High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.     

Robust estimation of protein expression ratios with lysate microarray technology

MOTIVATION: The protein lysate microarray is a developing proteomic technology for measuring protein expression levels in a large number of biological samples simultaneously. A challenge for accurate quantification is the relatively narrow dynamic range associated with the commonly used chromogenic signal detection system. To facilitate accurate measurement of the relative expression levels, each sample is serially diluted and each diluted version is spotted on a nitrocellulose-coated slide in triplicate. Thus, each sample yields multiple measurements in different dynamic ranges of the detection system. This study aims to develop suitable algorithms that yield accurate representations of the relative expression levels in different samples from multiple data points. RESULTS: We evaluated two algorithms for estimating relative protein expression in different samples on the lysate microarray by means of a cross-validation procedure. For this purpose as well as for quality control we designed a 1440-spot lysate microarray containing 80 identical samples of purified bovine serum albumin, printed in triplicate with six 2-fold dilutions. Our analysis showed that the algorithm based on a robust least squares estimator provided the most accurate quantification of the protein lysate microarray data. We also demonstrated our methods by estimating relative expression levels of p53 and p21 in either p53(+/+) or p53(-/-) HCT116 colon cancer cells after two drug treatments and their combinations on another lysate microarray. AVAILABILITY: http://www.cs.tut.fi/~mirceanc/lysate_array_bioinformatics.htm     

A framework for multidimensional modelling of activity and structure of multispecies biofilms

Concepts from previous biofilm models were integrated to create a framework for the implementation of multidimensional (2D and 3D) multispecies biofilm models. The framework is here described at three levels: (i) mathematical representation of the processes involved in biofilm formation, (ii) numerical implementation into a computer program (freely available from our website http://www.biofilms.bt.tudelft.nl/frameworkMaterial) and (iii) using the program for the creation of biofilm models with multiple bacterial and solute species. An improved version of the individual-based modelling (IbM) that allows structured biomass was used. In this approach biomass composition may be discriminated into any number of particulate species, including extracellular polymeric substances (EPS) for which specific functionality was included. Detachment is also included, described as occurring at the biofilm surface with variable local rates derived from functions of state variables. The application of this modelling framework to a multispecies system with structured biomass is illustrated in a case study where the competition between an organism capable of accumulating polyhydroxybutyrate (PHB, an internal storage compound) and an EPS-producing organism in a two-species biofilm is analysed. Results illustrate that biofilms enriched in PHB-producing organisms may be obtained by supplying substrate intermittently in feast/famine cycles.     

Mitochondrial DNA from prehistoric canids highlights relationships between dogs and South-East European wolves

The question of the origins of the dog has been much debated. The dog is descended from the wolf that at the end of the last glaciation (the archaeologically hypothesized period of dog domestication) was one of the most widespread among Holarctic mammals. Scenarios provided by genetic studies range from multiple dog-founding events to a single origin in East Asia. The earliest fossil dogs, dated approximately 17-12,000 radiocarbon ((14)C) years ago (YA), were found in Europe and in the Middle East. Ancient DNA (a-DNA) evidence could contribute to the identification of dog-founder wolf populations. To gain insight into the relationships between ancient European wolves and dogs we analyzed a 262-bp mitochondrial DNA control region fragment retrieved from five prehistoric Italian canids ranging in age from approximately 15,000 to approximately 3,000 (14)C YA. These canids were compared to a worldwide sample of 547 purebred dogs and 341 wolves. The ancient sequences were highly diverse and joined the three major clades of extant dog sequences. Phylogenetic investigations highlighted relationships between the ancient sequences and geographically widespread extant dog matrilines and between the ancient sequences and extant wolf matrilines of mainly East European origin. The results provide a-DNA support for the involvement of European wolves in the origins of the three major dog clades. Genetic data also suggest multiple independent domestication events. East European wolves may still reflect the genetic variation of ancient dog-founder populations.     

Study of the Villin headpiece folding dynamics by combining coarse-grained Monte Carlo evolution and all-atom molecular dynamics

The folding mechanism of the Villin headpiece (HP36) is studied by means of a novel approach which entails an initial coarse-grained Monte Carlo (MC) scheme followed by all-atom molecular dynamics (MD) simulations in explicit solvent. The MC evolution occurs in a simplified free-energy landscape and allows an efficient selection of marginally-compact structures which are taken as viable initial conformations for the MD. The coarse-grained MC structural representation is connected to the one with atomic resolution through a "fine-graining" reconstruction algorithm. This two-stage strategy is used to select and follow the dynamics of seven different unrelated conformations of HP36. In a notable case the MD trajectory rapidly evolves towards the folded state, yielding a typical root-mean-square deviation (RMSD) of the core region of only 2.4 A from the closest NMR model (the typical RMSD over the whole structure being 4.0 A). The analysis of the various MC-MD trajectories provides valuable insight into the details of the folding and mis-folding mechanisms and particularly about the delicate influence of local and nonlocal interactions in steering the folding process.     

Detection and partial characterization of simian immunodeficiency virus SIVsm strains from bush meat samples from rural Sierra Leone

Human immunodeficiency virus type 2 (HIV-2) originated from simian immunodeficiency viruses (SIVs) that naturally infect sooty mangabeys (SMs; Cercocebus atys). In order to further investigate the relationship between HIV-2 and SIVsm, the SIV specific to the SM, we characterized seven new SIVsm strains from SMs sold in Sierra Leone markets as bush meat. The gag, pol, and env sequences showed that, while the viruses of all seven SMs belonged to the SIVsm-HIV-2 lineage, they were highly divergent viruses, in spite of the fact that most of the samples originated from the same geographical region. They clustered in three lineages, two of which have been previously reported. Two of the new SIVsm strains clustered differently in gag and env phylogenetic trees, suggesting SIVsm recombination that had occurred in the past. In spite of the fact that our study doubles the number of known SIVsm strains from wild SMs, none of the simian strains were close to the groups in which HIV-2 was epidemic (groups A and B).     

Perp is a p63-regulated gene essential for epithelial integrity

p63 is a master regulator of stratified epithelial development that is both necessary and sufficient for specifying this multifaceted program. We show here that Perp, a tetraspan membrane protein originally identified as an apoptosis-associated target of the p53 tumor suppressor, is the first direct target of p63 clearly involved in mediating this developmental program in vivo. During embryogenesis, Perp is expressed in an epithelial pattern, and its expression depends on p63. Perp-/- mice die postnatally, with dramatic blistering in stratified epithelia symptomatic of compromised adhesion. Perp localizes specifically to desmosomes, adhesion junctions important for tissue integrity, and numerous structural defects in desmosomes are observed in Perp-deficient skin, suggesting a role for Perp in promoting the stable assembly of desmosomal adhesive complexes. These findings demonstrate that Perp is a key effector in the p63 developmental program, playing an essential role in an adhesion subprogram central to epithelial integrity and homeostasis.     

Impact of viral factors on very early in vivo replication profiles in simian immunodeficiency virus SIVagm-infected African green monkeys

To better understand which factors govern the levels of viral loads in early lentiviral infections of primates, we developed a model that allows distinguishing between the influences of host and viral factors on viremia. Herein we report that two species of African green monkeys (Chlorocebus sabaeus and C. pygerythrus) infected with their respective wild-type simian immunodeficiency virus SIVagm viruses (SIVagm.sab92018 and SIVagm.ver644) consistently showed reproducible differences in viremia during primary infection but not at later stages of infection. Cross-infections of SIVagm.sab92018 and SIVagm.ver644 into, respectively, C. pygerythrus and C. sabaeus revealed that the dynamics of viral replication during primary infection were dependent on the viral strain used for the infection but not on the host. Hence, the kinetics of SIVagm.sab92018 and SIVagm.ver644 were similar in both sabaeus and vervet animals, indicating that the difference in viremia levels between the two groups during the early phase of infection was not associated with the host. Coreceptor usage for these two strains showed a larger coreceptor repertoire for SIVagm.sab92018, which is able to efficiently use CXCR4 in addition to CCR5, than for SIVagm.ver644, which showed a classical CCR5 coreceptor usage pattern. These differences could not be explained by different charges of the V3 loop for SIVagm.sab92018 and for SIVagm.ver644. In conclusion, our study showed that the extent of virus replication during the primary infection is primarily dependent on viral determinants.     

Lack of functional estrogen receptor beta gene disrupts pubertal male sexual behavior

The estrogen receptor-beta (ERbeta) mediates estrogen action in the female gonads, reproductive tract, and central nervous system. In addition, in rats and mice, gonadotropin-releasing hormone (GnRH-I) neurons coexpress ERbeta. Here we asked if ERbeta plays a role in the onset of puberty and in hypothalamic-pituitary-gonadal (HPG) axis function in male mice. We examined mating behavior, testosterone concentrations, steroid negative feedback on gonadotropins, and GnRH-I function in male ERbeta knockout (ERbetaKO) and wild-type (WT) mice. Peripubertal ERbetaKO males displayed their first ejaculation at a significantly older age than WT littermates. Castrated, adult ERbetaKO mice had significantly higher plasma luteinizing hormone (LH) than WT counterparts. Estradiol (E2) treatment reduced LH and follicle stimulating hormone (FSH) concentrations to an equivalent degree in castrates of both genotypes. In three different measures of the adult GnRH-I system, no genotypic differences were observed. These data show that ERbeta plays an important role in the timing of male sexual behavior at puberty, but does not appear to be involved in adult HPG axis functioning. Furthermore, our data suggest that a primary role of ERbeta may be to regulate ejaculatory behavior.