In conclusion, genetic interaction by reassortment among cocirculating rotaviruses is not a rare event and contributes significantly to their overall diversity.

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates.

Abstrcat:We report an automated optically sectioning fluorescence lifetime imaging (FLIM) multiwell plate reader for high content analysis (HCA) in drug discovery and accelerated research in cell biology. The system utilizes a Nipkow disc confocal microscope and performs unsupervised FLIM with autofocus, automatic setting of acquisition parameters and automated localisation of cells in the field of view. We demonstrate its applications to test dye solutions, fixed and live cells and FLIM-FRET.Fluorescence lifetime imaging (FLIM) is a powerful and robust tool, able to contrast different chemical species and sense variations in fluorophore micro-environments [1]. In general, it is a robust quantitative imaging modality that is insensitive to artefacts arising from, e.g. fluorophore concentration, scattering, inner filter effects, etc, that can compromise intensity measurements. FLIM is widely applied to cell biology, particularly to study protein-protein interactions using Forster Resonance Energy Transfer (FRET) [2], as well as to read-out fluorescence-based sensors e.g. di-4-ANEPPDHQ to probe lipid order in the cellular membrane [3], and to provide labelfree contrast in biological tissue, e.g. [4]. These capabilities have many potential applications for high content analysis (HCA) but, until recently, a major obstacle to the application of FLIM in this context has been the speed of data acquisition. For cell biology experiments, the most commonly used commercial FLIM systems are based on time correlated single photon counting (TCSPC) implemented in a laser scanning confocal or multiphoton microscope. While this approach has been combined with a multiwell plate reader [5], this instrument did not acquire images but delivered a single lifetime measurement per well. The imaging speed of TCSPC FLIM is limited by the constraints of single photon counting detection and by the nonlinear photobleaching and photodamage that ensues as the power of the scanning laser beam is increased. Wide-field FLIM achieves much faster imaging rates with lower photobleaching due to the parallel pixel interrogation. An automated instrument for unsupervised FLIM was recently reported exploiting frequency domain fluorescence lifetime measurements, which provided an elegant demonstration of the application to FRET and the new opportunities afforded by statistical analysis of such FLIM array data [6]. This system did not, however, provide optical sectioning, which is important for quantitative imaging and is inherent in laser scanning confocal/multiphoton

Hepatitis C virus C, E1, E2 and p7 proteins are cleaved from a viral polyprotein by host signal peptidases. Cleavage at the E2/p7 site is incomplete in genotype 1a strain H (resulting in E2, p7 and E2p7 species), although it has been reported to be more efficient in genotype 1b strain BK. Here, the proteolytic processing and transmembrane topology of genotype 1a strain H77c p7 was investigated when expressed in the context of E2p7. Partial processing was seen at the E2/p7 site in mammalian cells, the efficiency of which improved in the presence of nucleotide sequences downstream of p7. In insect cells, no processing at the E2/p7 site occurred and the uncleaved E2p7 species was incorporated into virus-like particles when expressed in the context of CE1E2p7c-myc. E2p7c-myc formed a heterodimer with E1, indicating that, like the well-characterized E1-E2 complex, the E1-E2p7 heterodimer may also play a functional role in virus replication. Comparison of the p7 signal peptide sequences of strains BK and H77c revealed 3 aa differences (positions 720, 733 and 742). Mutational analysis showed that the V720L change in the H77c sequence substantially increased processivity at the E2/p7 site. The p7 protein adopts a double membrane-spanning topology with both its N and C termini orientated luminally in the endoplasmic reticulum. The transmembrane topology of E2p7 species was examined by two independent means. In both cases, the C terminus of p7 in E2p7 was found to be cytoplasmically orientated, indicating that p7 adopts a dual transmembrane topology. INTRODUCTIONHepatitis C virus (HCV), a member of the family Flaviviridae, is an enveloped virus containing a positive-strand genomic RNA encoding a single polyprotein of approximately 3010 aa that is processed co-and post-translationally by host and viral proteases into at least 10 different proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) (Lindenbach & Rice, 2001). The HCV structural proteins, core (C) and the two envelope glycoproteins E1 and E2, are located within the N terminus of the polyprotein, whilst the non-structural proteins (NS2 to NS5B) reside within the C-terminal part. The status of the p7 protein is currently unknown, although it has been shown to have an ionchannel activity (Griffin et al., 2003(Griffin et al., , 2004Pavlovic et al., 2003;Premkumar et al., 2004) and is essential for infectivity of HCV (Sakai et al., 2003).The C terminus of each structural protein is composed of a hydrophobic amino acid sequence, which acts as a signal peptide to target the proteins located downstream to the endoplasmic reticulum (ER). Cleavage at the C/E1, E1/E2, E2/p7 and p7/NS2 sites is mediated by ER-resident host signal peptidase(s) (Lindenbach & Rice, 2001;Rosenberg, 2001). Although cleavage at the C/E1 and E1/E2 sites proceeds to completion rapidly after translation, cleavage at E2/p7 and p7/NS2 is delayed, resulting in an E2p7NS2 species (Dubuisson et al., 1994). Furthermore, cleavage at the E2/p7 site has been shown to be incomplete, resulting in two E2-s...

BackgroundDuring aging, there is a decreased ability to maintain skeletal muscle mass and function (sarcopenia). Such changes in skeletal muscle are also co-morbidities of diseases including cancer, congestive heart failure and chronic obstructive pulmonary disease. The loss of muscle mass results in decreased strength and exercise tolerance and reduced ability to perform daily activities. Pharmacological agents addressing these pathologies could have significant clinical impact, but their identification requires understanding of mechanisms driving myotube formation (myogenesis) and atrophy and provision of relevant assays. The aim of this study was to develop robust in vitro methods to study human myogenesis.MethodsSatellite cells were isolated by digestion of post-mortem skeletal muscle and selection using anti-CD56 MicroBeads. CD56+ cell-derived myotubes were quantified by high content imaging of myosin heavy chains. TaqMan-polymerase chain reaction arrays were used to quantify expression of 41 selected genes during differentiation. The effects of activin receptor agonists and tumour necrosis factor alpha (TNFα) on myogenesis and gene expression were characterised.ResultsLarge-scale isolation of CD56+ cells enabled development of a quantitative myogenesis assay with maximal myotube formation 3 days after initiating differentiation. Gene expression analysis demonstrated expression of 19 genes changed substantially during myogenesis. TNFα and activin receptor agonists inhibited myogenesis and downregulated gene expression of muscle transcription factors, structural components and markers of oxidative phenotype, but only TNFα increased expression of pro-inflammatory markers.ConclusionsWe have developed methods for large-scale isolation of satellite cells from muscle and quantitative assays for studying human myogenesis. These systems may prove useful as part of a screening cascade designed to identify therapeutic agents for improving muscle function.Electronic supplementary materialThe online version of this article (doi:10.1007/s13539-012-0097-z) contains supplementary material, which is available to authorized users.

Phenotypic screening seeks to identify substances that modulate phenotypes in a desired manner with the aim of progressing first-in-class agents. Successful campaigns require physiological relevance, robust screening, and an ability to deconvolute perturbed pathways. High-content analysis (HCA) is increasingly used in cell biology and offers one approach to prosecution of phenotypic screens, but challenges exist in exploitation where data generated are high volume and complex. We combine development of an organotypic model with novel HCA tools to map phenotypic responses to pharmacological perturbations. We describe implementation for angiogenesis, a process that has long been a focus for therapeutic intervention but has lacked robust models that recapitulate more completely mechanisms involved. The study used human primary endothelial cells in co-culture with stromal fibroblasts to model multiple aspects of angiogenic signaling: cell interactions, proliferation, migration, and differentiation. Multiple quantitative descriptors were derived from automated microscopy using custom-designed algorithms. Data were extracted using a bespoke informatics platform that integrates processing, statistics, and feature display into a streamlined workflow for building and interrogating fingerprints. Ninety compounds were characterized, defining mode of action by phenotype. Our approach for assessing phenotypic outcomes in complex assay models is robust and capable of supporting a range of phenotypic screens at scale.

Modified messenger RNAs (mRNAs) hold great potential as therapeutics by using the body’s own processes for protein production. However, a key challenge is efficient delivery of therapeutic mRNA to the cell cytosol and productive protein translation. Lipid nanoparticles (LNPs) are the most clinically advanced system for nucleic acid delivery; however, a relatively narrow therapeutic index makes them unsuitable for many therapeutic applications. A key obstacle to the development of more potent LNPs is a limited mechanistic understanding of the interaction of LNPs with cells. To address this gap, we performed an arrayed CRISPR screen to identify novel pathways important for the functional delivery of MC3 lipid-based LNP encapsulated mRNA (LNP-mRNA). Here, we have developed and validated a robust, high-throughput screening–friendly phenotypic assay to identify novel targets that modulate productive LNP-mRNA delivery. We screened the druggable genome (7795 genes) and validated 44 genes that either increased (37 genes) or inhibited (14 genes) the productive delivery of LNP-mRNA. Many of these genes clustered into families involved with host cell transcription, protein ubiquitination, and intracellular trafficking. We show that both UDP-glucose ceramide glucosyltransferase and V-type proton ATPase can significantly modulate the productive delivery of LNP-mRNA, increasing and decreasing, respectively, with both genetic perturbation and by small-molecule inhibition. Taken together, these findings shed new light into the molecular machinery regulating the delivery of LNPs into cells and improve our mechanistic understanding of the cellular processes modulating the interaction of LNPs with cells.

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