Phosphoproteomics data classify hematological cancer cell lines according to tumor type and sensitivity to kinase inhibitors

Background

Tumor classification based on their predicted responses to kinase inhibitors is a major goal for advancing targeted personalized therapies. Here, we used a phosphoproteomic approach to investigate biological heterogeneity across hematological cancer cell lines including acute myeloid leukemia, lymphoma, and multiple myeloma.

Results

Mass spectrometry was used to quantify 2,000 phosphorylation sites across three acute myeloid leukemia, three lymphoma, and three multiple myeloma cell lines in six biological replicates. The intensities of the phosphorylation sites grouped these cancer cell lines according to their tumor type. In addition, a phosphoproteomic analysis of seven acute myeloid leukemia cell lines revealed a battery of phosphorylation sites whose combined intensities correlated with the growth-inhibitory responses to three kinase inhibitors with remarkable correlation coefficients and fold changes (> 100 between the most resistant and sensitive cells). Modeling based on regression analysis indicated that a subset of phosphorylation sites could be used to predict response to the tested drugs. Quantitative analysis of phosphorylation motifs indicated that resistant and sensitive cells differed in their patterns of kinase activities, but, interestingly, phosphorylations correlating with responses were not on members of the pathway being targeted; instead, these mainly were on parallel kinase pathways.

Conclusion

This study reveals that the information on kinase activation encoded in phosphoproteomics data correlates remarkably well with the phenotypic responses of cancer cells to compounds that target kinase signaling and could be useful for the identification of novel markers of resistance or sensitivity to drugs that target the signaling network.     

Traceless and Site-specific Attachment of Proteins onto Solid Supports

Many experimental approaches in biology and biophysics, as well as applications in diagnosis and drug discovery, require proteins to be immobilized on solid supports. Protein microarrays, for example, provide a high-throughput format to study biomolecular interactions. The technique employed for protein immobilization is a key to the success of these applications. Recent biochemical developments are allowing, for the first time, the selective and traceless immobilization of proteins generated by cell-free systems without the need for purification and/or reconcentration prior to the immobilization step.     

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.     

Evaluation of Candida species' susceptibility to fluconazole with the disk diffusion method

Infections caused by yeasts belonging to the genus Candida have increased dramatically in the last decades, especially in hospital settings. Concomittantly, antimycotic resistance has emerged, as well as the appearance of non-Candida albicans isolates. To standardize in vitro antifungal susceptibility tests, the agar diffusion test was developed using disks impregnated with the antimycotic compound. Electronic recording of the inhibition zone (BIOMIC), furnishes objective values for the minimal inhibitory concentration (MIC). The fluconazole susceptibility patterns were determined for Candida species isolated from 2.139 patients seen in outpatient clinics or in health-care centers in Colombia, Ecuador and Venezuela. Candida albicans was the species most frequently isolated (62%), followed at a distance by Candida parapsilosis (11%), Candida tropicalis (8.5%), Candida glabata (3.5%) and Candida krusei (2.2%). MIC determinations showed that 88.1% of these isolates were susceptible to fluconazole, 5.1% were susceptible-dose-dependant and 6.8% resistant. An important proportion (92.1%) of the C. albicans isolates proved susceptible while resistance predominated in the remaining species. These results indicate that the BIOMIC method is rapid and simple, constituting a suitable tool for the epidemiologic surveillance of resistance in Candida species.     

Distribution,habitat disturbance and pollination of the endangered orchid Broughtonia cubensis (Epidendrae: Laeliinae)

The geographical distribution, population structure and pollination ecology are key aspects in the conservation and management of rare orchids. Here, we address these aspects and the main threats affecting the endangered Cuban orchid Broughtonia cubensis. This rewardless orchid is self‐compatible, but pollinator dependent. However, seed production can be negatively affected by insect‐mediated selfing. Three species of small bee (genera Ceratina and Lasioglossum) act as pollinators. As in the case of other nectarless orchids, we detected two species of plant producing large amounts of nectar in the area, the floral morphology of which closely resembles that of B. cubensis. The simultaneous flowering of these species could positively affect the reproductive success of B. cubensis. Nonetheless, the fitness of this orchid in natural conditions is low, possibly related to strong pollen limitation. To the problems arising from reduced fitness is added the fact that its historical distribution range has been greatly reduced in recent years. Throughout this study, we have detected dramatic reductions in the population sizes, in some cases as a result of human plundering, but also as a consequence of hurricanes. Based on the results of this study, we propose some guidelines to manage and conserve this orchid. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 345–357.     

Macrolide use in the previous years is associated with failure to eradicate Helicobacter pylori with clarithromycin‐containing regimens

Background

There is some evidence that prior use of macrolide antibiotics is a useful predictor of the likelihood of standard triple therapy failure in Helicobacter pylori eradication. In this study, we have evaluated whether previous intake of macrolides correlates with failure to eradicate H. pylori using two different first‐line clarithromycin‐containing regimens.

Materials and Methods

Retrospective study of 212 patients with H. pylori infection treated with one of two first‐line clarithromycin‐containing regimens: 108 patients treated with triple therapy for 10 days and 104 patients treated with concomitant therapy for 10 days. The intake of macrolides (clarithromycin, azithromycin, and other macrolides) prior to the eradication therapy was obtained from the electronic medical record, which contains information regarding all the medication prescribed to the patients since the year 2004.

Results

One hundred of 212 patients (47.2%) had received at least one treatment with macrolides during the years prior to the eradication therapy. H. pylori eradication rates were significantly lower in patients with previous use compared to patients without previous use of macrolides, both with triple therapy (60.8% vs 92.9%; P < .0001) and with concomitant therapy (85.7% vs 98.2%; P = .024).

Conclusions

Previous use of macrolides correlates with a low H. pylori eradication rate with triple and concomitant clarithromycin‐containing regimens. In addition, our study shows that in patients without previous use of macrolides, triple therapy achieves per‐protocol eradication rates over 90%.     

Quantitative evaluation of mammalian skeletal muscle as a heterologous protein expression system

The production of mammalian proteins in sufficient quantity and quality for structural and functional studies is a major challenge in biology. Intrinsic limitations of yeast and bacterial expression systems preclude their use for the synthesis of a significant number of mammalian proteins. This creates the necessity of well-identified expression systems based on mammalian cells. In this paper, we demonstrate that adult mammalian skeletal muscle, transfected in vivo by electroporation with DNA plasmids, is an excellent heterologous mammalian protein expression system. By using the fluorescent protein EGFP as a model, it is shown that muscle fibers express, during the course of a few days, large amounts of authentic replicas of transgenic proteins. Yields of approximately 1mg/g of tissue were obtained, comparable to those of other expression systems. The involvement of adult mammalian cells assures an optimal environment for proper protein folding and processing. All these advantages complement a methodology that is universally accessible to biomedical investigators and simple to implement.     

Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes

Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.