Should it be funded through public-private partnerships to succeed?

In the face of a growing global burden of resistance to existing antibiotics, a combination of scientific and economic challenges has posed significant barriers to the development of novel antibacterials over the past few decades. Yet the bottlenecks at each stage of the pharmaceutical value chain-from discovery to post-marketing-present opportunities to reengineer an innovation pipeline that has fallen short. The upstream hurdles to lead identification and optimization may be eased with greater multi-sectoral collaboration, a growing array of alternatives to high-throughput screening, and the application of open source approaches. Product development partnerships and South-South innovation platforms have shown promise in bolstering the R&D efforts to tackle neglected diseases. Strategies that delink product sales from the firms' return on investment can help ensure that the twin goals of innovation and access are met. To effect these changes, both public and private sector stakeholders must show greater commitment to an R&D agenda that will address this problem, not only for industrialized countries but also globally.

Selective export of transmembrane proteins from the endoplasmic reticulum (ER) relies on recognition of cytosolic-domain-localized transport signals by the Sec24 subunit of the COPII vesicle coat. Human cells express four Sec24 isoforms, termed Sec24A, Sec24B, Sec24C and Sec24D that are differentially required for selective, signal-mediated ER export of transmembrane proteins. By contrast, luminally exposed glycosylphosphatidylinositol (GPI)-anchored membrane proteins cannot bind directly to Sec24 and must either use membrane-spanning cargo receptors or alternative mechanisms for ER export. Little is known about the mechanism underlying export of GPI-anchored proteins from the ER in higher eukaryotes. Using siRNA-based silencing, we identified that ER-to-Golgi transport of the human GPI-anchored protein CD59 requires Sec24, with preference for the Sec24C and Sec24D isoforms, and the recycling transmembrane protein complex p24-p23 that exhibited the same Sec24C-Sec24D isoform preference for ER export. Co-immunoprecipitation indicated unprecedented physical interaction of CD59 as well as a GFP-folate-receptor-GPI-anchor hybrid with a p24-p23 complex. Density gradient centrifugation revealed co-partitioning of CD59 and p24-p23 into biosynthetically early lipid raft fractions, and CD59 transport to the Golgi was cholesterol dependent. The results suggest that the 24p-23p complex acts as a cargo receptor for GPI-anchored proteins by facilitating their export from the ER in a Sec24-isoform-selective manner involving lipid rafts as early sorting platforms.

Sec24 of the COPII (coat protein complex II) vesicle coat mediates the selective export of membrane proteins from the endoplasmic reticulum (ER) in yeast.Human cells express four Sec24 isoforms, but their role is unknown.Here, we report the differential effects of Sec24 isoform-specific silencing on the transport of the membrane reporter protein ERGIC-53 (ER-Golgi intermediate compartment-53) carrying the cytosolic ER export signals di-phenylalanine, di-tyrosine, di-leucine, diisoleucine, di-valine or terminal valine.Knockdown of single Sec24 isoforms showed dependence of di-leucine-mediated transport on Sec24A, but transport mediated by the other signals was not affected.By contrast, double knockdown of Sec24A with one of the other three Sec24 isoforms impaired all aromatic/ hydrophobic signal-dependent transport.Double knockdown of Sec24B/C or Sec24B/D preferentially affected di-leucinemediated transport, whereas knockdown of Sec24C/D affected di-isoleucine-and valine-mediated transport.The isoformselective transport correlated with binding preferences of the signals for the corresponding isoforms in vitro.Thus, human Sec24 isoforms expand the repertoire of cargo for signal-mediated ER export, but are in part functionally redundant.

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using non-animal host models.

Export of membrane proteins from the ER is believed to be selective and require transport signals, but the identity of such signals has remained elusive. The recycling type I membrane protein ERGIC-53 carries a C-terminal diphenylalanine motif that is required for efficient ER export. Here we show that this motif can be functionally substituted by a single phenylalanine or tyrosine at position -2, two leucines or isoleucines at position -1 and -2 or a single valine at position -1. These motifs are common among mammalian type I membrane proteins. A single C-terminal valine, but none of the other motifs, accelerates transport of inefficiently exported reporter constructs and hence operates as an export signal. The valine signal is position, but not context, dependent. All transport motifs mediate COPII binding in vitro with distinct preferences for the COPII subunits Sec23p, Sec24Bp, Sec24Cp and p125. These results suggest that cytoplasmic C-terminal amino-acid motifs, either alone or in conjunction with other transport determinants, accelerate ER export of numerous type I and probably polytopic membrane proteins by mediating interaction with COPII coat components.

We show that the loss or inactivation of the polysialic acid (PSA) tail of neural cell adhesion molecule (NCAM) on rat cortical neurons in culture leads to reduced differentiation and survival. The mechanism by which this negative effect is mediated appears to involve the neuronal response to brain-derived neurotrophic factor (BDNF): (i) in the absence of PSA or in the presence of excess free PSA added to the culture medium, BDNF-induced cell signalling is reduced; (ii) the addition of exogenous BDNF to the medium reverses the effect of PSA loss or inactivation. These data suggest that PSA-NCAM, previously shown to modulate cell migration and plasticity, is needed for an adequate sensitivity of neurons to BDNF.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

The effects of the N-formyl methionyl peptide, formyl-methionyl-leucyl phenylalanine (fMLF) on the lateral mobility of the complement receptor type 1 (CR1/CD35) in glass-adherent human neutrophils were investigated, using fluorescence recovery after photobleaching (FRAP) and confocal microscopy (CSLM). It was found that addition of 0.1–1 μM fMLF increased the diffusion constant (D) of CR1/CD35 to 167–278% of controls. No effect was observed on the receptor distribution or the mobile fraction of receptors. The effect of fMLF on the lateral diffusion of CR1/CD35 could be totally inhibited by addition of pertussis toxin (PT, 250 ng/ml) or of the free radical scavenger enzymes superoxide dismutase (SOD, 2000 U/ml) and catalase (CAT, 200 U/ml), added together the results show that oxidative metabolites produced by neutrophils in response to fMLF can modulate CR1/CD35 diffusion, and indicate a regulatory role for oxygen radicals in phagocytosis.

Five mammalian members of the gp25L/ emp24/p24 family have been identified as major con- stituents of the cis -Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this cor- related with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surpris- ingly, upon expression of mutated members, steady- state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligo- meric properties.