Cell autonomous requirement for TGF-beta signaling during odontoblast differentiation and dentin matrix formation

TGF-β subtypes are expressed in tissues derived from cranial neural crest cells during early mouse craniofacial development. TGF-β signaling is critical for mediating epithelial-mesenchymal interactions, including those vital for tooth morphogenesis. However, it remains unclear how TGF-β signaling contributes to the terminal differentiation of odontoblast and dentin formation during tooth morphogenesis. Towards this end, we generated mice with conditional inactivation of the Tgfbr2 gene in cranial neural crest derived cells. Odontoblast differentiation was substantially delayed in the Tgfbr2^fl/fl;Wnt1-Cre mutant mice at E18.5. Following kidney capsule transplantation, Tgfbr2 mutant tooth germs expressed a reduced level of Col1a1 and Dspp and exhibited defects including decreased dentin thickness and absent dentinal tubules. In addition, the expression of the intermediate filament nestin was decreased in the Tgfbr2 mutant samples. Significantly, exogenous TGF-β2 induced nestin and Dspp expression in dental pulp cells in the developing tooth organ. Our data suggest that TGF-β signaling controls odontoblast maturation and dentin formation during tooth morphogenesis.     

Development of a semi-automatic system for pollen recognition

A semi-automatic system for pollen recognitionis studied for the european project ASTHMA. The goal of such a system is to provideaccurate pollen concentration measurements. This information can be used as well by thepalynologists, the clinicians or a forecastsystem to predict pollen dispersion. At first,our emphasis has been put on Cupressaceae, Olea, Poaceae and Urticaceae pollen types. The system is composed of two modules: pollengrain extraction and pollen grain recognition. In the first module, the pollen grains areobserved in light microscopy and are extractedautomatically from a pollen slide coloured withfuchsin and digitized in 3D. In the secondmodule, the pollen grain is analyzed forrecognition. To accomplish the recognition, itis necessary to work on 3D images and to usedetailed palynological knowledge. Thisknowledge describes the pollen types accordingto their main visible characteristerics and tothose which are important for recognition. Somepollen structures are identified like the porewith annulus in Poaceae, the reticulum in Oleaand similar pollen types or the cytoplasm inCupressaceae. The preliminary results show therecognition of some pollen types, likeUrticaceae or Poaceae or some groups of pollentypes, like reticulate group.     

Ultrasonic analysis of kinetic mechanism of hydrolysis of cellobiose by β-glucosidase

High-resolution ultrasonic spectroscopy (HR–US) was applied for real-time analysis of enzymatic hydrolysis of cellobiose by a β-glucosidase from Aspergillus niger (Novozyme 188) at 50 °C and pH 4.9. This technique is noninvasive, it does not require optical transparency and is suitable to continuously monitor the time dependence of the reaction progress in a broad range of experimental conditions. The time profiles of the amount of glucose released and the reaction rate were obtained from the time profile of ultrasonic velocity. The results are in good agreement with a discontinuous glucose assay (hexokinase method). The kinetic parameters of the reaction were estimated by fitting the ultrasonic time profiles of the reaction rates to several inhibition models. In addition, the equilibrium constant for the reaction of hydrolysis of cellobiose and the molar Gibbs free energy of hydrolysis were determined from the ultrasonic time profiles of concentration of glucose in the reverse reaction (glucose condensation). The results suggest the existence of more complex mechanisms regulating the activity of cellobiase than the combination of simple inhibitions. An extended kinetic model based on two sites for the competitive inhibitor (glucose) is proposed.     

Chemiluminescence of the Fenton reaction and a dihydroxybenzene-driven Fenton reaction

A dihydroxybenzenes(DHB)-driven Fenton reaction was found to be more efficient than a simple Fenton reaction based on OH radical and activated species production. The reason for this enhanced reactivity by [Fe DHB] complexes is not well understood, but results suggest that it may be explained by the formation of oxidation species different from those formed during the classic Fenton reactions. In previous work, greater concentrations, and more sustained production of OH over time were observed in DHB driven Fenton reactions versus neat Fenton and Fenton-like reactions. In this work, chemiluminescence (CL) was monitored, and compared to OH production kinetics. The CL of the DHB-driven Fenton reaction was shorter than that for sustained production of OH. CL appears to have been caused by excited Fe(IV) species stabilized by the DHB ligands initially formed in the reaction. Formation of this species would have to have occurred by the reaction between OH and Fe(III) in a DHB complex.     

4-1BB signaling synergizes with programmed death ligand 1 blockade to augment CD8 T cell responses during chronic viral infection

Previous studies have identified the inhibitory role that the programmed death 1 (PD-1) pathway plays during chronic infection. Blockade of this pathway results in rescue of viral-specific CD8 T cells, as well as reduction of viral loads in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). We tested the effect of combining PD ligand 1 (PD-L1) blockade with an agonistic regimen that induces 4-1BB costimulation during chronic LCMV infection. There is a boosting effect in the rescue of LCMV-specific CD8 T cell responses after dual treatment with PD-L1 blockade and 4-1BB agonistic Abs when the amount and timing of 4-1BB costimulation are carefully controlled. When PD-L1-blocking Abs are given together with a single low dose of anti-4-1BB agonistic Abs, there is an enhanced and stable expansion of viral-specific CD8 T cells. Conversely, when blocking Abs to PD-L1 are given with a repetitive high dose of anti-4-1BB, there is an initial synergistic expansion of viral-specific CD8 T cells by day 7, followed by dramatic apoptosis by day 14. Viral control paralleled CD8 T cell kinetics after dual treatment. By day 7 posttreatment, viral titers were lower in both of the combined regimens (compared with PD-L1 blockade alone). However, whereas the high dose of anti-4-1BB plus PD-L1 blockade resulted in rebound of viral titers to original levels, the low dose of anti-4-1BB plus PD-L1 blockade resulted in a stable reduction of viral loads. These findings demonstrate the importance of carefully manipulating the balance between activating and inhibitory signals to enhance T cell responses during chronic infection.     

The role of nurse plants in the establishment of shrub seedlings in the semi-arid subtropical Andes

In the nurse plant syndrome, or nurse association, seedlings (beneficiaries) are associated with adult shrubs/trees (benefactors). This phenomenon has been documented in several regions of the planet. Abiotic stress amelioration (one mechanism of facilitation) is one of the causes of this association. Most of the studies addressing the nurse syndrome have been conducted on spatial scales of a few hectares and have focused on only one or a few species. Moreover, there is an almost complete lack of studies addressing the incidence and characteristics of the nurse phenomenon in the arid Andes of South America. We undertook a first approximation to the study of facilitation in these ecosystems. The study was conducted at local and regional scales and involved the assessment of the spatial distribution of juveniles (seedlings and saplings) of 51 populations of 16 shrub and 12 cactus species in relation to shrub cover at 20 localities of the Prepuna (subtropical Andes of Bolivia and Argentina, 20–26°S). In terms of spatial distribution, the juveniles of most of the populations of shrubs studied were distributed both under the shrubs and in open spaces, thereby showing an apparent indifference to microhabitat. Globose and opuntioid cacti were preferentially distributed below the canopies of shrubs and were usually more associated with the dominant shrub species, which stood out as better potential nurses. The pattern was consistent throughout the region, including the more mesic and arid localities. The fact that Prepuna woody species are capable of establishing in open spaces would confer this region a greater resilience. Our findings further suggest that community dynamics in arid and semi-arid environments are more variable than previously thought.     

Effects of grazing on patch structure in a semi‐arid two‐phase vegetation mosaic

Abstract. Question: What are the grazing effects in the spatial organization and the internal structure of high and low cover patches from a two‐phase vegetation mosaic? Location: Patagonian steppe, Argentina. Methods: We mapped vegetation under three different grazing conditions: ungrazed, lightly grazed and heavily grazed. We analysed the spatial patterns of the dominant life forms. Also, in each patch type, we determined density, species composition, richness, diversity, size structure and dead biomass of grasses under different grazing conditions. Results: The vegetation was spatially organized in a two‐phase mosaic. High cover patches resulted from the association of grasses and shrubs and low cover patches were represented by scattered tussock grasses on bare ground. This spatial organization was not affected by grazing, but heavy grazing changed the grass species involved in high cover patches and reduced the density and cover of grasses in both patch types. Species richness and diversity in high cover patches decreased under grazing conditions, whereas in low cover patches it remained unchanged. Also, the decrease of palatable grasses was steeper in high cover patches than in low cover patches under grazing conditions. Conclusions: We suggest that although grazing promotes or inhibits particular species, it does not modify the mosaic structure of Patagonian steppe. The fact that the mosaic remained unchanged after 100 years of grazing suggests that grazing does not compromize population processes involved in maintaining patch structure, including seed dispersal, establishment or biotic interactions among life forms.