Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36^{intermediate/high} during adipocyte differentiation in vitro. The gradual increase of CD36^{intermediate/high/}NR^positive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression.     

Towards a physical description of habitat: quantifying environmental adversity (abiotic stress) in temperate forest and woodland ecosystems

1. Our aim was to develop a quantitative proxy for environmental adversity (abiotic stress) in temperate Eucalyptus and Nothofagus forest and woodland ecosystems.
2. Samples and measurements were collected at 42 sites across a rainfall gradient in southern Australia, an elevation gradient in south-eastern Australia, and a longitudinal transect (temperature gradient) in Patagonia, Argentina.
3. We compared the ability of (a) abiotic variables (14 soil and 21 climatic variables) and (b) the stable carbon isotope (δ¹³C) values of soil organic matter (SOM), to predict variation in leaf area index (LAI; a forest productivity variable).
4. The δ¹³C of SOM (soil aggregates) explained more variation (57%) in LAI than multivariate statistical models that integrated information on many abiotic variables. W* (a climatic water balance model) was also a powerful predictor variable, explaining 37% of the variability in LAI.
5 Synthesis . The stable carbon isotopic signature of soil aggregates is a powerful explanatory variable that may help us to quantify environmental adversity (abiotic stress) in temperate forest and woodland ecosystems.     

Identifying significant temporal variation in time course microarray data without replicates

Background  

An important component of time course microarray studies is the identification of genes that demonstrate significant time-dependent variation in their expression levels. Until recently, available methods for performing such significance tests required replicates of individual time points. This paper describes a replicate-free method that was developed as part of a study of the estrous cycle in the rat mammary gland in which no replicate data was collected.     

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.Extracellular matrix (ECM)¹ is a critical component of the tissue microenvironment. ECM plays a pivotal role in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years (7, 8). However, our ability to globally characterize ECM composition both in vitro and in vivo has been severely limited because of several unique attributes of ECM proteins such as high molecular weight glycans and the presence of covalent protein cross-links (6, 9, 10). Traditional proteomics approaches have proven to be ineffective for the identification of ECM proteins as demonstrated by the fact that collagens, despite being the most abundant protein in mammals, are significantly underrepresented in tissue-based proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for maximal and reproducible extraction and hence the accurate identification of small molecule and inorganic analytes (11). More recently, Capelo et al. (12) have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent mass spectrometry-based identification. Here we sought to determine whether this method could be optimized to prepare ECM samples for mass spectrometry-based analysis. For method development, we used rat tail collagen as a representative ECM protein for which current proteomics approaches have proven relatively unsuccessful. Type I collagen is defined as a right-handed triple helix heterotrimer comprising two identical α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion approach that improves our ability to perform proteomics analysis on a type I collagen preparation and then used this method to identify the protein composition of EHS murine chondrosarcoma matrix (10), herein referred to as Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion approach involves three cycles of ultrasonication for rapid initial trypsin digestion followed by overnight digestion using an acid-labile surfactant. This approach resulted in significant improvement in collagen peptide identification and the identification of numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The application of our ECM-optimized ultrasonic assisted trypsin digestion method is anticipated to significantly advance the identification of tissue- and disease state-specific ECM proteins.     

Rates of nausea and vomiting in pregnancy and dietary characteristics across populations

Nausea and vomiting in pregnancy (NVP) is a pervasive and debilitating phenomenon in humans. Several adaptive explanations for NVP occurrence have been recently proposed, the two most prominent of which predict associations with nutritional intake or specific dietary components. Here we extend previous cross-cultural analyses by analysing associations between NVP prevalence in 56 studies (21 countries) and quantitative estimates of per capita intake across major dietary categories, measured for the year of study by the Food and Agriculture Organisation (FAO). Rates of nausea and vomiting in pregnancy were correlated with high intake of macronutrients (kilocalories, protein, fat, carbohydrate), as well as sugars, stimulants, meat, milk and eggs, and with low intake of cereals and pulses. Restricting analyses to studies from North America and Europe caused relationships between macronutrient intake and NVP to disappear, suggesting that they might be influenced by non-dietary confounds associated with geographical region of study. However, factor analysis of dietary components revealed one factor significantly associated with NVP rate, which was characterized by low cereal consumption and high intake of sugars, oilcrops, alcohol and meat. The results provide further evidence for an association between diet and NVP prevalence across populations, and support for the idea that NVP serves an adaptive prophylactic function against potentially harmful foodstuffs.     

Evaluation of select sensors for real-time monitoring of Escherichia coli in water distribution systems

This study evaluated real-time sensing of Escherichia coli as a microbial contaminant in water distribution systems. Most sensors responded to increased E. coli concentrations, showing that select sensors can detect microbial water quality changes and be utilized as part of a contaminant warning system.     

Spontaneous Gac mutants of Pseudomonas biological control strains: cheaters or mutualists?

Bacteria rely on a range of extracellular metabolites to suppress competitors, gain access to resources, and exploit plant or animal hosts. The GacS/GacA two-component regulatory system positively controls the expression of many of these beneficial external products in pseudomonad bacteria. Natural populations often contain variants with defective Gac systems that do not produce most external products. These mutants benefit from a decreased metabolic load but do not appear to displace the wild type in nature. How could natural selection maintain the wild type in the presence of a mutant with enhanced growth? One hypothesis is that Gac mutants are "cheaters" that do not contribute to the public good, favored within groups but selected against between groups, as groups containing more mutants lose access to ecologically important external products. An alternative hypothesis is that Gac mutants have a mutualistic interaction with the wild type, so that each variant benefits by the presence of the other. In the biocontrol bacterium Pseudomonas chlororaphis strain 30-84, Gac mutants do not produce phenazines, which suppress competitor growth and are critical for biofilm formation. Here, we test the predictions of these alternative hypotheses by quantifying interactions between the wild type and the phenazine- and biofilm-deficient Gac mutant within growing biofilms. We find evidence that the wild type and Gac mutants interact mutualistically in the biofilm context, whereas a phenazine-defective structural mutant does not. Our results suggest that the persistence of alternative Gac phenotypes may be due to the stabilizing role of local mutualistic interactions.     

T cell receptor (TCR)-induced tyrosine phosphorylation dynamics identifies THEMIS as a new TCR signalosome component

Stimulation of the T cell antigen receptor (TCR) induces formation of a phosphorylation-dependent signaling network via multiprotein complexes, whose compositions and dynamics are incompletely understood. Using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we investigated the kinetics of signal propagation after TCR-induced protein tyrosine phosphorylation. We confidently assigned 77 proteins (of 758 identified) as a direct or indirect consequence of tyrosine phosphorylation that proceeds in successive "signaling waves" revealing the temporal pace at which tyrosine kinases activate cellular functions. The first wave includes thymocyte-expressed molecule involved in selection (THEMIS), a protein recently implicated in thymocyte development but whose signaling role is unclear. We found that tyrosine phosphorylation of THEMIS depends on the presence of the scaffold proteins Linker for activation of T cells (LAT) and SH2 domain-containing lymphocyte protein of 76 kDa (SLP-76). THEMIS associates with LAT, presumably via the adapter growth factor receptor-bound protein 2 (Grb2) and with phospholipase Cγ1 (PLC-γ1). RNAi-mediated THEMIS knock-down inhibited TCR-induced IL-2 gene expression due to reduced ERK and nuclear factor of activated T cells (NFAT)/activator protein 1 (AP-1) signaling, whereas JNK, p38, or nuclear factor κB (NF-κB) activation were unaffected. Our study reveals the dynamics of TCR-dependent signaling networks and suggests a specific role for THEMIS in early TCR signalosome function.     

Proterozoic phytoplankton and timing of Chlorophyte algae origins

Abstract: Morphological and reproductive features and cell wall ultrastructure and biochemistry of Proterozoic acritarchs are used to determine their affinity to modern algae. The first appearance datum of these microbiota is traced to infer a minimum age of the divergence of the algal classes to which they may belong. The chronological appearance of microfossils that represent phycoma‐like and zygotic cysts and vegetative cells and/or aplanospores, respectively, interpreted as prasinophyceaen and chlorophyceaen microalgae is related to the Viridiplantae phylogeny. An inferred minimum age of the Chlorophyte origin is before c. 1800 Ma, the Prasinophyceae at c. 1650 Ma and the Chlorophyceae at c. 1450 Ma. These divergence times differ from molecular clock estimates, and the palaeontological evidence suggests that they are older.