partie des différents éléments caractéristiques répertoriés puis analysés dans cette revue. 25 cures de pseudarthroses sont effectuées sur les 16 patients. Les techniques de fixations les plus utilisées sont la fixation par plaque après réduction ouverte (ORIF, 64%, 16 cures sur 25) et l'enclouage centromédullaire (24%, 6 cures sur 25). L'utilisation de substituts osseux est très souvent complémentaire à la refixation mécanique lors des cures de pseudarthrose (recours à la greffe osseuse dans 72% des cures). Le taux d'union après la première cure de pseudarthrose s'établit à 63% et il monte à 100% après l'ensemble des cures. Le retour à une fonction adéquate du membre inférieur est obtenu chez 14 des 16 patients (1 patient est traité par arthrodèse du genou sur pseudarthrose du tiers distal de la diaphyse fémorale et 1 patient tétraplégique est traité par résection de la tête et du col fémoral sur pseudarthrose du tiers proximal de la diaphyse fémorale). La revue quantifie la durée de l'invalidité causée par la pseudarthrose de la diaphyse fémorale : la longueur totale du traitement avant union est d'au minimum 12 mois pour 75% des patients. 8 patients sur 16 ont un temps avant union supérieur à 20 mois. La pseudarthrose de la diaphyse fémorale doit être considérée comme une complication grave. La localisation au niveau du fémur réduit voire abolit l'autonomie de marche du patient et limite ses activités de la vie quotidienne. Elle entrave le retour au travail et réduit terriblement la qualité de vie pendant souvent plus d'une année. L'ensemble des symptômes ont un effet dévastateur sur la rééducation et peuvent parfois laisser d'importantes séquelles physiques ou psychologiques sur le long terme. Pour ces raisons, le traitement doit être adapté du mieux possible à la pseudarthrose du patient afin d'augmenter les chances de réussite de la cure.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Phylogenetic analyses of C4 photosynthesis evolution in grasses: genetics of convergence and convergence of genes

Summary : During the evolutionary diversification of organisms, similar ecological constraints led to the recurrent appearances of the same traits (phenotypes) in distant lineages, a phenomenon called convergence. In most cases, the genetic origins of the convergent traits remain unknown, but recent studies traced the convergent phenotypes to recurrent alterations of the same gene or, in a few cases, to identical genetic changes. However, these cases remain anecdotal and there is a need for a study system that evolved several times independently and whose genetic determinism is well resolved and straightforward, such as C4 photosynthesis. This adaptation to warm environments, possibly driven by past atmospheric CO2 decreases, consists in a CO2-concentrating pump, created by numerous morphological and biochemical novelties. All genes encoding C4 enzymes already existed in C3 ancestors, and are supposed to have been recruited through gene duplication followed by neo-functionalization, to acquire the cell specific expression pattern and altered kinetic properties that characterize Ca-specific enzymes. These predictions have so far been tested only in species-poor and ecologically marginal C4 dicots. The monocots, and especially the grass family (Poaceae), the most important C4 family in terms of species number, ecological dominance and economical importance, have been largely under-considered as suitable study systems. This thesis aimed at understanding the evolution of the C4 trait in grasses at a molecular level and to use the genetics of C4 photosynthesis to infer the evolutionary history of the C4 phenotype and its driving selective pressures. A molecular phylogeny of grasses and affiliated monocots identified 17 to 18 independent acquisitions of the C4 pathway in the grass family. A relaxed molecular clock was used to date these events and the first C4 evolution was estimated in the Chloridoideae subfamily, between 32-25 million years ago, at a period when atmospheric CO2 abruptly declined. Likelihood models showed that after the COZ decline the probability of evolving the C4 pathway strongly increased, confirming low CO2 as a likely driver of C4 photosynthesis evolution. In order to depict the genetic changes linked to the numerous C4 origins, genes encoding phopshoenolpyruvate carboxylase (PEPC), the key-enzyme responsible for the initial fixation of atmospheric CO2 in the C4 pathway, were isolated from a large sample of C3 and C4 grasses. Phylogenetic analyses were used to reconstruct the evolutionary history of the PEPC multigene family and showed that the evolution of C4-specific PEPC had been driven by positive selection on 21 codons simultaneously in up to eight C4 lineages. These selective pressures led to numerous convergent genetic changes in many different C4 clades, highlighting the repeatability of some evolutionary processes, even at the molecular level. PEPC C4-adaptive changes were traced and used to show multiple appearances of the C, pathway in clades where species tree inferences were unable to differentiate multiple C4 appearances and a single appearance followed by C4 to C3 reversion. Further investigations of genes involved in some of the C4 subtypes only (genes encoding decarboxylating enzymes NADP-malic enzyme and phosphoenolpyruvate carboxykinase) showed that these C4-enzymes also evolved through strong positive selection and underwent parallel genetic changes during the different Ca origins. The adaptive changes on these subtype-specific C4 genes were used to retrace the history of the C4-subtypes phenotypes, which revealed that the evolution of C4-PEPC and C4-decarboxylating enzymes was in several cases disconnected, emphasizing the multiplicity of the C4 trait and the gradual acquisition of the features that create the CO2-pump. Finally, phylogenetic analyses of a gene encoding the Rubisco (the enzyme responsible for the fixation of CO2 into organic compounds in all photosynthetic organisms) showed that C4 evolution switched the selective pressures on this gene. Five codons were recurrently mutated to adapt the enzyme kinetics to the high CO2 concentrations of C4 photosynthetic cells. This knowledge could be used to introgress C4-like Rubisco in C3 crops, which could lead to an increased yield under predicted future high CO2 atmosphere. Globally, the phylogenetic framework adopted during this thesis demonstrated the widespread occurrence of genetic convergence on C4-related enzymes. The genetic traces of C4 photosynthesis evolution allowed reconstructing events that happened during the last 30 million years and proved the usefulness of studying genes directly responsible for phenotype variations when inferring evolutionary history of a given trait. Résumé Durant la diversification évolutive des organismes, des pressions écologiques similaires ont amené à l'apparition récurrente de certains traits (phénotypes) dans des lignées distantes, un phénomène appelé évolution convergente. Dans la plupart des cas, l'origine génétique des traits convergents reste inconnue mais des études récentes ont montré qu'ils étaient dus dans certains cas à des changements répétés du même gène ou, dans de rares cas, à des changements génétiques identiques. Malgré tout, ces cas restent anecdotiques et il y a un réel besoin d'un système d'étude qui ait évolué indépendamment de nombreuses fois et dont le déterminisme génétique soit clairement identifié. La photosynthèse dite en Ça répond à ces critères. Cette adaptation aux environnements chauds, dont l'évolution a pu être encouragé par des baisses passées de la concentration atmosphérique en CO2, est constituée de nombreuses nouveautés morphologiques et biochimiques qui créent une pompe à CO2. La totalité des gènes codant les enzymes Ç4 étaient déjà présents dans les ancêtres C3. Leur recrutement pour la photosynthèse Ç4 est supposé s'être fait par le biais de duplications géniques suivies par une néo-fonctionnalisation pour leur conférer l'expression cellule-spécifique et les propriétés cinétiques qui caractérisent les enzymes C4. Ces prédictions n'ont jusqu'à présent été testées que dans des familles C4 contenant peu d'espèces et ayant un rôle écologique marginal. Les graminées (Poaceae), qui sont la famille C4 la plus importante, tant en termes de nombre d'espèces que de dominance écologique et d'importance économique, ont toujours été considérés comme un système d'étude peu adapté et ont fait le sujet de peu d'investigations évolutives. Le but de cette thèse était de comprendre l'évolution de la photosynthèse en C4 chez les graminées au niveau génétique et d'utiliser les gènes pour inférer l'évolution du phénotype C4 ainsi que les pressions de sélection responsables de son évolution. Une phylogénie moléculaire de la famille des graminées et des monocotylédones apparentés a identifié 17 à 18 acquisitions indépendantes de la photosynthèse chez les graminées. Grâce à une méthode d'horloge moléculaire relâchée, ces évènements ont été datés et la première apparition C4 a été estimée dans la sous-famille des Chloridoideae, il y a 32 à 25 millions d'années, à une période où les concentrations atmosphériques de CO2 ont décliné abruptement. Des modèles de maximum de vraisemblance ont montré qu'à la suite du déclin de CO2, la probabilité d'évoluer la photosynthèse C4 a fortement augmenté, confirmant ainsi qu'une faible concentration de CO2 est une cause potentielle de l'évolution de la photosynthèse C4. Afin d'identifier les mécanismes génétiques responsables des évolutions répétées de la photosynthèse C4, un segment des gènes codant pour la phosphoénolpyruvate carboxylase (PEPC), l'enzyme responsable de la fixation initiale du CO2 atmosphérique chez les plantes C4, ont été séquencés dans une centaine de graminées C3 et C4. Des analyses phylogénétiques ont permis de reconstituer l'histoire évolutive de la famille multigénique des PEPC et ont montré que l'évolution de PEPC spécifiques à la photosynthèse Ça a été causée par de la sélection positive agissant sur 21 codons, et ce simultanément dans huit lignées C4 différentes. Cette sélection positive a conduit à un grand nombre de changements génétiques convergents dans de nombreux clades différents, ce qui illustre la répétabilité de certains phénomènes évolutifs, et ce même au niveau génétique. Les changements sur la PEPC liés au C4 ont été utilisés pour confirmer des évolutions indépendantes du phénotype C4 dans des clades où l'arbre des espèces était incapable de différencier des apparitions indépendantes d'une seule apparition suivie par une réversion de C4 en C3. En considérant des gènes codant des protéines impliquées uniquement dans certains sous-types C4 (deux décarboxylases, l'enzyme malique à NADP et la phosphoénolpyruvate carboxykinase), des études ultérieures ont montré que ces enzymes C4 avaient elles-aussi évolué sous forte sélection positive et subi des changements génétiques parallèles lors des différentes origines de la photosynthèse C4. Les changements adaptatifs sur ces gènes liés seulement à certains sous-types C4 ont été utilisés pour retracer l'histoire des phénotypes de sous-types C4, ce qui a révélé que les caractères formant le trait C4 ont, dans certains cas, évolué de manière déconnectée. Ceci souligne la multiplicité du trait C4 et l'acquisition graduelle de composants participant à la pompe à CO2 qu'est la photosynthèse C4. Finalement, des analyses phylogénétiques des gènes codant pour la Rubisco (l'enzyme responsable de la fixation du CO2 en carbones organiques dans tous les organismes photosynthétiques) ont montré que l'évolution de la photosynthèse Ça a changé les pressions de sélection sur ce gène. Cinq codons ont été mutés de façon répétée afin d'adapter les propriétés cinétiques de la Rubisco aux fortes concentrations de CO2 présentes dans les cellules photosynthétiques des plantes C4. Globalement, l'approche phylogénétique adoptée durant cette thèse de doctorat a permis de démontré des phénomène fréquents de convergence génétique sur les enzymes liées à la photosynthèse C4. Les traces génétiques de l'évolution de la photosynthèse C4 ont permis de reconstituer des évènements qui se sont produits durant les derniers 30 millions d'années et ont prouvé l'utilité d'étudier des gènes directement responsables des variations phénotypiques pour inférer l'histoire évolutive d'un trait donné.

The rate-limiting step for glucose transport into the hypothalamus is across the blood-hypothalamus interface.

Specialized glucosensing neurons are present in the hypothalamus, some of which neighbor the median eminence, where the blood-brain barrier has been reported leaky. A leaky blood-brain barrier implies high tissue glucose levels and obviates a role for endothelial glucose transporters in the control of hypothalamic glucose concentration, important in understanding the mechanisms of glucose sensing We therefore addressed the question of blood-brain barrier integrity at the hypothalamus for glucose transport by examining the brain tissue-to-plasma glucose ratio in the hypothalamus relative to other brain regions. We also examined glycogenolysis in hypothalamus because its occurrence is unlikely in the potential absence of a hypothalamus-blood interface. Across all regions the concentration of glucose was comparable at a given plasma glucose concentration and was a near linear function of plasma glucose. At steady-state, hypothalamic glucose concentration was similar to the extracellular hypothalamic glucose concentration reported by others. Hypothalamic glycogen fell at a rate of approximately 1.5 micromol/g/h and remained present in substantial amounts. We conclude for the hypothalamus, a putative primary site of brain glucose sensing that: the rate-limiting step for glucose transport into brain cells is at the blood-hypothalamus interface, and that glycogenolysis is consistent with a substantial blood -to- intracellular glucose concentration gradient.

Mise au point et validation d'un fantôme spécifique pour asssurer le suivi des mesures quantitatives en tomographie par émission de positons.

L'index de consommation du glucose, SUV pour standardized uptake value, est largement utilisé en tomographie par émission de positons (TEP) pour quantifier la concentration relative de [18F]2-fluoro-2-désoxy-D-glucose (18F-FDG) dans les tissus. Cependant, cet indice dépend de nombreux facteurs méthodologiques et biologiques et son utilisation fait débat. Il est donc primordial d'instaurer un contrôle qualité régulier permettant d'assurer la stabilité de la mesure des indices quantitatifs. Dans cette optique, un fantôme spécifique avec inserts cylindriques de différentes tailles a été développé. L'objectif de cette étude est de montrer la sensibilité et l'intérêt de ce fantôme. Méthodes. - La sensibilité du fantôme a été étudiée à travers la mesure des SUV et des coefficients de recouvrement (RC). Plusieurs méthodes de mesure ont été utilisées. Les données ont été reconstruites en utilisant les algorithmes de routine clinique. Nous avons étudié la variation des RC en fonction de la taille des cylindres et le changement relatif de fixation, en utilisant des activités différentes. Le fantôme a ensuite été testé sur l'appareil d'un autre centre. Résultats. - Pour toutes les méthodes de mesure, une forte variation des RC avec la taille des cylindres, de l'ordre de 50 %, a été obtenue. Ce fantôme a également permis de mesurer un changement relatif de fixation qui s'est révélé être indépendant de la méthode de mesure. Malgré un étalonnage des deux systèmes TEP/TDM, des différences de quantification d'environ 20 % ont subsisté. Conclusion. - Les résultats obtenus montrent l'intérêt de ce fantôme dans le cadre du suivi des mesures quantitatives.

Substance P-like-immunoreactive sensory neurons in dorsal root ganglia of the chick embryo: ontogenesis and influence of peripheral targets.

The expression of substance P (SP) was studied in sensory neurons of developing chick lumbosacral dorsal root ganglia (DRG) by using a mixture of periodic acid, lysine and paraformaldehyde as fixative and a monoclonal antibody for SP-like immunostaining. The first SP-like-immunoreactive DRG cells appeared first at E5, then rapidly increased in number to reach a peak (88% of ganglion cells) at E8, and finally declined (59% at E12, 51% after hatching). The fall of the SP-like-positive DRG cells resulted from two concomitant events affecting a subset of small B-neurons: a loss of neuronal SP-like immunoreactivity and cell death. After one hindlimb resection at an early (E6) or late (E12) stage of development (that is before or after establishment of peripheral connections), the DRG were examined 6 days later. In both cases, a drastic neuronal death occurred in the ispilateral DRG. However, the resection at E6 did not change the percentage of SP-like-positive neurons, while the resection at E12 severely reduced the proportion of SP-like-immunoreactive DRG cells (25%). In conclusion, connections established between DRG and peripheral target tissues not only promote the survival of sensory neurons, but also control the maintenance of SP-like-expression. Factors issued from innervated targets such as NGF would support the survival of SP-expressing DRG cells and enhance their SP content while other factors present in skeletal muscle or skin would hinder SP expression and therefore lower SP levels in a subset of primary sensory neurons.

Multicentre validation study of nucleic acids extraction from FFPE tissues.

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

Comparison of fundus autofluorescence between fundus camera (Topcon) and confocal scanning laser ophthalmoscope (HRA) in various pathologies

Purpose: To investigate the differences between Fundus Camera (Topcon TRC-50X) and Confocal Scanning Laser Ophthalmoscope (Heidelberg retina angiogram (HRA)) on the fundus autofluorescence (FAF) imaging (resolution and FAF characteristics). Methods: 105 eyes of 56 patients with various retinal diseases underwent FAF imaging with HRA (488nm exciter/500nm barrier filter) before fluorescein angiography (FFA) and Topcon Fundus Camera (580nm exciter/695nm barrier filter) before and after FFA. The quality of the FAF images was compared for their resolution and analysed for the influence of fixation stability and cataracts. Hypo-and hyper-FAF behaviour was analysed for the healthy disc, healthy fovea, and a variety of pathological features. Results: HRA images were found to be of superior resolution in 18, while Topcon images were estimated superior in 29 eyes. No difference was found in 58 eyes. Both poor fixation (p=0.009) and more advanced cataract (p=0.013) were found associated with better image quality by Topcon. Images acquired by Topcon before and after FFA were identical (100%). The healthy disc was usually dark on HRA (72%), but showed mild autofluorescence on Topcon (85%). The healthy fovea showed in 100% Hypo-FAF on HRA, while Topcon showed in 53% Iso-FAF, in 43% mild Hypo-FAF, and in 4% Hypo-FAF as on HRA. No difference of FAF was found for geographic atrophy, pigment changes, and drusen, although Topcon images were often more detailed. Hyper-FAF due to serous exudation showed better on HRA. Cystic edema was visible only on HRA in a petaloid hyper-FAF pattern in 83%, while only two eyes (17%) showed similar behavior in both HRA- and Topcon images. Hard exudates caused Hypo-FAF only on HRA, hardly visible on Topcon. Blockage phenomenon by blood however was identical. Conclusions: The filter set of Topcon and the single image acquisition appear to be an advantage for patients with cataract and poor fixation respectively. Preceding FFA does not alter the Topcon FAF image. Regarding the FAF behavior, there are differences between the 2 systems which need to be taken into account when interpreting the images.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.

Crescent and star shapes of members of the Chlamydiales order: impact of fixative methods.

Members of the Chlamydiales order all share a biphasic lifecycle alternating between small infectious particles, the elementary bodies (EBs) and larger intracellular forms able to replicate, the reticulate bodies. Whereas the classical Chlamydia usually harbours round-shaped EBs, some members of the Chlamydia-related families display crescent and star-shaped morphologies by electron microscopy. To determine the impact of fixative methods on the shape of the bacterial cells, different buffer and fixative combinations were tested on purified EBs of Criblamydia sequanensis, Estrella lausannensis, Parachlamydia acanthamoebae, and Waddlia chondrophila. A linear discriminant analysis was performed on particle metrics extracted from electron microscopy images to recognize crescent, round, star and intermediary forms. Depending on the buffer and fixatives used, a mixture of alternative shapes were observed in varying proportions with stars and crescents being more frequent in C. sequanensis and P. acanthamoebae, respectively. No tested buffer and chemical fixative preserved ideally the round shape of a majority of bacteria and other methods such as deep-freezing and cryofixation should be applied. Although crescent and star shapes could represent a fixation artifact, they certainly point towards a diverse composition and organization of membrane proteins or intracellular structures rather than being a distinct developmental stage.