Functional Microdomains Control Glutamate Exocytosis from Astrocytes

The discovery that astrocytes possess a non-electrical form of excitability (Ca21-excitability) that leads to the release of chemical transmitters, an activity called ''gliotransmission'', indicates that these cells may have additional important roles in brain function. Elucidating the stimulus-secretion coupling leading to the exocytic release of chemical transmitters (such as glutamate, Bezzi et al., Nature Neurosci, 2004) may therefore clarify i) whether astrocytes represent in full a new class of secretory cells in the brain and ii) whether they can participate to the fast brain signaling in the brain. We have recently discovered the existence in astrocytes of functional sub-membrane microdomains of Ca21 release from the internal stores in response to mGluR5 activation (Marchaland et al., J of Neurosci., 2008). Such Ca21 microdomains control exocytosis of astrocytic glutamate signalling to neurons. Homer proteins are scaffold proteins controlling Ca21 signalling in different cellular microdomains, including dendritic spines in neurons (Sala et al., J of Neurosci., 2005). Thus, similarly to dendritic pines, Homer1 could be implicated in the coupling between astrocytic mGluR5 and IP3Rs on the ER. Here, by using a recently developed approach for studying vesicle recycling dynamics at synapses (Voglmaier et al., Neuron, 2006; Balaji and Ryan, PNAS, 2007) combined with epifluorescence and total internal reflection fluorescence (TIRF) imaging, we have investigated the involvement of Homer1 proteins in the Ca21-dependent stimulus-secretion coupling leading glutamate exocytosis of synaptic-like microvesicles (SLMVs) in astrocytes.

Falls efficacy predicts functional status on month after discharge in elderly patients undergoing post-accute rehabilitation

Objective: To investigate the relationship between falls efficacy at admission and functional status reported one month after discharge from post-acute rehabilitation in a cohort of elderly patients. Methods: Participants were elderly patients admitted to postacute rehabilitation in an academic geriatric facility. Data on demographics and affective status were collected upon admission; functional status and gait speed were measured at admission and at discharge; self-reported functional status and history of falls since discharge were collected one month after discharge (follow-up). Falls efficacy was measured using the Fall Efficacy Scale, that assesses confidence in performing 12 activities of daily living without falling (range 0 to 100, higher score indicating higher confidence). Patients were classified using the median FES score at baseline (95) as cut-off to divide the population into "confident" and "fearful" groups. Results: Participants' (N=180, mean age 81.3±7.1 years, 75.6% women) mean FES score was 92.3±8.7 at baseline (range 60-100). Basic ADL score averaged 3.5±1.6 at baseline, 4.7±1.3 at discharge, and 5.5±0.7 at follow-up (self-reported). Baseline FES score was positively correlated with basic ADL at follow-up (rho=0.35, p<.001). At follow-up, 58.7% of the patients were fully independent in basic ADL, this proportion being significantly higher in confident than fearful patients (70.7% vs 42.4%, p<.001). Compared to confident patients, those fearful had significantly lower odds (OR 0.3, 95%CI 0.2-0.6, p<.001) to report full independence at follow-up. This relationship remained (adjOR = 0.4, 95%CI 0.2-0.8, p=.01) after controlling for demographics, baseline gait speed, depressive symptoms, functional status at discharge, and history of falls since discharge. Conclusion: In this cohort of older rehab patients, poor falls efficacy at admission was associated with lower function reported one month after discharge even after controlling for initial mobility performance and functional status at discharge. Further studies should determine whether interventions aiming at falls efficacy improvement will also result in improved function in fearful subjects undergoing rehabilitation.

Functional analysis of cis- and trans-acting elements of the Candida albicans CDR2 promoter with a novel promoter reporter system.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P((CDR2))-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

Revisiting sodium and water reabsorption with functional genomics tools.

PURPOSE OF REVIEW: The kidney plays an essential role in maintaining sodium and water balance, thereby controlling the volume and osmolarity of the extracellular body fluids, the blood volume and the blood pressure. The final adjustment of sodium and water reabsorption in the kidney takes place in cells of the distal part of the nephron in which a set of apical and basolateral transporters participate in vectorial sodium and water transport from the tubular lumen to the interstitium and, finally, to the general circulation. According to a current model, the activity and/or cell-surface expression of these transporters is/are under the control of a gene network composed of the hormonally regulated, as well as constitutively expressed, genes. It is proposed that this gene network may include new candidate genes for salt- and water-losing syndromes and for salt-sensitive hypertension. A new generation of functional genomics techniques have recently been applied to the characterization of this gene network. The purpose of this review is to summarize these studies and to discuss the potential of the different techniques for characterization of the renal transcriptome. RECENT FINDINGS: Recently, DNA microarrays and serial analysis of gene expression have been applied to characterize the kidney transcriptome in different in-vivo and in-vitro models. In these studies, a set of new interesting genes potentially involved in the regulation of sodium and water reabsorption by the kidney have been identified and are currently under detailed investigation. SUMMARY: Characterization of the kidney transcriptome is greatly expanding our knowledge of the gene networks involved in multiple kidney functions, including the maintenance of sodium and water homeostasis.

Absence of association between a functional polymorphism of ALOX15 gene and infertility in endometriosis.

The aim of the present study, involving 463 women of reproductive age, was to evaluate for the first time the relationship between endometriosis, endometriosis-related infertility, and a recently described functional polymorphism in the ALOX15 gene, reported to be essential for implantation. In our study population, ALOX15 -292 C/T was not correlated either with the risk of developing an endometriosis or with the risk of infertility.

Functional GacS in Pseudomonas DSS73 prevents digestion by Caenorhabditis elegans and protects the nematode from killer flagellates.

The success of biocontrol bacteria in soil depends in part on their ability to escape predation. We explored the interactions between Pseudomonas strain DSS73 and two predators, the nematode Caenorhabditis elegans and the flagellate Cercomonas sp. Growth of the nematode in liquid culture was arrested when it was feeding on DSS73 or a DSS73 mutant (DSS73-15C2) unable to produce the biosurfactant amphisin, whereas a regulatory gacS mutant (DSS73-12H8) that produces no exoproducts supported fast growth of the nematode. The flagellate Cercomonas sp. was able to grow on all three strains. The biosurfactant-deficient DSS73 mutant caused severe dilation of the nematode gut. In three-species systems (DSS73, Cercomonas and C. elegans), the nematodes fed on the flagellates, which in turn grazed the bacteria and the number of C. elegans increased. The flagellates Cercomonas sp. usually kill C. elegans. However, DSS73 protected the nematodes from flagellate killing. Soil microcosms inoculated with six rhizobacteria and grazed by nematodes were colonized more efficiently by DSS73 than similar systems grazed by flagellates or without grazers. In conclusion, our results suggest that C. elegans and DSS73 mutually increase the survival of one another in complex multispecies systems and that this interaction depends on the GacS regulator.

Information retention during competitive interactions : siblings need to constantly repeat vocal displays

Memory is essential to adjust behaviour according to past experience. In societies where animals interact on numerous occasions, memory of previous social interactions may help optimise investment in competition. How long information about the resource holding potential and motivation to compete of conspecifics is retained depends on how fast the value of this information fades, but also on the cost and benefit of retaining information. Information retention has never been investigated in the context of interactions prevailing within the family and more specifically sibling competition. In the absence of parents, barn owl (Tyto alba) nestlings vocally compete for priority of access to the next indivisible food item brought by a parent. The finding that owlets eavesdrop on vocal interactions between siblings to adjust investment in vocalization once competing with them suggests that they memorize siblings' vocal interactions. Playback experiments showed that owlets take into account the past siblings' vocal performance that signals hunger for at least 15 min, but only if the performance was witnessed during a sufficiently long period of time (30 min). Moreover, using natural vocal exchanges in another set of individuals, we showed that sibling signalling was no more taken into account after a few minutes. This suggests that young barn owls need to continuously display their motivation to trigger siblings' withdrawal from the current competition. Repeating a vocal display may ensure its honesty. Studying the extent to which individuals retain past information is important to understand how individuals adjust their competitive investment over resources.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Functional plasticity of the LACK-reactive Vbeta4-Valpha8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice.

Early production of IL-4 by LACK-reactive Vbeta4-Valpha8 CD4(+) T cells instructs aberrant Th2 cell development and susceptibility to Leishmania major in BALB / c mice. This was demonstrated using Vbeta4(+)-deficient BALB / c mice as a result of chronic infection with MMTV (SIM), a mouse mammary tumor virus expressing a Vbeta4-specific superantigen. The early IL-4 response was absent in these mice which develop a Th1 response to L. major. Here, we studied the functional plasticity of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells using BALB/ c mice inoculated with L. major shortly after infection with MMTV (SIM), i. e. before deletion of Vbeta4(+) cells. These mice fail to produce the early IL-4 response to L. major and instead exhibit an IFN-gamma response that occurs within LACK-reactive Vbeta4-Valpha8 CD4(+) T cells. Neutralization of IFN-gamma restores the production of IL-4 by these cells. These data suggest that the functional properties of LACK-reactive Vbeta4-Valpha8 CD4(+) T cells are not irreversibly fixed.

Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation.

Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [(3)H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [(3)H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release.

Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.

Functional and morphological evidence for a ventricular conduction system in zebrafish and Xenopus hearts.

Zebrafish and Xenopus have become popular model organisms for studying vertebrate development of many organ systems, including the heart. However, it is not clear whether the single ventricular hearts of these species possess any equivalent of the specialized ventricular conduction system found in higher vertebrates. Isolated hearts of adult zebrafish (Danio rerio) and African toads (Xenopus laevis) were stained with voltage-sensitive dye and optically mapped in spontaneous and paced rhythms followed by histological examination focusing on myocardial continuity between the atrium and the ventricle. Spread of the excitation wave through the atria was uniform with average activation times of 20 +/- 2 and 50 +/- 2 ms for zebrafish and Xenopus toads, respectively. After a delay of 47 +/- 8 and 414 +/- 16 ms, the ventricle became activated first in the apical region. Ectopic ventricular activation was propagated significantly more slowly (total ventricular activation times: 24 +/- 3 vs. 14 +/- 2 ms in zebrafish and 74 +/- 14 vs. 35 +/- 9 ms in Xenopus). Although we did not observe any histologically defined tracts of specialized conduction cells within the ventricle, there were trabecular bands with prominent polysialic acid-neural cell adhesion molecule staining forming direct myocardial continuity between the atrioventricular canal and the apex of the ventricle; i.e., the site of the epicardial breakthrough. We thus conclude that these hearts are able to achieve the apex-to-base ventricular activation pattern observed in higher vertebrates in the apparent absence of differentiated conduction fascicles, suggesting that the ventricular trabeculae serve as a functional equivalent of the His-Purkinje system.

T cell differentiation in chronic infection and cancer: functional adaptation or exhaustion?

Chronic viral infections and malignant tumours induce T cells that have a reduced ability to secrete effector cytokines and have upregulated expression of the inhibitory receptor PD1 (programmed cell death protein 1). These features have so far been considered to mark terminally differentiated 'exhausted' T cells. However, several recent clinical and experimental observations indicate that phenotypically exhausted T cells can still mediate a crucial level of pathogen or tumour control. In this Opinion article, we propose that the exhausted phenotype results from a differentiation process in which T cells stably adjust their effector capacity to the needs of chronic infection. We argue that this phenotype is optimized to cause minimal tissue damage while still mediating a critical level of pathogen control. In contrast to the presently held view of functional exhaustion, this new concept better reflects the pathophysiology and clinical manifestations of persisting infections, and it provides a rationale for emerging therapies that enhance T cell activity in chronic infection and cancer by blocking inhibitory receptors.

A random linear functional approach to efficiency bounds

This paper suggests a method for obtaining efficiency bounds in models containing either only infinite-dimensional parameters or both finite- and infinite-dimensional parameters (semiparametric models). The method is based on a theory of random linear functionals applied to the gradient of the log-likelihood functional and is illustrated by computing the lower bound for Cox's regression model

Mouse Grueneberg ganglion neurons share molecular and functional features with C. elegans amphid neurons.

The mouse Grueneberg ganglion (GG) is an olfactory subsystem located at the tip of the nose close to the entry of the naris. It comprises neurons that are both sensitive to cold temperature and play an important role in the detection of alarm pheromones (APs). This chemical modality may be essential for species survival. Interestingly, GG neurons display an atypical mammalian olfactory morphology with neurons bearing deeply invaginated cilia mostly covered by ensheathing glial cells. We had previously noticed their morphological resemblance with the chemosensory amphid neurons found in the anterior region of the head of Caenorhabditis elegans (C. elegans). We demonstrate here further molecular and functional similarities. Thus, we found an orthologous expression of molecular signaling elements that was furthermore restricted to similar specific subcellular localizations. Calcium imaging also revealed a ligand selectivity for the methylated thiazole odorants that amphid neurons are known to detect. Cellular responses from GG neurons evoked by chemical or temperature stimuli were also partially cGMP-dependent. In addition, we found that, although behaviors depending on temperature sensing in the mouse, such as huddling and thermotaxis did not implicate the GG, the thermosensitivity modulated the chemosensitivity at the level of single GG neurons. Thus, the striking similarities with the chemosensory amphid neurons of C. elegans conferred to the mouse GG neurons unique multimodal sensory properties.