Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.     

Proteoglycan 4 deficiency protects against glucose intolerance and fatty liver disease in diet-induced obese mice

Objective

Proteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we investigated the causal role of Prg4 in controlling lipid and glucose metabolism in mice.

Residues of the human metapneumovirus fusion (F) protein critical for its strain-related fusion phenotype: implications for the virus replication cycle

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891-8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511-1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.     

Time to wake up: No impact of COMT Val158Met gene variation on circadian preferences,arousal regulation and sleep

Dopamine has been implicated in the regulation of sleep–wake states and the circadian rhythm. However, there is no consensus on the impact of two established dopaminergic gene variants: the catechol-O-methyltransferase Val158Met (COMT Val158Met; rs4680) and the dopamine D4 receptor Exon III variable-number-of-tandem-repeat polymorphism (DRD4 VNTR). Pursuing a multi-method approach, we examined their potential effects on circadian preferences, arousal regulation and sleep. Subjects underwent a 7-day actigraphy assessment (SenseWear Pro3), a 20-minute resting EEG (analyzed using VIGALL 2.0) and a body mass index (BMI) assessment. Further, they completed the Morningness–Eveningness Questionnaire (MEQ), the Epworth Sleepiness Scale (ESS) and the Pittsburgh Sleep Quality Index (PSQI). The sample comprised 4625 subjects (19–82 years) genotyped for COMT Val158Met, and 689 elderly subjects (64–82 years) genotyped for DRD4 VNTR. The number of subjects varied across phenotypes. Power calculations revealed a minimum required phenotypic variance explained by genotype ranging between 0.5% and 1.5% for COMT Val158Met and between 3.3% and 6.0% for DRD4 VNTR. Analyses did not reveal significant genotype effects on MEQ, ESS, PSQI, BMI, actigraphy and EEG variables. Additionally, we found no compelling evidence in sex- and age-stratified subsamples. Few associations surpassed the threshold of nominal significance (p < .05), providing some indication for a link between DRD4 VNTR and daytime sleepiness. Taken together, in light of the statistical power obtained in the present study, our data particularly suggest no impact of the COMT Val158Met polymorphism on circadian preferences, arousal regulation and sleep. The suggestive link between DRD4 VNTR and daytime sleepiness, on the other hand, might be worth investigation in a sample enriched with younger adults.     

Economic Appraisal of Ontario's Universal Influenza Immunization Program: A Cost-Utility Analysis

Background

In July 2000, the province of Ontario, Canada, initiated a universal influenza immunization program (UIIP) to provide free seasonal influenza vaccines for the entire population. This is the first large-scale program of its kind worldwide. The objective of this study was to conduct an economic appraisal of Ontario''s UIIP compared to a targeted influenza immunization program (TIIP).

Methods and Findings

A cost-utility analysis using Ontario health administrative data was performed. The study was informed by a companion ecological study comparing physician visits, emergency department visits, hospitalizations, and deaths between 1997 and 2004 in Ontario and nine other Canadian provinces offering targeted immunization programs. The relative change estimates from pre-2000 to post-2000 as observed in other provinces were applied to pre-UIIP Ontario event rates to calculate the expected number of events had Ontario continued to offer targeted immunization. Main outcome measures were quality-adjusted life years (QALYs), costs in 2006 Canadian dollars, and incremental cost-utility ratios (incremental cost per QALY gained). Program and other costs were drawn from Ontario sources. Utility weights were obtained from the literature. The incremental cost of the program per QALY gained was calculated from the health care payer perspective. Ontario''s UIIP costs approximately twice as much as a targeted program but reduces influenza cases by 61% and mortality by 28%, saving an estimated 1,134 QALYs per season overall. Reducing influenza cases decreases health care services cost by 52%. Most cost savings can be attributed to hospitalizations avoided. The incremental cost-effectiveness ratio is Can$10,797/QALY gained. Results are most sensitive to immunization cost and number of deaths averted.

Conclusions

Universal immunization against seasonal influenza was estimated to be an economically attractive intervention. Please see later in the article for the Editors'' Summary     

Perturbation Biology: Inferring Signaling Networks in Cellular Systems

We present a powerful experimental-computational technology for inferring network models that predict the response of cells to perturbations, and that may be useful in the design of combinatorial therapy against cancer. The experiments are systematic series of perturbations of cancer cell lines by targeted drugs, singly or in combination. The response to perturbation is quantified in terms of relative changes in the measured levels of proteins, phospho-proteins and cellular phenotypes such as viability. Computational network models are derived de novo, i.e., without prior knowledge of signaling pathways, and are based on simple non-linear differential equations. The prohibitively large solution space of all possible network models is explored efficiently using a probabilistic algorithm, Belief Propagation (BP), which is three orders of magnitude faster than standard Monte Carlo methods. Explicit executable models are derived for a set of perturbation experiments in SKMEL-133 melanoma cell lines, which are resistant to the therapeutically important inhibitor of RAF kinase. The resulting network models reproduce and extend known pathway biology. They empower potential discoveries of new molecular interactions and predict efficacious novel drug perturbations, such as the inhibition of PLK1, which is verified experimentally. This technology is suitable for application to larger systems in diverse areas of molecular biology.     

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.     

Major remodelling of the murine stem cell kinome following differentiation in the hematopoietic compartment

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells, and myeloid cells. The LT-HSC kinome is characterized by noncanonical Wnt, Ca(2+) and classical protein kinase C (PKC)-driven signaling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signaling cassette. Further differentiation to iNK maintains signaling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signaling, which becomes the dominant trait in the kinase signature following full differentiation toward NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signaling cassette. T cells, however, deactivate signaling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature.