A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

Slow rates of habituation predict greater zBMI gains over 12 months in lean children.

Slow rates of habituation are cross-sectionally related to greater energy intake and body weight. The present study is designed to assess whether slow rates of habituation are prospectively related to zBMI change over a 12 month period in 66 lean 8-12 year-old children, and whether the rate of habituation is a stable behavioral phenotype. Results showed that slower rates of habituation predicted greater zBMI change, controlling for child sex, age, initial zBMI, dietary awareness and minority status. In addition, the rate of habituation was stable over the year of observation. These data suggest that slow rates of habituation may be a risk factor for weight gain and the development of obesity. Future research is needed to understand the mechanism for this effect, and assess whether the habituation phenotype interacts with other behavioral phenotypes, such as food reinforcement, to influence increases in zBMI.

Wolbachia-mediated resistance to dengue virus infection and death at the cellular level.

BACKGROUND: Dengue is currently the most important arthropod-borne viral disease of humans. Recent work has shown dengue virus displays limited replication in its primary vector, the mosquito Aedes aegypti, when the insect harbors the endosymbiotic bacterium Wolbachia pipientis. Wolbachia-mediated inhibition of virus replication may lead to novel methods of arboviral control, yet the functional and cellular mechanisms that underpin it are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Using paired Wolbachia-infected and uninfected Aedes-derived cell lines and dengue virus, we confirm the phenomenon of viral inhibition at the cellular level. Although Wolbachia imposes a fitness cost to cells via reduced proliferation, it also provides a significant degree of protection from virus-induced mortality. The extent of viral inhibition is related to the density of Wolbachia per cell, with highly infected cell lines showing almost complete protection from dengue infection and dramatically reduced virus titers compared to lines not infected with the bacteria. CONCLUSIONS/SIGNIFICANCE: We have shown that cells infected with Wolbachia display inhibition of dengue virus replication, that the extent of inhibition is related to bacterial density and that Wolbachia infection, although costly, will provide a fitness benefit in some circumstances. Our results parallel findings in mosquitoes and flies, indicating that cell line models will provide useful and experimentally tractable models to study the mechanisms underlying Wolbachia-mediated protection from viruses.

What constitutes food variety? Stimulus specificity of food.

Variety is a major influence of energy intake, but it is not known how much foods have to vary to influence eating. Using a stimulus specificity habituation paradigm we assessed the influence of varying the texture and appearance of nutritionally identical foods on responding for food and energy intake, and whether sensitization, or an increase in responding prior to habituation, was related to the rate of habituation or recovery of responding. Children responded for elbow macaroni and cheese until they habituated, then were provided either more elbow macaroni and cheese, spiral macaroni and cheese, or chicken nuggets. Children provided chicken nuggets or spiral macaroni and cheese recovered responding in comparison to more elbow macaroni and cheese. Children who sensitized showed slower habituation and consumed more food and more energy than those who did not sensitize, but did not differ in recovery of responding to the chicken nuggets or spiral macaroni and cheese. Results show small variations in food characteristics lead to recovery of responding and increased intake after children have habituated.

Variety influences habituation of motivated behavior for food and energy intake in children.

BACKGROUND: Research has shown that variety reduces the rate of habituation, or a general reduction in the rate of responding, for low-energy-density (LED) and high-energy-density (HED) foods. OBJECTIVE: We assessed whether the effects of variety on habituation of motivation to eat are different in overweight and lean children. DESIGN: Overweight and lean children (n = 84) were randomly assigned to groups that varied as to whether they received their favorite or a variety of LED or HED foods. RESULTS: Habituation was slower for overweight than for nonoverweight children (P = 0.008), for a variety of foods than for the same foods (P < 0.001), and for LED than for HED foods (P < 0.001). Energy intake was greater for overweight than for nonoverweight children provided with variety (P = 0.004) and was greater for overweight or nonoverweight children provided with the same food (P < 0.001). A variety of HED foods increased energy intake more than did the same HED foods (P < 0.001); this increase was greater than energy intake with the same or a variety of LED foods (P < 0.001). Children who sensitized, or showed an increase in responding before habituating, showed slower habituation (P < 0.001) and consumed more energy (P = 0.039) than did children who did not sensitize. CONCLUSIONS: Habituation is influenced by variety of foods, and overweight children increase energy intake more with variety than do leaner children. Research is needed to evaluate mechanisms of how variety influences the motivation to eat and energy intake, and how the variety effect can be used to influence intake across multiple eating occasions in children.

Identification of a non-purple tartrate-resistant acid phosphatase: an evolutionary link to Ser/Thr protein phosphatases?

BACKGROUND: Tartrate-resistant acid phosphatases (TRAcPs), also known as purple acid phosphatases (PAPs), are a family of binuclear metallohydrolases that have been identified in plants, animals and fungi. The human enzyme is a major histochemical marker for the diagnosis of bone-related diseases. TRAcPs can occur as a small form possessing only the ~35 kDa catalytic domain, or a larger ~55 kDa form possessing both a catalytic domain and an additional N-terminal domain of unknown function. Due to its role in bone resorption the 35 kDa TRAcP has become a promising target for the development of anti-osteoporotic chemotherapeutics. FINDINGS: A new human gene product encoding a metallohydrolase distantly related to the ~55 kDa plant TRAcP was identified and characterised. The gene product is found in a number of animal species, and is present in all tissues sampled by the RIKEN mouse transcriptome project. Construction of a homology model illustrated that six of the seven metal-coordinating ligands in the active site are identical to that observed in the TRAcP family. However, the tyrosine ligand associated with the charge transfer transition and purple color of TRAcPs is replaced by a histidine. CONCLUSION: The gene product identified here may represent an evolutionary link between TRAcPs and Ser/Thr protein phosphatases. Its biological function is currently unknown but is unlikely to be associated with bone metabolism.

Overview of the pipeline for structural and functional characterization of macrophage proteins at the university of queensland.

This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.

A randomized trial of the effects of reducing television viewing and computer use on body mass index in young children.

OBJECTIVE: To assess the effects of reducing television viewing and computer use on children's body mass index (BMI) as a risk factor for the development of overweight in young children. DESIGN: Randomized controlled clinical trial. SETTING: University children's hospital. PARTICIPANTS: Seventy children aged 4 to 7 years whose BMI was at or above the 75th BMI percentile for age and sex. INTERVENTIONS: Children were randomized to an intervention to reduce their television viewing and computer use by 50% vs a monitoring control group that did not reduce television viewing or computer use. MAIN OUTCOME MEASURES: Age- and sex-standardized BMI (zBMI), television viewing, energy intake, and physical activity were monitored every 6 months during 2 years. RESULTS: Children randomized to the intervention group showed greater reductions in targeted sedentary behavior (P < .001), zBMI (P < .05), and energy intake (P < .05) compared with the monitoring control group. Socioeconomic status moderated zBMI change (P = .01), with the experimental intervention working better among families of low socioeconomic status. Changes in targeted sedentary behavior mediated changes in zBMI (P < .05). The change in television viewing was related to the change in energy intake (P < .001) but not to the change in physical activity (P =.37). CONCLUSIONS: Reducing television viewing and computer use may have an important role in preventing obesity and in lowering BMI in young children, and these changes may be related more to changes in energy intake than to changes in physical activity.

Sensitization and habituation of motivated behavior in overweight and non-overweight children.

The rate of habituation to food is inversely related to energy intake, and overweight children may habituate slower to food and consume more energy. This study compared patterns of sensitization, as defined by an initial increase in operant or motivated responding for food, and habituation, defined by gradual reduction in responding, for macaroni and cheese and pizza in overweight and non-overweight 8-12 year-old children. Non-overweight children habituated faster to both foods than overweight children (p = 0.03). All children recovered motivated responding for a new food (chocolate). Overweight children consumed more energy than non-overweight children (p = 0.0004). Children who showed a sensitization in responding consumed more food (p = 0.001), and sensitization moderated the effect of overweight on habituation, with slower habituation for overweight children who sensitized (p < 0.0001). This study replicates previous data on overweight/non-overweight differences in habituation of food and of energy intake, and provides new information that individual differences in sensitization and habituation of motivated responding to food cues may be associated with a sustained motivation to eat, resulting in greater energy intake.

Structural basis for recruitment of tandem hotdog domains in acyl-CoA thioesterase 7 and its role in inflammation.

Acyl-CoA thioesterases (Acots) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA and thereby regulate lipid metabolism and cellular signaling. We present a comprehensive structural and functional characterization of mouse acyl-CoA thioesterase 7 (Acot7). Whereas prokaryotic homologues possess a single thioesterase domain, mammalian Acot7 contains a pair of domains in tandem. We determined the crystal structures of both the N- and C-terminal domains of the mouse enzyme, and inferred the structure of the full-length enzyme using a combination of chemical cross-linking, mass spectrometry, and molecular modeling. The quaternary arrangement in Acot7 features a trimer of hotdog fold dimers. Both domains of Acot7 are required for activity, but only one of two possible active sites in the dimer is functional. Asn-24 and Asp-213 (from N- and C-domains, respectively) were identified as the catalytic residues through site-directed mutagenesis. An enzyme with higher activity than wild-type Acot7 was obtained by mutating the residues in the nonfunctional active site. Recombinant Acot7 was shown to have the highest activity toward arachidonoyl-CoA, suggesting a function in eicosanoid metabolism. In line with the proposal, Acot7 was shown to be highly expressed in macrophages and up-regulated by lipopolysaccharide. Overexpression of Acot7 in a macrophage cell line modified the production of prostaglandins D2 and E2. Together, the results link the molecular and cellular functions of Acot7 and identify the enzyme as a candidate drug target in inflammatory disease.

Cost effectiveness of recruitment methods in an obesity prevention trial for young children.

BACKGROUND: Recruitment of participants for clinical trials requires considerable effort and cost. There is no research on the cost effectiveness of recruitment methods for an obesity prevention trial of young children. METHODS: This study determined the cost effectiveness of recruiting 70 families with a child aged 4 to 7 (5.9+/-1.3) years in Western New York from February 2003 to November 2004, for a 2-year randomized obesity prevention trial to reduce television watching in the home. RESULTS: Of the 70 randomized families, 65.7% (n=46) were obtained through direct mailings, 24.3% (n=17) were acquired through newspaper advertisements, 7.1% (n=5) from other sources (e.g., word of mouth), and 2.9% (n=2) through posters and brochures. Costs of each recruitment method were computed by adding the cost of materials, staff time, and media expenses. Cost effectiveness (money spent per randomized participant) was US $0 for other sources, US $227.76 for direct mailing, US $546.95 for newspaper ads, and US $3,020.84 for posters and brochures. CONCLUSION: Of the methods with associated costs, direct mailing was the most cost effective in recruiting families with young children, which supports the growing literature of the effectiveness of direct mailing.

Relationship between parental estimate and an objective measure of child television watching.

Many young children have televisions in their bedrooms, which may influence the relationship between parental estimate and objective measures of child television usage/week. Parental estimates of child television time of eighty 4-7 year old children (6.0 +/- 1.2 years) at the 75th BMI percentile or greater (90.8 +/- 6.8 BMI percentile) were compared to an objective measure of television time obtained from TV Allowance devices attached to every television in the home over a three week period. Results showed that parents overestimate their child's television time compared to an objective measure when no television is present in the bedroom by 4 hours/week (25.4 +/- 11.5 vs. 21.4 +/- 9.1) in comparison to underestimating television time by over 3 hours/week (26.5 +/- 17.2 vs. 29.8 +/- 14.4) when the child has a television in their bedroom (p = 0.02). Children with a television in their bedroom spend more objectively measured hours in television time than children without a television in their bedroom (29.8 +/- 14.2 versus 21.4 +/- 9.1, p = 0.003). Research on child television watching should take into account television watching in bedrooms, since it may not be adequately assessed by parental estimates.

Association of access to parks and recreational facilities with the physical activity of young children.

OBJECTIVE: To determine associations of the neighborhood and home television environments with young children's physical activity. METHOD: 32 boys and 27 girls age 4 to 7 years wore accelerometers for 3 weekdays and 1 weekend day. The number of televisions in the home and television watching of the child were monitored using TV Allowance units for 3 weeks. A geographic information system was used to measure neighborhood environment variables. RESULTS: Hierarchical regression analysis was used to predict physical activity, initially controlling for sex, age, socioeconomic status, adiposity, and child television watching in step 1. In step 2, the number of televisions did not significantly increase the amount of variability accounted for in the prediction of physical activity. In step 3, housing density and the interaction of housing density by sex accounted for an incremental 12% (p < 0.05) of the variability and in step 4 percentage park plus recreation area accounted for a further 10% (p < 0.05) of the variability. Greater housing density predicted increased physical activity of boys, but not girls. CONCLUSION: Neighborhoods with increased proximity between homes and a greater proportion of park area are associated with greater physical activity in young children.

Anti-proliferative effects of novel glyco-lipid-arsenicals (III) on MCF-7 human breast cancer cells.

Arsenic trioxide appears to be effective in the treatment of pro-myelocytic leukaemia. The substituted phenylarsen(III)oxides are highly polar, they have a high tendency to undergo oxidation to As (V) and to form oligomers, to prevent this we protected the As-(OH)(2) group as cyclic dithiaarsanes. To increase the compound's biological stability and passive diffusion we conjugated the compound of interest with lipoamino acids (Laas). Alternatively, we further conjugated the dithiaarsane derivative with a carbohydrate to utilize active transport systems and to target compound. We investigated two novel glyco-lipid arsenicals (III) (compounds 9 and 11) for their ability to initiate MCF-7 breast cancer cell death and characterized the mechanism by which death was initiated. A significant decrease in MCF-7 cell proliferation was observed using 1 microM and 10 microM compound (11) and 10 microM of compound (9). Treatment with compound (11) triggered apoptosis of MFC-7 cells while compound (9) induced inhibition of cellular proliferation was not via rapid induction of apoptosis and more likely reflected necrosis and/or alterations in the cell cycle. Differences in the anti-proliferative potency of the two compounds indicate that structural modifications influence effectiveness.

Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate.

Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor alpha mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor alpha positive, MCF-7 cells increased peroxisome proliferator-activated receptor alpha mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4h of treatment. Peroxisome proliferator-activated receptor alpha modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor alpha negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors.

Mono(2-ethylhexyl)phthalate and mono-n-butyl phthalate activation of peroxisome proliferator activated-receptors alpha and gamma in breast.

The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are environmental contaminants with significant human exposures. Both compounds are known reproductive toxins in rodents and DEHP also induces rodent hepatocarcinogenesis in a process believed to be mediated via the peroxisome proliferator-activated receptor alpha (PPARalpha). DEHP and DBP are metabolised to their respective monoesters, mono-(2-ethylhexyl)phthalate (MEHP) and mono-n-butyl phthalate (MBP), which are the active metabolites. MEHP also activates another member of the PPAR subfamily, PPARgamma. The effects of PPARalpha and PPARgamma activation in human breast cells appears to be opposing; PPARalpha activators in breast cells cause an increase in proliferation, while PPARgamma activation in breast cells is associated with differentiation and an inhibition of cell proliferation. Further to this the activation of the PPARs is cell and ligand specific, suggesting the importance of examining the effect of MEHP and MBP on the activation of PPARalpha, PPARbeta and PPARgamma in human breast. We used the common model of human breast cancer MCF-7 and examined the ability of MEHP and MBP to activate human PPARs in this system. The ability of MBP and MEHP to block PPAR responses was also assessed. We found that both human PPARalpha and PPARgamma were activated by MEHP whereas MEHP could not activate PPARbeta. MBP was unable to activate any PPAR isoforms in this breast model, despite being a weak peroxisome proliferator in liver, although MBP was an antagonist for both PPARgamma and PPARbeta. Our results suggest that the toxicological consequences of MEHP in the breast could be complex given the opposing effects of PPARalpha and PPARgamma in human breast cells.

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures.

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action.

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-1R rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK1/2 phosphorylation. Using toll-like receptor (tlr)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R.

Antisense-mediated Inhibition of the plasma membrane calcium-ATPase suppresses proliferation of MCF-7 cells.

Alterations in Ca2+ signaling may contribute to tumorigenesis and the mechanism of action of some anti-cancer drugs. The plasma membrane calcium-ATPase (PMCA) is a crucial controller of intracellular Ca2+ signaling. Altered PMCA expression occurs in the mammary gland during lactation and in breast cancer cell lines. Despite this, the consequences of PMCA inhibition in breast cancer cell lines have not been investigated. In this work, we used Tet-off PMCA antisense-expressing MCF-7 cells to assess the effects of PMCA inhibition in a human breast cancer cell line. At a level of PMCA inhibition that did not completely prevent PMCA-mediated Ca2+ efflux and did not induce cell death, a dramatic inhibition of cellular proliferation was observed. Fluorescence-activated cell sorting analysis indicated that PMCA antisense involves changes in cell cycle kinetics but not cell cycle arrest. We concluded that modulation of PMCA has important effects in regulating the proliferation of human breast cancer MCF-7 cells.

Effect of the peroxisome proliferator-activated receptor beta activator GW0742 in rat cultured cerebellar granule neurons.

The ligand-activated transcription factor peroxisome proliferator-activated receptor beta (PPARbeta) is present in the brain and is implicated in the regulation of genes with potential roles in neurotoxicity. We sought to examine the role of PPARbeta in neuronal cell death by using the PPARbeta ligand GW0742. Primary cultures of rat cerebellar granule neurons were prepared from 7-day-old pups. Reverse transcriptase-polymerase chain reaction and in situ hybridization were used to verify that PPARbeta mRNA was present in neurons. After 10-12 days in culture, the neuronal cells were incubated in the presence of GW0742, and cell death was measured with a lactate dehydrogenase release (LDH) assay. After 24 hr of exposure, PPARbeta activation by GW0742 was not inherently toxic to cerebellar granule neurons. However, toxicity was observed after 48 hr, with cell death mediated via an apoptotic mechanism. In an effect opposite to that observed with PPARalpha-activating ligands, PPARbeta activation exhibited neuroprotective properties. Treatment with GW0742 significantly reduced cell death during a 12-hr exposure to low-KCl media. These results clearly reinforce very specific roles for the PPAR isoforms in neurons and suggest that PPARbeta is worthy of further investigation regarding its potential role as a therapeutic target in neurodegenerative states.