Invader immunology: invasion history alters immune system function in cane toads (Rhinella marina) in tropical Australia.

Because an individual's investment into the immune system may modify its dispersal rate, immune function may evolve rapidly in an invader. We collected cane toads (Rhinella marina) from sites spanning their 75-year invasion history in Australia, bred them, and raised their progeny in standard conditions. Evolved shifts in immune function should manifest as differences in immune responses among the progeny of parents collected in different locations. Parental location did not affect the offspring's cell-mediated immune response or stress response, but blood from the offspring of invasion-front toads had more neutrophils, and was more effective at phagocytosis and killing bacteria. These latter measures of immune function are negatively correlated with rate of dispersal in free-ranging toads. Our results suggest that the invasion of tropical Australia by cane toads has resulted in rapid genetically based compensatory shifts in the aspects of immune responses that are most compromised by the rigours of long-distance dispersal.

Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria.

The sensor kinase GacS and the response regulator GacA are members of a two-component system that is present in a wide variety of gram-negative bacteria and has been studied mainly in enteric bacteria and fluorescent pseudomonads. The GacS/GacA system controls the production of secondary metabolites and extracellular enzymes involved in pathogenicity to plants and animals, biocontrol of soilborne plant diseases, ecological fitness, or tolerance to stress. A current model proposes that GacS senses a still-unknown signal and activates, via a phosphorelay mechanism, the GacA transcription regulator, which in turn triggers the expression of target genes. The GacS protein belongs to the unorthodox sensor kinases, characterized by an autophosphorylation, a receiver, and an output domain. The periplasmic loop domain of GacS is poorly conserved in diverse bacteria. Thus, a common signal interacting with this domain would be unexpected. Based on a comparison with the transcriptional regulator NarL, a secondary structure can be predicted for the GacA sensor kinases. Certain genes whose expression is regulated by the GacS/GacA system are regulated in parallel by the small RNA binding protein RsmA (CsrA) at a posttranscriptional level. It is suggested that the GacS/GacA system operates a switch between primary and secondary metabolism, with a major involvement of posttranscriptional control mechanisms.

Compressibility of Oxisol aggregates under no-till in response to soil water potential

The system of no-till sowing stands out as being a technology that suits the objectives of more rational use of the soil and greater protection against the erosion. However, through till, any of it, occurs modifications of the soil's structure. This current work aims to study the influence of the energy state of the water and of the organic matter on the mechanism of compaction of Red Oxisol under no-till management system. Humid and non-deformed sample were collected in horizon AP of two agricultural areas under no-till, with and without rotation of cultures. In the laboratory, these samples were broken into fragments and sifted to obtain aggregates of 4 to 5 mm sized, which were placed in equilibrium under four matrix potentials. Thereafter, they were exposed to uni-dimensional compression with pressures varying from 32 to 1,000 kPa. The results in such a way show that the highest compressibility of aggregates both for the tilling with rotation of cultures as for the tilling without rotation of cultures, occurred for matrix potential -32 kPa (humidity of 0.29-0.32 kg kg-1, respectively), while the minor occurred for the potentials of -1 and -1,000 kPa (humidity of 0.35 and 0.27 kg kg-1, respectively), indicating that this soil should not be worked with humidity ranging around 0.29 to 0.32 kg kg-1 and the highest reduction of volume of aggregates was obtained for the mechanical pressures lower than 600 inferior kPa, indicating that these soils showed to be very influenced by compression, when exposed to mechanical work. Also, the aggregates of soil under no-till and rotation of crops presented higher sensitivity to the compression than the aggregates of soil under no-till and without rotation of crops, possibly for having better structural conditions given to a higher content of organic matter.

The wound watch: an objective staging system for wounds in the diabetic (db/db) mouse model.

As in cancer biology, in wound healing there is a need for objective staging systems to decide for the best treatment and predictors of outcome. We developed in the diabetic (db/db) wound healing model, a staging system, the "wound watch," based on the quantification of angiogenesis and cell proliferation in open wounds. In chronic wounds, there is often a lack of cellular proliferation and angiogenesis that leads to impaired healing. The wound watch addresses this by quantifying the proliferative phase of wound healing in two dimensions (cellular division and angiogenesis). The results are plotted in a two-dimensional graph to monitor the course of healing and compare the response to different treatments.

Nutrient supply by mass flow and diffusion to maize plants in response to soil aggregate size and water potential

Nutrients are basically transported to the roots by mass flow and diffusion. The aim of this study was to quantify the contribution of these two mechanisms to the acquisition of macronutrients (N, P, K, Ca, Mg, and S) and cationic micronutrients (Fe, Mn, Zn, and Cu) by maize plants as well as xylem exudate volume and composition in response to soil aggregate size and water availability. The experiment was conducted in a greenhouse with samples of an Oxisol, from under two management systems: a region of natural savanna-like vegetation (Cerradão, CER) and continuous maize under conventional management for over 30 years (CCM). The treatments were arranged in a factorial [2 x (1 + 2) x 2] design, with two management systems (CER and CCM), (1 + 2) soil sifted through a 4 mm sieve and two aggregate classes (< 0.5 mm and 0.5 - 4.0 mm) and two soil matric potentials (-40 and -10 kPa). These were evaluated in a randomized block design with four replications. The experiment was conducted for 70 days after sowing. The influence of soil aggregate size and water potential on the nutrient transport mechanisms was highest in soil samples with higher nutrient concentrations in solution, in the CER system; diffusion became more relevant when water availability was higher and in aggregates < 0.5 mm. The volume of xylem exudate collected from maize plants increased with the decrease in aggregate size and the increased availability of soil water in the CER system. The highest Ca and Mg concentrations in the xylem exudate of plants grown on samples from the CER system were related to the high concentrations of these nutrients in the soil solution of this management system.

"In vitro" study of the molecular mechanism of the "E.Coli" Hsp 70/Hsp40/NEF chaperone system and its interactions with ?-sinuclein

SUMMARY Under stressful conditions, mutant or post-translationally modified proteins may spontaneously misfold and form toxie species, which may further assemble into a continuum of increasingly large and insoluble toxic oligomers that may further condense into less toxic, compact amyloids in the cell Intracellular accumulation of aggregated proteins is a common denominator of several neurodegenerative diseases. To cope with the cytotoxicity induced by abnormal, aggregated proteins, cells have evolved various defence mechanisms among which, the molecular chaperones Hsp70. Hsp70 (DnaK in E. coii) is an ATPase chaperone involved in many physiological processes in the cell, such as assisting de novo protein folding, dissociating native protein oligomers and serving as pulling motors in the import of polypeptides into organelles. In addition, Hsp70 chaperones can actively solubilize and reactivate stable protein aggregates, such as heat- or mutation-induced aggregates. Hsp70 requires the cooperation of two other co-chaperones: Hsp40 and NEF (Nucleotide exchange factor) to fulfil its unfolding activity. In the first experimental section of this thesis (Chapter II), we studied by biochemical analysis the in vitro interaction between recombinant human aggregated α-synuclein (a-Syn oligomers) mimicking toxic a-Syn oligomers species in PD brains, with a model Hsp70/Hsp40 chaperone system (the E. coii DnaK/DnaJ/GrpE). We found that chaperone-mediated unfolding of two denatured model enzymes were strongly affected by α-Syn oligomers but, remarkably, not by monomers. This in vitro observed dysfunction of the Hsp70 chaperone system resulted from the sequestration of the Hsp40 proteins by the oligomeric α-synuclein species. In the second experimental part (Chapter III), we performed in vitro biochemical analysis of the co-chaperone function of three E. coii Hsp40s proteins (DnaJ, CbpA and DjlA) in the ATP-fuelled DnaK-mediated refolding of a model DnaK chaperone substrate into its native state. Hsp40s activities were compared using dose-response approaches in two types of in vitro assays: refolding of heat-denatured G6PDH and DnaK-mediated ATPase activity. We also observed that the disaggregation efficiency of Hsp70 does not directly correlate with Hsp40 binding affinity. Besides, we found that these E. coii Hsp40s confer substrate specificity to DnaK, CbpA being more effective in the DnaK-mediated disaggregation of large G6PDH aggregates than DnaJ under certain conditions. Sensibilisées par différents stress ou mutations, certaines protéines fonctionnelles de la cellule peuvent spontanément se convertir en formes inactives, mal pliées, enrichies en feuillets bêta, et exposant des surfaces hydrophobes favorisant l'agrégation. Cherchant à se stabiliser, les surfaces hydrophobes peuvent s'associer aux régions hydrophobes d'autres protéines mal pliées, formant des agrégats protéiques stables: les amyloïdes. Le dépôt intracellulaire de protéines agrégées est un dénominateur commun à de nombreuses maladies neurodégénératives. Afin de contrer la cytotoxicité induite par les protéines agrégées, les cellules ont développé plusieurs mécanismes de défense, parmi lesquels, les chaperonnes moléculaires Hsp70. Hsp70 nécessite la collaboration de deux autres co-chaperonnes : Hsp40 et NEF pour accomplir son activité de désagrégation. Hsp70 (DnaK, chez E. coli) est impliquée par ailleurs dans d'autres fonctions physiologiques telles que l'assistanat de protéines néosynthétisées à la sortie du ribosome, ou le transport transmembranaire de polypeptides. Par ailleurs, les chaperonnes Hsp70 peuvent également solubiliser et réactiver des protéines agrégées à la suite d'un stress ou d'une mutation. Dans la première partie expérimentale de cette thèse (Chapter II), nous avons étudié in vitro l'interaction entre les oligomères d'a-synucleine, responsables entre autres, de la maladie de Parkinson, et le système chaperon Hsp70/Hsp40 (système Escherichia coli DnaK/DnaJ/GrpE). Nous avons démontré que contrairement aux monomères, les oligomères d'a-synucleine inhibaient le système chaperon lors du repliement de protéines agrégées. Cette dysfonction du système chaperon résulte de la séquestration des chaperonnes Hsp40 par les oligomères d'a-synucleine. La deuxième partie expérimentale (Chapitre III) est consacrée à une étude in vitro de la fonction co-chaperonne de trois Hsp40 d'is. coli (DnaJ, CbpA, et DjlA) lors de la désagrégation par DnaK d'une protéine pré-agrégée. Leurs activités ont été comparées par le biais d'une approche dose-réponse au niveau de deux analyses enzymatiques: le repliement de la protéine agrégée et l'activité ATPase de DnaK. Par ailleurs, nous avons mis en évidence que l'efficacité de désagrégation d'Hsp70 et l'affinité des chaperonnes Hsp40 vis-à-vis de leur substrat n'étaient pas corrélées positivement. Nous avons également montré que ces trois chaperonnes Hsp40 étaient directement impliquées dans la spécificité des fonctions accomplies par les chaperonnes Hsp70. En effet, DnaK en présence de CbpA assure la désagrégation de large agrégats protéiques avec une efficacité nettement plus accrue qu'en présence de DnaJ.

Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

Hysteresis in a system driven by either generalized force or displacement variables: martensitic phase transition in single-crystalline Cu-Zn-Al

We report on experiments aimed at comparing the hysteretic response of a Cu-Zn-Al single crystal undergoing a martensitic transition under strain-driven and stress-driven conditions. Strain-driven experiments were performed using a conventional tensile machine while a special device was designed to perform stress-driven experiments. Significant differences in the hysteresis loops were found. The strain-driven curves show reentrant behavior yield point which is not observed in the stress-driven case. The dissipated energy in the stress-driven curves is larger than in the strain-driven ones. Results from recently proposed models qualitatively agree with experiments.

Soil tillage systems: changes in soil structure and crop response

The introduction and intensification of no-tillage systems in Brazilian agriculture in recent decades have created a new scenario, increasing concerns about soil physical properties. The objective of this study was to assess the effects of different tillage systems on some physical properties of an Ultisol previously under native grassland. Five tillage methods were tested: no-tillage (NT), chiseling (Ch), no-tillage with chiseling every two years (NTCh2), chiseling using an equipment with a clod-breaking roller (ChR) and chiseling followed by disking (ChD). The bulk density, macroporosity, microporosity and total porosity, mechanical resistance to penetration, water infiltration into the soil and crop yields were evaluated. The values of soil bulk density, mechanical resistance to penetration and microporosity increased as macroporosity decreased. Soil bulk density was lower in tillage systems with higher levels of tillage/soil mobilization; highest values were observed in NT and the lowest in the ChD system. The water infiltration rate was highest in the ChR system, followed by the systems ChD, NT and NTCh2, while crop yields were higher in systems with less soil mobilization.

Non-invasive detection of coronary endothelial response to sequential handgrip exercise in coronary artery disease patients and healthy adults.

OBJECTIVES: Our objective is to test the hypothesis that coronary endothelial function (CorEndoFx) does not change with repeated isometric handgrip (IHG) stress in CAD patients or healthy subjects. BACKGROUND: Coronary responses to endothelial-dependent stressors are important measures of vascular risk that can change in response to environmental stimuli or pharmacologic interventions. The evaluation of the effect of an acute intervention on endothelial response is only valid if the measurement does not change significantly in the short term under normal conditions. Using 3.0 Tesla (T) MRI, we non-invasively compared two coronary artery endothelial function measurements separated by a ten minute interval in healthy subjects and patients with coronary artery disease (CAD). METHODS: Twenty healthy adult subjects and 12 CAD patients were studied on a commercial 3.0 T whole-body MR imaging system. Coronary cross-sectional area (CSA), peak diastolic coronary flow velocity (PDFV) and blood-flow were quantified before and during continuous IHG stress, an endothelial-dependent stressor. The IHG exercise with imaging was repeated after a 10 minute recovery period. RESULTS: In healthy adults, coronary artery CSA changes and blood-flow increases did not differ between the first and second stresses (mean % change ±SEM, first vs. second stress CSA: 14.8%±3.3% vs. 17.8%±3.6%, p = 0.24; PDFV: 27.5%±4.9% vs. 24.2%±4.5%, p = 0.54; blood-flow: 44.3%±8.3 vs. 44.8%±8.1, p = 0.84). The coronary vasoreactive responses in the CAD patients also did not differ between the first and second stresses (mean % change ±SEM, first stress vs. second stress: CSA: -6.4%±2.0% vs. -5.0%±2.4%, p = 0.22; PDFV: -4.0%±4.6% vs. -4.2%±5.3%, p = 0.83; blood-flow: -9.7%±5.1% vs. -8.7%±6.3%, p = 0.38). CONCLUSION: MRI measures of CorEndoFx are unchanged during repeated isometric handgrip exercise tests in CAD patients and healthy adults. These findings demonstrate the repeatability of noninvasive 3T MRI assessment of CorEndoFx and support its use in future studies designed to determine the effects of acute interventions on coronary vasoreactivity.

Systemic antibodies can inhibit mouse mammary tumor virus-driven superantigen response in mucosa-associated lymphoid tissues.

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer's patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer's patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer's patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer's patches or NALT.

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

Fertilization response likelihood for the interpretation of leaf analyses

Leaf analysis is the chemical evaluation of the nutritional status where the nutrient concentrations found in the tissue reflect the nutritional status of the plants. Thus, a correct interpretation of the results of leaf analysis is fundamental for an effective use of this tool. The purpose of this study was to propose and compare the method of Fertilization Response Likelihood (FRL) for interpretation of leaf analysis with that of the Diagnosis and Recommendation Integrated System (DRIS). The database consisted of 157 analyses of the N, P, K, Ca, Mg, S, Cu, Fe, Mn, Zn, and B concentrations in coffee leaves, which were divided into two groups: low yield (< 30 bags ha-1) and high yield (> 30 bags ha-1). The DRIS indices were calculated using the method proposed by Jones (1981). The fertilization response likelihood was computed based on the approximation of normal distribution. It was found that the Fertilization Response Likelihood (FRL) allowed an evaluation of the nutritional status of coffee trees, coinciding with the DRIS-based diagnoses in 84.96 % of the crops.

A colorectal cancer classification system that associates cellular phenotype and responses to therapy.

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise, individualized treatment strategies are needed. To that end, we analyzed gene expression profiles from 1,290 CRC tumors using consensus-based unsupervised clustering. The resultant clusters were then associated with therapeutic response data to the epidermal growth factor receptor-targeted drug cetuximab in 80 patients. The results of these studies define six clinically relevant CRC subtypes. Each subtype shares similarities to distinct cell types within the normal colon crypt and shows differing degrees of 'stemness' and Wnt signaling. Subtype-specific gene signatures are proposed to identify these subtypes. Three subtypes have markedly better disease-free survival (DFS) after surgical resection, suggesting these patients might be spared from the adverse effects of chemotherapy when they have localized disease. One of these three subtypes, identified by filamin A expression, does not respond to cetuximab but may respond to cMET receptor tyrosine kinase inhibitors in the metastatic setting. Two other subtypes, with poor and intermediate DFS, associate with improved response to the chemotherapy regimen FOLFIRI in adjuvant or metastatic settings. Development of clinically deployable assays for these subtypes and of subtype-specific therapies may contribute to more effective management of this challenging disease.

Crop response to organic fertilization with supplementary mineral nitrogen

Animal manure is applied to the soil as a nutrient source, especially of nitrogen, to plants. However, manure application rates can be reduced with the use of N fertilizer in topdressing. The aim of this study was to evaluate crop responses to different application rates of animal manure sources, used alone and supplemented with mineral N topdressing, in a no-tillage system. The study was carried out from 2005 to 2008 on a Hapludalf soil. The treatments consisted of rates of 10, 20 and 30 m³ ha-1 of pig slurry (PS), and of 1 and 2 t ha-1 of turkey manure (TM), applied alone and supplemented with topdressed N fertilizer (TNF), as well as two controls, mineral fertilization (NPK) and one control without fertilizer application. Grain yield in common bean and maize, and dry matter yield and nutrient accumulation in common bean, maize and black oat crops were evaluated. Nitrogen application in topdressing in maize and common bean, especially when PS was used at rates of 20 and 30 m³ ha-1, and TM, at 2 t ha-1, proved effective in increasing the crop grain yields, showing the viability of the combined use of organic and industrialized mineral sources. Nitrogen accumulation in maize and common bean tissues was the indicator most strongly related to grain yield, in contrast with the apparent nutrient recovery, which was not related to the N, P and K quantities applied in the organic sources. No clear residual effect of N topdressing of maize and common bean was observed on the dry matter yield of black oat grown in succession to the main crops with PS and TM applications.