Phenotypic characterization of transgenic mice harboring Nf1+/- or Nf1-/- osteoclasts in otherwise Nf1+/+ background

Skeletal abnormalities in neurofibromatosis type 1 syndrome (NF1) are observed in ～50% of patients. Here, we describe the phenotype of Nf1_Ocl mouse model with Nf1‐deficient osteoclasts. Nf1_Ocl mice with Nf1^+/? or Nf1^?/? osteoclasts in otherwise Nf1^+/+ background were successfully generated by mating parental Nf1flox/flox and TRAP‐Cre mice. Contrary to our original hypothesis, osteoporotic or fragile bone phenotype was not observed. The µCT analysis revealed that tibial bone marrow cavity, trabecular tissue volume, and the perimeter of cortical bone were smaller in Nf1 mice compared to Nf1 control mice. Nf1 mice also a displayed narrowed growth plate in the proximal tibia. In vitro analysis showed increased bone resorption capacity and cytoskeletal changes including irregular cell shape and abnormal actin ring formation in Nf1^?/? osteoclasts. Surprisingly, the size of spleen in Nf1 mice was two times larger than in controls and histomorphometric analysis showed splenic megakaryocytosis. In summary, Nf1Ocl mouse model presented with a mild but specific bone phenotype. This study shows that NF1‐deficiency in osteoclasts may have a role in the development of NF1‐related skeletal abnormalities, but Nf1‐deficiency in osteoclasts in Nf1^+/+ background is not sufficient to induce skeletal abnormalities analogous to those observed in patients with NF1. J. Cell. Biochem. 113: 2136–2146, 2012. © 2012 Wiley Periodicals, Inc.     

Not4 enhances JAK/STAT pathway-dependent gene expression in Drosophila and in human cells

The JAK/STAT pathway is essential for organogenesis, innate immunity, and stress responses in Drosophila melanogaster. The JAK/STAT pathway and its associated regulators have been highly conserved in evolution from flies to humans. We have used a genome-wide RNAi screen in Drosophila S2 cells to identify regulators of the JAK/STAT pathway, and here we report the characterization of Not4 as a positive regulator of the JAK/STAT pathway. Overexpression of Not4 enhanced Stat92E-mediated gene responses in vitro and in vivo in Drosophila. Specifically, Not4 increased Stat92E-mediated reporter gene activation in S2 cells; and in flies, Not4 overexpression resulted in an 8-fold increase in Turandot M (TotM) and in a 4-fold increase in Turandot A (TotA) stress gene activation when compared to wild-type flies. Drosophila Not4 is structurally related to human CNOT4, which was found to regulate interferon-γ- and interleukin-4-induced STAT-mediated gene responses in human HeLa cells. Not4 was found to coimmunoprecipitate with Stat92E but not to affect tyrosine phosphorylation of Stat92E in Drosophila cells. However, Not4 is required for binding of Stat92E to its DNA recognition sequence in the TotM gene promoter. In summary, Not4/CNOT4 is a novel positive regulator of the JAK/STAT pathway in Drosophila and in humans.     

Biochemical and pharmacological characterization of human α/β-hydrolase domain containing 6 (ABHD6) and 12 (ABHD12)

In the central nervous system, three enzymes belonging to the serine hydrolase family are thought to regulate the life time of the endocannabinoid 2-arachidonoylglycerol (C20:4) (2-AG). From these, monoacylglycerol lipase (MAGL) is well characterized and, on a quantitative basis, is the main 2-AG hydrolase. The postgenomic proteins α/β-hydrolase domain containing (ABHD)6 and ABHD12 remain poorly characterized. By applying a sensitive fluorescent glycerol assay, we delineate the substrate preferences of human ABHD6 and ABHD12 in comparison with MAGL. We show that the three hydrolases are genuine MAG lipases; medium-chain saturated MAGs were the best substrates for hABHD6 and hMAGL, whereas hABHD12 preferred the 1 (3)- and 2-isomers of arachidonoylglycerol. Site-directed mutagenesis of the amino acid residues forming the postulated catalytic triad (ABHD6: S148-D278-H306, ABHD12: S246-D333-H372) abolished enzymatic activity as well as labeling with the active site serine-directed fluorophosphonate probe TAMRA-FP. However, the role of D278 and H306 as residues of the catalytic core of ABHD6 could not be verified because none of the mutants showed detectable expression. Inhibitor profiling revealed striking potency differences between hABHD6 and hABHD12, a finding that, when combined with the substrate profiling data, should facilitate further efforts toward the design of potent and selective inhibitors, especially those targeting hABHD12, which currently lacks such inhibitors.     

No effects of short-term GSM mobile phone radiation on cerebral blood flow measured using positron emission tomography

The present study investigated the effects of 902.4 MHz Global System for Mobile Communications (GSM) mobile phone radiation on cerebral blood flow using positron emission tomography (PET) with the ¹⁵O‐water tracer. Fifteen young, healthy, right‐handed male subjects were exposed to phone radiation from three different locations (left ear, right ear, forehead) and to sham exposure to test for possible exposure effects on brain regions close to the exposure source. Whole‐brain [¹⁵O]H₂O–PET images were acquired 12 times, 3 for each condition, in a counterbalanced order. Subjects were exposed for 5 min in each scan while performing a simple visual vigilance task. Temperature was also measured in the head region (forehead, eyes, cheeks, ear canals) during exposure. The exposure induced a slight temperature rise in the ear canals but did not affect brain hemodynamics and task performance. The results provided no evidence for acute effects of short‐term mobile phone radiation on cerebral blood flow. Bioelectromagnetics 33:247–256, 2012. © 2011 Wiley Periodicals, Inc.     

Contrasting growth strategies of pond versus marine populations of nine-spined stickleback (Pungitius pungitius): a combined effect of predation and competition?

Gigantism in isolated ponds in the absence of sympatric fish species has previously been observed in nine-spined sticklebacks (Pungitius pungitius). Patterns in sexual size dimorphism suggested that fecundity selection acting on females might be responsible for the phenomenon. However, the growth strategy behind gigantism in pond sticklebacks has not been studied yet. Here, we compared von Bertalanffy growth parameters of four independent nine-spined stickleback populations reared in a common laboratory environment: two coastal marine (typical size) and two pond (giant size) populations. We found that both pond populations had larger estimated final size than marine populations, which in turn exhibited higher intrinsic growth rates than the pond populations. Female growth strategies were more divergent among marine and pond populations than those of males. Asymptotic body size and intrinsic growth rate were strongly negatively correlated. Hence, pond versus marine populations exhibited different growth strategies along a continuum. Our data suggest that quick maturation—even with the cost of being small (low fecundity)—is favoured in marine environments. On the contrary, growth to a giant final size (high fecundity)—even if it entails extended growth period—is favoured in ponds. We suggest that the absence (ponds) versus presence (marine environment) of sympatric predatory fish species, and the consequent change in the importance of intraspecific competition are responsible for the divergence in growth strategies. The sex-dependence of the patterns further emphasizes the role of females in the body size divergence in the species. Possible alternative hypotheses are also discussed.     

Radiation-induced genomic instability in Caenorhabditis elegans

Currently, the cosmetics industry relies on the results of in vitro genotoxicity tests to assess the safety of chemicals. Although the cytokinesis-block micronucleus (CBMN) test for the detection of cells that have divided once is routinely used and currently accepted by regulatory agencies, it has some limitations. Reconstituted human epidermis (RHE) is widely used in safety assessments because its physiological properties resemble those of the skin, and because it allows testing of substances such as hydrophobic compounds. Thus, the micronucleus test is being adapted for application in RHE-reconstructed tissues. Here we investigated whether two different reconstructed epidermis models (EPI/001 from Straticell, and RHE/S/17 from Skinethic) are suitable for application of the micronucleus test. We found that acetone does not modify micronucleus frequency, cell viability, and model structure, compared with non-treated RHE. Treatment of the EPI/001 model with mitomycin C and vinblastine resulted in a dose-dependent increase of micronucleus frequency as well as a decrease of tissue viability and of binucleated cell rate, while no changes of the epidermal structure were observed. The number of binucleated cells obtained with the RHE/S/17 model was too small to permit micronucleus testing. These results indicate that the proliferative rate of the tissue used is a critical parameter in performing the micronucleus test on a 3D model.     

Morphological anti‐predator defences in the nine‐spined stickleback: constitutive,induced or both?

Environmental differences among populations are expected to lead to local adaptation, while spatial or temporal environmental variation within a population will favour evolution of phenotypic plasticity. As plasticity itself can be under selection, locally adapted populations can vary in levels of plasticity. Nine‐spined stickleback (Pungitius pungitius) originating from isolated ponds (low piscine predation risk, high competition) vs. lake and marine populations (high piscine predation risk, low competition) are known to be morphologically adapted to their respective environments. However, nothing is known about their ability to express phenotypic plasticity in morphology in response to perceived predation risk or food availability/competition. We studied predator‐induced phenotypic plasticity in body shape and armour of marine and pond nine‐spined stickleback in a factorial common garden experiment with two predator treatments (present vs. absent) and two feeding regimes (low vs. high). The predation treatment did not induce any morphological shifts in fish from either habitat or food regime. However, strong habitat‐dependent differences between populations as well as strong sexual dimorphism in both body shape and armour were found. The lack of predator‐induced plasticity in development of the defence traits (viz. body armour and body depth) suggests that morphological anti‐predator traits in nine‐spined stickleback are strictly constitutive, rather than inducible. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.     

Genes involved in systemic and arterial bed dependent atherosclerosis--Tampere Vascular study

Background

Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed.

Methodology/Principal Findings

We characterized the genes generally involved in human advanced atherosclerotic (AHA type V–VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25).

Conclusions

This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.     

Improved classification of Alzheimer's disease data via removal of nuisance variability

Diagnosis of Alzheimer's disease is based on the results of neuropsychological tests and available supporting biomarkers such as the results of imaging studies. The results of the tests and the values of biomarkers are dependent on the nuisance features, such as age and gender. In order to improve diagnostic power, the effects of the nuisance features have to be removed from the data. In this paper, four types of interactions between classification features and nuisance features were identified. Three methods were tested to remove these interactions from the classification data. In stratified analysis, a homogeneous subgroup was generated from a training set. Data correction method utilized linear regression model to remove the effects of nuisance features from data. The third method was a combination of these two methods. The methods were tested using all the baseline data from the Alzheimer's Disease Neuroimaging Initiative database in two classification studies: classifying control subjects from Alzheimer's disease patients and discriminating stable and progressive mild cognitive impairment subjects. The results show that both stratified analysis and data correction are able to statistically significantly improve the classification accuracy of several neuropsychological tests and imaging biomarkers. The improvements were especially large for the classification of stable and progressive mild cognitive impairment subjects, where the best improvements observed were 6% units. The data correction method gave better results for imaging biomarkers, whereas stratified analysis worked well with the neuropsychological tests. In conclusion, the study shows that the excess variability caused by nuisance features should be removed from the data to improve the classification accuracy, and therefore, the reliability of diagnosis making.