In vivo imaging of T cell delivery to tumors after adoptive transfer therapy.

Adoptive transfer therapy of in vitro-expanded tumor-specific cytolytic T lymphocytes (CTLs) can mediate objective cancer regression in patients. Yet, technical limitations hamper precise monitoring of posttherapy T cell responses. Here we show in a mouse model that fused single photon emission computed tomography and x-ray computed tomography allows quantitative whole-body imaging of (111)In-oxine-labeled CTLs at tumor sites. Assessment of CTL localization is rapid, noninvasive, three-dimensional, and can be repeated for longitudinal analyses. We compared the effects of lymphodepletion before adoptive transfer on CTL recruitment and report that combined treatment increased intratumoral delivery of CTLs and improved antitumor efficacy. Because (111)In-oxine is a Food and Drug Administration-approved clinical agent, and human SPECT-CT systems are available, this approach should be clinically translatable, insofar as it may assess the efficacy of immunization procedures in individual patients and lead to development of more effective therapies.

Clearance of a hirano body-like F-actin aggresome generated by jasplakinolide

We have reported in a variety of mammalian cells the reversible formation of a filamentous actin (F-actin)-enriched aggresome generated by the actin toxin jasplakinolide (Lázaro-Diéguez et al., J Cell Sci 2008; 121:1415-25). Notably, this F-actin aggresome (FAG) resembles in many aspects the pathological Hirano body, which frequently appears in some diseases such as Alzheimer's and alcoholism. Using selective inhibitors, we examined the molecular and subcellular mechanisms that participate in the clearance of the FAG. Chaperones, microtubules, proteasomes and autophagosomes all actively participate to eliminate the FAG. Here we compile and compare these results and discuss the involvement of each process. Because of its simplicity and high reproducibility, our cellular model could help to test pharmacological agents designed to interfere with the mechanisms involved in the clearance of intracellular bodies and, in particular, of those enriched in F-actin.

Dynamic and Regulated Association of Caveolin with Lipid Bodies: Modulation of Lipid Body Motility and Function by a Dominant Negative Mutant

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Therapeutic drug monitoring and metabolites profil analysis of antiviral drugs

The widespread use of combination antiretroviral therapy (ARVs) has considerably improved the prognosis of patients infected with HIV. Conversely, considerable advances have been recently realized for the therapy of hepatitis C infection with the recent advent of potent new anti-HCV drugs that allow an increasing rate HCV infection cure. Despite their overall efficacy, a significant number of patients do not achieve or maintain adequate clinical response, defined as an undetectable viral load for HIV, and a sustained virological response (or cure) in HCV infection. Treatment failure therefore still remains an important issue besides drugs toxicities and viral resistance which is not uncommon in a significant percentage of patients who do not reach adequate virological suppression. The reasons of variability in drug response are multifactorial and apart from viral genetics, other factors such as environmental factors, drug- drug interactions, and imperfect compliance may have profound impact on antiviral drugs' clinical response. The possibility of measuring plasma concentration of antiviral drugs enables to guide antiviral drug therapy and ensure optimal drug exposure. The overall objective of this research was to widen up the current knowledge on pharmacokinetic and pharmacogenetic factors that influence the clinical response and toxicity of current and newly approved antiretroviral and anti-HCV drugs. To that endeavour, analytical methods using liquid chromatography coupled with tandem mass spectrometry have been developed and validated for the precise and accurate measurement of new antiretroviral and anti-HCV drugs . These assays have been applied for the TDM of ARVs and anti-HCV in patients infected with either HIV or HCV respectively, and co-infected with HIV- HCV. A pharmacokinetic population model was developed to characterize inter and intra-patient variability of rilpivirine, the latest marketed Non Nucleoside Reverse transcriptase (NNRTI) Inhibitor of HIVand to identify genetic and non genetic covariates influencing rilpivirine exposure. None of the factors investigated so far showed however any influence of RPV clearance. Importantly, we have found that the standard daily dosage regimen (25 mg QD) proposed for rilpivirine results in concentrations below the proposed therapeutic target in about 40% of patients. In these conditions, virologie escape is a potential risk that remains to be further investigated, notably via the TDM approach that can be a useful tool to identify patients who are at risk for being exposed to less than optimal levels of rilpivirine in plasma. Besides the last generation NNRTI rilpivirine, we have studied efavirenz, the major NNRTI clinically used so far. Namely for efavirenz, we aimed at identifying a potential new marker of toxicity that may be incriminated for the neuropsychological sides effects and hence discontinuation of efavirenz therapy. To that endeavour, a comprehensive analysis of phase I and phase II metabolites profiles has been performed in plasma, CSF and in urine from patients under efavirenz therapy. We have found that phase II metabolites of EFV constitute the major species circulating in blood, sometimes exceeding the levels of the parent drug efavirenz. Moreover we have identified a new metabolite of efavirenz in humans, namely the 8-OH-EFV- sulfate which is present at high concentrations in all body compartments from patients under efavirenz therapy. These investigations may open the way to possible alternate phenotypic markers of efavirenz toxicity. Finally, the specific influence of P-glycoprotein on the cellular disposition of a series ARVs (NNRTIs and Pis] has been studies in in vitro cell systems using the siRNA silencing approach. -- Depuis l'introduction de la thérapie antirétrovirale (ARVs) la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Une réponse imparfaite ou la toxicité du traitement est certainement multifactorielle. Le suivi thérapeutique des médicaments [Therapeutic Drug Monitoring TDM) à travers la mesure des concentrations plasmatiques constitue une approche importante pour guider le traitement médicamenteux et de s'assurer que les patients sont exposés à des concentrations optimales des médicaments dans le sang, et puissent tirer tout le bénéfice potentiel du traitement. L'objectif global de cette thèse était d'étudier les facteurs pharmacocinétiques et pharmacogénétiques qui influencent l'exposition des médicaments antiviraux (ARVs et anti- VHC) récemment approuvés. A cet effet, des méthodes de quantification des concentrations plasmatiques des médicaments antirétroviraux, anti-VHC ainsi que pour certains métabolites ont été développées et validées en utilisant la Chromatographie liquide couplée à la spectrométrie de masse tandem. Ces méthodes ont été utilisées pour le TDM des ARVs et pour les agents anti-VHC chez les patients infectés par le VIH, et le VHC, respectivement, mais aussi chez les patients co-infectés par le VIH-VHC. Un modèle de pharmacocinétique de population a été développé pour caractériser la variabilité inter-et intra-patient du médicament rilpivirine, un inhibiteur non nucléosidique de la transcriptase de VIH et d'identifier les variables génétiques et non génétiques influençant l'exposition au médicament. Aucun des facteurs étudiés n'a montré d'influence notable sur la clairance de la rilpivirine. Toutefois, la concentration résiduelle extrapolée selon le modèle de pharmacocinétique de population qui a été développé, a montré qu'une grande proportion des patients présente des concentrations minimales inférieures à la cible thérapeutique proposée. Dans ce contexte, la relation entre les concentrations minimales et l'échappement virologique nécessite une surveillance étroite des taux sanguins des patients recevant de la rilpivirine. A cet effet, le suivi thérapeutique est un outil important pour l'identification des patients à risque soient sous-exposés à lai rilpivirine. Pour identifier de nouveaux marqueurs de la toxicité qui pourraient induire l'arrêt du traitement, le profil des métabolites de phase I et de phase II a été étudié dans différentes matrices [plasma, LCR et urine) provenant de patients recevant de l'efavirenz. Les métabolites de phase II, qui n'avaient à ce jour jamais été investigués, constituent les principales espèces présentes dans les matrices étudiées. Au cours de ces investigations, un nouveau métabolite 8- OH-EFV-sulfate a été identifié chez l'homme, et ce dernier est. présent à des concentrations importantes. L'influence de certains facteurs pharmacogénétique des patients sur le profil des métabolites a été étudiée et ouvre la voie à de possibles nouveaux marqueurs phénotypiques alternatifs qui pourraient possiblement mieux prédire la toxicité associée au traitement par l'efavirenz. Finalement, nous nous sommes intéressés à étudier dans un modèle in vitro certains facteurs, comme la P-glycoprotéine, qui influencent la disposition cellulaire de certains médicaments antirétroviraux, en utilisant l'approche par la technologie du siRNA permettant de bloquer sélectivement l'expression du gène de cette protéine d'efflux des médicaments. -- Depuis l'introduction de la thérapie antiretrovirale (ARVs] la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Il a pu être démontré que la concentration de médicament présente dans l'organisme est corrélée avec l'efficacité clinique pour la plupart des médicaments agissant contre le VIH et contre le VHC. Les médicaments antiviraux sont généralement donnés à une posologie fixe et standardisée, à tous les patients, il existe cependant une importante variabilité entre les concentrations sanguines mesurées chez les individus. Cette variabilité peut être expliquée par plusieurs facteurs démographiques, environnementaux ou génétiques. Dans ce contexte, le suivi des concentrations sanguines (ou Therapeutic Drug Monitoring, TDM) permet de contrôler que les patients soient exposés à des concentrations suffisantes (pour bloquer la réplication du virus dans l'organisme) et éviter des concentrations excessives, ce qui peut entraîner l'apparition d'intolérence au traitement. Le but de ce travail de thèse est d'améliorer la compréhension des facteurs pharmacologiques et génétiques qui peuvent influencer l'efficacité et/ou la toxicité des médicaments antiviraux, dans le but d'améliorer le suivi des patients. A cet effet, des méthodes de dosage très sensibles et ont été mises au point pour permettre de quantifier les médicaments antiviraux dans le sang et dans d'autres liquides biologiques. Ces méthodes de dosage sont maintenant utilisées d'une part dans le cadre de la prise en charge des patients en routine et d'autre part pour diverses études cliniques chez les patients infectés soit par le HIV, le HCV ou bien coinfectés par les deux virus. Une partie de ce travail a été consacrée à l'investigation des différents facteurs démographiques, génétiques et environnementaux qui pourraient l'influencer la réponse clinique à la rilpivirine, un nouveau médicament contre le VIH. Toutefois, parmi tous les facteurs étudiés à ce jour, aucun n'a permis d'expliquer la variabilité de l'exposition à la rilpivirine chez les patients. On a pu cependant observer qu'à la posologie standard recommandée, un pourcentage relativement élevé de patients pourrait présenter des concentrations inférieures à la concentration sanguine minimale actuellement proposée. Il est donc utile de surveiller étroitement les concentrations de rilpivirine chez les patients pour identifier sans délai ceux qui risquent d'être sous-exposés. Dans l'organisme, le médicament subit diverses transformations (métabolisme) par des enzymes, notamment dans le foie, il est transporté dans les cellules et tissus par des protéines qui modulent sa concentration au site de son action pharmacologique. A cet effet, différents composés (métabolites) produits dans l'organisme après l'administration d'efavirenz, un autre médicament anti-VIH, ont été étudiés. En conclusion, nous nous sommes intéressés à la fois aux facteurs pharmacologiques et génétiques des traitements antiviraux, une approche qui s'inscrit dans l'optique d'une stratégie globale de prise en charge du patient. Dans ce contexte, le suivi des concentrations sanguines de médicaments constitue une des facettes du domaine émergent de la Médecine Personnalisée qui vise à maximiser le bénéfice thérapeutique et le profil de tolérance des médicaments antiviraux

Vascular continuity, cell axialization and auxin.

Phytohormones have been implicated in vascular development in various ways, but their precise function and the extent of their influence is still controversial. Recent results from experimental manipulation of developing organs and Arabidopsis developmental genetics support a role for polar auxin flow in cell axis formation within the vascular system and, interestingly, also in the embryonic establishment of the plant body axis. Vascular responses to auxin transport inhibition indicate patterns of auxin distribution during leaf development and new technologies may enable these predictions to be tested within the near future. Moreover, recently discovered Arabidopsis axialisation mutants seem to identify essential genes that relay auxin signals in vascular development. A first gene in this class, MONOPTEROS (MP) has been cloned and encodes a transcription factor capable of binding to auxin response elements in the control regions of auxin regulated genes. Molecular identification of further axialisation genes may provide access to a mechanistic understanding of plant cell axis formation.

Identification of novel and cell type enriched cofactors of the transcription activation domain of RelA (p65 NF-kappaB).

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.

An unusual uterine tumour with signet ring cell features

Objective: Lymphomas with signet ring cell features are rare, as is uterine dissemination of lymphomas. We report an exceptional case of a uterine tumor combining these two characteristics. Method: A 61-year-old female was diagnosed in 2004 with localized nodal grade 2 follicular lymphoma (stage IA). She received local radiation therapy, experienced total remission, and did well until 2009 when a systematic CT scan evidenced a pelvic anterior-lateral mass. Total enlarged hysterectomy was performed. Results: The anterior uterine wall contained a 4.8-cm fish flesh well-delineated mass corresponding to a mostly diffuse and focally nodular proliferation of medium to large cells with extensive signet ring cell changes. Tumor cells were CD20-, CD10-, Bcl2-, and Bcl6-positive with a low proliferation rate (<10-15%); CD21 underlined a focal follicular architecture. The vacuoles were PAS-negative and did not stain for immunoglobulin; ultrastructural analysis revealed nonspecific degenerative vacuoles. No lymph nodes were identified isolated from the surgical specimen. The tumor was considered as a secondary localization of the systemic follicular lymphoma, though no signet ring cells were evidenced in the cervical lymph node biopsy (reviewed). Follow-up showed retroperitoneal tissue infiltration (PET-CT) and normal medullar biopsy. She recently started R-CHOP chemotherapy. Conclusion: This case illustrates both an unusual site of dissemination and challenging cytological characteristics in a follicular lymphoma. The signet ring cell changes challenged the adequate classification of this lymphoma as either a large B cell or a follicular B cell lymphoma.

Cell polarity in plants: a PARspective on PINs.

Plants have acquired the ability for organized multicellular development independent from animals. Because of this, they represent an independent example in nature for the development of coordinated, complex cell polarity from the simple polarity found in unicellular eukaryotes. Plants display a striking array of polarized cell types, with different axes of polarity being defined in one cell. The most investigated and best understood aspect of plant polarity is the apical-basal polarity of the PIN family of auxin efflux facilitators, which are of crucial importance for the organization of the entire plant body. Striking differences exist between the PAR-polarity modules known in animals and the ways PINs polarize plant cells. Nonetheless, a common regulatory logic probably applies to all polarizing eukaryotic cells, which includes self-reinforcing, positive feedback loops, intricate interactions between membrane-attached proteins, lipid signatures, and the targeting of transmembrane proteins to the correct domains of the plasma membrane.

Increased body fat is independently and negatively related with cardiorespiratory fitness levels in children and adolescents with normal weight.

Body mass index (BMI) is related with cardiorespiratory fitness (CRF), but less is known regarding the combined relationships between BMI and body fat (BF) on CRF. Cross-sectional study included 2361 girls and 2328 boys aged 10–18 years living in the area of Lisbon, Portugal. BMI was calculated by measuring height and weight, and obesity was assessed by international criteria. BF was assessed by bioimpedance. CRF was assessed by the 20-m shuttle run and the participants were classified as normal-to-high or low-CRF level according to Fitness gram criterion-referenced standards. The prevalence of low CRF was 47 and 39% in girls and boys, respectively. The corresponding values for the prevalence of obesity were 4.8 and 5.6% (not significant) and of excess BF of 12.1 and 25.1% (P <0.001), respectively. In both sexes, BMI and BF were inversely related with CRF: r = – 0.53 and – 0.45 for BMI and % BF, respectively, in boys and the corresponding values in girls were – 0.50 and – 0.33 (all P <0.01). When compared with a participant with normal BMI and BF, the odds ratios (95% confidence interval) for low CRF were 1.94 (1.46–2.58) for a participant with normal BMI and high BF, and 6.19 (5.02–7.63) for a participant with high BMI and high BF. The prevalence of low-CRF levels is high in Portuguese youths. BF negatively influences CRF levels among children/adolescents with normal BMI.

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Cell differentiation to "mating bodies" induced by an integrating and conjugative element in free-living bacteria.

Lateral gene transfer (LGT) is one of the most important processes leading to prokaryotic genome innovation. LGT is typically associated with conjugative plasmids and bacteriophages, but recently, a new class of mobile DNA known as integrating and conjugative elements (ICE) was discovered, which is abundant and widespread among bacterial genomes. By studying at the single-cell level the behavior of a prevalent ICE type in the genus Pseudomonas, we uncover the remarkable way in which the ICE orchestrates host cell differentiation to ensure horizontal transmission. We find that the ICE induces a state of transfer competence (tc) in 3%-5% of cells in a population under nongrowing conditions. ICE factors control the development of tc cells into specific assemblies that we name "mating bodies." Interestingly, cells in mating bodies undergo fewer and slower division than non-tc cells and eventually lyse. Mutations in ICE genes disrupting mating-body formation lead to 5-fold decreased ICE transfer rates. Hence, by confining the tc state to a small proportion of the population, ICE horizontal transmission is achieved with little cost in terms of vertical transmission. Given the low transfer frequencies of most ICE, we anticipate regulation by subpopulation differentiation to be widespread.

Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight.

Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.

Low viscosity contrast fingering in a rotating Hele-Shaw cell

We study the fingering instability of a circular interface between two immiscible liquids in a radial Hele-Shaw cell. The cell rotates around its vertical symmetry axis, and the instability is driven by the density difference between the two fluids. This kind of driving allows studying the interfacial dynamics in the particularly interesting case of an interface separating two liquids of comparable viscosity. An accurate experimental study of the number of fingers emerging from the instability reveals a slight but systematic dependence of the linear dispersion relation on the gap spacing. We show that this result is related to a modification of the interface boundary condition which incorporates stresses originated from normal velocity gradients. The early nonlinear regime shows nearly no competition between the outgrowing fingers, characteristic of low viscosity contrast flows. We perform experiments in a wide range of experimental parameters, under conditions of mass conservation (no injection), and characterize the resulting patterns by data collapses of two characteristic lengths: the radius of gyration of the pattern and the interface stretching. Deep in the nonlinear regime, the fingers which grow radially outwards stretch and become gradually thinner, to a point that the fingers pinch and emit drops. We show that the amount of liquid emitted in the first generation of drops is a constant independent of the experimental parameters. Further on there is a sharp reduction of the amount of liquid centrifugated, punctuated by periods of no observable centrifugation.

Origin-related, environmental, sex, and age determinants of immunocompetence, susceptibility to ectoparasites, and disease symptoms in the barn owl

Knowledge of the role of origin-related, environmental, sex, and age factors on host defence mechanisms is important to understand variation in parasite intensity. Because alternative components of parasite defence may be differently sensitive to various factors, they may not necessarily covary. Many components should therefore be considered to tackle the evolution of host-parasite interactions. In a population of barn owls (Tyto alba), we investigated the role of origin-related, environmental (i.e. year, season, nest of rearing, and body condition), sex, and age factors on 12 traits linked to immune responses [humoral immune responses towards sheep red blood cells (SRBC), human serum albumin (HSA) and toxoid toxin TT, T-cell mediated immune response towards the mitogen phytohemagglutinin (PHA)], susceptibility to ectoparasites (number and fecundity of Carnus haemapterus, number of Ixodes ricinus), and disease symptoms (size of the bursa of Fabricius and spleen, proportion of proteins that are immunoglobulins, haematocrit and blood concentration in leucocytes). Cross-fostering experiments allowed us to detect a heritable component of variation in only four out of nine immune and parasitic parameters (i.e. SRBC- and HSA-responses, haematocrit, and number of C. haemapterus). However, because nestlings were not always cross-fostered just after hatching, the finding that 44% of the immune and parasitic parameters were heritable is probably an overestimation. These experiments also showed that five out of these nine parameters were sensitive to the nest environment (i.e. SRBC- and PHA-responses, number of C. haemapterus, haematocrit and blood concentration in leucocytes). Female nestlings were more infested by the blood-sucking fly C. haemapterus than their male nestmates, and their blood was less concentrated in leucocytes. The effect of year, season, age (i.e. reflecting the degree of maturation of the immune system), brood size, position in the within-brood age hierarchy, and body mass strongly differed between the 12 parameters. Different components of host defence mechanisms are therefore not equally heritable and sensitive to environmental, sex, and age factors, potentially explaining why most of these components did not covary.