A Quantitative Assessment of Costimulation and Phosphatase Activity on Microclusters in Early T Cell Signaling

T cell signaling is triggered through stimulation of the T cell receptor and costimulatory receptors. Receptor activation leads to the formation of membrane-proximal protein microclusters. These clusters undergo tyrosine phosphorylation and organize multiprotein complexes thereby acting as molecular signaling platforms. Little is known about how the quantity and phosphorylation levels of microclusters are affected by costimulatory signals and the activity of specific signaling proteins. We combined micrometer-sized, microcontact printed, striped patterns of different stimuli and simultaneous analysis of different cell strains with image processing protocols to address this problem. First, we validated the stimulation protocol by showing that high expression levels CD28 result in increased cell spreading. Subsequently, we addressed the role of costimulation and a specific phosphotyrosine phosphatase in cluster formation by including a SHP2 knock-down strain in our system. Distinguishing cell strains using carboxyfluorescein succinimidyl ester enabled a comparison within single samples. SHP2 exerted its effect by lowering phosphorylation levels of individual clusters while CD28 costimulation mainly increased the number of signaling clusters and cell spreading. These effects were observed for general tyrosine phosphorylation of clusters and for phosphorylated PLCγ1. Our analysis enables a clear distinction between factors determining the number of microclusters and those that act on these signaling platforms.     

Interpreting Metabolomic Profiles using Unbiased Pathway Models

Human disease is heterogeneous, with similar disease phenotypes resulting from distinct combinations of genetic and environmental factors. Small-molecule profiling can address disease heterogeneity by evaluating the underlying biologic state of individuals through non-invasive interrogation of plasma metabolite levels. We analyzed metabolite profiles from an oral glucose tolerance test (OGTT) in 50 individuals, 25 with normal (NGT) and 25 with impaired glucose tolerance (IGT). Our focus was to elucidate underlying biologic processes. Although we initially found little overlap between changed metabolites and preconceived definitions of metabolic pathways, the use of unbiased network approaches identified significant concerted changes. Specifically, we derived a metabolic network with edges drawn between reactant and product nodes in individual reactions and between all substrates of individual enzymes and transporters. We searched for “active modules”—regions of the metabolic network enriched for changes in metabolite levels. Active modules identified relationships among changed metabolites and highlighted the importance of specific solute carriers in metabolite profiles. Furthermore, hierarchical clustering and principal component analysis demonstrated that changed metabolites in OGTT naturally grouped according to the activities of the System A and L amino acid transporters, the osmolyte carrier SLC6A12, and the mitochondrial aspartate-glutamate transporter SLC25A13. Comparison between NGT and IGT groups supported blunted glucose- and/or insulin-stimulated activities in the IGT group. Using unbiased pathway models, we offer evidence supporting the important role of solute carriers in the physiologic response to glucose challenge and conclude that carrier activities are reflected in individual metabolite profiles of perturbation experiments. Given the involvement of transporters in human disease, metabolite profiling may contribute to improved disease classification via the interrogation of specific transporter activities.     

ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha

In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking. To investigate the role of these molecules in axonogenesis, we expressed ARNO and ARF6 in cultured rat hippocampal neurons. Expression of catalytically inactive ARNO or dominant negative ARF6 resulted in enhanced axonal extension and branching and this effect was abrogated by coexpression of constitutively active ARF6. We sought to identify the downstream effectors of ARF6 during neurite extension by coexpressing phosphatidyl-inositol-4-phosphate 5-Kinase alpha [PI(4)P 5-Kinase alpha] with catalytically inactive ARNO and dominant negative ARF6. We found that PI(4)P 5-Kinase alpha plays a role in neurite extension and branching downstream of ARF6. Also, expression of inactive ARNO/ARF6 depleted the actin binding protein mammalian ena (Mena) from the growth cone leading edge, indicating that these effects on axonogenesis may be mediated by changes in cytoskeletal dynamics. These results suggest that ARNO and ARF6, through PI(4)P 5-Kinase alpha, regulate axonal elongation and branching during neuronal development.     

Does the Bicoid gradient matter?

The generation and interpretation of positional information are key processes in developmental systems. In this issue, Chen et?al. report discoveries made in the Drosophila embryo that give new insights into how positional information can be produced by patterning gradients.     

Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.     

Role of Wnt-5a in the Determination of Human Mesenchymal Stem Cells into Preadipocytes

Increasing adipocyte size as well as numbers is important in the development of obesity and type 2 diabetes, with adipocytes being generated from mesenchymal precursor cells. This process includes the determination of mesenchymal stem cells (MSC) into preadipocytes (PA) and the differentiation of PA into mature fat cells. Although the process of differentiation has been highly investigated, the determination in humans is poorly understood. In this study, we compared human MSC and human committed PA on a cellular and molecular level to gain further insights into the regulatory mechanisms in the determination process. Both cell types showed similar morphology and expression patterns of common mesenchymal and hematopoietic surface markers. However, although MSC were able to differentiate into adipocytes and osteocytes, PA were only able to undergo adipogenesis, indicating that PA lost their multipotency during determination. WNT-5a expression showed significantly higher levels in MSC compared with PA suggesting that WNT-5a down-regulation might be important in the determination process. Indeed, incubation of human MSC in medium containing neutralizing WNT-5a antibodies abolished their ability to undergo osteogenesis, although adipogenesis was still possible. An opposite effect was achieved using recombinant WNT-5a protein. On a molecular level, WNT-5a was found to promote c-Jun N-terminal kinase-dependent intracellular signaling in MSC. Activation of this noncanonical pathway resulted in the induction of osteopontin expression further indicating pro-osteogenic effects of WNT-5a. Our data suggest that WNT-5a is necessary to maintain osteogenic potential of MSC and that inhibition of WNT-5a signaling therefore plays a role in their determination into PA in humans.     

Is bigger always better? A critical appraisal of the use of volumetric analysis in the study of the hippocampus

A well-developed spatial memory is important for many animals, but appears especially important for scatter-hoarding species. Consequently, the scatter-hoarding system provides an excellent paradigm in which to study the integrative aspects of memory use within an ecological and evolutionary framework. One of the main tenets of this paradigm is that selection for enhanced spatial memory for cache locations should specialize the brain areas involved in memory. One such brain area is the hippocampus (Hp). Many studies have examined this adaptive specialization hypothesis, typically relating spatial memory to Hp volume. However, it is unclear how the volume of the Hp is related to its function for spatial memory. Thus, the goal of this article is to evaluate volume as a main measurement of the degree of morphological and physiological adaptation of the Hp as it relates to memory. We will briefly review the evidence for the specialization of memory in food-hoarding animals and discuss the philosophy behind volume as the main currency. We will then examine the problems associated with this approach, attempting to understand the advantages and limitations of using volume and discuss alternatives that might yield more specific hypotheses. Overall, there is strong evidence that the Hp is involved in the specialization of spatial memory in scatter-hoarding animals. However, volume may be only a coarse proxy for more relevant and subtle changes in the structure of the brain underlying changes in behaviour. To better understand the nature of this brain/memory relationship, we suggest focusing on more specific and relevant features of the Hp, such as the number or size of neurons, variation in connectivity depending on dendritic and axonal arborization and the number of synapses. These should generate more specific hypotheses derived from a solid theoretical background and should provide a better understanding of both neural mechanisms of memory and their evolution.     

Learning capabilities enhanced in harsh environments: a common garden approach

Previous studies have suggested that the ability to inhabit harsh environments may be linked to advanced learning traits. However, it is not clear if individuals express such traits as a consequence of experiencing challenging environments or if these traits are inherited. To assess the influence of differential selection pressures on variation in aspects of cognition, we used a common garden approach to examine the response to novelty and problem-solving abilities of two populations of black-capped chickadees (Poecile atricapillus). These populations originated from the latitudinal extremes of the species''s range, where we had previously demonstrated significant differences in memory and brain morphology in a multi-population study. We found that birds from the harsh northern population, where selection for cognitive abilities is expected to be high, significantly outperformed conspecifics from the mild southern population. Our results imply differences in cognitive abilities that may be inherited, as individuals from both populations were raised in and had experienced identical environmental conditions from 10 days of age. Although our data suggest an effect independent of experience, we cannot rule out maternal effects or experiences within the nest prior to day 10 with our design. Nevertheless, our results support the idea that environmental severity may be an important factor in shaping certain aspects of cognition.     

Dietary preference for methionine sources in weaned pigs

The objective of the current study was to investigate the preference of weaned pigs given the choice of diets supplemented with dl-methionine (DLM) or liquid dl-methionine hydroxy analog-free acid (MHA-FA). A basal diet (BD) was formulated to contain 2.5 g methionine (Met) per kilogram of diet. The experimental diets included: (1) BD, (2) BD + 1 g DLM/kg, (3) BD + 2 g DLM/kg, (4) BD + 1.13 g MHA-FA/kg, (5) BD + 1.52 g MHA-FA/kg, (6) BD + 2.25 g MHA-FA/kg and (7) BD + 3.05 g MHA-FA/kg. Sixty weaned mixed-sex pigs were allotted to 5 treatment groups with 12 pig replicates per treatment in a randomized complete block design. During a 35-day experimental period, pigs in treatment group 1 received the BD whereas pigs in the other 4 treatment groups were allowed to choose between a pair of diets with either added 1 g DLM/kg in combination with either 1.13 or 1.52 g MHA-FA/kg or 2 g DLM/kg in combination with 2.25 or 3.05 g MHA-FA/kg, respectively. Pigs were housed in individual pens and had free access to feed and water. Daily feed intake (FI) was used as an indicator of diet preference. Cumulatively, pigs showed a preference (% of total FI) for the diet added with 1 g/kg DLM at 74% (P<0.05) in group 2, and 65% in group 3. Irrespective of the level of MHA-FA supplementation (2.25 or 3.05 g/kg), a preference for the diet supplemented with 2 g DLM/kg was 84% (P<0.05) in groups 4 and 5. During the entire period, pigs consistently (P<0.05) consumed more and preferred the diets supplemented with DLM more than the diets supplemented with MHA-FA in groups 2, 4 and 5. The preference for diets supplemented with DLM was more pronounced at higher Met supplementation levels. Feeding pigs a pair of diets supplemented with DLM or MHA-FA improved (P<0.05) the final body weight, daily weight gain, and FCR. The performance of pigs among the Met-supplemented groups was not different. In conclusion, when given a choice pigs preferred the diets supplemented with DLM more than the diets supplemented with liquid MHA-FA.