Detecting the oxidative reactivity of nanoparticles: a new protocol for reducing artifacts

Understanding the oxidative reactivity of nanoparticles (NPs; <100 nm) could substantially contribute to explaining their toxicity. We attempted to refine the use of 2′7-dichlorodihydrofluorescein (DCFH) to characterize NP generation of reactive oxygen species (ROS). Several fluorescent probes have been applied to testing oxidative reactivity, but despite DCFH being one of the most popular for the detection of ROS, when it has been applied to NPs there have been an unexplainably wide variability in results. Without a uniform methodology, validating even robust results is impossible. This study, therefore, identified sources of conflicting results and investigated ways of reducing occurrence of artificial results. Existing techniques were tested and combined (using their most desirable features) to form a more reliable method for the measurement of NP reactivity in aqueous dispersions. We also investigated suitable sample ranges necessary to determine generation of ROS. Specifically, ultrafiltration and time-resolved scan absorbance spectra were used to study possible optical interference when using high sample concentrations. Robust results were achieved at a 5 µM DCFH working solution with 0.5 unit/mL horseradish peroxidase (HRP) dissolved in ethanol. Sonication in DCFH-HRP working solution provided more stable data with a relatively clean background. Optimal particle concentration depends on the type of NP and in general was in the µg/mL range. Major reasons for previously reported conflicting results due to interference were different experimental approaches and NP sample concentrations. The protocol presented here could form the basis of a standardized method for applying DCFH to detect generation of ROS by NPs.

Interobserver and intraobserver variability of apparent diffusion coefficient measurements in treated malignant hepatic lesions: measurements in the whole lesion versus in the area with the most restricted diffusion

Purpose: The increase of apparent diffusion coefficient (ADC) in treated hepatic malignancies compared to pre-therapeutic values has been interpreted as treatment success; however, the variability of ADC measurements remains unknown. Furthermore, ADC has been usually measured in the whole lesion, while measurements should be probably centered on the area with the most restricted diffusion (MRDA) as it represents potential tumoral residue. Our objective was to compare the inter/intraobserver variability of ADC measurements in the whole lesion and in MRDA. Material and methods: Forty patients previously treated with chemoembolization or radiofrequency were evaluated (20 on 1.5T and 20 on 3.0T). After consensual agreement on the best ADC image, two readers measured the ADC values using separate regions of interest that included the whole lesion and the whole MRDA without exceeding their borders. The same measurements were repeated two weeks later. Spearman test and the Bland-Altman method were used. Results: Interobserver correlation in ADC measurements in the whole lesion and MRDA was as follows: 0.962 and 0.884. Intraobserver correlation was, respectively, 0.992 and 0.979. Interobserver limits of variability (mm2/sec*10-3) were between -0.25/+0.28 in the whole lesion and between -0.51/+0.46 in MRDA. Intraobserver limits of variability were, respectively: -0.25/+0.24 and -0.43/+0.47. Conclusion: We observed a good inter/intraobserver correlation in ADC measurements. Nevertheless, a limited variability does exist, and it should be considered when interpreting ADC values of hepatic malignancies.

Long-term and long-range migration of radioactive fallout in a Karst system.

Mountainous areas are often covered by little evolved soils from which deposited radionuclides can potentially leak into the vadose zone. In the Swiss Jura mountains, we observed unusual isotopic ratios of nuclear weapon test (NWTs) fallout with an apparent loss of NWTs plutonium relative to &supl;³⁷Cs of Chernobyl origin in thinner soils. Here, we studied the karstic watershed of a vauclusian spring to determine the residence times of plutonium, ²⁴&supl;Am, and ⁹⁰Sr deposited by global fallout and their respective mobility in carbonaceous soils. The results show that ⁹⁰Sr is washed most efficiently from the watershed with a residence time of several hundred years. The estimated plutonium residence time is more than 10 times higher (in the range of 5000-10,000 years), and the ²⁴&supl;Am residence time is double that of plutonium. The spring water ²⁴&supl;Am/²³⁹+²⁴⁰Pu isotopic ratio is lower (0.12 - 0.28) than found in watershed soils (0.382 ± 0.077). Similar differences are found in aquatic mosses (²⁴&supl;Am/²³⁹+²⁴⁰Pu isotopic ratio 0.05-0.12), which are permanently submerged in spring waters. In contrast to plutonium, ⁹⁰Sr is leached from these mosses with 0.5M HCl, demonstrating that strontium is probably associated with calcium carbonate precipitations on the mosses. The higher plutonium to americium isotopic ratio found in the samples of spring water and mosses at the outlet of the karst shows that plutonium mobility is enhanced.

Genes, environment and sex : perspectives from ectothermic vertebrates

RESUMELes modèles classiques sur l'évolution des chromosomes sexuels supposent que des gènes sexe- antagonistes s'accumulent sur les chromosomes sexuels, entraînant ainsi l'apparition d'une région non- recombinante, qui se répand progressivement en favorisant l'accumulation de mutations délétères. En accord avec cette théorie, les chromosomes sexuels que l'on observe aujourd'hui chez les mammifères et les oiseaux sont considérablement différenciés. En revanche, chez la plupart des vertébrés ectothermes, les chromosomes sexuels sont indifférenciés et il existe une impressionnante diversité de mécanismes de détermination du sexe. Au cours de cette thèse, j'ai étudié l'évolution des chromosomes sexuels chez les vertébrés ectothermes, en outre pour mieux comprendre ce contraste avec les vertébrés endothermes. L'hypothèse « high-turnover » postule que les chromosomes sexuels sont remplacés régulièrement à partir d'autosomes afin d'éviter leur dégénérescence. L'hypothèse « fountain-of-youth » propose que la recombinaison entre le chromosome X et le chromosome Y au sein de femelles XY empêche la dégénérescence. Les résultats de ma thèse, basés sur des études théoriques et empiriques, suggèrent que les deux processus peuvent être entraînés par l'environnement et ainsi jouent un rôle important dans l'évolution des chromosomes sexuels chez les vertébrés ectothermes.SUMMARYClassical models of sex-chromosome evolution assume that sexually antagonistic genes accumulate on sex chromosomes leading to a non-recombining region, which progressively expands and favors the accumulation of deleterious mutations. Concordant with this theory, sex chromosomes in extant mammals and birds are considerably differentiated. In most ectothermic vertebrates, such as frogs, however, sex chromosomes are undifferentiated and a striking diversity of sex determination systems is observed. This thesis was aimed to investigate this apparent contrast of sex chromosome evolution between endothermic and ectothermic vertebrates. The "high-turnover" hypothesis holds that sex chromosomes arose regularly from autosomes preventing decay. The "fountain-of-youth" hypothesis posits that sex chromosomes undergo episodic X-Y recombination in sex-reversed XY females, thereby purging ("rejuvenating") the Y chromosome. We suggest that both processes likely played an important role in sex chromosome evolution of ectothermic vertebrates. The literature largely views sex determination as a dichotomous process: individual sex is assumed to be determined either by genetic (genotypic sex determination, GSD) or by environmental factors (environmental sex determination, ESD), most often temperature (temperature sex determination, TSD). We endorsed an alternative view, which sees GSD and TSD as the ends of a continuum. The conservatism of molecular processes among different systems of sex determination strongly supports the continuum view. We proposed to define sex as a threshold trait underlain by a liability factor, and reaction norms allowing modeling interactions between genotypic and temperature effects. We showed that temperature changes (due to e.g., climatic changes or range expansions) are expected to provoke turnovers in sex-determination mechanisms maintaining homomorphic sex chromosomes. The balanced lethal system of crested newts might be the result of such a sex determination turnover, originating from two variants of ancient Y-chromosomes. Observations from a group of tree frogs, on the other hand, supported the 'fountain of youth' hypothesis. We then showed that low rates of sex- reversals in species with GSD might actually be adaptive considering joint effects of deleterious mutation purging and sexually antagonistic selection. Ongoing climatic changes are expected to threaten species with TSD by biasing population sex ratios. In contrast, species with GSD are implicitly assumed immune against such changes, because genetic systems are thought to necessarily produce even sex ratios. We showed that this assumption may be wrong and that sex-ratio biases by climatic changes may represent a previously unrecognized extinction threat for some GSD species.

Improvement in gross motor performance between 3 and 5 years of age.

This study examined gross motor performance of 101 typically developing children between 3 and 5 years of age (48 boys, 53 girls, M age = 3.9 yr., SD = 0.5). All children performed 7 different gross motor tasks which were rated on a 5-point scale. Age and sex were assessed by an ordinal-logistic model, and odds ratios were calculated for each task using age and sex as covariates. For standing on one leg, walking on a beam, hopping on one leg, running, and taking stairs, statistically significant age differences were found, while for rising and jumping down, none were apparent. Mean motor performance did not differ between boys and girls on the tasks. The older the children were, the better they performed on the tasks.

Persistent unequal sex ratio in a population of grayling (salmonidae) and possible role of temperature increase.

In some fishes, water chemistry or temperature affects sex determination or creates sex-specific selection pressures. The resulting population sex ratios are hard to predict from laboratory studies if the environmental triggers interact with other factors, whereas in field studies, singular observations of unusual sex ratios may be particularly prone to selective reporting. Long-term monitoring largely avoids these problems. We studied a population of grayling (Thymallus thymallus) in Lake Thun, Switzerland, that has been monitored since 1948. Samples of spawning fish have been caught about 3 times/week around spawning season, and water temperature at the spawning site has been continuously recorded since 1970. We used scale samples collected in different years to determine the average age of spawners (for life-stage specific analyses) and to identify the cohort born in 2003 (an extraordinarily warm year). Recent tissue samples were genotyped on microsatellite markers to test for genetic bottlenecks in the past and to estimate the genetically effective population size (N(e) ). Operational sex ratios changed from approximately 65% males before 1993 to approximately 85% males from 1993 to 2011. Sex ratios correlated with the water temperatures the fish experienced in their first year of life. Sex ratios were best explained by the average temperature juvenile fish experienced during their first summer. Grayling abundance is declining, but we found no evidence of a strong genetic bottleneck that would explain the apparent lack of evolutionary response to the unequal sex ratio. Results of other studies show no evidence of endocrine disruptors in the study area. Our findings suggest temperature affects population sex ratio and thereby contributes to population decline. Persistencia de Proporción de Sexos Desigual en una Población de Tímalos (Salmonidae) y el Posible Papel del Incremento de la Temperatura.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

AltitudeOmics: enhanced cerebrovascular reactivity and ventilatory response to CO2 with high-altitude acclimatization and reexposure.

The present study is the first to examine the effect of high-altitude acclimatization and reexposure on the responses of cerebral blood flow and ventilation to CO2. We also compared the steady-state estimates of these parameters during acclimatization with the modified rebreathing method. We assessed changes in steady-state responses of middle cerebral artery velocity (MCAv), cerebrovascular conductance index (CVCi), and ventilation (V(E)) to varied levels of CO2 in 21 lowlanders (9 women; 21 ± 1 years of age) at sea level (SL), during initial exposure to 5,260 m (ALT1), after 16 days of acclimatization (ALT16), and upon reexposure to altitude following either 7 (POST7) or 21 days (POST21) at low altitude (1,525 m). In the nonacclimatized state (ALT1), MCAv and V(E) responses to CO2 were elevated compared with those at SL (by 79 ± 75% and 14.8 ± 12.3 l/min, respectively; P = 0.004 and P = 0.011). Acclimatization at ALT16 further elevated both MCAv and Ve responses to CO2 compared with ALT1 (by 89 ± 70% and 48.3 ± 32.0 l/min, respectively; P < 0.001). The acclimatization gained for V(E) responses to CO2 at ALT16 was retained by 38% upon reexposure to altitude at POST7 (P = 0.004 vs. ALT1), whereas no retention was observed for the MCAv responses (P > 0.05). We found good agreement between steady-state and modified rebreathing estimates of MCAv and V(E) responses to CO2 across all three time points (P < 0.001, pooled data). Regardless of the method of assessment, altitude acclimatization elevates both the cerebrovascular and ventilatory responsiveness to CO2. Our data further demonstrate that this enhanced ventilatory CO2 response is partly retained after 7 days at low altitude.

Elemental (C/N ratios) and isotopic (δ15N(org), δ13C(org)) compositions of sedimentary organic matter from a high-altitude mountain lake (Meidsee, 2661 m a.s.l., Switzerland): Implications for Lateglacial and Holocene Alpine landscape evolution

The deposition of Late Pleistocene and Holocene sediments in the high-altitude lake Meidsee (located at an altitude of 2661 m a.s.l. in the Southwestern Alps) strikingly coincided with global ice-sheet and mountain-glacier decay in the Alpine forelands and the formation of perialpine lakes. Radiocarbon ages of bottom-core sediments point out (pre-) Holocene ice retreat below 2700 m a.s.l., at about 16, 13, 10, and 9 cal. kyr BP. The Meidsee sedimentary record therefore provides information about the high-altitude Alpine landscape evolution since the Late Pleistocene/Holocene deglaciation in the Swiss Southwestern Alps. Prior to 5 cal. kyr BP, the C/N ratio and the isotopic composition of sedimentary organic matter (delta N-15(org), delta C-13(org)) indicate the deposition of algal-derived organic matter with limited input of terrestrial organic matter. The early Holocene and the Holocene climatic optimum (between 7.0 and 5.5 cal. kyr BP) were characterized by low erosion (decreasing magnetic susceptibility, chi) and high content of organic matter (C-org > 13 wt.%), enriched in C-13(org) (>-18 parts per thousand) with a low C/N (similar to 10) ratio, typical of modern algal matter derived from in situ production. During the late Holocene, there was a long-term increasing contribution of terrestrial organic matter into the lake (C/N > 11), with maxima between 2.4 and 0.9 cal. kyr BP. A major environmental change took place 800 years ago, with an abrupt decrease in the relative contribution of terrestrial organic material into the lake compared with aquatic organic material which subsequently largely dominated (C/N drop from 16 to 10). Nonetheless, this event was marked by a rise in soil erosion (chi), in nutrients input (N and P contents) and in anthropogenic lead deposition, suggesting a human disturbance of Alpine ecosystems 800 years ago. Indeed, this time period coincided with the migration of the Walser Alemannic people in the region, who settled at relatively high altitude in the Southwestern Alps for farming and maintaining Alpine passes.

Chemical and isotopic equilibrium between CO2 and CH4 in fumarolic gas discharges: Generation of CH4 in arc magmatic-hydrothermal systems

The chemical and isotopic composition of fumarolic gases emitted from Nisyros Volcano, Greece, and of a single gas sample from Vesuvio, Italy, was investigated in order to determine the origin of methane (CH,) within two subduction-related magmatic-hydrothermal environments. Apparent temperatures derived from carbon isotope partitioning between CH4 and CO2 of around 340degreesC for Nisyros and 470degreesC for Vesuvio correlate well with aquifer temperatures as measured directly and/or inferred from compositional data using the H2O-H-2-CO2-CO-CH4 geothermometer. Thermodynamic modeling reveals chemical equilibrium between CH4, CO2 and H2O implying that carbon isotope partitioning between CO2 and CH, in both systems is controlled by aquifer temperature. N-2/(3) He and CH4/(3) He ratios of Nisyros fumarolic gases are unusually low for subduction zone gases and correspond to those of midoceanic ridge environments. Accordingly, CH4 may have been primarily generated through the reduction of CO, by H, in the absence of any organic matter following a Fischer-Tropsch-type reaction. However, primary occurrence of minor amounts of thermogenic CH4 and subsequent re-equilibration with co-existing CO2 cannot be ruled out entirely- CO2/He-3 ratios and delta(13)C(CO2) values imply that the evolved CO2 either derives from a metasomatized mantle or is a mixture between two components, one outgassing from an unaltered mantle and the other released by thermal breakdown of marine carbonates. The latter may contain traces of organic matter possibly decomposing to CH4 during thermometamorphism. Copyright (C) 2004 Elsevier Ltd.

Apparent stable-isotope heterogeneities in gangue carbonates of the Mississippi Valley-type Zn-Pb deposits of San-Vicente, Central Peru

The aim of the present communication is to emphasize that some variations of the measured delta(13)C and delta(18)O values are apparent, and due to analytical interferences caused by the presence of sulfur and organosulfur compounds in the analyzed carbonates. This is particularly relevant for isotopic studies on carbonate-hosted mineral deposits, where the nearly ubiquitous association of the host carbonates with organic matter and sulfides can certainly affect the metallogenetic interpretations. In this work two methods were used to overcome the disturbing effects of sulfides and organic matter: (1) sample pretreatment following the method proposed by Charef and Sheppard (1984), combining the oxidation of organic matter with sodium hypochlorite and trapping of the sulfur species with silver phosphate; and (2) laser-based microprobe extraction. Apparent isotopic variations in sparry dolomite from a single hand sample of zebra ore from the MVT Zn-Pb deposit, San Vicente, central Peru, are as large as 6 parts per thousand delta(13)C and 4 parts per thousand delta(18)O. These variations are reduced to several tenths of a per mil when the samples are pretreated. A careful examination of the effects of treatment with NaOCl and/or Ag3PO4 in relation to the concentration of sulfide inclusions indicates that the main disturbing effects for delta(13)C values are the presence of sulfur species and organic matter, whereas the delta(18)O values are mainly affected by the presence of sulfides. Fine- and medium-grained replacement carbonates from MVT and other sediment-hosted base metal deposits are potentially the most affected during isotope analysis, due to the common presence of organic matter and sulfides. Using in situ laser microprobe techniques, it is possible to determine isotopic variations at a sub-millimeter scale. Our results show that laser extraction analysis allows a more precise sampling of the carbonate minerals, and minimizes contamination of the sample with sulfides and to some extent with intergrown organic matter. However, there is an isotopic shift associated with the laser extraction technique, of the order of 0.5-1 parts per thousand for delta(13)C and delta(18)O values.

Multi-scale systems analysis of plant development : beyond cellular signaling and tissue morphodynamics

Les plantes sont essentielles pour les sociétés humaines. Notre alimentation quotidienne, les matériaux de constructions et les sources énergétiques dérivent de la biomasse végétale. En revanche, la compréhension des multiples aspects développementaux des plantes est encore peu exploitée et représente un sujet de recherche majeur pour la science. L'émergence des technologies à haut débit pour le séquençage de génome à grande échelle ou l'imagerie de haute résolution permet à présent de produire des quantités énormes d'information. L'analyse informatique est une façon d'intégrer ces données et de réduire la complexité apparente vers une échelle d'abstraction appropriée, dont la finalité est de fournir des perspectives de recherches ciblées. Ceci représente la raison première de cette thèse. En d'autres termes, nous appliquons des méthodes descriptives et prédictives combinées à des simulations numériques afin d'apporter des solutions originales à des problèmes relatifs à la morphogénèse à l'échelle de la cellule et de l'organe. Nous nous sommes fixés parmi les objectifs principaux de cette thèse d'élucider de quelle manière l'interaction croisée des phytohormones auxine et brassinosteroïdes (BRs) détermine la croissance de la cellule dans la racine du méristème apical d'Arabidopsis thaliana, l'organisme modèle de référence pour les études moléculaires en plantes. Pour reconstruire le réseau de signalement cellulaire, nous avons extrait de la littérature les informations pertinentes concernant les relations entre les protéines impliquées dans la transduction des signaux hormonaux. Le réseau a ensuite été modélisé en utilisant un formalisme logique et qualitatif pour pallier l'absence de données quantitatives. Tout d'abord, Les résultats ont permis de confirmer que l'auxine et les BRs agissent en synergie pour contrôler la croissance de la cellule, puis, d'expliquer des observations phénotypiques paradoxales et au final, de mettre à jour une interaction clef entre deux protéines dans la maintenance du méristème de la racine. Une étude ultérieure chez la plante modèle Brachypodium dystachion (Brachypo- dium) a révélé l'ajustement du réseau d'interaction croisée entre auxine et éthylène par rapport à Arabidopsis. Chez ce dernier, interférer avec la biosynthèse de l'auxine mène à la formation d'une racine courte. Néanmoins, nous avons isolé chez Brachypodium un mutant hypomorphique dans la biosynthèse de l'auxine qui affiche une racine plus longue. Nous avons alors conduit une analyse morphométrique qui a confirmé que des cellules plus anisotropique (plus fines et longues) sont à l'origine de ce phénotype racinaire. Des analyses plus approfondies ont démontré que la différence phénotypique entre Brachypodium et Arabidopsis s'explique par une inversion de la fonction régulatrice dans la relation entre le réseau de signalisation par l'éthylène et la biosynthèse de l'auxine. L'analyse morphométrique utilisée dans l'étude précédente exploite le pipeline de traitement d'image de notre méthode d'histologie quantitative. Pendant la croissance secondaire, la symétrie bilatérale de l'hypocotyle est remplacée par une symétrie radiale et une organisation concentrique des tissus constitutifs. Ces tissus sont initialement composés d'une douzaine de cellules mais peuvent aisément atteindre des dizaines de milliers dans les derniers stades du développement. Cette échelle dépasse largement le seuil d'investigation par les moyens dits 'traditionnels' comme l'imagerie directe de tissus en profondeur. L'étude de ce système pendant cette phase de développement ne peut se faire qu'en réalisant des coupes fines de l'organe, ce qui empêche une compréhension des phénomènes cellulaires dynamiques sous-jacents. Nous y avons remédié en proposant une stratégie originale nommée, histologie quantitative. De fait, nous avons extrait l'information contenue dans des images de très haute résolution de sections transverses d'hypocotyles en utilisant un pipeline d'analyse et de segmentation d'image à grande échelle. Nous l'avons ensuite combiné avec un algorithme de reconnaissance automatique des cellules. Cet outil nous a permis de réaliser une description quantitative de la progression de la croissance secondaire révélant des schémas développementales non-apparents avec une inspection visuelle classique. La formation de pôle de phloèmes en structure répétée et espacée entre eux d'une longueur constante illustre les bénéfices de notre approche. Par ailleurs, l'exploitation approfondie de ces résultats a montré un changement de croissance anisotropique des cellules du cambium et du phloème qui semble en phase avec l'expansion du xylème. Combinant des outils génétiques et de la modélisation biomécanique, nous avons démontré que seule la croissance plus rapide des tissus internes peut produire une réorientation de l'axe de croissance anisotropique des tissus périphériques. Cette prédiction a été confirmée par le calcul du ratio des taux de croissance du xylème et du phloème au cours de développement secondaire ; des ratios élevés sont effectivement observés et concomitant à l'établissement progressif et tangentiel du cambium. Ces résultats suggèrent un mécanisme d'auto-organisation établi par un gradient de division méristématique qui génèrent une distribution de contraintes mécaniques. Ceci réoriente la croissance anisotropique des tissus périphériques pour supporter la croissance secondaire. - Plants are essential for human society, because our daily food, construction materials and sustainable energy are derived from plant biomass. Yet, despite this importance, the multiple developmental aspects of plants are still poorly understood and represent a major challenge for science. With the emergence of high throughput devices for genome sequencing and high-resolution imaging, data has never been so easy to collect, generating huge amounts of information. Computational analysis is one way to integrate those data and to decrease the apparent complexity towards an appropriate scale of abstraction with the aim to eventually provide new answers and direct further research perspectives. This is the motivation behind this thesis work, i.e. the application of descriptive and predictive analytics combined with computational modeling to answer problems that revolve around morphogenesis at the subcellular and organ scale. One of the goals of this thesis is to elucidate how the auxin-brassinosteroid phytohormone interaction determines the cell growth in the root apical meristem of Arabidopsis thaliana (Arabidopsis), the plant model of reference for molecular studies. The pertinent information about signaling protein relationships was obtained through the literature to reconstruct the entire hormonal crosstalk. Due to a lack of quantitative information, we employed a qualitative modeling formalism. This work permitted to confirm the synergistic effect of the hormonal crosstalk on cell elongation, to explain some of our paradoxical mutant phenotypes and to predict a novel interaction between the BREVIS RADIX (BRX) protein and the transcription factor MONOPTEROS (MP),which turned out to be critical for the maintenance of the root meristem. On the same subcellular scale, another study in the monocot model Brachypodium dystachion (Brachypodium) revealed an alternative wiring of auxin-ethylene crosstalk as compared to Arabidopsis. In the latter, increasing interference with auxin biosynthesis results in progressively shorter roots. By contrast, a hypomorphic Brachypodium mutant isolated in this study in an enzyme of the auxin biosynthesis pathway displayed a dramatically longer seminal root. Our morphometric analysis confirmed that more anisotropic cells (thinner and longer) are principally responsible for the mutant root phenotype. Further characterization pointed towards an inverted regulatory logic in the relation between ethylene signaling and auxin biosynthesis in Brachypodium as compared to Arabidopsis, which explains the phenotypic discrepancy. Finally, the morphometric analysis of hypocotyl secondary growth that we applied in this study was performed with the image-processing pipeline of our quantitative histology method. During its secondary growth, the hypocotyl reorganizes its primary bilateral symmetry to a radial symmetry of highly specialized tissues comprising several thousand cells, starting with a few dozens. However, such a scale only permits observations in thin cross-sections, severely hampering a comprehensive analysis of the morphodynamics involved. Our quantitative histology strategy overcomes this limitation. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with an automated cell type recognition algorithm, it allows precise quantitative characterization of vascular development and reveals developmental patterns that were not evident from visual inspection, for example the steady interspace distance of the phloem poles. Further analyses indicated a change in growth anisotropy of cambial and phloem cells, which appeared in phase with the expansion of xylem. Combining genetic tools and computational modeling, we showed that the reorientation of growth anisotropy axis of peripheral tissue layers only occurs when the growth rate of central tissue is higher than the peripheral one. This was confirmed by the calculation of the ratio of the growth rate xylem to phloem throughout secondary growth. High ratios are indeed observed and concomitant with the homogenization of cambium anisotropy. These results suggest a self-organization mechanism, promoted by a gradient of division in the cambium that generates a pattern of mechanical constraints. This, in turn, reorients the growth anisotropy of peripheral tissues to sustain the secondary growth.

Partial protection and intrathecal invasion of CD8(+) T cells in acute canine distemper virus infection.

Initial non-inflammatory demyelination in canine distemper virus infection (CDV) develops against a background of severe immunosuppression and is therefore, thought to be virus-induced. However, recently we found a marked invasion of T cells throughout the central nervous system (CNS) in dogs with acute distemper despite drastic damage to the immune system. In the present study, this apparent paradox was further investigated by immunophenotyping of lymphocytes, following experimental CDV challenge in vaccinated and non-vaccinated dogs. In contrast to CDV infected, unprotected dogs, vaccinated dogs did not become immunosuppressed and exhibited a strong antiviral immune response following challenge with virulent CDV. In unprotected dogs rapid and drastic lymphopenia was initially due to depletion of T cells. In peripheral blood, CD4(+) T cells were more sensitive and depleted earlier and for a longer time than CD8(+) cells which recovered soon. In the cerebrospinal fluid (CSF) we could observe an increase in the T cell to B cell and CD8(+) to CD4(+) ratios. Thus, partial protection of the CD8(+) cell population could explain why part of the immune function in acute distemper is preserved. As found earlier, T cells invaded the CNS parenchyma in these dogs but also in the protected challenged dogs, which did not develop any CNS disease at all. Since markers of T cell activation were upregulated in both groups of animals, this phenomenon could in part be related to non-specific penetration of activated T cells through the blood brain barrier. However, in diseased animals much larger numbers of T cells were found in the CNS than in the protected dogs, suggesting that massive invasion of T cells in the brain requires CDV expression in the CNS.

Monoclonal antibody against a "Pan-T-cell" antigen expressed by thymocytes, peripheral T-lymphocytes and T-cell leukemias.

A monoclonal antibody, LAU-A1, which selectively reacts with all cells of the T-lineage, was derived from a fusion between spleen cells of a mouse immunized with paediatric thymocytes and mouse myeloma P X 63/Ag8 cells. As shown by an antibody-binding radioimmunoassay and analysis by flow microfluorometry of cells labelled by indirect immunofluorescence, the LAU-A1 antibody reacted with all six T-cell lines but not with any of the B-cell lines or myeloid cell lines tested from a panel of 17 human hematopoietic cell lines. The LAU-A1 antibody was also shown to react with the majority of thymocytes and E-rosette-enriched peripheral blood lymphocytes. Among the malignant cell populations tested, the blasts from all 20 patients with acute T-cell lymphoblastic leukemia (T-ALL) were found to react with the LAU-A1 antibody, whereas blasts from 85 patients with common ALL and 63 patients with acute myeloid leukemias were entirely negative. Examination of frozen tissue sections from fetal and adult thymuses stained by an indirect immunoperoxidase method revealed that cells expressing the LAU-A1 antigen were localized in both the cortex and the medulla. From the very broad reactivity spectrum of LAU-A1 antibody, we conclude that this antibody is directed against a T-cell antigen expressed throughout the T-cell differentiation lineage. SDS-PAGE analysis of immunoprecipitates formed by LAU-A1 antibody with detergent lysates of radiolabeled T-cells showed that the LAU-A1 antigen had an apparent mol. wt of 76,000 under non-reducing conditions. Under reducing conditions a single band with an apparent mol. wt of 40,000 was observed. Two-dimensional SDS-PAGE analysis confirmed that the 76,000 mol. wt component consisted of an S-S-linked dimeric complex. The surface membrane expression of LAU-A1 antigen on HSB-2 T-cells was modulated when these cells were cultured in the presence of LAU-A1 antibody. Re-expression of LAU-A1 antigen occurred within 24 hr after transfer of the modulated cells into antibody-free medium.