Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts

Background

Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells.

Methods

In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC–MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells.

Results

We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication.

Conclusions

Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

     

Studies on the conversion of 60s yeast ribosomes into a 50s component

The process of conversion of the larger (60s) subunit of yeast ribosomes into a 50s component was studied. The release of any RNA or protein material during conversion was assayed by using (32)P- or (35)S-labelled ribosomes; ribosomal RNA distributions of the particles were examined and protein/RNA ratios of subunits were determined. The change in sedimentation coefficients was found to be due, not to loss of material, but to a structural change. The change in structure was shown by electron microscopy.     

Demographic characteristics of cladocerans subject to predation by the flatworm Stenostomum leucops

We present data on the population dynamics and life table demography of four common cladoceran taxa Ceriodaphnia dubia, Ceriodaphnia cornuta, Macrothrix triserialis, and Moina macrocopa at varying densities (0.04 and 0.16 ind ml^?1) of the predatory flatworm Stenostomum leucops. We also studied the impact of S. leucops on competition between C. dubia, M. triserialis, and M. macrocopa. Experiments, with four replicates for each treatment, were conducted in 200 ml recipients with 50 ml of moderately hard water and the green alga Scenedesmus acutus at a concentration of 0.5 × 10⁶ cells ml^?1. We conducted all the experiments with single clones of each taxa. We found that Ceriodaphnia cornuta, regardless of the presence of its beak, was adversely affected to a greater degree than C. dubia due to the presence of the flatworms. Moina macrocopa and Macrothrix triserialis were adversely affected in the competition experiments due to the presence of the flatworms whereas C. dubia was not. The spines of Macrothrix triserialis were not an effective defense against predation by the worms. The population growth rate of Moina macrocopa was significantly higher (0.45 d^?1) in the presence of S. leucops infochemicals than in controls (0.3 d^?1).     

Allelopathic effects of male and female calanoids and cyclopoids (Copepoda) on the demographic response of Brachionus havanaensis (Rotifera)

We hypothesized that cyclopoids, being predators, have a stronger allelopathic influence than calanoids on life-history variables of herbivorous rotifers. To test this hypothesis, we quantified the allelopathic effects of conditioned medium obtained separately from males and females of a calanoid (Arctodiaptomus dorsalis) and a cyclopoid (Mesocyclops longisetus) on the life table demography of the rotifer Brachionus havanaensis. The conditioned medium of the male and female A. dorsalis and male M. longisetus caused a significant reduction (14–18%) in the life expectancy at birth of B. havanaensis. Gross and net reproductive rates of rotifers reduced significantly on the conditioned medium of male or female A. dorsalis; however, the conditioned medium from either sex of M. longisetus had no significant effect on these variables. The population growth rate, r, varied from 0.53 to 0.64 d^?1 depending on the treatment; the r was significantly lower when cultured on the conditioned medium from A. dorsalis (males or females) and female M. longisetus Contrary to our hypothesis, calanoid allelochemicals adversely affected the life history variables of rotifers more than those produced by the cyclopoids.

     

Sessile rotifers (Rotifera) exhibit strong seasonality in a shallow,eutrophic Ramsar site in Mexico

To better understand the sessile rotifer community of a shallow, eutrophic lake, we monitored over a 1‐year period the monthly changes in the density and diversity of rotifers on the roots of the common water hyacinth, Eichhornia crassipes, which served as a representative hydrophyte. Selected physical and chemical parameters of the water were also determined. Mean annual density of all sessile species was 630 individuals per ml. Species of Collothecacea (n = 7) were most abundant. Of the 12 species of Flosculariaceae (Flosculariidae), Sinantherina socialis occurred seasonally, while Ptygura beauchampi was most common across all seasons. The Shannon–Wiener diversity index was highest (H′ = 2.8) in April, but decreased in January (H′ = 1.3). Applying the concept of the guild ratio (functional‐based analysis) we found that raptorial species (Collothecacea) were dominant over most of the year (June–February), while microphagous Flosculariidae were dominant during the warm, drier season (March–May). BEST (optimal matching of biota to environment, including stepwise search) analysis and canonical correspondence revealed that temperature, dissolved oxygen, pH, turbidity, and transparency strongly influenced sessile rotifer dynamics. We also found significant correlations between the density of some species and physicochemical variables. These species included Collotheca ambigua, C. coronetta, C. ornata, C. campanulata, C. tenuilobata, and Stephanoceros millsii in the Collothecacea; and Beauchampia crucigere, Limnias melicerta, L. ceratophylli, Ptygura crystallina, P. melicerta, and Sinantherina ariprepes in the Flosculariidae. Our study shows that the communities of sessile rotifers were structured by abiotic factors and that the guild ratio is useful for understanding the relationship between sessile rotifers and their substrata.     

The high-level similarity of some disparate gene expression measures

Probe-level data from Affymetrix GeneChips can be summarized in many ways to produce probe-set level gene expression measures (GEMs). Disturbingly, the different approaches not only generate quite different measures but they could also yield very different analysis results. Here, we explore the question of how much the analysis results really do differ, first at the gene level, then at the biological process level. We demonstrate that, even though the gene level results may not necessarily match each other particularly well, as long as there is reasonably strong differentiation between the groups in the data, the various GEMs do in fact produce results that are similar to one another at the biological process level. Not only that the results are biologically relevant. As the extent of differentiation drops, the degree of concurrence weakens, although the biological relevance of findings at the biological process level may yet remain.     

Combined effects of zinc and algal food on the competition between planktonic rotifers, <Emphasis Type="Italic">Anuraeopsis fissa</Emphasis> and <Emphasis Type="Italic">Brachionus rubens</Emphasis> (Rotifera)

We evaluated the combined effects of algal (Chlorella vulgaris) food levels (low, 0.5 × 10⁶ (or 2.9 μg C ml⁻¹); and high, 1 × 10⁶ cells ml⁻¹ (or 5.8 μg C ml⁻¹)) and zinc concentrations (0, 0.125, and 0.250 mg l⁻¹ of ZnCl₂) on the competition between two common planktonic rotifers Anuraeopsis fissa and Brachionus rubens using their population growth. Median lethal concentration data (LC₅₀) (mean ± 95% confidence intervals) showed that B. rubens was more resistant to zinc (0.554 ± 0.08 mg l⁻¹) than A. fissa (0.315 ± 0.07 mg l⁻¹). A. fissa when grown alone or with Zn was always numerically more abundant than B. rubens. When grown in the absence of zinc, under low- and high-food levels, the peak abundances of A. fissa varied from 251 ± 24 to 661 ± 77 ind. ml⁻¹, respectively, and the corresponding maxima for B. rubens were 52 ± 3 and 102 ± 18 ind. ml⁻¹. At a given food level, competition for food reduced the peak abundances of both rotifers considerably. Increase in Zn concentration also lowered the rotifer abundances. The impact of zinc on competition between the two-rotifer species was evident at low-food level, mainly for A. fissa. At zinc concentrations of 0 and 0.125 mg l⁻¹, the populations of both rotifers continued to grow for about 10 days, but thereafter B. rubens began to decline. Role of zinc on the competitive outcome of the two species is discussed in relation to the changing algal densities in natural water bodies.     

Progression of early phase diabetic nephropathy in streptozotocin-induced diabetic rats: evaluation of various kidney-related parameters

Diabetic nephropathy (DN) is one of the serious secondary complications of diabetes, which results in end-stage renal failure. Reports on the progressive nature of early phase DN especially with respect to kidney parameters such as kidney weight, type IV collagen excretion, total kidney and urinary glycosaminoglycans (GAGs) are few. This work was undertaken to determine systematically the progression of early phase DN in relation to various kidney-related parameters for a period of four months. Experimentally-induced diabetic rats were grouped based on fasting blood glucose levels. Various basic and kidney-related parameters such as kidney weight, microalbuminuria, urinary excretion of GAGs and type IV collagen, total kidney GAGs, histopathology, glomerular area and glomerular volume were examined in control and diabetic rats. There was a progressive increase in fasting blood sugar, urine sugar, kidney weight, microalbuminuria, urine glycosaminoglycans, urine type IV collagen, glomerular area and glomerular volume but there was a progressive decrease in kidney glycosaminoglycans. Glomerular sclerotic condition was aggravated with the increase in duration of diabetes from 1 to 4 months. Onset of DN in rats begins subtly after one month of diabetes but gets vitiated and more pronounced at the end of four months.