Corticosterone inhibits the lipid-mobilizing effects of oleoyl-estrone in adrenalectomized rats

Oleoyl-estrone (OE) is an adipose-derived signal that decreases energy intake and body lipid, maintaining energy expenditure and glycemic homeostasis. Glucocorticoids protect body lipid and the metabolic status quo. We studied the combined effects of OE and corticosterone in adrenalectomized female rats: daily OE gavages (0 or 10 nmol/g) and slow-release corticosterone pellets at four doses (0, 0.5, 1.7, and 4.8 mg/d). Intact and sham-operated controls were also included. After 8 d, body composition and plasma metabolites and hormones were measured. OE induced a massive lipid mobilization (in parallel with decreased food intake and maintained energy expenditure). Corticosterone increased fat deposition and inhibited the OE-elicited mobilization of body energy, even at the lowest dose. OE enhanced the corticosterone-induced rise in plasma triacylglycerols, and corticosterone blocked the OE-induced decrease in leptin. High corticosterone and OE increased insulin resistance beyond the effects of corticosterone alone. The presence of corticosterone dramatically affected OE effects, reversing its decrease of body energy (lipid) content, with little or no change on food intake or energy expenditure. The maintenance of glycemia and increasing insulin in parallel to the dose of corticosterone indicate a decrease in insulin sensitivity, which is enhanced by OE. The reversal of OE effects on lipid handling, insulin resistance, can be the consequence of a corticosterone-induced OE resistance. Nevertheless, OE effects on cholesterol were largely unaffected. In conclusion, corticosterone administration effectively blocked OE effects on body lipid and energy balance as well as insulin sensitivity and glycemia.

Cortistain is expressed in a distinct subset of cortical interneurons

Cortistatin is a presumptive neuropeptide that shares 11 of its 14 amino acids with somatostatin. In contrast to somatostatin, administration of cortistatin into the rat brain ventricles specifically enhances slow wave sleep, apparently by antagonizing the effects of acetylcholine on cortical excitability. Here we show that preprocortistatin mRNA is expressed in a subset of GABAergic cells in the cortex and hippocampus that partially overlap with those containing somatostatin. A significant percentage of cortistatin-positive neurons is also positive for parvalbumin. In contrast, no colocalization was found between cortistatin and calretinin, cholecystokinin, or vasoactive intestinal peptide. During development there is a transient increase in cortistatin-expressing cells in the second postnatal week in all cortical areas and in the dentate gyrus. A transient expression of preprocortistatin mRNA in the hilar region at P16 is paralleled by electrophysiological changes in dentate granule cells. Together, these observations suggest mechanisms by which cortistatin may regulate cortical activity.

Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway.

We previously reported that glucose can be released from GLUT2-null hepatocytes through a membrane traffic-based pathway issued from the endoplasmic reticulum. Here, we further characterized this glucose release mechanism using biosynthetic labeling protocols. In continuous pulse-labeling experiments, we determined that glucose secretion proceeded linearly and with the same kinetics in control and GLUT2-null hepatocytes. In GLUT2-deficient hepatocytes, however, a fraction of newly synthesized glucose accumulated intracellularly. The linear accumulation of glucose in the medium was inhibited in mutant, but not in control, hepatocytes by progesterone and low temperature, as previously reported, but, importantly, also by microtubule disruption. The intracellular pool of glucose was shown to be present in the cytosol, and, in pulse-chase experiments, it was shown to be released at a relatively slow rate. Release was not inhibited by S-4048 (an inhibitor of glucose-6-phosphate translocase), cytochalasin B, or progesterone. It was inhibited by phloretin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and low temperature. We conclude that the major release pathway segregates glucose away from the cytosol by use of a membrane traffic-based, microtubule-dependent mechanism and that the release of the cytosolic pool of newly synthesized glucose, through an as yet unidentified plasma membrane transport system, cannot account for the bulk of glucose release.

Differential effects of sodium oxybate and baclofen on EEG, sleep, neurobehavioral performance, and memory.

STUDY OBJECTIVES: Sodium oxybate (SO) is a GABA(B) agonist used to treat the sleep disorder narcolepsy. SO was shown to increase slow wave sleep (SWS) and EEG delta power (0.75-4.5 Hz), both indexes of NREM sleep (NREMS) intensity and depth, suggesting that SO enhances recuperative function of NREM. We investigated whether SO induces physiological deep sleep. DESIGN: SO was administered before an afternoon nap or before the subsequent experimental night in 13 healthy volunteers. The effects of SO were compared to baclofen (BAC), another GABA(B) receptor agonist, to assess the role of GABA(B) receptors in the SO response. MEASUREMENTS AND RESULTS: As expected, a nap significantly decreased sleep need and intensity the subsequent night. Both drugs reversed this nap effect on the subsequent night by decreasing sleep latency and increasing total sleep time, SWS during the first NREMS episode, and EEG delta and theta (0.75-7.25 Hz) power during NREMS. The SO-induced increase in EEG delta and theta power was, however, not specific to NREMS and was also observed during REM sleep (REMS) and wakefulness. Moreover, the high levels of delta power during a nap following SO administration did not affect delta power the following night. SO and BAC taken before the nap did not improve subsequent psychomotor performance and subjective alertness, or memory consolidation. Finally, SO and BAC strongly promoted the appearance of sleep onset REM periods. CONCLUSIONS: The SO-induced EEG slow waves seem not to be functionally similar to physiological slow waves. Our findings also suggest a role for GABA(B) receptors in REMS generation. CITATION: Vienne J; Lecciso G; Constantinescu I; Schwartz S; Franken P; Heinzer R; Tafti M. Differential effects of sodium oxybate and baclofen on EEG, sleep, neurobehavioral performance, and memory. SLEEP 2012;35(8):1071-1084.

Dynamics of sweeping through an instability: Passage-time statistics for colored noise

A calculation of passage-time statistics is reported for the laser switch-on problem, under the influence of colored noise, when the net gain is continuously swept from below to above threshold. Cases of fast and slow sweeping are considered. In the weak-noise limit, asymptotic results are given for small and large correlation times of the noise. The mean first passage time increases with the correlation time of the noise. This effect is more important for fast sweeping than for slow sweeping.

Enhanced pulse propagation in non-linear arrays of oscillators

The propagation of a pulse in a nonlinear array of oscillators is influenced by the nature of the array and by its coupling to a thermal environment. For example, in some arrays a pulse can be speeded up while in others a pulse can be slowed down by raising the temperature. We begin by showing that an energy pulse (one dimension) or energy front (two dimensions) travels more rapidly and remains more localized over greater distances in an isolated array (microcanonical) of hard springs than in a harmonic array or in a soft-springed array. Increasing the pulse amplitude causes it to speed up in a hard chain, leaves the pulse speed unchanged in a harmonic system, and slows down the pulse in a soft chain. Connection of each site to a thermal environment (canonical) affects these results very differently in each type of array. In a hard chain the dissipative forces slow down the pulse while raising the temperature speeds it up. In a soft chain the opposite occurs: the dissipative forces actually speed up the pulse, while raising the temperature slows it down. In a harmonic chain neither dissipation nor temperature changes affect the pulse speed. These and other results are explained on the basis of the frequency vs energy relations in the various arrays

Periodic forcing of scroll rings and control of Winfree turbulence in excitable media

By simulations of the Barkley model, action of uniform periodic nonresonant forcing on scroll rings and wave turbulence in three-dimensional excitable media is investigated. Sufficiently strong rapid forcing converts expanding scroll rings into the collapsing ones and suppresses the Winfree turbulence caused by the negative tension of wave filaments. Slow strong forcing has an opposite effect, leading to expansion of scroll rings and induction of the turbulence. These effects are explained in the framework of the phenomenological kinematic theory of scroll waves.

Tous égaux devant Dieu ? Réflexions sur les logiques d'éligibilité des miraculés

Le pentecôtisme a fait du miracle le coeur de sa théologie et l'élément central de ses activités d'évangélisation. Le catholicisme, par contre, a toujours voulu contrôler l'ensemble des déclarations de manifestations divines. Apparitions et guérisons miraculeuses ont donc systématiquement, et de plus en plus, été soumises à de lentes et rigoureuses procédures d'authentification. Les pentecôtistes voient Dieu comme un être extérieur qui surgit sur la terre pour chasser le mal qui l'envahit. Tous les convertis ont donc droit à la libération et personne ne doit accepter sagement la souffrance. Or, les pèlerins catholiques que nous avons étudiés ne partagent pas ces convictions pentecôtistes. Dieu agit de l'intérieur, non pas en les délivrant, mais en les soutenant dans leurs épreuves quotidiennes. Rare et peu recherchée, la guérison physique cède la place à la guérison spirituelle, accessible à tous. Il nous semble que ces deux types de représentations placent les fidèles dans des dispositions d'esprit très divergentes suscitant, dans un cas ou dans l'autre, des espoirs adaptés à la capacité du groupe à produire des miracles. Pentecostalism placed miracles at the centre of its theology as a key element of its evangelization activities. Catholicism, on the other hand, has always tried to control all declarations of divine demonstrations. Miraculous appearances and recoveries have been more and more systematically subjected to slow and rigorous procedures of verification. The Pentecostals see God as an external force which manifests itself on earth to drive out the evil which invades it. All believers have the right to be free from evil, and nobody should have to accept pain meekly. But the Catholic pilgrims we studied do not share these Pentecostal convictions. God acts from inside, not by delivering them but by supporting them in their daily tests. Physical recovery is rare and not very sought after so it takes second place to spiritual recovery which is accessible to everyone. It seems to us that these two types of representation place believers in very divergent frames of mind giving rise, in one group or the other, to hopes that correspond to the group's capacity to produce miracles.

Eye movements in the blind

Objectives : Eye movements are necessary to stabilize the retinal picture and to find a new object. This seems useless for blind people, so why do they nevertheless have them. We report on an EOG study on 29 blind volunteers and 5 volunteers with closed eyes. Material and methods: We recorded eye movements by EOG and let the volunteers fulfill different exercises by following an acoustic running point by gaze, pointing, imagining in the room, listing words that begin with the vocal U and a finger labyrinth. Results: We found slow eye movements as well as pathological eye movements in the blind subjects. We found that blind subjects have a horizontal preferency.The duration of fixation of pictures is shorter in the blind subjects. The blind could even modulate saccade amplitudes . Discussion: Eye movements seem to be structural properties of the brain which prepare the organism for certain situations-even if they do not take place. We think that eye movements are partially independent of the experience of view. We did not expect that the blind subjects could modify gaze according to the subject. This leads to the hypothesis of a preformed dimensional system.

Sleep and vigilance linked to melanism in wild barn owls.

Understanding the function of variation in sleep requires studies in the natural ecological conditions in which sleep evolved. Sleep has an impact on individual performance and hence may integrate the costs and benefits of investing in processes that are sensitive to sleep, such as immunity or coping with stress. Because dark and pale melanic animals differentially regulate energy homeostasis, immunity and stress hormone levels, the amount and/or organization of sleep may covary with melanin-based colour. We show here that wild, cross-fostered nestling barn owls (Tyto alba) born from mothers displaying more black spots had shorter non-REM (rapid eye movement) sleep bouts, a shorter latency until the occurrence of REM sleep after a bout of wakefulness and more wakefulness bouts. In male nestlings, the same sleep traits also correlated with their own level of spotting. Because heavily spotted male nestlings and the offspring of heavily spotted biological mothers switched sleep-wakefulness states more frequently, we propose the hypothesis that they could be also behaviourally more vigilant. Accordingly, nestlings from mothers displaying many black spots looked more often towards the nest entrance where their parents bring food and towards their sibling against whom they compete. Owlets from heavily spotted mothers might invest more in vigilance, thereby possibly increasing associated costs due to sleep fragmentation. We conclude that different strategies of the regulation of brain activity have evolved and are correlated with melanin-based coloration.

Characterization of surface functional groups present on laboratory-generated and ambient aerosol particles by means of heterogeneous titration reactions

A Knudsen flow reactor has been used to quantify surface functional groups on aerosols collected in the field. This technique is based on a heterogeneous titration reaction between a probe gas and a specific functional group on the particle surface. In the first part of this work, the reactivity of different probe gases on laboratory-generated aerosols (limonene SOA, Pb(NO3)2, Cd(NO3)2) and diesel reference soot (SRM 2975) has been studied. Five probe gases have been selected for the quantitative determination of important functional groups: N(CH3)3 (for the titration of acidic sites), NH2OH (for carbonyl functions), CF3COOH and HCl (for basic sites of different strength), and O3 (for oxidizable groups). The second part describes a field campaign that has been undertaken in several bus depots in Switzerland, where ambient fine and ultrafine particles were collected on suitable filters and quantitatively investigated using the Knudsen flow reactor. Results point to important differences in the surface reactivity of ambient particles, depending on the sampling site and season. The particle surface appears to be multi-functional, with the simultaneous presence of antagonistic functional groups which do not undergo internal chemical reactions, such as acid-base neutralization. Results also indicate that the surface of ambient particles was characterized by a high density of carbonyl functions (reactivity towards NH2OH probe in the range 0.26-6 formal molecular monolayers) and a low density of acidic sites (reactivity towards N(CH3)3 probe in the range 0.01-0.20 formal molecular monolayer). Kinetic parameters point to fast redox reactions (uptake coefficient ?0>10-3 for O3 probe) and slow acid-base reactions (?0<10-4 for N(CH3)3 probe) on the particle surface. [Authors]

Régulation du canal à sodium épithélial par le sodim extracellulaire et développement d'un microsystème intégré pour l'étude électrophysiologique sur ovocytes de "Xenopus laevis"

Résumé : La première partie de ce travail de thèse est consacrée au canal à sodium épithélial (ENaC), l'élément clé du transport transépithélial de Na+ dans le néphron distal, le colon et les voies aériennes. Ce canal est impliqué dans certaines formes génétiques d'hypo- et d'hypertension (PHA I, syndrome de Liddle), mais aussi, indirectement, dans la mucoviscidose. La réabsorption transépithéliale de Na+ est principalement régulée par des hormones (aldostérone, vasopressine), mais aussi directement par le Na+, via deux phénomènes distincts, la « feedback inhibition » et la « self-inhibition » (SI). Ce second phénomène est dépendant de la concentration de Na+ extracellulaire, et montre une cinétique rapide (constante de temps d'environ 3 s). Son rôle physiologique serait d'assurer l'homogénéité de la réabsorption de Na+ et d'empêcher que celle-ci soit excessive lorsque les concentrations de Na+ sont élevées. Différents éléments appuient l'hypothèse de la présence d'un site de détection de la concentration du Na+ extracellulaire sur ENaC, gouvernant la SI. L'objectif de ce premier projet est de démontrer l'existence du site de détection impliqué dans la SI et de déterminer ses propriétés physiologiques et sa localisation. Nous avons montré que les caractéristiques de la SI (en termes de sélectivité et affinité ionique) sont différentes des propriétés de conduction du canal. Ainsi, nos résultats confirment l'hypothèse de l'existence d'un site de détection du Na+ (responsable de la transmission de l'information au mécanisme de contrôle de l'ouverture du canal), différent du site de conduction. Par ailleurs, ce site présente une affinité basse et indépendante du voltage pour le Na+ et le Li+ extracellulaires. Le site semble donc être localisé dans le domaine extracellulaire, plutôt que transmembranaire, de la protéine. L'étape suivante consiste alors à localiser précisément le site sur le canal. Des études précédentes, ainsi que des résultats préliminaires récemment obtenus, mettent en avant le rôle dans la self-inhibition du premiers tiers des boucles extracellulaires des sous-unités α et γ du canal. Le second projet tire son origine des limitations de la méthode classique pour l'étude des canaux ioniques, après expression dans les ovocytes de Xenopus laevis, par la méthode du voltage-clamp à deux électrodes, en particulier les limitations dues à la lenteur des échanges de solutions. En outre, cette méthode souffre de nombreux désavantages (manipulations délicates et peu rapides, grands volumes de solution requis). Plusieurs systèmes améliorés ont été élaborés, mais aucun ne corrige tous les désavantages de la méthode classique Ainsi, l'objectif ici est le développement d'un système, pour l'étude électrophysiologique sur ovocytes, présentant les caractéristiques suivantes : manipulation des cellules facilitée et réduite, volumes de solution de perfusion faibles et vitesse rapide d'échange de la perfusion. Un microsystème intégré sur une puce a été élaboré. Ces capacités de mesure ont été testées en utilisant des ovocytes exprimant ENaC. Des résultats similaires (courbes IV, courbes dose-réponse au benzamil) à ceux obtenus avec le système traditionnel ont été enregistrés avec le microsystème. Le temps d'échange de solution a été estimé à ~20 ms et des temps effectifs de changement ont été déterminés comme étant 8 fois plus court avec le nouveau système comparé au classique. Finalement, la SI a été étudiée et il apparaît que sa cinétique est 3 fois plus rapide que ce qui a été estimé précédemment avec le système traditionnel et son amplitude de 10 à 20 % plus importante. Le nouveau microsystème intégré apparaît donc comme adapté à la mesure électrophysiologique sur ovocytes de Xenopus, et possèdent des caractéristiques appropriées à l'étude de phénomènes à cinétique rapide, mais aussi à des applications de type « high throughput screening ». Summary : The first part of the thesis is related to the Epithelial Sodium Channel (ENaC), which is a key component of the transepithelial Na+ transport in the distal nephron, colon and airways. This channel is involved in hypo- and hypertensive syndrome (PHA I, Liddle syndrome), but also indirectly in cystic fibrosis. The transepithelial reabsorption of Na+ is mainly regulated by hormones (aldosterone, vasopressin), but also directly by Na+ itself, via two distinct phenomena, feedback inhibition and self-inhibition. This latter phenomenon is dependant on the extracellular Na+ concentration and has rapid kinetics (time constant of about 3 s). Its physiological role would be to prevent excessive Na+ reabsorption and ensure this reabsorption is homogenous. Several pieces of evidence enable to propose the hypothesis of an extracellular Na+ sensing site on ENaC, governing self-inhibition. The aim of this first project is to demonstrate the existence of the sensing site involved in self-inhibition and to determine its physiological properties and localization. We show self-inhibition characteristics (ionic selectivity and affinity) are different from the conducting properties of the channel. Our results support thus the hypothesis that the Na+ sensing site (responsible of the transmission of the information about the extracellular Na+ concentration to the channel gating mechanism), is different from the channel conduction site. Furthermore, the site has a low and voltage-insensitive affinity for extracellular Na+ or Li+. This site appears to be located in the extracellular domain rather than in the transmembrane part of the channel protein. The next step is then to precisely localize the site on the channel. Some previous studies and preliminary results we recently obtained highlight the role of the first third of the extracellular loop of the α and γ subunits of the channel in self-inhibition. The second project originates in the limitation of the classical two-electrode voltageclamp system classically used to study ion channels expressed in Xenopus /aevis oocytes, in particular limitations related to the slow solution exchange time. In addition, this technique undergoes several drawbacks (delicate manipulations, time consumption volumes). Several improved systems have been built up, but none corrected all these detriments. The aim of this second study is thus to develop a system for electrophysiological study on oocytes featuring an easy and reduced cell handling, small necessary perfusion volumes and fast fluidic exchange. This last feature establishes the link with the first project, as it should enable to improve the kinetics analysis of self-inhibition. A PDMS chip-based microsystem has been elaborated. Its electrophysiological measurement abilities have been tested using oocytes expressing ENaC. Similar measurements (IV curves of benzamil-sensitive currents, benzamil dose-response curves) have been obtained with this system, compared to the traditional one. The solution exchange time has been estimated at N20 ms and effective exchange times (on inward currents) have been determined as 8 times faster with the novel system compared to the classical one. Finally, self-inhibition has been studied and it appears its kinetics is 3 times faster and its amplitude 10 to 20 % higher than what has been previously estimated with the traditional system. The novel integrated microsystem appears therefore to be convenient for electrophysiological measurement on Xenopus oocytes, and displays features suitable for the study of fast kinetics phenomenon, but also high throughput screening applications. Résumé destiné large public : Le corps humain est composé d'organes, eux-mêmes constitués d'un très grand nombre de cellules. Chaque cellule possède une paroi appelée membrane cellulaire qui sépare l'intérieur de cette cellule (milieu intracellulaire) du liquide (milieu extracellulaire) dans lequel elle baigne. Le maintien de la composition stable de ce milieu extracellulaire est essentiel pour la survie des cellules et donc de l'organisme. Le sodium est un des composants majeurs du milieu extracellulaire, sa quantité dans celui-ci doit être particulièrement contrôlée. Le sodium joue en effet un rôle important : il conditionne le volume de ce liquide extracellulaire, donc, par la même, du sang. Ainsi, une grande quantité de sodium présente dans ce milieu va de paire avec une augmentation du volume sanguin, ce qui conduit l'organisme à souffrir d'hypertension. On se rend donc compte qu'il est très important de contrôler la quantité de sodium présente dans les différents liquides de l'organisme. Les apports de sodium dans l'organisme se font par l'alimentation, mais la quantité de sodium présente dans le liquide extracellulaire est contrôlée de manière très précise par le rein. Au niveau de cet organe, on appelle urine primaire le liquide résultant de la filtration du sang. Elle contient de nombreuses substances, des petites molécules, dont l'organisme a besoin (sodium, glucose...), qui sont ensuite récupérées dans l'organe. A la sortie du rein, l'urine finale ne contient plus que l'excédent de ces substances, ainsi que des déchets à éliminer. La récupération du sodium est plus ou moins importante, en fonction des ajustements à apporter à la quantité présente dans le liquide extracellulaire. Elle a lieu grâce à la présence de protéines, dans les membranes des cellules du rein, capables de le transporter et de le faire transiter de l'urine primaire vers le liquide extracellulaire, qui assurera ensuite sa distribution dans l'ensemble de l'organisme. Parmi ces protéines « transporteurs de sodium », nous nous intéressons à une protéine en particulier, appelée ENaC. Il a été montré qu'elle jouait un rôle important dans cette récupération de sodium, elle est en effet impliquée dans des maladies génétiques conduisant à l'hypo- ou à l'hypertension. De précédents travaux ont montré que lorsque le sodium est présent en faible quantité dans l'urine primaire, cette protéine permet d'en récupérer une très grande partie. A l'inverse, lorsque cette quantité de sodium dans l'urine primaire est importante, sa récupération par le biais d'ENaC est réduite. On parle alors d'autorégulation : la protéine elle-même est capable d'adapter son activité de transport en fonction des conditions. Ce phénomène d'autorégulation constitue a priori un mécanisme préventif visant à éviter une trop grande récupération de sodium, limitant ainsi les risques d'hypertension. La première partie de ce travail de thèse a ainsi consisté à clarifier le mécanisme d'autorégulation de la protéine ENaC. Ce phénomène se caractérise en particulier par sa grande vitesse, ce qui le rend difficile à étudier par les méthodes traditionnelles. Nous avons donc, dans une deuxième partie, développé un nouveau système permettant de mieux décrire et analyser cette « autorégulation » d'ENaC. Ce second projet a été mené en collaboration avec l'équipe de Martin Gijs de l'EPFL.

Light-induced racemization: artifacts in the analysis of the diastereoisomeric pairs of thioridazine 5-sulfoxide in the plasma and urine of patients treated with thioridazine.

The ring sulfoxidation of thioridazine (THD), a widely used neuroleptic agent, yields two diastereoisomeric pairs, fast- and slow-eluting (FE and SE) thioridazine 5-sulfoxide (THD 5-SO). Until now, studies in which concentrations of these metabolites were measured in THD-treated patients have revealed no significant differences in their concentrations. Preliminary experiments in our laboratory had shown that sunlight and, to a lesser extent, dim daylight led to racemization and probably also to photolysis of the diastereoisomeric pairs as measured by high-performance liquid chromatography. Similar results were also obtained with direct UV light (UV lamp). In appropriate light-protected conditions, THD, northioridazine, mesoridazine, sulforidazine, and FE and SE THD 5-SO were measured in 11 patients treated with various doses of THD for at least 1 week. Significantly higher concentrations of the FE stereoisomeric pair were found. The concentration ratios THD 5-SO (FE)/THD 5-SO (SE) ranged from 0.89 to 1.75 in plasma and from 1.15 to 2.05 in urine. Because it is known that the ring sulfoxide contributes to the cardiotoxicity of the drug even more potently than the parent compound does, these results justify further studies to determine whether there is stereoselectivity in the cardiotoxicity of THD 5-SO.

Determination of four sequential stages during microautophagy in vitro.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

Evaluation of Variable Advisory Speed Limits in Work Zones, TPF-5(081), 2013

Variable advisory speed limit (VASL) systems could be effective at both urban and rural work zones, at both uncongested and congested sites. At uncongested urban work zones, the average speeds with VASL were lower than without VASL. But the standard deviation of speeds with VASL was higher. The increase in standard deviation may be due to the advisory nature of VASL. The speed limit compliance with VASL was about eight times greater than without VASL. At the congested sites, the VASL were effective in making drivers slow down gradually as they approached the work zone, reducing any sudden changes in speeds. Mobility-wise the use of VASL resulted in a decrease in average queue length, throughput, number of stops, and an increase in travel time. Several surrogate safety measures also demonstrated the benefits of VASL in congested work zones. VASL deployments in rural work zones resulted in reductions in mean speed, speed variance, and 85th percentile speeds downstream of the VASL sign. The study makes the following recommendations based on the case studies investigated: 1. The use of VASL is recommended for uncongested work zones to achieve better speed compliance and lower speeds. Greater enforcement of regulatory speed limits could help to decrease the standard deviation in speeds; 2. The use of VASL to complement the static speed limits in rural work zones is beneficial even if the VASL is only used to display the static speed limits. It leads to safer traffic conditions by encouraging traffic to slow down gradually and by reminding traffic of the reduced speed limit. A well-designed VASL algorithm, like the P5 algorithm developed in this study, can significantly improve the mobility and safety conditions in congested work zones. The use of simulation is recommended for optimizing the VASL algorithms before field deployment.