The ROSA26-iPSC Mouse: A Conditional,Inducible, and Exchangeable Resource for Studying Cellular (De)Differentiation

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Highlights? Conditional and inducible reprogrammable mouse model presented ? Allows removal of the Yamanaka factors after reprogramming ? Fully and partially reprogrammed endothelial cells generated ? Lineage-directed differentiation of iPSCs toward beating cardiomyocytes     

A comparative analysis of leaf shape of wheat, barley and maize using an empirical shape model

Background and Aims

The phenotypes of grasses show differences depending on growth conditions and ontogenetic stage. Understanding these responses and finding suitable mathematical formalizations are an essential part of the development of plant and crop models. Usually, a marked change in architecture between juvenile and adult plants is observed, where dimension and shape of leaves are likely to change. In this paper, the plasticity of leaf shape is analysed according to growth conditions and ontogeny.

Methods

Leaf shape of Triticum aestivum, Hordeum vulgare and Zea mays cultivars grown under varying conditions was measured using digital image processing. An empirical leaf shape model was fitted to measured shape data of single leaves. Obtained values of model parameters were used to analyse the patterns in leaf shape.

Key Results

The model was able to delineate leaf shape of all studied species. The model error was small. Differences in leaf shape between juvenile and adult leaves in T. aestivum and H. vulgare were observed. Varying growth conditions impacted leaf dimensions but did not impact leaf shape of the respective species.

Conclusions

Leaf shape of the studied T. aestivum and H. vulgare cultivars was remarkably stable for a comparable ontogenetic stage (leaf rank), but differed between stages. Along with other aspects of grass architecture, leaf shape changed during the transition from juvenile to adult growth phase. Model-based analysis of leaf shape is a method to investigate these differences. Presented results can be integrated into architectural models of plant development to delineate leaf shape for different species, cultivars and environmental conditions.     

Unbinding forces of single pertussis toxin-antibody complexes measured by atomic force spectroscopy correlate with their dissociation rates determined by surface plasmon resonance

An inactivated form of pertussis toxin (PTX) is the primary component of currently available acellular vaccines against Bordetella pertussis, the causative agent of whooping cough. The PTX analyzed here is purified at industrial scale and is subsequently inactivated using glutaraldehyde. The influence of this treatment on antibody recognition is of crucial importance and is analyzed in this study. Surface plasmon resonance (SPR) experiments using PTX and its inactivated form (toxoid) with 10 different monoclonal antibodies were conducted. PTX was found to recognize the antibodies with an average affinity of 1.34 ± 0.50 nM, and chemical inactivation caused only a modest decrease in affinity by a factor of approximately 4.5. However, glutaraldehyde treatment had contrary effects on the kinetic association constant k(a) and the dissociation constant k(d) . A significant reduction in k(a) was observed, whereas the dissociation of the toxoid from the bound antibody occurred slower than PTX. These data indicate that the chemical inactivation of PTX not only reduces the velocity of antibody recognition but also stabilizes the interaction with antibodies as shown by a reduction in k(d) . The same interactions were also studied by dynamic force spectroscopy (DFS). Data reveal a correlation between the k(d) values determined by SPR and the mean unbinding force as measured by DFS. The unbinding forces of one complex were determined as a function of the loading rate to directly estimate the k(d) value. Several interactions were impossible to be analyzed using SPR because of ultratight binding. Using DFS, the unbinding forces of these interactions were determined, which in turn could be used to estimate k(d) values. The use of DFS as a technique to study ultratight binding is discussed.     

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 μM (for Mg(2+)) to 3 μM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed.     

The importance of marine vs. human-induced subsidies in the maintenance of an expanding mesocarnivore in the arctic tundra

1. Most studies addressing the causes of the recent increases and expansions of mesopredators in many ecosystems have focused on the top-down, releasing effect of extinctions of large apex predators. However, in the case of the northward expansion of the red fox into the arctic tundra, a bottom-up effect of increased resource availability has been proposed, an effect that can counteract prey shortage in the low phase of the multi-annual rodent cycle. Resource subsidies both with marine and with terrestrial origins could potentially be involved. 2. During different phases of a multi-annual rodent cycle, we investigated the seasonal dynamics and spatial pattern of resource use by red foxes across a coast to inland low arctic tundra gradient, Varanger Peninsula, Norway. We employed two complementary methods of diet analyses: stomach contents and stable isotope analysis. 3. We found that inland red foxes primarily subsisted on reindeer carrions during the low phase of a small rodent population cycle. Lemmings became the most important food item towards the peak phase of the rodent cycle, despite being less abundant than sympatric voles. Isotopic signatures of tissue from both predator and prey also revealed that red foxes near the coast used marine-derived subsidies in the winter, but these allochthonous resources did not spillover to adult foxes living beyond 20-25 km from the coast. 4. Although more needs to be learned about the link between increasing primary productivity due to climatic warming and trophic dynamics in tundra ecosystems, we suggest that changes in reindeer management through a bottom-up effect, at least regionally, may have paved the way towards the establishment of a new mesopredator in the tundra biome.     

Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron

As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells--probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term.     

The plasticity of global proteome and genome expression analyzed in closely related W3110 and MG1655 strains of a well-studied model organism, Escherichia coli-K12

The use of Escherichia coli as a model organism has provided a great deal of basic information in biomolecular sciences. Examining trait differences among closely related strains of the same species addresses a fundamental biological question: how much diversity is there at the single species level? The main aim of our research was to identify significant differences in the activities of groups of genes between two laboratory strains of an organism closely related in genome structure. We demonstrate that despite strict and controlled growth conditions, there is high plasticity in the global proteome and genome expression in two closely related E. coli K12 sub-strains (W3110 and MG1655), which differ insignificantly in genome structure. The growth patterns of these two sub-strains were very similar in a well-equipped bioreactor, and their genome structures were shown to be almost identical by DNA microarray. However, detailed profiling of protein and gene expression by 2-dimensional gel electrophoresis and microarray analysis showed many differentially expressed genes and proteins, combinations of which were highly correlated. The differentially regulated genes and proteins belonged to the following functional categories: genes regulated by sigma subunit of RNA polymerase (RpoS), enterobactin-related genes, and genes involved in central metabolism. Genes involved in central cell metabolism - the glycolysis pathway, the tricarboxylic acid cycle and the glyoxylate bypass - were differentially regulated at both the mRNA and proteome levels. The strains differ significantly in central metabolism and thus in the generation of precursor metabolites and energy. This high plasticity probably represents a universal feature of metabolic activities in closely related species, and has the potential to reveal differences in regulatory networks. We suggest that unless care is taken in the choice of strains for any validating experiment, the results might be misleading.     

Synthesis and biological evaluation of novel 5(H)-phenanthridin-6-ones, 5(H)-phenanthridin-6-one diketo acid, and polycyclic aromatic diketo acid analogs as new HIV-1 integrase inhibitors

A new series of phenanthridinone derivatives, and diketo acid analogs, as well as related phenanthrene and anthracene diketo acids have been synthesized and evaluated as HIV integrase (IN) inhibitors. Several new beta-diketo acid analogs with the phenanthridinone scaffold replaced by phenanthrene, anthracene or pyrene exhibited the highest IN inhibitory potency. There is a general selectivity against the integrase strand transfer step. The most potent IN was 2,4-dioxo-4-phenanthren-9-yl-butyric acid (27f) with an IC(50) of 0.38microM against integrase strand transfer. The phenanthrene diketo acids 27d-f were more potent (IC(50)=2.7-0.38microM) than the corresponding phenanthridinone diketo acid 16 (IC(50)=65microM), suggesting that the polar amide bridge in the phenanthridinone system decreases inhibitory activity relative to the more lipophilic phenanthrene system. This might have to do with the possible binding of the aryl group of the compounds binding to a lipophilic pocket at the integrase active site as suggested by the docking simulations. Molecular modeling also suggested that effectiveness of chelation of the active site Mg(2+) contributes to IN inhibitory potency. Finally, some of the potent compounds inhibited HIV-1 replication in human peripheral blood mononuclear cells (PBMC) with EC(50) down to 8microM for phenanthrene-3-(2,4-dioxo)butyric acid (27d), with a selectivity index of 10 against PBMCs.