TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

Determination of ferric heme-human serum albumin by 1H NMR relaxometry

A new method for the accurate determination of ferric heme-human serum albumin (heme-HSA) at concentrations down to the physiological level, i.e., in the micromolar concentration range, is proposed. This method is based on the (1)H NMR relaxometric properties of heme-HSA. Actually, the binding of the paramagnetic ferric heme to the primary binding site of HSA determines a strong paramagnetic enhancement of the water (1)H NMR relaxation rate. Although a linear relationship may be seen by operating at 20 MHz on conventional electromagnets, the method here reported is improved by working at 0.02 MHz on a field-cycling instrument. This (1)H NMR relaxometric method does not suffer from the presence in serum of heme catabolites (e.g., bilirubin) that affect significantly the optical determination of ferric heme-HSA in the micromolar concentration range. Paramagnetic ferric hemoglobin contribution may be selectively quenched by cyanide binding.     

Epidemiology of CKD Regression in Patients under Nephrology Care

Chronic Kidney Disease (CKD) regression is considered as an infrequent renal outcome, limited to early stages, and associated with higher mortality. However, prevalence, prognosis and the clinical correlates of CKD regression remain undefined in the setting of nephrology care. This is a multicenter prospective study in 1418 patients with established CKD (eGFR: 60–15 ml/min/1.73m²) under nephrology care in 47 outpatient clinics in Italy from a least one year. We defined CKD regressors as a ΔGFR ≥0 ml/min/1.73 m²/year. ΔGFR was estimated as the absolute difference between eGFR measured at baseline and at follow up visit after 18–24 months, respectively. Outcomes were End Stage Renal Disease (ESRD) and overall-causes Mortality.391 patients (27.6%) were identified as regressors as they showed an eGFR increase between the baseline visit in the renal clinic and the follow up visit. In multivariate regression analyses the regressor status was not associated with CKD stage. Low proteinuria was the main factor associated with CKD regression, accounting per se for 48% of the likelihood of this outcome. Lower systolic blood pressure, higher BMI and absence of autosomal polycystic disease (PKD) were additional predictors of CKD regression. In regressors, ESRD risk was 72% lower (HR: 0.28; 95% CI 0.14–0.57; p<0.0001) while mortality risk did not differ from that in non-regressors (HR: 1.16; 95% CI 0.73–1.83; p = 0.540). Spline models showed that the reduction of ESRD risk associated with positive ΔGFR was attenuated in advanced CKD stage. CKD regression occurs in about one-fourth patients receiving renal care in nephrology units and correlates with low proteinuria, BP and the absence of PKD. This condition portends better renal prognosis, mostly in earlier CKD stages, with no excess risk for mortality.     

Echocardiographic detection of congestive heart failure in postinfarction rats

In studies of congestive heart failure (CHF) treatment, it is essential to select animals with a similar degree of cardiac dysfunction. However, this is difficult to establish without hemodynamic evaluation in rat postinfarction-induced CHF. This study aimed to diagnose CHF in long-term follow-up postinfarction rats using only echocardiographic criteria through a J-tree cluster analysis and Fisher's linear discriminant function. Two sets of sham and infarcted rats were studied. The first was used to perform cluster analysis and the second to prospectively validate the results. Six months after inducing myocardial infarction (MI), rats were subjected to transthoracic echocardiography. Infarct size was measured by histological analysis. Six echocardiographic variables were used in the cluster analysis: left ventricular (LV) systolic dimension, LV diastolic dimension-to-body weight ratio, left atrial diameter-to-body weight ratio, LV posterior wall shortening velocity, E wave, and isovolumetric relaxation time. Cluster analysis joined the rats into one sham and two MI groups. One MI cluster had more severe anatomical and echocardiographic changes and was called MI with heart failure (MI/HF+, n = 24, infarct size: 42.7 ± 5.8%). The other had less severe changes and was called MI without heart failure (MI/HF-, n = 11, infarct size: 32.3 ± 9.9%; P < 0.001 vs. MI/HF+). Three rats with small infarct size (21.6 ± 2.2%) presenting mild cardiac alterations were misallocated in the sham group. Fisher's linear discriminant function was built using these groups and used to prospectively classify additional groups of sham-operated (n = 20) and infarcted rats (n = 57) using the same echocardiographic parameters. The discriminant function therefore detected CHF with 100% specificity and 80% sensitivity considering allocation in MI/HF+ and sham group, and 100% specificity and 58.8% sensitivity considering MI/HF+ and MI/HF- groups, taking into account pathological criteria of CHF diagnosis. Echocardiographic analysis can be used to accurately predict congestive heart failure in postinfarction rats.     

Development and Validation of a 20K Single Nucleotide Polymorphism (SNP) Whole Genome Genotyping Array for Apple (Malus × domestica Borkh)

High-density SNP arrays for genome-wide assessment of allelic variation have made high resolution genetic characterization of crop germplasm feasible. A medium density array for apple, the IRSC 8K SNP array, has been successfully developed and used for screens of bi-parental populations. However, the number of robust and well-distributed markers contained on this array was not sufficient to perform genome-wide association analyses in wider germplasm sets, or Pedigree-Based Analysis at high precision, because of rapid decay of linkage disequilibrium. We describe the development of an Illumina Infinium array targeting 20K SNPs. The SNPs were predicted from re-sequencing data derived from the genomes of 13 Malus × domestica apple cultivars and one accession belonging to a crab apple species (M. micromalus). A pipeline for SNP selection was devised that avoided the pitfalls associated with the inclusion of paralogous sequence variants, supported the construction of robust multi-allelic SNP haploblocks and selected up to 11 entries within narrow genomic regions of ±5 kb, termed focal points (FPs). Broad genome coverage was attained by placing FPs at 1 cM intervals on a consensus genetic map, complementing them with FPs to enrich the ends of each of the chromosomes, and by bridging physical intervals greater than 400 Kbps. The selection also included ∼3.7K validated SNPs from the IRSC 8K array. The array has already been used in other studies where ∼15.8K SNP markers were mapped with an average of ∼6.8K SNPs per full-sib family. The newly developed array with its high density of polymorphic validated SNPs is expected to be of great utility for Pedigree-Based Analysis and Genomic Selection. It will also be a valuable tool to help dissect the genetic mechanisms controlling important fruit quality traits, and to aid the identification of marker-trait associations suitable for the application of Marker Assisted Selection in apple breeding programs.     

Changes in the ascorbate system in the response of pumpkin (Cucurbita pepo L.) roots to aluminium stress

The involvement of the ascorbate (AsA) system in the response of pumpkin (Cucurbita pepo L.) roots to aluminium stress was studied. The treatment of 5-day-old pumpkin seedlings with 50 microM aluminium sulphate resulted in approximately 60% inhibition of root growth within 48-60 h of treatment, while aluminium accumulated in the roots reaching a maximum within 48h. During the same period, the hydrogen peroxide content of the roots was strongly enhanced. The increased level of hydrogen peroxide was matched by both increased ascorbate peroxidase (APX) (EC 1.11.1.11) activity and ascorbate free radical reductase (AFRR) (EC 1.1.5.4) activity, while dehydroascorbate reductase (DHAR) (EC 1.8.5.1) and glutathione reductase (GR) (EC 1.6.4.2) did not change. The levels of AsA in the roots were also increased by the Al treatment. It was concluded that an oxidative burst is probably involved in the toxicity of Al in pumpkin roots and that plants react to the enhanced production of reactive oxygen species by expressing higher levels of scavenging systems such as the AsA-APX system.     

How can we possibly resolve the planet's nitrogen dilemma?

Nitrogen is the most crucial element in the production of nutritious feeds and foods. The production of reactive nitrogen by means of fossil fuel has thus far been able to guarantee the protein supply for the world population. Yet, the production and massive use of fertilizer nitrogen constitute a major threat in terms of environmental health and sustainability. It is crucial to promote consumer acceptance and awareness towards proteins produced by highly effective microorganisms, and their potential to replace proteins obtained with poor nitrogen efficiencies from plants and animals. The fact that reactive fertilizer nitrogen, produced by the Haber Bosch process, consumes a significant amount of fossil fuel worldwide is of concern. Moreover, recently, the prices of fossil fuels have increased the cost of reactive nitrogen by a factor of 3 to 5 times, while international policies are fostering the transition towards a more sustainable agro-ecology by reducing mineral fertilizers inputs and increasing organic farming. The combination of these pressures and challenges opens opportunities to use the reactive nitrogen nutrient more carefully. Time has come to effectively recover used nitrogen from secondary resources and to upgrade it to a legal status of fertilizer. Organic nitrogen is a slow-release fertilizer, it has a factor of 2.5 or higher economic value per unit nitrogen as fertilizer and thus adequate technologies to produce it, for instance by implementing photobiological processes, are promising. Finally, it appears wise to start the integration in our overall feed and food supply chains of the exceptional potential of biological nitrogen fixation. Nitrogen produced by the nitrogenase enzyme, either in the soil or in novel biotechnology reactor systems, deserves to have a ‘renaissance’ in the context of planetary governance in general and the increasing number of people who desire to be fed in a sustainable way in particular.     

High Meiofaunal and Nematodes Diversity around Mesophotic Coral Oases in the Mediterranean Sea

Although the mesophotic zone of the Mediterranean Sea has been poorly investigated, there is an increasing awareness about its ecological importance for its biodiversity, as fish nursery and for the recruitment of shallow water species. Along with coastal rocky cliffs, isolated coralligenous concretions emerging from muddy bottoms are typical structures of the Mediterranean Sea mesophotic zone. Coralligenous concretions at mesophotic depths in the South Tyrrhenian Sea were investigated to assess the role of these coralligenous oases in relation to the biodiversity of surrounding soft sediments. We show here that the complex structures of the coralligenous concretions at ca. 110 m depth influence the trophic conditions, the biodiversity and assemblage composition in the surrounding sediments even at considerable distances. Coral concretions not only represent deep oases of coral biodiversity but they also promote a higher biodiversity of the fauna inhabiting the surrounding soft sediments. Using the biodiversity of nematodes as a proxy of the total benthic biodiversity, a high turnover biodiversity within a 200 m distance from the coralligenous concretions was observed. Such turnover is even more evident when only rare taxa are considered and seems related to specific trophic conditions, which are influenced by the presence of the coralligenous structures. The presence of a high topographic complexity and the trophic enrichment make these habitats highly biodiverse, nowadays endangered by human activities (such as exploitation of commercial species such as Corallium rubrum, or trawling fisheries, which directly causes habitat destruction or indirectly causes modification in the sedimentation and re-suspension rates). We stress that the protection of the coralligenous sea concretions is a priority for future conservation policies at the scale of large marine ecosystems and that a complete census of these mesophotic oases of biodiversity should be a priority for future investigations in the Mediterranean Sea.     

Regulation of G protein-linked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase.

A recently identified family of guanine nucleotide exchange factors for Rho that includes PDZ-RhoGEF, LARG, and p115RhoGEF exhibits a unique structural feature consisting in the presence of area of similarity to regulators of G protein signaling (RGS). This RGS-like (RGL) domain provides a structural motif by which heterotrimeric G protein alpha subunits of the Galpha(12) family can bind and regulate the activity of RhoGEFs. Hence, these newly discovered RGL domain-containing RhoGEFs provide a direct link from Galpha(12) and Galpha(13) to Rho. Recently available data suggest, however, that tyrosine kinases can regulate the ability of G protein-coupled receptors (GPCRs) to stimulate Rho, although the underlying molecular mechanisms are still unknown. Here, we found that the activation of thrombin receptors endogenously expressed in HEK-293T cells leads to a remarkable increase in the levels of GTP-bound Rho within 1 min (11-fold) and a more limited but sustained activation (4-fold) thereafter, which lasts even for several hours. Interestingly, tyrosine kinase inhibitors did not affect the early phase of Rho activation, immediately after thrombin addition, but diminished the levels of GTP-bound Rho during the delayed phase. As thrombin receptors stimulate focal adhesion kinase (FAK) potently, we explored whether this non-receptor tyrosine kinase participates in the activation of Rho by GPCRs. We obtained evidence that FAK can be activated by thrombin, Galpha(12), Galpha(13), and Galpha(q) through both Rho-dependent and Rho-independent mechanisms and that PDZ-RhoGEF and LARG can in turn be tyrosine-phosphorylated through FAK in response to thrombin, thereby enhancing the activation of Rho in vivo. These data indicate that FAK may act as a component of a positive feedback loop that results in the sustained activation of Rho by GPCRs, thus providing evidence of the existence of a novel biochemical route by which tyrosine kinases may regulate the activity of Rho through the tyrosine phosphorylation of RGL-containing RhoGEFs.