The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell- and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates.

Using a genome-wide screen of 9.6 million genetic variants achieved through 1000 Genomes imputation in 62,166 samples, we identify association to lipids in 93 loci including 79 previously identified loci with new lead-SNPs, 10 new loci, 15 loci with a low-frequency and 10 loci with missense lead-SNPs, and, 2 loci with an accumulation of rare variants. In six loci, SNPs with established function in lipid genetics (CELSR2, GCKR, LIPC, and APOE), or candidate missense mutations with predicted damaging function (CD300LG and TM6SF2), explained the locus associations. The low-frequency variants increased the proportion of variance explained, particularly for LDL-C and TC. Altogether, our results highlight the impact of low-frequency variants in complex traits and show that imputation offers a cost-effective alternative to resequencing.Genome-wide association (GWA) studies have been successful in identifying genetic loci associated with complex diseases and traits. Due to the design of genotyping arrays, most of the associated variants have been common in population samples. While thousands of loci have been associated with complex diseases and traits, they so far typically explain only a fraction of the heritability 1 .It has now become possible to search for associations with variants that are less frequent than in previous GWA studies by the analysis of large numbers of samples using whole genome or exome sequencing approaches. However, costs have so far limited the possibility for sequencing of tens of thousands of samples likely needed to detect significant associations for low-frequency variants.Stochastic imputation to individuals genotyped using genotyping arrays in large enough samples offers an alternative and cost-effective design to study associations of lowfrequency and rare variants at a genome-wide level. GWA studies of circulating lipids have been highly successful in identifying loci with common variants with small effects 2,3 . In

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor ␣2␤1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, ␣2␤1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.

Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the ␣2␤1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways. INTRODUCTIONViruses that enter host cells by endocytosis are important tools in studies of membrane traffic in animal cells. Recently, they have been used to identify novel endocytic mechanisms that bypass the classical clathrin-mediated uptake processes (Pelkmans et al., 2001;Meier et al., 2002). We have previously reported echovirus 1 (EV1) to be one of the viruses entering host cells via caveolar endocytosis (Marjomäki et al., 2002;Upla et al., 2004); however, the cellular mechanisms involved in the process are still incompletely defined.Echoviruses are human pathogens that can cause meningitis, encephalitis, rash, and mild respiratory and enteric infections (Grist et al., 1978). Echoviruses belong to the Picornavirus family of small (30 nm), nonenveloped, positivestranded RNA viruses. They include such significant pathogens of humans and livestock as polioviruses (PV), rhinoviruses (HRV), hepatitis A virus (HAV), and foot-andmouth disease viruses (FMDV). In spite of their close structural relationships, a variety of entry routes into host cells, including clathrin-mediated endocytosis, caveolae, and lipid rafts, are used by different picornaviruses (DeTulleo and Kirchhausen, 1998;Joki-Korpela et al., 2001;Marjomäki et al., 2002;Stuart et al., 2002).EV1 recognizes the ␣2I domain of the ␣2␤1 integrin, a collagen receptor, on the cell surface (Bergelson et al., 1994), leading to uptake of the receptor and the virus through caveolae-mediated endocytosis (Marjomäki et al., 2002). Caveolae are invaginations of the plasma membrane ϳ50 -80 nm in size (Palade, 1953), and their main protein component is caveolin-1 (Rothberg et al., 1992). They are involved in various signaling processes and in uptake of chol...