Effects of body size, trophic mode and larval habitat on Diptera stoichiometry: a regional comparison

Ecological stoichiometry has emerged as a tool for exploring nutrient demand and evolutionary responses to nutrient limitation. Previous studies of insects have found predictable variability in stoichiometry, both in relation to body size and trophic mode, at ordinal levels or higher. Our study further examines the evolutionary and ecological lability in these traits by comparing the effects of body size, trophic mode (larval and adult) and larval habitat on the stoichiometry of insects within one order (Diptera). The study also expands on previous work by analyzing trophic mode both at coarse (detritivore, herbivore, predator) and fine (high- vs low- nutrient quality resources within trophic categories) scales and by comparing nutrient stoichiometry in two geographical regions, Sweden and Arizona. As predicted, adults feeding on nectar or pollen had the lowest body N content in the dataset. Additionally, for Diptera with predatory larvae, species low N diets had lower body N content than those with high N diets. However, body N content was not consistently lower for all species with low N resources, as species feeding on plant material were indistinguishable in stoichiometry from predators with high N diets. We suggest that these results emerge because larval resource exploitation is poorly understood in herbivorous Diptera species. Body P content for Swedish Diptera decreased with body size for all trophic modes, and the only difference among trophic modes was that blood feeders had higher P content than other groups. The regional comparison further showed that the allometry of body P content is a labile trait that may vary at regional scales, as there was no allometric scaling of body P content in the Arizona data set, in contrast to the Swedish data set. These results are not easily explained by existing theoretical frameworks, but instead point to a general context-dependence of P stoichiometry, which should now be a focus for future work.     

Synthesis and biological evaluation of novel (4 or 5-aryl)pyrazolyl-indoles as inhibitors of interleukin-2 inducible T-cell kinase (ITK)

Interleukin-2 inducible T-cell kinase (ITK) is one of five kinases that belong to the Tec kinase family that plays an important role in T-cell and mast cell signaling. Various reports point to a role of ITK in the treatment of allergic asthma. For example, it was shown that mice lacking ITK have reduced airway hyperresponsiveness, inflammation and tracheal responses in an allergic asthma model. In this article, we disclose novel ITK inhibitors based on (4 or 5-aryl)pyrazolyl-indole scaffold that were also found to be selective for ITK over other kinases like IRK, CDK2, GSK3ß and PKA.     

Ecological Stoichiometry and Density Responses of Plant-Arthropod Communities on Cormorant Nesting Islands

Seabirds deposit large amounts of nutrient rich guano on their nesting islands. The increased nutrient availability strongly affects plants and consumers. Consumer response differs among taxonomic groups, but mechanisms causing these differences are poorly understood. Ecological stoichiometry might provide tools to understand these mechanisms. ES suggests that nutrient rich taxa are more likely to be nutrient limited than nutrient poorer taxa and are more favored under nutrient enrichment. Here, we quantified differences in the elemental composition of soil, plants, and consumers between islands with and without nesting cormorant colonies and tested predictions made based on ES by relating the elemental composition and the eventual mismatch between consumer and resource stoichiometry to observed density differences among the island categories. We found that nesting cormorants radically changed the soil nutrient content and thereby indirectly plant nutrient content and resource quality to herbivores. In contrast, consumers showed only small differences in their elemental composition among the island categories. While we cannot evaluate the cause of the apparent homeostasis of invertebrates without additional data, we can conclude that from the perspective of the next trophic level, there is no difference in diet quality (in terms of N and P content) between island categories. Thus, bottom-up effects seemed mainly be mediated via changes in resource quantity not quality. Despite a large potential trophic mismatch we were unable to observe any relation between the invertebrate stoichiometry and their density response to nesting cormorant colonies. We conclude that in our system stoichiometry is not a useful predictor of arthropod responses to variation in resource nutrient content. Furthermore, we found no strong evidence that resource quality was a prime determinant of invertebrate densities. Other factors like resource quantity, habitat structure and species interactions might be more important or masked stoichiometric effects.     

Continuous cultivation of human hamstring tenocytes on microcarriers in a spinner flask bioreactor system

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering‐based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon‐derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 × 10⁶ cells/1000 cm² surface) on Cytodex? type 3 microcarriers. Tenocytes' vitality, growth kinetics and glucose/lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD‐PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead‐to‐bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:142–151, 2014     

Anaphylatoxin receptors and complement regulatory proteins in human articular and non-articular chondrocytes: interrelation with cytokines

Tissue trauma induces an inflammatory response associated with a cytokine release that may engage complement pathways. Cytokine-mediated complement expression may contribute to cartilage degradation. Hence, we analysed the complement expression profile in primary articular and non-articular chondrocytes and its interrelation with cytokines. The expression of the anaphylatoxin receptors (C3aR and C5aR) and the complement regulatory proteins (CPRs) CD35, CD46, CD55 and CD59 was studied in cultured articular, auricular and nasoseptal chondrocytes using RTD-PCR and immunofluorescence labelling. The complement profile of peripheral blood mononuclear cells (PBMCs) was opposed to the expression in articular chondrocytes. The time-dependent regulation (6 and 24?h) of these complement factors was assessed in articular chondrocytes in response to the cytokines TNF??, IL-10 or TNF?? combined with IL-10 (each 10?ng/mL). C3aR, C5aR, CD46, CD55 and CD59 but almost no CD35 mRNA was expressed in any of chondrocyte types studied. The anaphylatoxin receptor expression was lower and that of the CRPs was higher in chondrocytes when compared with PBMCs. The majority of the studied complement factors were expressed at a significantly lower level in non-articular chondrocytes compared with the articular chondrocytes. TNF?? significantly increased the C3aR expression in chondrocytes after 6 and 24?h. TNF?? + IL-10 significantly downregulated C5aR and IL-10 significantly inhibited the CD46 and CD55 gene expression after 24?h. C5aR and CD55 could be localised in cartilage in situ. Anaphylatoxin receptors and CRPs are regulated differentially by TNF?? and IL-10. Whether cytokine-induced complement activation occurs in response to cartilage trauma has to be further identified.     

Fam60a defines a variant Sin3a‐Hdac complex in embryonic stem cells required for self‐renewal

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a‐Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3‐positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1‐phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.     

Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and nonmethylotrophic conditions

In order to validate a gel free quantitative proteomics assay for the model methylotrophic bacterium Methylobacterium extorquens AM1, we examined the M. extorquens AM1 proteome under single carbon (methanol) and multicarbon (succinate) growth, conditions that have been studied for decades and for which extensive corroborative data have been compiled. In total, 4447 proteins from a database containing 7556 putative ORFs from M. extorquens AM1 could be identified with two or more peptide sequences, corresponding to a qualitative proteome coverage of 58%. Statistically significant nonzero (log(2) scale) differential abundance ratios of methanol/succinate could be detected for 317 proteins using summed ion intensity measurements and 585 proteins using spectral counting, at a q-value cut-off of 0.01, a measure of false discovery rate. The results were compared to recent microarray studies performed under equivalent chemostat conditions. The M. extorquens AM1 studies demonstrated the feasibility of scaling up the multidimensional capillary HPLC MS/MS approach to a prokaryotic organism with a proteome more than three times the size of microbes we have investigated previously, while maintaining a high degree of proteome coverage and reliable quantitative abundance ratios.