Statistics for proteomics: experimental design and 2-DE differential analysis

Proteomics relies on the separation of complex protein mixtures using bidimensional electrophoresis. This approach is largely used to detect the expression variations of proteins prepared from two or more samples. Recently, attention was drawn on the reliability of the results published in literature. Among the critical points identified were experimental design, differential analysis and the problem of missing data, all problems where statistics can be of help. Using examples and terms understandable by biologists, we describe how a collaboration between biologists and statisticians can improve reliability of results and confidence in conclusions.     

The size of the plasmacytoid dendritic cell compartment is a multigenic trait dominated by a locus on mouse chromosome 7

Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.     

Synthesis and biological characterisation of sirtuin inhibitors based on the tenovins

The tenovins are small molecule inhibitors of the NAD(+)-dependent family of protein deacetylases known as the sirtuins. There remains considerable interest in inhibitors of this enzyme family due to possible applications in both cancer and neurodegenerative disease therapy. Through the synthesis of novel tenovin analogues, further insights into the structural requirements for activity against the sirtuins in vitro are provided. In addition, the activity of one of the analogues in cells led to an improved understanding of the function of SirT1 in cells.     

Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.     

Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

S-Adenosyl-N-decyl-aminoethyl, a Potent Bisubstrate Inhibitor of Mycobacterium tuberculosis Mycolic Acid Methyltransferases

S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC₅₀ values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s. The inhaled pathogenic bacilli are taken up by phagocytosis by pulmonary macrophages, which, together with lymphocytes and dendritic cells, form granulomas. The bacilli persist in the granuloma until their reactivation, dissemination into the lungs, and the triggering of disease. The natural resistance of persistent tubercle bacilli to drugs and the emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains are two main concerns in the treatment of the disease. A survey carried out by the Centers for Disease Control and Prevention and the World Health Organization between 2000 and 2004 reported that 20% of 17,690 M. tuberculosis isolates from 49 countries were multidrug-resistant, and 2% were extensively drug-resistant (1). The development of new drugs effective against persistent and drug-resistant bacilli has therefore become a priority.The thick lipid-rich envelope of the Mycobacterium genus is characterized by the presence of mycolic acids (MAs),⁴ very long chain (C₆₀–C₉₀) α-alkylated β-hydroxylated fatty acids (2). MAs are the major components of the mycomembrane (3, 4) lipid bilayer, which plays a key role in both the architecture and permeability of the mycobacterial envelope. The MA biosynthetic pathway is essential for mycobacterial survival. MAs are generated by Claisen condensation between two fatty acyl chains as follows: the very long meromycoloyl chain (C₄₀–C₆₀) and a shorter saturated chain (C₂₂–C₂₆) (2). The different types of MAs are defined by the presence of decorations introduced at proximal and distal positions of the meromycolic chain (Fig. 1A) by a family of paralogous S-adenosylmethionine-dependent methyltransferases (AdoMet-MTs), the mycolic acid methyltransferases (MA-MTs). These chemical modifications are known to be important for the pathogenicity, virulence, and persistence of M. tuberculosis. For example, the cis-cyclopropane introduced at the proximal position of α-MAs by PcaA has an impact on the persistence of the tubercle bacillus within infected organisms (5). Furthermore, the keto and methoxy groups, with a vicinal methyl ramification at the distal position of oxygenated MAs, play a role in M. tuberculosis virulence in the mouse model of infection (6) and have recently been reported to be involved in host-pathogen interplay. Indeed, oxygenated MAs have been shown to modulate IL-12p40 production by macrophages (7) and to trigger the in vitro differentiation of monocyte-derived macrophages into foamy macrophages, which house the bacillus in a dormant state, within granulomas (8). Oxygenated MA biosynthesis requires the Hma (MmaA4) methyltransferase (Fig. 1B), as demonstrated by the absence of the oxygenated form in an M. tuberculosis hma knock-out mutant (6, 9). These results suggest that the enzymes responsible for adding the decorations to MAs, including oxygenated groups in particular, may be relevant pharmacological targets for the development of new antituberculous drugs (10).Open in a separate windowFIGURE 1.A, structures of MAs from M. tuberculosis and M. smegmatis. D, distal position; P, proximal position. Enzymes involved in the introduction of decorations on the meromycolic chain are indicated. B, proposed reaction scheme for the introduction of oxygenated groups. m = 17, 19; n, unknown; X, unknown carrier.Based on the essential role played by MA-MTs in the physiopathology of tuberculosis, several studies have investigated the possible inhibition of this family of enzymes. A recent study revealed that the antituberculous drug thiacetazone and its chemical analogs inhibited MA cyclopropanation at concentrations in the micromolar range (11). Another study, based on mixtures of crude extracts of heat-inactivated mycobacteria and recombinant Escherichia coli overproducing MA-MTs, suggested that the incorporation of [³H]AdoMet into growing meromycolic chains is inhibited by a high concentration (1 mg/ml, i.e. 2.6 mm) of S-adenosyl-l-homocysteine (AdoHcy) or sinefungin (12), the demethylated reaction product and a natural structural analog of AdoMet, respectively (Fig. 2). By contrast, AdoHcy and sinefungin are strong inhibitors of other AdoMet-MTs in vitro, including the cyclopropane fatty-acid synthase (CFAS) from E. coli (K_i of 30 and 0.22 μm, respectively) (13, 14). However, they are active only against the isolated enzyme, whereas S-adenosyl-N-decyl-aminoethyl (SADAE), a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain (Fig. 2), is active against CFAS both in vitro (K_i,app = 6 μm) and in vivo (complete inhibition at 150 μm) (15). The broad screening of possible inhibitors of MA-MTs with an in vitro mini-assay poses a major challenge, as these enzymes most likely use very long meromycolic chains as substrates. In this context, the similarity between CFAS and Hma in terms of their sequences (31% sequence identity) and substrates may be useful, as it suggests that SADAE may inhibit MA-MTs (15).Open in a separate windowFIGURE 2.Structure of AdoMet and of the AdoHcy, sinefungin, and SADAE analogs.We report here our investigations of the interactions between Hma and SADAE, as compared with those between Hma and AdoHcy or sinefungin, and the potential impact of these interactions on the activities of Hma and other MA-MTs and mycobacterial growth. Our high resolution crystallographic characterization of the Hma-SADAE interaction illustrates the bisubstrate nature of the ligand, which is strongly correlated with its strong inhibitory properties.     

Chemicals Possessing a Neurotrophin-Like Activity on Dopaminergic Neurons in Primary Culture

Background

Neurotrophic factors have been shown to possess strong neuroprotective and neurorestaurative properties in Parkinson''s disease patients. However the issues to control their delivery into the interest areas of the brain and their surgical administration linked to their unability to cross the blood brain barrier are many drawbacks responsible of undesirable side effects limiting their clinical use. A strategy implying the use of neurotrophic small molecules could provide an interesting alternative avoiding neurotrophin administration and side effects. In an attempt to develop drugs mimicking neurotrophic factors, we have designed and synthesized low molecular weight molecules that exhibit neuroprotective and neuritogenic potential for dopaminergic neurons.

Principal Findings

A cell-based screening of an in-house quinoline-derived compound collection led to the characterization of compounds exhibiting both activities in the nanomolar range on mesencephalic dopaminergic neurons in spontaneous or 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurodegeneration. This study provides evidence that rescued neurons possess a functional dopamine transporter and underlines the involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in these processes.

Conclusion

Cell-based screening led to the discovery of a potent neurotrophic compound possessing expected physico-chemical properties for blood brain barrier penetration as a serious candidate for therapeutic use in Parkinson disease.     

Heterotrophy in Tropical Scleractinian Corals

The dual character of corals, that they are both auto- and heterotrophs, was recognized early in the twentieth Century. It is generally accepted that the symbiotic association between corals and their endosymbiotic algae (called zooxanthellae) is fundamental to the development of coral reefs in oligotrophic tropical oceans because zooxanthellae transfer the major part of their photosynthates to the coral host (autotrophic nutrition). However, numerous studies have confirmed that many species of corals are also active heterotrophs, ingesting organisms ranging from bacteria to mesozooplankton. Heterotrophy accounts for between 0 and 66% of the fixed carbon incorporated into coral skeletons and can meet from 15 to 35% of daily metabolic requirements in healthy corals and up to 100% in bleached corals. Apart from this carbon input, feeding is likely to be important to most scleractinian corals, since nitrogen, phosphorus, and other nutrients that cannot be supplied from photosynthesis by the coral's symbiotic algae must come from zooplankton capture, particulate matter or dissolved compounds. A recent study showed that during bleaching events some coral species, by increasing their feeding rates, are able to maintain and restore energy reserves.
This review assesses the importance and effects of heterotrophy in tropical scleractinian corals. We first provide background information on the different food sources (from dissolved organic matter to meso- and macrozooplankton). We then consider the nutritional inputs of feeding. Finally, we review feeding effects on the different physiological parameters of corals (tissue composition, photosynthesis and skeletal growth).