A dual-color fluorescence-based platform to identify selective inhibitors of Akt signaling

Background

Inhibition of Akt signaling is considered one of the most promising therapeutic strategies for many cancers. However, rational target-orientated approaches to cell based drug screens for anti-cancer agents have historically been compromised by the notorious absence of suitable control cells.

Methodology/Principal Findings

In order to address this fundamental problem, we have developed BaFiso, a live-cell screening platform to identify specific inhibitors of this pathway. BaFiso relies on the co-culture of isogenic cell lines that have been engineered to sustain interleukin-3 independent survival of the parental Ba/F3 cells, and that are individually tagged with different fluorescent proteins. Whilst in the first of these two lines cell survival in the absence of IL-3 is dependent on the expression of activated Akt, the cells expressing constitutively-activated Stat5 signaling display IL-3 independent growth and survival in an Akt-independent manner. Small molecules can then be screened in these lines to identify inhibitors that rescue IL-3 dependence.

Conclusions/Significance

BaFiso measures differential cell survival using multiparametric live cell imaging and permits selective inhibitors of Akt signaling to be identified. BaFiso is a platform technology suitable for the identification of small molecule inhibitors of IL-3 mediated survival signaling.     

RNA-containing cytoplasmic inclusion bodies in ciliated bronchial epithelium months to years after acute Kawasaki disease

Background

Kawasaki Disease (KD) is the most common cause of acquired heart disease in children in developed nations. The KD etiologic agent is unknown but likely to be a ubiquitous microbe that usually causes asymptomatic childhood infection, resulting in KD only in genetically susceptible individuals. KD synthetic antibodies made from prevalent IgA gene sequences in KD arterial tissue detect intracytoplasmic inclusion bodies (ICI) resembling viral ICI in acute KD but not control infant ciliated bronchial epithelium. The prevalence of ICI in late-stage KD fatalities and in older individuals with non-KD illness should be low, unless persistent infection is common.

Methods and Principal Findings

Lung tissue from late-stage KD fatalities and non-infant controls was examined by light microscopy for the presence of ICI. Nucleic acid stains and transmission electron microscopy (TEM) were performed on tissues that were strongly positive for ICI. ICI were present in ciliated bronchial epithelium in 6/7 (86%) late-stage KD fatalities and 7/27 (26%) controls ages 9–84 years (p = 0.01). Nucleic acid stains revealed RNA but not DNA within the ICI. ICI were also identified in lung macrophages in some KD cases. TEM of bronchial epithelium and macrophages from KD cases revealed finely granular homogeneous ICI.

Significance

These findings are consistent with a previously unidentified, ubiquitous RNA virus that forms ICI and can result in persistent infection in bronchial epithelium and macrophages as the etiologic agent of KD.     

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.     

Design, synthesis and biological evaluation of new tryptamine and tetrahydro-beta-carboline-based selective inhibitors of CDK4

We present the design, synthesis and biological activity of a library of substituted (biphenylcarbonyl)-tryptamine and (biphenylcarbonyl)-tetrahydro-beta-carboline compounds related to the natural product fascaplysin, as novel inhibitors of CDK4/cyclin D1. We show all these molecules, prepared using the Suzuki-Miyaura reaction, being selective inhibitors of CDK4 over CDK2. The most active compounds have a CDK4 IC(50) in the range 9-11 microM, three of them containing the para-biphenyl plus para-substituents supporting the existence of a pi-stacking pocket within the active site of CDK4.     

Energetics during reproduction: a doubly labeled water study of lactating baboons

Understanding the costs and regulation of reproduction in primates requires understanding the separate but linked effects of energy availability and total energy expenditure (TEE). We compared variation in TEE and energy intake (EI) between two periods, early lactation and after the resumption of sexual cycling, for eight females from two groups of normally reproducing colony-living baboons (Papio h. anubis). Total energy expenditure was assessed using the doubly labeled water method. TEE was correlated with maternal mass both during early lactation and after the resumption of cycling. TEE after the resumption of cycling was positively related to infant growth rates; mothers with rapidly growing infants had higher energy expenditure. TEE was however unrelated to maternal rank and only weakly associated with reproductive parameters such as delay to conception. EI in early lactation was related to infant mass and interbirth intervals, but unrelated to infant growth or reproductive parameters once cycling had resumed. Energy availability (EA; the difference between intake and expenditure) differed significantly between subordinate and dominant females during early lactation, was highly variable among individuals as a function of body composition, and is suggested to follow a nonlinear relationship as a complex function of social status, lactation stage, infant growth, and female fertility. Thus, as a consequence of reduced energy availability, subordinate females in this captive context may experience reproductive delays even though the total energy expenditure after the return of cycling was similar between high and low ranking females.     

Structural basis of specificity and cross-reactivity in T cell receptors specific for cytochrome c-I-E(k)

T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.     

Information Integration in Molecular Bioscience

Integrating information in the molecular biosciences involves more than the cross-referencing of sequences or structures. Experimental protocols, results of computational analyses, annotations and links to relevant literature form integral parts of this information, and impart meaning to sequence or structure. In this review, we examine some existing approaches to integrating information in the molecular biosciences. We consider not only technical issues concerning the integration of heterogeneous data sources and the corresponding semantic implications, but also the integration of analytical results. Within the broad range of strategies for integration of data and information, we distinguish between platforms and developments. We discuss two current platforms and six current developments, and identify what we believe to be their strengths and limitations. We identify key unsolved problems in integrating information in the molecular biosciences, and discuss possible strategies for addressing them including semantic integration using ontologies, XML as a data model, and graphical user interfaces as integrative environments.