Gene Literature Dashboard

Breast cancer is a complex disease resulting from a combination of genetic and environmental factors. Among environmental factors, body composition and intake of specific dietary components like total fat are associated with increased incidence of breast cancer and metastasis. We previously showed that mice fed a high-fat diet have shorter mammary cancer latency, increased tumor growth and more pulmonary metastases than mice fed a standard diet. Subsequent genetic analysis identified several modifiers of metastatic mammary cancer along with widespread interactions between cancer modifiers and dietary fat. To elucidate diet-dependent genetic modifiers of mammary cancer and metastasis risk, global gene expression profiles and copy number alterations from mammary cancers were measured and expression quantitative trait loci (eQTL) identified. Functional candidate genes that colocalized with previously detected metastasis modifiers were identified. Additional analyses, such as eQTL by dietary fat interaction analysis, causality and database evaluations, helped to further refine the candidate loci to produce an enriched list of genes potentially involved in the pathogenesis of metastatic mammary cancer.

<h4>Background</h4>Carbonic anhydrases (CAs) are found in many organisms, in which they contribute to several important biological processes. The vertebrate alpha-CA family consists of 16 subfamilies, three of which (VIII, X and XI) consist of acatalytic proteins. These are named carbonic anhydrase related proteins (CARPs), and their inactivity is due to absence of one or more Zn-binding histidine residues. In this study, we analyzed and evaluated the distribution of genes encoding CARPs in different organisms using bioinformatic methods, and studied their expression in mouse tissues using immunohistochemistry and real-time quantitative PCR.<h4>Results</h4>We collected 84 sequences, of which 22 came from novel or improved gene models which we created from genome data. The distribution of CARP VIII covers vertebrates and deuterostomes, and CARP X appears to be universal in the animal kingdom. CA10-like genes have had a separate history of duplications in the tetrapod and fish lineages. Our phylogenetic analysis showed that duplication of CA10 into CA11 has occurred only in tetrapods (found in mammals, frogs, and lizards), whereas an independent duplication of CA10 was found in fishes. We suggest the name CA10b for the second fish isoform. Immunohistochemical analysis showed a high expression level of CARP VIII in the mouse cerebellum, cerebrum, and also moderate expression in the lung, liver, salivary gland, and stomach. These results also demonstrated low expression in the colon, kidney, and Langerhans islets. CARP X was moderately expressed in the cerebral capillaries and the lung and very weakly in the stomach and heart. Positive signals for CARP XI were observed in the cerebellum, cerebrum, liver, stomach, small intestine, colon, kidney, and testis. In addition, the results of real-time quantitative PCR confirmed a wide distribution for the Car8 and Car11 mRNAs, whereas the expression of the Car10 mRNA was restricted to the frontal cortex, parietal cortex, cerebellum, midbrain, and eye.<h4>Conclusions</h4>CARP sequences have been strongly conserved between different species, and all three CARPs show high expression in the mouse brain and CARP VIII is also expressed in several other tissues. These findings suggest an important functional role for these proteins in mammals.

Phosphorylation of the C-terminal end of histone H2A.X is the most characterized histone post-translational modification in DNA double-stranded breaks (DSB). DNA-dependent protein kinase (DNA-PK) is one of the three phosphatidylinositol 3 kinase-like family of kinase members that is known to phosphorylate histone H2A.X during DNA DSB repair. There is a growing body of evidence supporting a role for histone acetylation in DNA DSB repair, but the mechanism or the causative relation remains largely unknown. Using bacterially expressed recombinant mutants and stably and transiently transfected cell lines, we find that DNA-PK can phosphorylate Thr-136 in addition to Ser-139 both in vitro and in vivo. Furthermore, the phosphorylation reaction is not inhibited by the presence of H1, which in itself is a substrate of the reaction. We also show that, in contrast to previous reports, the ability of the enzyme to phosphorylate these residues is not affected by the extent of acetylation of the core histones. In vitro assembled nucleosomes and HeLa S3 native oligonucleosomes consisting of non-acetylated and acetylated histones are equally phosphorylated by DNA-PK. We demonstrate that the apparent differences in the extent of phosphorylation previously observed can be accounted for by the differential chromatin solubility under the MgCl(2) concentrations required for the phosphorylation reaction in vitro. Finally, we show that although H2A.X does not affect nucleosome conformation, it has a de-stabilizing effect that is enhanced by the DNA-PK-mediated phosphorylation and results in an impaired histone H1 binding.

Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559-32573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3'-terminal exon, a 217-residue protein termed Dfa(A). Other Dfa mRNAs also contain this exon. Dfa(A) is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse Dfa(A) that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa(A). Furthermore, Dfa(A) was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa(A) acts as a repressor of TATA-box transcriptional promoters.

Defects in cardiac neural crest lead to congenital heart disease through failure of cardiac outflow tract and ventricular septation. In this report, we demonstrate a previously unappreciated role for the transcription factor Ets1 in the regulation of cardiac neural crest development. When bred onto a C57BL/6 genetic background, Ets1(-/-) mice have a nearly complete perinatal lethality. Histologic examination of Ets1(-/-) embryos revealed a membranous ventricular septal defect and an abnormal nodule of cartilage within the heart. Lineage-tracing experiments in Ets1(-/-) mice demonstrated that cells of the neural crest lineage form this cartilage nodule and do not complete their migration to the proximal aspects of the outflow tract endocardial cushions, resulting in the failure of membranous interventricular septum formation. Given previous studies demonstrating that the MEK/ERK pathway directly regulates Ets1 activity, we cultured embryonic hearts in the presence of the MEK inhibitor U0126 and found that U0126 induced intra-cardiac cartilage formation, suggesting the involvement of a MEK/ERK/Ets1 pathway in blocking chondrocyte differentiation of cardiac neural crest. Taken together, these results demonstrate that Ets1 is required to direct the proper migration and differentiation of cardiac neural crest in the formation of the interventricular septum, and therefore could play a role in the etiology of human congenital heart disease.

Osteoporosis is a complex human disease that results in increased susceptibility to fragility fractures. It can be phenotypically characterized using several traits, including bone mineral density, bone size, bone strength, and bone turnover markers. The identification of gene variants that contribute to osteoporosis phenotypes, or responses to therapy, can eventually help individualize the prognosis, treatment, and prevention of fractures and their adverse outcomes. Our previously published reviews have comprehensively summarized the progress of molecular genetic studies of gene identification for osteoporosis and have covered the data available to the end of September 2007. This review represents our continuing efforts to summarize the important and representative findings published between October 2007 and November 2009. The topics covered include genetic association and linkage studies in humans, transgenic and knockout mouse models, as well as gene-expression microarray and proteomics studies. Major results are tabulated for comparison and ease of reference. Comments are made on the notable findings and representative studies for their potential influence and implications on our present understanding of the genetics of osteoporosis.

The translation of genetic information according to the sequence of the mRNA template occurs with high accuracy and fidelity. Critical events in each single step of translation are selection of transfer RNA (tRNA), codon reading and tRNA-regeneration for a new cycle. We developed a model that accurately describes the dynamics of single elongation steps, thus providing a systematic insight into the sensitivity of the mRNA translation rate to dynamic environmental conditions. Alterations in the concentration of the aminoacylated tRNA can transiently stall the ribosomes during translation which results, as suggested by the model, in two outcomes: either stress-induced change in the tRNA availability triggers the premature termination of the translation and ribosomal dissociation, or extensive demand for one tRNA species results in a competition between frameshift to an aberrant open-reading frame and ribosomal drop-off. Using the bacterial Escherichia coli system, we experimentally draw parallels between these two possible mechanisms.

Endothelial cells (ECs) form extensive, highly branched and hierarchically organized tubular networks in vertebrates to ensure the proper distribution of molecular and cellular cargo in the vertebrate body. The growth of this vascular system during development, tissue repair or in disease conditions involves the sprouting, migration and proliferation of endothelial cells in a process termed angiogenesis. Surprisingly, specialized ECs, so-called tip cells, which lead and guide endothelial sprouts, share many feature with another guidance structure, the axonal growth cone. Tip cells are motile, invasive and extend numerous filopodial protrusions sensing growth factors, extracellular matrix and other attractive or repulsive cues in their tissue environment. Axonal growth cones and endothelial tip cells also respond to signals belonging to the same molecular families, such as Slits and Roundabouts, Netrins and UNC5 receptors, Semaphorins, Plexins and Neuropilins, and Eph receptors and ephrin ligands. Here we summarize fundamental principles of angiogenic growth, the selection and function of tip cells and the underlying regulation by guidance cues, the Notch pathway and vascular endothelial growth factor signaling.

Genetic variation in the cholinergic muscarinic-2 (M(2)) receptor gene (CHRM2) has been associated with the risk for developing depression. We previously reported that M(2)-receptor distribution volume (V(T)) was reduced in depressed subjects with bipolar disorder (BD) relative to depressed subjects with major depressive disorder (MDD) and healthy controls (HCs). In this study, we investigated the effects of six single-nucleotide polymorphisms (SNPs) for CHRM2 on M(2)-receptor binding to test the hypotheses that genetic variation in CHRM2 influences M(2)-receptor binding and that a CHRM2 polymorphism underlies the deficits in M(2)-receptor V(T) observed in BD. The M(2)-receptor V(T) was measured using positron emission tomography and [(18)F]FP-TZTP in unmedicated, depressed subjects with BD (n=16) or MDD (n=24) and HCs (n=25), and the effect of genotype on V(T) was assessed. In the controls, one SNP (with identifier rs324650, in which the ancestral allele adenine (A) is replaced with one or two copies of thymine (T), showed a significant allelic effect on V(T) in the pregenual and subgenual anterior cingulate cortices in the direction AA<AT<TT. In contrast, in BD subjects with the TT genotype, V(T) was significantly lower than in BD subjects with the AT genotype in these regions. The BD subjects homozygous for the T -allele also showed markedly lower V(T) (by 27 to 37% across regions) than HCs of the same genotype. Post hoc analyses suggested that T homozygosity was associated with a more severe illness course, as manifested by lower socioeconomic function, poorer spatial recognition memory and a greater likelihood of having attempted suicide. These data represent novel preliminary evidence that reduced M(2)-receptor V(T) in BD is associated with genetic variation within CHRM2. The differential impact of the M(2)-receptor polymorphism at rs324650 in the BD and HC samples suggests interactive effects with an unidentified vulnerability factor for BD.

The effect of folate-targeted liposomal doxorubicin (FTL-Dox) has been well characterized in folate receptor (FR) overexpressing tumors in vitro, particularly in KB human carcinoma cells. However, there are few studies evaluating the in vivo efficacy of FTL-Dox in KB murine xenograft models. In this study, we investigated the antitumor activity of FTL-Dox injected intravenously in mice bearing KB tumors. Folate ligands comprising of folate-polyethyleneglycol-distearoylphosphatidylethanolamine (FA-PEG-DSPE) were synthesized with different MW PEG. To design an optimum FTL-Dox formulation for therapeutic studies, we prepared various FTLs and characterized their in vitro targeting and in vivo tissue biodistribution. Mice were administered a single intravenous injection of free Dox, nontargeted PEGylated liposomal Dox (PL-Dox), or FTL-Dox. FTLs and PLs accumulated similarly in tumor tissue, despite FTLs' faster clearance from circulation. Mice treated with FTL-Dox 20 mg/kg had a slightly greater tumor growth inhibition and almost a 50% increase in life span than mice receiving PL-Dox 20 mg/kg (P = 0.0121; log-rank test). We conclude that FTLs administered systemically have the potential to enhance the delivery of anticancer drugs in vivo; however, their removal by FR expressing normal tissues may have to be blocked if the benefits of tumor targeting are to be realized.

An expanded polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt) causes misfolding and accumulation of htt in neuronal cells and the subsequent neurodegeneration of Huntington's disease (HD). Clearing the misfolded htt is critical for preventing neuropathology, and this process is mediated primarily by both the ubiquitin-proteasome system (UPS) and autophagy. Although overexpression of mutant htt can inhibit UPS activity in cultured cells, mutant htt does not inhibit global UPS activity in the brains of HD transgenic mice. These findings underscore the importance of investigating the function of the UPS and autophagy in the brain when mutant proteins are not overexpressed. When cultured PC12 cells were treated with either UPS or autophagy inhibitors, more N-terminal mutant htt fragments accumulated via inhibition of the UPS. Furthermore, in HD CAG repeat knock-in mouse brain, inhibiting the UPS also resulted in a greater accumulation of N-terminal, but not full-length, mutant htt than inhibiting autophagy did. Our findings suggest that impairment of the UPS may be more important for the accumulation of N-terminal mutant htt and might therefore make an attractive therapeutic target.

Peroxiredoxins (PRDX) are a family of thiol-dependent peroxidases. Among the six mammalian members of this family, PRDX6 is the only protein that additionally exhibits phospholipase A(2) (PLA(2)) activity. The physiologic role of this interesting PRDX6 feature is largely unknown at present. In this study, we show that PRDX6 increases the metastatic potential of lung cancer cells. Functional analyses of the enzymatic activities of PRDX6, using specific pharmacologic inhibitors and mutagenesis studies, reveal that both peroxidase and PLA(2) activities are required for metastasis. Specifically, peroxidase activity facilitates the growth of cancer cells, and PLA(2) activity promotes invasiveness. Further investigation of the latter event discloses that PLA(2) activity promotes accumulation of arachidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and urokinase-type plasminogen activator. This study is the first to define the functions of the enzymatic activities of PRDX6 in metastasis and to show the involvement of arachidonic acid in PRDX6 action in intact cells. These novel findings provide a significant step toward elucidating the role of PRDX6 in cancer and the mechanism of its action. Mol Cancer Ther; 9(4); 825-32. (c)2010 AACR.

Iron overload can cause insulin deficiency, but in some cases this may be insufficient to result in diabetes. We hypothesized that the protective effects of decreased iron would be more significant with increased beta-cell demand and stress. Therefore, we treated the ob/ob mouse model of type 2 diabetes with an iron-restricted diet (35 mg/kg iron) or with an oral iron chelator. Control mice were fed normal chow containing 500 mg/kg iron. Neither treatment resulted in iron deficiency or anemia. The low-iron diet significantly ameliorated diabetes in the mice. The effect was long lasting and reversible. Ob/ob mice on the low-iron diet exhibited significant increases in insulin sensitivity and beta-cell function, consistent with the phenotype in mouse models of hereditary iron overload. The effects were not accounted for by changes in weight or feeding behavior. Treatment with iron chelation had a more dramatic effect, allowing the ob/ob mice to maintain normal glucose tolerance for at least 10.5 wk despite no effect on weight. Although dietary iron restriction preserved beta-cell function in ob/ob mice fed a high-fat diet, the effects on overall glucose levels were less apparent due to a loss of the beneficial effects of iron on insulin sensitivity. Beneficial effects of iron restriction were minimal in wild-type mice on normal chow but were apparent in mice on high-fat diets. We conclude that, even at "normal" levels, iron exerts detrimental effects on beta-cell function that are reversible with dietary restriction or pharmacotherapy.

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. (45)Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of (45)Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.

<h4>Background</h4>The ability to reside and proliferate in macrophages is characteristic of several infectious agents that are of major importance to public health, including the intracellular parasites Trypanosoma cruzi (the etiological agent of Chagas disease) and Leishmania species (etiological agents of Kala-Azar and cutaneous leishmaniasis). Although recent studies have elucidated some of the ways macrophages respond to these pathogens, the relationships between activation programs elicited by these pathogens and the macrophage activation programs elicited by bacterial pathogens and cytokines have not been delineated.<h4>Methodology/principal findings</h4>To provide a global perspective on the relationships between macrophage activation programs and to understand how certain pathogens circumvent them, we used transcriptional profiling by genome-wide microarray analysis to compare the responses of mouse macrophages following exposure to the intracellular parasites T. cruzi and Leishmania mexicana, the bacterial product lipopolysaccharide (LPS), and the cytokines IFNG, TNF, IFNB, IL-4, IL-10, and IL-17. We found that LPS induced a classical activation state that resembled macrophage stimulation by the Th1 cytokines IFNG and TNF. However, infection by the protozoan pathogen L. mexicana produced so few transcriptional changes that the infected macrophages were almost indistinguishable from uninfected cells. T. cruzi activated macrophages produced a transcriptional signature characterized by the induction of interferon-stimulated genes by 24 h post-infection. Despite this delayed IFN response by T. cruzi, the transcriptional response of macrophages infected by the kinetoplastid pathogens more closely resembled the transcriptional response of macrophages stimulated by the cytokines IL-4, IL-10, and IL-17 than macrophages stimulated by Th1 cytokines.<h4>Conclusions/significance</h4>This study provides global gene expression data for a diverse set of biologically significant pathogens and cytokines and identifies the relationships between macrophage activation states induced by these stimuli. By comparing macrophage activation programs to pathogens and cytokines under identical experimental conditions, we provide new insights into how macrophage responses to kinetoplastids correlate with the overall range of macrophage activation states.

The metacaspase Mca1 from Saccharomyces cerevisiae displays a Q/N-rich region at its N-terminus reminiscent of yeast prion proteins. In this study, we show that the ability of Mca1 to form insoluble aggregates is modulated by a peptide stretch preceding its putative prion-forming domain. Based on its genomic locus, three potential translational start sites of Mca1 can give rise to two slightly different long Mca1 proteins or a short version, Mca1(451/453) and Mca1(432,) respectively, although under normal physiological conditions Mca1(432) is the predominant form expressed. All Mca1 variants exhibit the Q/N-rich regions, while only the long variants Mca1(451/453) share an extra stretch of 19 amino acids at their N-terminal end. Strikingly, only long versions of Mca1 but not Mca1(432) revealed pronounced aggregation in vivo and displayed prion-like properties when fused to the C-terminal domain of Sup35 suggesting that the N-terminal peptide element promotes the conformational switch of Mca1 protein into an insoluble state. Transfer of the 19 N-terminal amino acid stretch of Mca1(451) to the N-terminus of firefly luciferase resulted in increased aggregation of luciferase, suggesting a protein destabilizing function of the peptide element. We conclude that the aggregation propensity of the potential yeast prion protein Mca1 in vivo is strongly accelerated by a short peptide segment preceding its Q/N-rich region and we speculate that such a conformational switch might occur in vivo via the usage of alternative translational start sites.

The atmospheric chemistry of several gases used in industrial applications, C(4)F(9)OC(2)H(5) (HFE-7200), C(4)F(9)OCH(3) (HFE-7100), C(3)F(7)OCH(3) (HFE-7000) and C(3)F(7)CH(2)OH, has been studied. The discharge flow technique coupled with mass-spectrometric detection has been used to study the kinetics of their reactions with OH radicals as a function of temperature. The infrared spectra of the compounds have also been measured. The following Arrhenius expressions for the reactions were determined (in units of cm(3) molecule(-1) s(-1)): k(OH + HFE-7200) = (6.9(-1.7)(+2.3)) x 10(-11) exp(-(2030 +/- 190)/T); k(OH + HFE-7100) = (2.8(-1.5)(+3.2)) x 10(-11) exp(-(2200 +/- 490)/T); k(OH + HFE-7000) = (2.0(-0.7)(+1.2)) x 10(-11) exp(-(2130 +/- 290)/T); and k(OH + C(3)F(7)CH(2)OH) = (1.4(-0.2)(+0.3)) x 10(-11) exp(-(1460 +/- 120)/T). From the infrared spectra, radiative forcing efficiencies were determined and compared with earlier estimates in the literature. These were combined with the kinetic data to estimate 100-year time horizon global warming potentials relative to CO(2) of 69, 337, 499 and 36 for HFE-7200, HFE-7100, HFE-7000 and CF(3)CF(2)CF(2)CH(2)OH, respectively.

No abstract available.

DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.

Humans differing in the amount of serotonin transporter (5-HTT) are known to be differentially prone to neuropsychiatric disorders. Genetically modified mice eliciting abrogated transporter function display a number of corresponding phenotypic changes in behavioural tests. However, a characterisation of the effects of serotonergic malfunction on the day-to-day life is still missing. Yet, this is precisely what an animal model is needed for in order to be meaningful for translation into human anxiety disorders. Homozygous 5-HTT knockout mice, heterozygous 5-HTT mice, and wild-type controls were housed in groups of males of the same genotype in spacious and richly structured cages. This enriched environment allowed the animals to show a wide variety of spontaneous behavioural patterns quantified by a trained experimenter. Additionally the mice could emigrate from the cages through a tunnel and a water basin. The results revealed unaltered daily behaviour in heterozygous mice. In knockouts, however, reduced locomotion, increased socio-positive behaviour, and reduced aggressive behaviour were observed. Nevertheless, all groups showed a significant amount of aggressive behaviour and there were no differences regarding the establishment of dominance relationships, emigration, and the number of animals remaining in their groups. In a second step, pairs of heterozygous and wild-type males and pairs of knockout and wild-type males were brought together in order to assess their ability to obtain a dominant social position in a direct encounter. Heterozygous mice did not differ from wild-type mice but knockout mice were significantly inferior in obtaining the dominant position. In addition to confirming multiple effects of abolished 5-HTT function in a real life situation, this study supports the central role of the 5-HTT in the control of social interactions.

Considering the wide variety of effects that have been reported to occur in the developmental neurotoxicity of chlorpyrifos (CP) and the lack of consensus on their dependence of brain acetylcholinesterase (AChE) activity inhibition, we applied microarray technology to explore dose-dependent alterations in transcriptional response in the fetal and maternal C57BL/6 mouse brain after daily gestational exposure (days 6 to 17) to CP (2, 4, 10, 12 or 15 mg/kg, sc). We identified significantly altered genes across doses and assessed for overrepresentation of Gene Ontology (GO) biological processes and KEGG pathways. We further clustered genes based on their expression profiles across doses and repeated the GO/pathways analysis for each cluster. The dose-effect relationship of CP on gene expression, both at the gene and pathway levels was non-monotonic and not necessarily related to brain AChE inhibition. The largest impact was observed in the 10mg/kg dose group which was also the LOAEL for brain AChE inhibition. In the maternal brain, lower doses (4 mg/kg) influenced GO categories and pathways such as cell adhesion, behavior, lipid metabolism, long-term potentiation, nervous system development, neurogenesis, synaptic transmission. In the fetal brain, lower doses (2 and/or 4 mg/kg) significantly altered cell division, translation, transmission of nerve impulse, chromatin modification, long-term potentiation. In addition, some genes involved in nervous system development and signaling were shown to be specifically influenced by these lower CP doses. Our approach was sensitive and reflected the diversity of responses known to be disrupted by CP and highlighted possible additional consequences of CP neurotoxicity, such as disturbance of the ubiquitin proteasome system.

<h4>Background</h4>Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFdelta/delta mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease.<h4>Results</h4>Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFdelta/delta mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFdelta/delta mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFdelta/delta mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFdelta/delta mice, axon outgrowth is significantly reduced compared to wild-type littermates.<h4>Conclusions</h4>Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Chemoprevention has become an important area in cancer research due to the failure of current therapeutic modalities. Epidemiological and preclinical studies have demonstrated that nutrition plays a vital role in the etiology of cancer. This study was conducted to determine the chemopreventive effects of germinated brown rice (GBR) in rats induced with colon cancer. GBR is brown rice that has been claimed to be richer in nutrients compared to the common white rice. The male Sprague Dawley rats (6 weeks of age) were randomly divided into 5 groups: (G1) positive control (with colon cancer, unfed with GBR), (G2) fed with 2.5 g/kg of GBR (GBR (g)/weight of rat (kg)), (G3) fed with 5 g/kg of GBR, (G4) fed with 10 g/kg of GBR and (G5) negative control (without colon cancer, unfed with GBR). GBR was administered orally once daily via gavage after injection of 15 mg/kg of body weight of azoxymethane (AOM) once a week for two weeks, intraperitonially. After 8 weeks of treatment, animals were sacrificed and colons were removed. Colonic aberrant crypt foci (ACF) were evaluated histopathologically. Total number of ACF and AC, and multicrypt of ACF, and the expression of beta-catenin and COX-2 reduced significantly (p < 0.05) in all the groups treated with GBR (G2, G3 and G4) compared to the control group (G1). Spearman rank correlation test showed significant positive linear relationship between total beta-catenin and COX-2 score (Spearman's rho = 0.616, p = 0.0001). It is demonstrated that GBR inhibits the development of total number of ACF and AC, and multicrypt of ACF, reduces the expression of beta-catenin and COX-2, and thus can be a promising dietary supplement in prevention of colon cancer.

<h4>Background</h4>The human umbilical cord contains mucoid connective tissue and fibroblast-like cells. These cells named Wharton's jelly cells, (WJCs) display properties similar to mesenchymal stem cells therefore representing a rich source of primitive cells to be potentially used in regenerative medicine.<h4>Results</h4>To better understand their self-renewal and potential in vitro expansion capacity, a reference 2D map was constructed as a proteomic data set. 158 unique proteins were identified. More than 30% of these proteins belong to cytoskeleton compartment. We also found that several proteins including Shootin1, Adenylate kinase 5 isoenzyme and Plasminogen activator-inhibitor 2 are no longer expressed after the 2nd passage of in vitro replication. This indicates that the proliferative potency of these cells is reduced after the initial stage of in vitro growing. At the end of cellular culturing, new synthesized proteins, including, ERO1-like protein alpha, Aspartyl-tRNA synthetase and Prolyl-4-hydroxylase were identified. It is suggested that these new synthesized proteins are involved in the impairment of cellular surviving during replication and differentiation time.<h4>Conclusions</h4>Our work represents an essential step towards gaining knowledge of the molecular properties of WJCs so as to better understand their possible use in the field of cell therapy and regenerative medicine.

<h4>Background and methods</h4>Data on the association between the SLC6A4 5-HTTLPR polymorphism and migraine are conflicting. We performed a systematic review and meta-analysis among studies published up to September 2009. For each study with genotype information, we calculated odds ratios (OR) and 95% confidence intervals (CI) assuming additive, dominant, and recessive genetic models. We then calculated pooled ORs and 95% CIs.<h4>Results</h4>Among the ten studies identified there was no overall association between the polymorphism and any migraine for Europeans or Asians. However, European women carrying the S allele had an increased risk for any migraine (dominant model: pooled OR=2.02; 95% CI 1.24-3.28). Results among Europeans further suggested an increased risk for migraine with aura among carriers of the S/S genotype (recessive model: pooled OR=1.41; 95% CI 0.83-2.40).<h4>Conclusions</h4>While our results indicate no overall association between the SLC6A4 5-HTTLPR polymorphism and migraine among Europeans and Asians, gender and migraine aura status may have modifying roles among Europeans.

Overstimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors accounts, at least in part, for excitotoxic neuronal damage, potentially contributing to a wide range of acute and chronic neurologic diseases. Neurodegenerative disorders including Alzheimer's disease (AD) and Parkinson's disease (PD), manifest deposits of misfolded or aggregated proteins, and result from synaptic injury and neuronal death. Recent studies have suggested that nitrosative stress due to generation of excessive nitric oxide (NO) can mediate excitotoxicity in part by triggering protein misfolding and aggregation, and mitochondrial fragmentation in the absence of genetic predisposition. S-Nitrosylation, or covalent reaction of NO with specific protein thiol groups, represents a convergent signal pathway contributing to NO-induced protein misfolding and aggregation, compromised dynamics of mitochondrial fission-fusion process, thus leading to neurotoxicity. Here, we review the effect of S-nitrosylation on protein function under excitotoxic conditions, and present evidence suggesting that NO contributes to protein misfolding and aggregation via S-nitrosylating protein-disulfide isomerase or the E3 ubiquitin ligase parkin, and mitochondrial fragmentation through beta-amyloid-related S-nitrosylation of dynamin-related protein-1. Moreover, we also discuss that inhibition of excessive NMDA receptor activity by memantine, an uncompetitive/fast off-rate (UFO) drug can ameliorate excessive production of NO, protein misfolding and aggregation, mitochondrial fragmentation, and neurodegeneration.

<h4>Background</h4>Pattern formation of the limb skeleton is regulated by a complex interplay of signaling centers located in the ectodermal sheath and mesenchymal core of the limb anlagen, which results, in the forelimb, in the coordinate array of humerus, radius, ulna, carpals, metacarpals and digits. Much less understood is why skeletal elements form only in the central mesenchyme of the limb, whereas muscle anlagen develop in the peripheral mesenchyme ensheathing the chondrogenic center. Classical studies have suggested a role of the limb ectoderm as a negative regulator of limb chondrogenesis.<h4>Results</h4>In this paper, we investigated the molecular nature of the inhibitory influence of the ectoderm on limb chondrogenesis in the avian embryo in vivo. We show that ectoderm ablation in the early limb bud leads to increased and ectopic expression of early chondrogenic marker genes like Sox9 and Collagen II, indicating that the limb ectoderm inhibits limb chondrogenesis at an early stage of the chondrogenic cascade. To investigate the molecular nature of the inhibitory influence of the ectoderm, we ectopically expressed Wnt6, which is presently the only known Wnt expressed throughout the avian limb ectoderm, and found that Wnt6 overexpression leads to reduced expression of the early chondrogenic marker genes Sox9 and Collagen II.<h4>Conclusion</h4>Our results suggest that the inhibitory influence of the ectoderm on limb chondrogenesis acts on an early stage of chondrogenesis upsteam of Sox9 and Collagen II. We identify Wnt6 as a candidate mediator of ectodermal chondrogenic inhibition in vivo. We propose a model of Wnt-mediated centripetal patterning of the limb by the surface ectoderm.

<h4>Background</h4>Transport of molecules from one subcellular compartment to another involves the recruitment of cytosolic coat protein complexes to a donor membrane to concentrate cargo, deform the membrane and ultimately to form an independent carrier. Small-GTP-binding proteins of the Arf family are central to many membrane trafficking events. Arfs are activated by guanine nucleotide exchange factors (GEFs) which results in their recruitment to membranes and subsequent engagement with Arf-effectors, many of which are coat proteins. Among the human BFA-sensitive large Arf-GEFs, the function of the two closely related BIG1 and BIG2 is still not clear, and recent studies have raised the question of functional redundancy between the two proteins.<h4>Methodology/principal findings</h4>Here we have used small-interfering RNA on human cells and a combination of fixed and live-cell imaging to investigate the differential functions of BIG1 and BIG2 in endomembrane organization and function. Importantly, in this direct comparative study, we show discrete functions for BIG1 and BIG2. Our results show that depletion of BIG2 but not of BIG1 induces a tubulation of the recycling endosomal compartment, consistent with a specific role for BIG2 here. In contrast, suppression of BIG1 induces the formation of Golgi mini-stacks still polarized and functional in terms of cargo export.<h4>Conclusions</h4>A key finding from our work is that suppression of BIG1 expression results in a fragmentation of the Golgi apparatus. Our data indicate that the human BFA-sensitive large Arf-GEFs have non-redundant functions in cell organization and membrane trafficking. BIG1 is required to maintain the normal morphology of the Golgi; BIG2 is important for endosomal compartment integrity and cannot replace the function of BIG1 in Golgi organization.

Alcohol dependence is believed to be a multifactorial, polygenic disorder involving complex gene-gene and gene-environment interactions and confounded by heterogeneity and sociocultural factors. Serotonin (5-Hydroxytryptamine, 5-HT) is thought to be involved in many aspects of alcohol consumption, abuse, and dependence. There was some evidence that serotonin-related genes might interact with the alcohol dehydrogenase (ADH) and the aldehyde dehydrogenase (ALDH) genes in the development of alcohol dependence. In the current review, we discuss the role of serotonin and possible interaction of serotonin-related genes with ADH and ALDH genes in alcohol dependence.

Human aldo-keto reductase (AKR) 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSC)/ type 5 17β-HSD, is known to be involved in steroids, prostaglandins, and lipid aldehydes metabolism. The expression of AKR1C3 has been demonstrated in hormone-dependent normal tissues such as breast, endometrium, prostate, and testis; and de -regulated AKR1C3 expression has been shown in breast carcinoma, endometrial hyperplasia, endometrial carcinoma, and prostate carcinoma. AKR1C3 expression has also been demonstrated in hormone-independent normal tissues (renal tubules and urothelium) and neoplastic tissues (renal cell carcinoma, Wilm's tumor, and urothelial cell carcinoma). Extensive expression of AKR1C3 in normal and neoplastic as well as hormone-dependent and hormone-independent tissues indicates that AKR1C3 may have functions beyond steroid hormone metabolism. In this report, we describe a widespread expression of AKR1C3 in glial neoplasms and meningiomas, with limited expression in medulloblastoma and no expression in Schwannoma. These tumors, except meningioma, are not classically considered to be sex hormone-dependent or related brain tumors. The current results corroborate our earlier observations that AKR1C3 is expressed in both sex hormone-dependent and hormone-independent malignancies. Similar to AKR1C3 distribution in Wilm's tumor, we also demonstrate that expression of AKR1C3 is reduced in tumors with embryonic phenotypes.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms in the world. Genetic polymorphism has been reported to be a factor increasing the risk of HCC. Phase II enzymes such as glutathione s-transferases (GSTP1, GSTA1) play important roles in protecting cells against damage induced by carcinogens. The aim of this study was to estimate the relationship of the GSTP1 and GSTA1 gene polymorphisms to HCC risk and clinico-pathological status.<h4>Methods</h4>Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to measure GSTP1 (A-->G) and GSTA1 (C-->T) gene polymorphisms in 386 healthy controls and 177 patients with HCC.<h4>Results</h4>Neither gene polymorphism was associated with the clinico-pathological status of HCC and serum expression of liver-related clinico-pathological markers. No association between the GSTA1 gene polymorphism and HCC susceptibility was found. However, in the younger group, aged < or = 57 years, individuals with AG or GG alleles of GSTP1 had a 2.18-fold (95%CI = 1.09-4.36; p = 0.02) and 5.64-fold (95%CI = 1.02-31.18; p = 0.04) risk, respectively, of developing HCC compared to individuals with AA alleles, after adjusting for other confounders.<h4>Conclusion</h4>AG and GG alleles of GSTP1 gene polymorphisms may be considered as factors increasing the susceptibility to and risk of HCC in Taiwanese aged < or = 57 years.

Fanconi anemia (FA) and dyskeratosis congenita (DC) are rare inherited syndromes that cause head and neck squamous cell cancer (HNSCC). Prior studies of inherited forms of cancer have been extremely important in elucidating tumor suppressor genes inactivated in sporadic tumors. Here, we studied whether sporadic tumors have epigenetic silencing of the genes causing the inherited forms of HNSCC. Using bisulfite sequencing, we investigated the incidence of promoter hypermethylation of the 17 Fanconi- and DC-associated genes in sporadic HNSCC. Genes that only showed methylation in the tumor patients were chosen for quantitative methylation-specific PCR (qMSP) in a set of 45 tumor and 16 normal patients. Three gene promoters showed differences in methylation: FancB (FAAP95, FA core complex), FancJ (BRIP1, DNA Helicase/ATPase), and DKC1 (dyskeratin). Bisulfite sequencing revealed that only FancB and DKC1 showed no methylation in normal patients, yet the presence of promoter hypermethylation in tumor patients. On qMSP, 1/16 (6.25%) of the normal mucosal samples from non-cancer patients and 14/45 (31.1%) of the tumor patients demonstrated hypermethylation of the FancB locus (p < 0.05). These results suggest that inactivation of FancB may play a role in the pathogenesis of sporadic HNSCC.

The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of various plant diseases. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including cysteine-based peroxidases named peroxiredoxins. This work is a comprehensive analysis of the catalysis performed by PrxQ from X. fastidiosa (XfPrxQ) that belongs to a peroxiredoxin class still poorly characterized and previously considered as moderately reactive toward hydroperoxides. Contrary to these assumptions, our competitive kinetics studies have shown that the second-order rate constants of the peroxidase reactions of XfPrxQ with hydrogen peroxide and peroxynitrite are in the order of 10(7) and 10(6) M(-1) S(-1), respectively, which are as fast as the most efficient peroxidases. The XfPrxQ disulfides were only slightly reducible by dithiothreitol; therefore, the identification of a thioredoxin system as the probable biological reductant of XfPrxQ was a relevant finding. We also showed by site-specific mutagenesis and mass spectrometry that an intramolecular disulfide bond between Cys-47 and Cys-83 is generated during the catalytic cycle. Furthermore, we elucidated the crystal structure of XfPrxQ C47S in which Ser-47 and Cys-83 lie approximately 12.3 A apart. Therefore, significant conformational changes are required for disulfide bond formation. In fact, circular dichroism data indicated that there was a significant redox-dependent unfolding of alpha-helices, which is probably triggered by the peroxidatic cysteine oxidation. Finally, we proposed a model that takes data from this work as well data as from the literature into account.

Iron is essential in the brain, yet too much iron can be toxic. Tight regulation of iron in the brain may involve intrinsic mechanisms that control internal homeostasis independent of systemic iron status. Iron abnormalities occur in various neurological disorders, usually with symptoms or neuropathology associated with movement impairment or behavioral disturbances rather than cognitive impairment or dementia. Consistent with this, polymorphisms in the HFE gene, associated with the iron overload disorder hemochromatosis, show stronger associations with the movement disorder amyotrophic lateral sclerosis (motor neuron disease) than with cognitive impairment. Such associations may arise because certain brain regions involved in movement or executive control are particularly iron-rich, notably the basal ganglia, and may be highly reliant on iron. Various mechanisms, including iron redistribution causing functional iron deficiency, lysosomal and mitochondrial abnormalities or oxidative damage, could underlie iron-related neuropathogenesis. Clarifying how iron contributes causatively to neurodegeneration may improve treatment options in a range of neurodegenerative disorders. This review considers how modern molecular genetic approaches can be applied to resolve the complex molecular systems and pathways by which brain iron homeostasis is regulated and the molecular changes that occur with iron dyshomeostasis and neuropathogenesis.

<h4>Background & aims</h4>Hepatopulmonary syndrome (HPS) affects 10%-30% of patients with cirrhosis and portal hypertension and significantly increases mortality. Studies in experimental models indicate that pulmonary angiogenesis contributes to the development of HPS, but pathogenesis in humans is poorly understood. We investigated genetic risk factors for HPS in patients with advanced liver disease.<h4>Methods</h4>We performed a multicenter case-control study of patients with cirrhosis being evaluated for liver transplantation. Cases had an alveolar-arterial oxygen gradient > or = 15 mm Hg (or > or =20 mm Hg if age > 64 years) and contrast echocardiography with late appearance of microbubbles after venous injection of agitated saline (intrapulmonary vasodilatation); controls did not meet both criteria for case status. The study sample included 59 cases and 126 controls. We genotyped 1086 common single nucleotide polymorphisms (SNPs) in 94 candidate genes.<h4>Results</h4>Forty-two SNPs in 21 genes were significantly associated with HPS after adjustments for race and smoking. Eight genes had at least 2 SNPs associated with disease: CAV3, ENG, NOX4, ESR2, VWF, RUNX1, COL18A1, and TIE1. For example, rs237872 in CAV3 showed an odds ratio of 2.75 (95% confidence interval: 1.65-4.60, P = .0001) and rs4837192 in ENG showed an odds ratio of 0.35 (95% confidence interval: 0.14-0.89, P = .027). Furthermore, variation in CAV3 and RUNX1 was associated with HPS in gene-based analyses.<h4>Conclusions</h4>Polymorphisms in genes involved in the regulation of angiogenesis are associated with the risk of HPS. Further investigation of these biologic pathways might elucidate the mechanisms that mediate the development of HPS in certain patients with severe liver disease.

<h4>Background</h4>Isolated fucoidans from brown marine algae have been shown to have a range of anti-inflammatory effects.<h4>Purpose</h4>This present study tested a Maritech((R)) extract formulation, containing a blend of extracts from three different species of brown algae, plus nutrients in an open label combined phase I and II pilot scale study to determine both acute safety and efficacy in osteoarthritis of the knee.<h4>Patients and methods</h4>Participants (n = 12, five females [mean age, 62 +/- 11.06 years] and seven males [mean age, 57.14 +/- 9.20 years]) with a confirmed diagnosis of osteoarthritis of the knee were randomized to either 100 mg (n = 5) or 1000 mg (n = 7) of a Maritech((R)) extract formulation per day. The formulation contained Maritech((R)) seaweed extract containing Fucus vesiculosis (85% w/w), Macrocystis pyrifera (10% w/w) and Laminaria japonica (5% w/w) plus vitamin B6, zinc and manganese. Primary outcome was the average comprehensive arthritis test (COAT) score which is comprised of four sub-scales: pain, stiffness, difficulty with physical activity and overall symptom severity measured weekly. Safety measures included full blood count, serum lipids, liver function tests, urea, creatinine and electrolytes determined at baseline and week 12. All adverse events were recorded.<h4>Results</h4>Eleven participants completed 12 weeks and one completed 10 weeks of the study. Using a multilevel linear model, the average COAT score was reduced by 18% for the 100 mg treatment and 52% for the 1000 mg dose at the end of the study. There was a clear dose response effect seen between the two treatments (P </= 0.0005) on the average COAT score and each of the four COAT subscales (pain, stiffness, difficulty with physical activity and overall symptom severity) (P </= 0.05). The preparation was well tolerated and the few adverse events were unlikely to be related to the study medication. There were no changes in blood parameters measured over the course of the study with the exception of an increase in serum albumin which was not clinically significant.<h4>Conclusion</h4>The seaweed extract nutrient complex when taken orally over twelve weeks decreased the symptoms of osteoarthritis in a dose-dependent manner. It was demonstrated to be safe to use over the study period at the doses tested. The efficacy of the preparation now needs to be demonstrated in a phase III randomized controlled trial (RCT). AUSTRALIAN AND NEW ZEALAND CLINICAL TRIALS REGISTER: ACTRN12607000229471.

This study reports two novel 5-fluorouracil-substituted ampelopsin derivatives. The structures of two new derivatives were characterized by elemental analysis, 1H-NMR, 13C-NMR, IR and MS. Their anticancer activities in vitro against two cancer cell lines, K562 and K562/ADR, were investigated using the MTT assay, and the results showed that the two new compounds were more effective than reference drugs such as ampelopsin and verapamil.

Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues--netrins, semaphorins, ephrins, and slits--have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.

Perturbations of gene regulatory networks are essentially responsible for oncogenesis. Therefore, inferring the gene regulatory networks is a key step to overcoming cancer. In this work, we propose a method for inferring directed gene regulatory networks based on soft computing rules, which can identify important cause-effect regulatory relations of gene expression. First, we identify important genes associated with a specific cancer (colon cancer) using a supervised learning approach. Next, we reconstruct the gene regulatory networks by inferring the regulatory relations among the identified genes, and their regulated relations by other genes within the genome. We obtain two meaningful findings. One is that upregulated genes are regulated by more genes than downregulated ones, while downregulated genes regulate more genes than upregulated ones. The other one is that tumor suppressors suppress tumor activators and activate other tumor suppressors strongly, while tumor activators activate other tumor activators and suppress tumor suppressors weakly, indicating the robustness of biological systems. These findings provide valuable insights into the pathogenesis of cancer.

<h4>Background</h4>Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat and mouse post-myocardial infarction (post-MI) heart failure and across developmental stages in fetal and neonatal rat myocardium.<h4>Results</h4>The abundance of Arbp, Rpl32, Rpl4, Tbp, Polr2a, Hprt1, Pgk1, Ppia and Gapdh mRNA and 18S ribosomal RNA in myocardial samples was quantified by RT-qPCR. The expression variability of these transcripts was evaluated by the geNorm and Normfinder algorithms and by a variance component analysis method. Biological variability was a greater contributor to sample variability than either repeated reverse transcription or PCR reactions.<h4>Conclusions</h4>The most stable reference genes were Rpl32, Gapdh and Polr2a in mouse post-infarction heart failure, Polr2a, Rpl32 and Tbp in rat post-infarction heart failure and Rpl32 and Pgk1 in human heart failure (ischemic disease and cardiomyopathy). The overall most stable reference genes across all three species was Rpl32 and Polr2a. In rat myocardium, all reference genes tested showed substantial variation with developmental stage, with Rpl4 as was most stable among the tested genes.

Monoamine oxidase A (MAOA) and the transporters for serotonin (5-HTT) and norepinephrine (NET) may play important roles in regulating maternal monoamine neurotransmitters transferred across the placenta to the fetus. We investigated whether promoter polymorphisms in MAOA (uVNTR), 5-HTT (5-HTTLPR), and NET (NETpPR AAGG(4)) could influence gene expression and protein activity in human placentas. Normal term human placentas (n = 73) were collected, and placental MAOA, 5-HTT, and NET mRNA levels and protein activity were determined. The mRNA levels or protein activities were compared between different genotype groups. Placentas hemizygous (male fetus) or homozygous (female fetus) for MAOA uVNTR 4-repeat allele had significantly higher MAOA mRNA levels than those hemizygous or homozygous for the 3-repeat allele (P = 0.001). However, no significant difference in MAOA enzyme activity was found for these two groups of genotypes (P = 0.161). Placentas with the 5-HTTLPR short (S)-allele (S/S+S/L) had significantly lower 5-HTT mRNA levels and serotonin uptake rate than those homozygous for the long (L)-allele (L/L) (mRNA: P < 0.001; serotonin transporting activity: P < 0.001). Placentas homozygous for the NET AAGG(4) L(4) allele had significantly higher NET mRNA levels, as well as dopamine and norepinephrine uptake rates, than those with the S(4)/L(4) genotype (mRNA: P < 0.001; dopamine transporting activity: P = 0.012; norepinephrine transporting activity: P = 0.011). These findings suggest that the three promoter polymorphisms of MAOA, 5-HTT, and NET influence gene expression levels and protein activity of these genes in human placentas, potentially leading to different fetal levels of maternal monoamine neurotransmitters, which may have an impact on fetal neurodevelopment.

"Reactive" or activated stroma characterizes many malignancies including breast cancers. Recently, we isolated a reactive mouse mammary gland stromal cell line called BJ3Z. These cells express alpha-smooth muscle actin and stromal cell-derived factor 1 (SDF-1) and are tumorigenic when injected into mice. Here we show that, in vivo, BJ3Z cells influence the angiogenesis and proliferation of xenografted estrogen receptor (ER)-positive MCF-7 human breast cancer cell-derived solid tumors. The growth-promoting effects of BJ3Z cells are equivalent to those of estradiol (E(2)). BJ3Z cells also increase the proliferation of normal mouse mammary luminal cells adjacent to tumors. In vitro, BJ3Z cells reorganize and increase the proliferation of cocultured malignant MCF-7 and normal human breast MCF10A cells grown as organoids in three-dimensional culture. The effects of BJ3Z cells on MCF-7 cells are equivalent to those of E(2). In contrast, BJ3Z cells do not alter the growth of highly aggressive ER-negative MDA-MB-231 human breast cancer cells. We show that BJ3Z cells secrete vascular endothelial growth factor (VEGF). The growth of MCF-7 organoids induced by BJ3Z can be inhibited by antagonists of VEGF and SDF-1. Conversely, recombinant VEGF stimulates the proliferation of MCF-7, but not MDA-MB-231, organoids. We conclude that, in addition to angiogenesis, VEGF released by activated stroma increases the growth of ER-positive malignant epithelial cells and of adjacent normal epithelium. Because activated stroma can substitute for E(2) and fosters hormone-independent growth of ER-positive tumors, we suggest that breast cancers exhibiting intrinsic hormone resistance may respond to antiangiogenic therapies.

<h4>Purpose</h4>Patients with mixed lineage leukemia (MLL)-rearranged B-lymphoblastic leukemias (B-ALL) have an unfavorable prognosis and require intensified treatment. Multiple MLL fusion partners have been identified, complicating the diagnostic evaluation of MLL rearrangements. We analyzed molecular markers of MLL rearrangement for use in rapid diagnostic assays and found the immunomodulatory protein, Galectin-1 (Gal-1), to be selectively expressed in MLL-rearranged B-ALL.<h4>Experimental design</h4>Transcriptional profiling of ALL subtypes revealed selective overexpression of Gal-1 in MLL-rearranged ALLs. For this reason, we analyzed Gal-1 protein expression in MLL-germline and MLL-rearranged adult and infant pediatric B-ALLs and cell lines by immunoblotting, immunohistochemistry, and intracellular flow cytometry of viable tumor cell suspensions. Because deregulated gene expression in MLL-rearranged leukemias may be related to the altered histone methyltransferase activity of the MLL fusion protein complex, we also analyzed histone H3 lysine 79 (H3K79) dimethylation in the LGALS1 promoter region using chromatin immunoprecipitation.<h4>Results</h4>Gal-1 transcripts were significantly more abundant in MLL-rearranged B-ALLs. All 32 primary MLL-rearranged B-ALLs exhibited abundant Gal-1 immunostaining, regardless of the translocation partner, whereas only 2 of 81 germline-MLL B-ALLs expressed Gal-1. In addition, Gal-1 was selectively detected in newly diagnosed MLL-rearranged B-ALLs by intracellular flow cytometry. The LGALS1 promoter H3K79 was significantly hypermethylated in MLL-rearranged B-ALLs compared with MLL-germline B-ALLs and normal pre-B cells.<h4>Conclusion</h4>In B-ALL, Gal-1 is a highly sensitive and specific biomarker of MLL rearrangement that is likely induced by a MLL-dependent epigenetic modification.

Mucopolysaccharidosis IVA (MPS IVA, Morquio A disease) is an inherited lysosomal storage disorder that features skeletal chondrodysplasia caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Human GALNS was bioengineered with the N-terminus extended by the hexaglutamate sequence (E6) to improve targeting to bone (E6-GALNS). We initially assessed blood clearance and tissue distribution. Next, to assess the effectiveness of storage clearance and reversal of pathological phenotype, a dose of 250 U/g of enzyme was given weekly to Morquio A mice (adults: 12 or 24 weeks, newborn: 8 weeks). Sulfatase modifier factor 1 (SUMF1) was co-transfected to activate the enzyme fully. The E6-GALNS tagged enzyme had markedly prolonged clearance from circulation, giving over 20 times exposure time in blood, compared to untagged enzyme. The tagged enzyme was retained longer in bone, with residual enzyme activity demonstrable at 48 hours after infusion. The pathological findings in adult mice treated with tagged enzyme showed substantial clearance of the storage materials in bone, bone marrow, and heart valves, especially after 24 weekly infusions. Mice treated from the newborn period showed marked reduction of storage materials in tissues investigated. These findings indicate the feasibility of using tagged enzyme to enhance delivery and pathological effectiveness in Morquio A mice.

<h4>Background</h4>In the Drosophila embryonic nerve cord, the formation of commissures require both the chemoattractive Netrin receptor Frazzled (Fra) and the Abelson (Abl) cytoplasmic tyrosine kinase. Abl binds to the cytoplasmic domain of Fra and loss-of-function mutations in abl enhance fra-dependent commissural defects. To further test Abl's role in attractive signaling, we over-expressed Abl in Fra mutants anticipating rescue of commissures.<h4>Methodology/principal findings</h4>The Gal4-UAS system was used to pan-neurally over-express Abl in homozygous fra embryos. Surprisingly, this led to a significant decrease in both posterior and anterior commissure formation and induced some commissural and longitudinal axons to project beyond the CNS/PNS border. Re-expressing wild-type Fra, or Fra mutants with a P-motif deleted, revert both commissural and exiting phenotypes, indicating that Fra is required but not a specific P-motif. This is supported by S2 cell experiments demonstrating that Abl binds to Fra independent of any specific P-motif and that Fra continues to be phosphorylated when individual P-motifs are removed. Decreasing midline repulsion by reducing Robo signaling had no effect on the Abl phenotype and the phenotypes still occur in a Netrin mutant. Pan-neural over-expression of activated Rac or Cdc42 in a fra mutant also induced a significant loss in commissures, but axons did not exit the CNS.<h4>Conclusion/significance</h4>Taken together, these data suggest that Fra activity is required to correctly regulate Abl-dependent cytoskeletal dynamics underlying commissure formation. In the absence of Fra, increased Abl activity appears to be incorrectly utilized downstream of other guidance receptors resulting in a loss of commissures and the abnormal projections of some axons beyond the CNS/PNS border.

T-regulatory cells (Treg) and mast cells (MC) are abundant in colorectal cancer (CRC) tumors. Interaction between the two is known to promote immune suppression or loss of Treg functions and autoimmunity. Here, we demonstrate that in both human CRC and murine polyposis the outcome of this interaction is the generation of potently immune suppressive but proinflammatory Treg (DeltaTreg). These Treg shut down IL10, gain potential to express IL17, and switch from suppressing to promoting MC expansion and degranulation. This change is also brought about by direct coculture of MC and Treg, or culture of Treg in medium containing IL6 and IL2. IL6 deficiency in the bone marrow of mice susceptible to polyposis eliminated IL17 production by the polyp infiltrating Treg, but did not significantly affect the growth of polyps or the generation of proinflammatory Treg. IL6-deficient MC could generate proinflammatory Treg. Thus, MC induce Treg to switch function and escalate inflammation in CRC without losing T-cell-suppressive properties. IL6 and IL17 are not needed in this process.

<h4>Background & aims</h4>Hemochromatosis is a common hereditary disease caused by mutations in HFE and characterized by increased absorption of iron in the intestine. However, the intestine does not appear to be the site of mutant HFE activity in the disease; we investigated the role of the liver-the source of the iron regulatory hormone hepcidin-in pathogenesis in mice.<h4>Methods</h4>We exchanged livers between Hfe wild-type (+/+) and Hfe null (-/-) mice by orthotopic liver transplantation (OLT) and assessed histopathology, serum and tissue iron parameters, and hepatic hepcidin messenger RNA expression.<h4>Results</h4>At 6-8 months after OLT, Hfe(-/-) mice that received Hfe(-/-) livers maintained the hemochromatosis phenotype: iron accumulation in hepatocytes but not Kupffer cells (KC), increased transferrin levels, and low levels of iron in the spleen. Hfe(+/+) mice that received Hfe(-/-) livers had increased levels of iron in serum and liver and low levels of iron in spleen. However, they did not develop the iron-poor KCs that characterize hemochromatosis: KCs appeared iron rich, although hepatic hepcidin expression was low. Transplantation of Hfe(+/+) livers into Hfe(-/-) mice prevented hepatic iron accumulation but did not return spleen and plasma levels of iron to normal; KCs still appeared to be iron poor, despite normal hepcidin expression.<h4>Conclusions</h4>In Hfe(-/-) mice, transplantation of livers from Hfe(+/+) mice reversed the iron-loading phenotype associated with hemochromatosis (regardless of Hfe expression in intestine). However, KCs still had low levels of iron that were not affected by hepatic hepcidin expression. These findings indicate an independent, iron-modifying effect of HFE in KCs.

<h4>Background</h4>Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by an increase in pulmonary artery pressure leading to right ventricular (RV) hypertrophy, RV failure, and ultimately death. Current treatments can improve symptoms and reduce severity of the hemodynamic disorder but gradual deterioration in their condition often necessitates a lung transplant.<h4>Methods and results</h4>In experimental models of PAH, particularly the model of monocrotaline-induced pulmonary hypertension, efficacious treatment options tested so far include a spectrum of pharmacologic agents with actions such as anti-mitogenic, proendothelial function, proangiogenic, antiinflammatory and antioxidative. Emerging trends in PAH treatment are gene and cell therapy and their combination, like (progenitor) cells enriched with eNOS or VEGF gene. More animal data should be collected to investigate optimal cell type, in vitro cell transduction, route of administration, and number of cells to inject. Several recently discovered and experimentally tested interventions bear potential for therapeutic purposes in humans or have been shown already to be effective in PAH patients leading to improved life expectation and better quality of life.<h4>Conclusion</h4>Since many patients remain symptomatic despite therapy, we should encourage research in animal models of PAH and implement promising treatments in homogeneous groups of PAH patients.

In 181 healthy Japanese volunteers we examined the relationship between personality, sensitivity to pain and a single nucleotide polymorphism (rs3813034) in the 3' untranslated region (3' UTR) of the serotonin transporter (5-HTT) gene (SLC6A4). Pain sensitivity was assessed by using cold and pressure thresholds. Personality was assessed by the Temperament and Character Inventory (TCI). Males without the T allele (G/G) showed a significantly higher spiritual acceptance (ST3) score than those who had the T allele (T/T and T/G). Females with the T allele (T/T and T/G) showed significantly higher transpersonal identification (ST2) and self-transcendence (ST) scores than those without the T allele (G/G). As for pain sensitivity and its relationship with TCI, we found a low negative correlation between cold water stimulation, disorderliness (NS4) and novelty seeking (NS) in males, whereas in females we found a low positive correlation between cold water stimulation, self-acceptance (SD4) and pure-hearted principles (C5), as well as pressure stimulation and SD4. It is possible that the 5-HTT 3' UTR gene polymorphism affects the character dimensions of Cloniger's theory, and that there might be a low correlation between pain and a part of the personality.

<h4>Background</h4>Serum ferritin was discovered in the 1930s, and was developed as a clinical test in the 1970s. Many diseases are associated with iron overload or iron deficiency. Serum ferritin is widely used in diagnosing and monitoring these diseases.<h4>Scope of review</h4>In this chapter, we discuss the role of serum ferritin in physiological and pathological processes and its use as a clinical tool.<h4>Major conclusions</h4>Although many aspects of the fundamental biology of serum ferritin remain surprisingly unclear, a growing number of roles have been attributed to extracellular ferritin, including newly described roles in iron delivery, angiogenesis, inflammation, immunity, signaling and cancer.<h4>General significance</h4>Serum ferritin remains a clinically useful tool. Further studies on the biology of this protein may provide new biological insights.

<h4>Background</h4>Lung cancer in individuals who have never smoked tobacco products is an increasing medical and public-health issue. We aimed to unravel the genetic basis of lung cancer in never smokers.<h4>Methods</h4>We did a four-stage investigation. First, a genome-wide association study of single nucleotide polymorphisms (SNPs) was done with 754 never smokers (377 matched case-control pairs at Mayo Clinic, Rochester, MN, USA). Second, the top candidate SNPs from the first study were validated in two independent studies among 735 (MD Anderson Cancer Center, Houston, TX, USA) and 253 (Harvard University, Boston, MA, USA) never smokers. Third, further replication of the top SNP was done in 530 never smokers (UCLA, Los Angeles, CA, USA). Fourth, expression quantitative trait loci (eQTL) and gene-expression differences were analysed to further elucidate the causal relation between the validated SNPs and the risk of lung cancer in never smokers.<h4>Findings</h4>44 top candidate SNPs were identified that might alter the risk of lung cancer in never smokers. rs2352028 at chromosome 13q31.3 was subsequently replicated with an additive genetic model in the four independent studies, with a combined odds ratio of 1.46 (95% CI 1.26-1.70, p=5.94x10(-6)). A cis eQTL analysis showed there was a strong correlation between genotypes of the replicated SNPs and the transcription level of the gene GPC5 in normal lung tissues (p=1.96x10(-4)), with the high-risk allele linked with lower expression. Additionally, the transcription level of GPC5 in normal lung tissue was twice that detected in matched lung adenocarcinoma tissue (p=6.75x10(-11)).<h4>Interpretation</h4>Genetic variants at 13q31.3 alter the expression of GPC5, and are associated with susceptibility to lung cancer in never smokers. Downregulation of GPC5 might contribute to the development of lung cancer in never smokers.<h4>Funding</h4>US National Institutes of Health; Mayo Foundation.

Huntingtin-associated protein-1 (Hap1) is a neuronal protein that associates with huntingtin, the Huntington disease protein. Although Hap1 and huntingtin are known to be involved in intracellular trafficking, whether and how the impairment of Hap1-associated trafficking leads to neurological pathology and symptoms remain to be seen. As Hap1 is enriched in neuronal cells in the brain, addressing this issue is important in defining the role of defective intracellular trafficking in the selective neuropathology associated with Hap1 dysfunction. Here, we find that Hap1 is abundantly expressed in orexin (hypocretin)-producing neurons (orexin neurons), which are distinctly distributed in the hypothalamus and play an important role in the regulation of feeding and behavior. We created conditional Hap1 knock-out mice to selectively deplete Hap1 in orexin neurons via the Cre-loxP system. These mice show process fragmentation of orexin neurons and reductions in food intake, body weight, and locomotor activity. Sucrose density gradient fractionation reveals that loss of Hap1 in the mouse brain also reduces the distribution of trafficking protein complexes and cargo proteins in the fractions that are enriched in synaptosomes. These results suggest that Hap1 is critical for the transport of multiple proteins to the nerve terminals to maintain the integrity of neuronal processes and that selective disruption of the processes of orexin neurons can cause abnormal feeding and locomotor activity.

Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M(2) muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M(2) receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M(2) receptor to enhanced green fluorescent protein-M(2) receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n = 2, 0.495 +/- 0.019; n = 4, 0.202 +/- 0.010; n = 6, 0.128 +/- 0.006; n = 8, 0.093 +/- 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20-0.24, identifying the M(2) receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.

Fundamental to neural organization during development, the netrin-1 guidance cue and its receptor, deleted in colorectal cancer (DCC), continue to be expressed in the adult brain. We have shown recently that adult dcc heterozygous mice do not develop sensitization to the stimulant drug of abuse amphetamine (AMPH) and that repeated exposure to AMPH upregulates DCC expression in adult rats. This upregulation is selective to the ventral tegmental area (VTA), a site critical for the initiation of behavioral plasticity induced by stimulant drugs, and is glutamate-dependent. Here we demonstrate that the lack of AMPH-induced sensitization in dcc heterozygotes is associated with a failure of AMPH to upregulate DCC receptor expression in the VTA. Further, we show that, in wild-type mice, repeated AMPH induces increases in VTA expression of the dendritic spine-associated protein, spinophilin. Significantly, however, this effect is not observed in dcc heterozygotes. In parallel experiments conducted in adult rats, we show that VTA DCC receptor activation, at the time of AMPH pretreatment, is critical for sensitization to AMPH. Together, these results demonstrate that the DCC netrin-1 receptor, a protein traditionally known for its role in organizing brain development, plays a critical function in adult brain plasticity, possibly via orchestration of neuronal circuitry reorganization. We propose VTA DCC receptor signaling as a novel mechanism in the series of glutamate-dependent cellular processes that lead to enduring plasticity by drugs of abuse.

MicroRNAs (miRNAs) are a major class of small endogenous RNA molecules that post-transcriptionally inhibit gene expression. Many miRNAs have been implicated in several human cancers, including breast cancer. Here we describe the association between altered miRNA signatures and breast cancer tumorigenesis and metastasis. The loss of several tumor suppressor miRNAs (miR-206, miR-17-5p, miR-125a, miR-125b, miR-200, let-7, miR-34 and miR-31) and the overexpression of certain oncogenic miRNAs (miR-21, miR-155, miR-10b, miR-373 and miR-520c) have been observed in many breast cancers. The gene networks orchestrated by these miRNAs are still largely unknown, although key targets have been identified that may contribute to the disease phenotype. Here we report how the observed perturbations in miRNA expression profiles may lead to disruption of key pathways involved in breast cancer.

Many studies showed abnormal serotonin transporter (5-HTT) function and heart rate variability (HRV) in panic disorder patients. The present study investigated the relationship between HRV power spectral analysis findings and platelet serotonin uptake in panic disorder patients. Short-term HRV over 5 min and platelet serotonin transporter uptake parameters (V(max) and K(m)) were measured both in 45 patients with panic disorder and in 30 age-matched normal healthy control subjects. Low frequency power (LF) normalized unit (nu) and LF/high frequency power (HF) were significantly higher, whereas HF and HF nu were lower in the patient group than in the control group. V(max) and K(m) were all significantly lower (i.e., reflects decreased 5-HTT function) in patients with panic disorder than in normal controls. In the patient group, K(m) was negatively correlated with LF/HF and LF nu whereas no such correlations between them were found in the control group. By multivariate analysis based on multiple hierarchical linear regression, a low K(m) independently predicted an increased LF nu even after controlling for age, sex, and body mass index in the patient group. These results suggest that impaired 5-HTT function is closely related to dysregulation of autonomic nervous system in panic disorder.

It was reported that C-reactive protein (CRP) levels increase in parallel with the progression of chronic liver diseases, such as chronic hepatitis and liver cirrhosis. Inflammatory markers, such as high sensitive C-reactive protein (hsCRP), ferritin, transferrin, albumin, alpha-1 acid glycoprotein (AAG), alpha-2 macroglobulin (AMG), alpha-1 anti-trypsin (AAT) and lipoprotein a [Lp(a)] were measured in coronary artery disease patients (CAD) and CAD patients with non-alcoholic steatohepatitis (NASH). In the present preliminary study an attempt was made to study whether there is an increase in the levels of CRP in CAD patients associated with NASH. CAD patients showed an increase in CRP and serum ferritin levels. In CAD patients with NASH along with an increase in the levels of serum ferittin (p<0.001), the levels of serum AMG and ceruloplasmin (CP) were also increased (p<0.01). The CAD patients with NASH had a higher proportion of diabetes, hypertension and dyslipidaemia compared to CAD patients. But how this difference contributes to the elevation in acute inflammatory markers particularly AMG and CP levels in CAD patients with NASH cannot be explained. This study shows that a substantial number of CAD patients may be associated with NASH. Non-invasive simple parameters that reflect the degree of inflammation and fibrosis of the liver in patients with NASH would facilitate improved understanding and treatment of the disease. Further studies may be necessary to evaluate the percentage of NASH patients progressing to CAD.

Existing molecular knowledge base of cardiovascular diseases is rudimentary because of lack of specific attribution to cell type and function. The aim of this study was to investigate cell-specific molecular remodeling in human atrial and ventricular myocytes associated with ischemic cardiomyopathy. Our strategy combines two technological innovations, laser-capture microdissection of identified cardiac cells in selected anatomical regions of the heart and splice microarray of a narrow catalog of the functionally most important genes regulating ion homeostasis. We focused on expression of a principal family of genes coding for ion channels, exchangers and pumps (CE&P genes) that are involved in electrical, mechanical and signaling functions of the heart and constitute the most utilized drug targets. We found that (1) CE&P genes remodel in a cell-specific manner: ischemic cardiomyopathy affected 63 CE&P genes in ventricular myocytes and 12 essentially different genes in atrial myocytes. (2) Only few of the identified CE&P genes were previously linked to human cardiac disfunctions. (3) The ischemia-affected CE&P genes include nuclear chloride channels, adrenoceptors, cyclic nucleotide-gated channels, auxiliary subunits of Na(+), K(+) and Ca(2+) channels, and cell-surface CE&Ps. (4) In both atrial and ventricular myocytes ischemic cardiomyopathy reduced expression of CACNG7 and induced overexpression of FXYD1, the gene crucial for Na(+) and K(+) homeostasis. Thus, our cell-specific molecular profiling defined new landmarks for correct molecular modeling of ischemic cardiomyopathy and development of underlying targeted therapies.

Although red blood cell (RBC) transfusions can be lifesaving, they are not without risk. In critically ill patients, RBC transfusions are associated with increased morbidity and mortality, which may increase with prolonged RBC storage before transfusion. The mechanisms responsible remain unknown. We hypothesized that acute clearance of a subset of damaged, stored RBCs delivers large amounts of iron to the monocyte/macrophage system, inducing inflammation. To test this in a well-controlled setting, we used a murine RBC storage and transfusion model to show that the transfusion of stored RBCs, or washed stored RBCs, increases plasma nontransferrin bound iron (NTBI), produces acute tissue iron deposition, and initiates inflammation. In contrast, the transfusion of fresh RBCs, or the infusion of stored RBC-derived supernatant, ghosts, or stroma-free lysate, does not produce these effects. Furthermore, the insult induced by transfusion of stored RBC synergizes with subclinical endotoxinemia producing clinically overt signs and symptoms. The increased plasma NTBI also enhances bacterial growth in vitro. Taken together, these results suggest that, in a mouse model, the cellular component of leukoreduced, stored RBC units contributes to the harmful effects of RBC transfusion that occur after prolonged storage. Nonetheless, these findings must be confirmed by prospective human studies.

Alzheimer's disease (AD), the most common age-associated dementing disorder, is pathologically manifested by progressive cognitive dysfunction concomitant with the accumulation of senile plaques consisting of amyloid-beta (Abeta) peptide aggregates in the brain of affected individuals. Abeta is derived from a type I transmembrane protein, amyloid precursor protein (APP), by the sequential proteolytic events mediated by beta-site APP cleaving enzyme 1 (BACE1) and gamma-secretase. Multiple lines of evidence have implicated cholesterol and cholesterol-rich membrane microdomains, termed lipid rafts in the amyloidogenic processing of APP. In this review, we summarize the cell biology of APP, beta- and gamma-secretases and the data on their association with lipid rafts. Then, we will discuss potential raft targeting signals identified in the secretases and their importance on amyloidogenic processing of APP.

Accumulation of abnormal proteins and endoplasmic reticulum stress accompany neurodegenerative diseases including Huntington's disease. We show that the expression of mutant huntingtin proteins with extended polyglutamine repeats differentially affected endoplasmic reticulum signaling cascades linked to the inositol-requiring enzyme-1 (IRE1) pathway. Thus, the p38 and c-Jun N-terminal kinase pathways were activated, while the levels of the nuclear factor-kappaB-p65 (NF-kappaB-p65) protein decreased. Downregulation of NF-kappaB signaling was linked to decreased antioxidant levels, increased oxidative stress, and enhanced cell death. Concomitantly, calpain was activated, and treatment with calpain inhibitors restored NF-kappaB-p65 levels and increased cell viability. The calpain regulator, calpastatin, was low in cells expressing mutant huntingtin, and overexpression of calpastatin counteracted the deleterious effects caused by N-terminal mutant huntingtin proteins. These results show that calpastatin and an altered NF-kappaB-p65 signaling are crucial factors involved in oxidative stress and cell death mediated by mutant huntingtin proteins.

Elevated concentrations of neurotoxic metabolites of the kynurenine pathway (KP) of tryptophan degradation may play a causative role in Huntington's disease (HD). The brain levels of one of these compounds, 3-hydroxykynurenine (3-HK), are increased in both HD and several mouse models of the disease. In the present study, we examined this impairment in greater detail using the R6/2 mouse, a well-established animal model of HD. Initially, mutant and age-matched wild-type mice received an intrastriatal injection of (3)H-tryptophan to assess the acute, local de novo production of kynurenine, the immediate bioprecursor of 3-HK, in vivo. No effect of genotype was observed between 4 and 12 weeks of age. In contrast, intrastriatally applied (3)H-kynurenine resulted in significantly increased neosynthesis of (3)H-3-HK, but not other tritiated KP metabolites, in the R6/2 striatum. Subsequent ex vivo studies in striatal, cortical and cerebellar tissue revealed substantial increases in the activity of the biosynthetic enzyme of 3-HK, kynurenine 3-monooxygenase and significant reductions in the activity of its degradative enzyme, kynureninase, in HD mice starting at 4 weeks of age. Decreased kynureninase activity was most evident in the cortex and preceded the increase in kynurenine 3-monooxygenase activity. The activity of other KP enzymes showed no consistent brain abnormalities in the mutant mice. These findings suggest that impairments in its immediate metabolic enzymes jointly account for the abnormally high brain levels of 3-HK in the R6/2 model of HD.

Abnormal expansion of a polyglutamine tract in huntingtin (Htt) protein results in Huntington's disease (HD), an autosomal dominant neurodegenerative disorder involving progressive loss of motor and cognitive function. Contrasting with the ubiquitous tissue expression of polyglutamine-expanded Htt, HD pathology is characterized by the increased vulnerability of specific neuronal populations within the striatum and the cerebral cortex. Morphological, biochemical, and functional characteristics of neurons affected in HD that might render these cells more vulnerable to the toxic effects of polyglutamine-Htt are covered in this review. The differential vulnerability of neurons observed in HD is discussed in the context of various major pathogenic mechanisms proposed to date, and in line with evidence showing a 'dying-back' pattern of degeneration in affected neuronal populations.

It is now generally accepted that RNA localization in the central nervous system conveys important roles both during development and in the adult brain. Of special interest is protein synthesis located at the synapse, as this potentially confers selective synaptic modification and has been implicated in the establishment of memories. However, the underlying molecular events are largely unknown. In this review, we will first discuss novel findings that highlight the role of RNA localization in neurons. We will focus on the role of RNA localization in neurotrophin signaling, axon outgrowth, dendrite and dendritic spine morphogenesis as well as in synaptic plasticity. Second, we will briefly present recent work on the role of microRNAs in translational control in dendrites and its implications for learning and memory. Finally, we discuss recent approaches to visualize RNAs in living cells and their employment for studying RNA trafficking in neurons.

We report a case of a 6-month-old boy with a mixed phenotype acute leukemia (MPAL), bilineal and biphenotypic immunophenotype (B-lymphoid lineage and combined B-lymphoid and monocytic lineage) with t(10;11)(p12;q23);MLL-MLLT10. He was treated with acute myeloid leukemia protocol and in complete remission at 7-month follow-up. To the best of our knowledge, this is the first reported MLL-MLLT10 rearranged case presenting as MPAL in an infant. From a clinical practice standpoint, this case illustrates the importance of detection of MLL rearrangement due to its prognostic implication and the effectiveness of flow cytometry immunophenotyping in diagnosing MPAL and monitoring minimal residual disease.

Bioinformatic studies on a revised hierarchy of hematopoietic progenitors have provided a genome-wide view of lineage-affiliated transcriptional programs directing early hematopoiesis. Unexpectedly, lymphoid, myeloid, and erythroid gene expression programs were primed with similar frequency at the multipotent progenitor stage indicating a stochastic nature to this process. Multilineage transcriptional priming is quickly resolved upon erythroid lineage restriction with both lymphoid and myeloid transcriptional programs rapidly extinguished. However, expression of lymphoid and myeloid factors remains active past nominal lymphoid and myeloid lineage restrictions, revealing a common genetic network utilized by both pathways. Priming and resolution of multilineage potential is dependent on the activity of the DNA binding factor Ikaros. Ikaros primes the lymphoid transcriptional program in the HSC and represses the stem cell and other disparate transcriptional programs downstream of the HSC. Loss of Ikaros removes the lymphoid leg of the immune system and may confer aberrant self-renewing properties to myeloid progenitors.

Resistance to endocrine therapy is a major clinical problem in breast cancer. The role of ERalpha splice variants in endocrine resistance is largely unknown. We observed reduced protein expression of an N-terminally truncated ERalpha46 in endocrine-resistant LCC2, LCC9, and LY2 compared to MCF-7 breast cancer cells. Transfection of LCC9 and LY2 cells with hERalpha46 partially restored growth inhibition by TAM. Overexpression of hERalpha46 in MCF-7 cells reduced estradiol (E(2))-stimulated endogenous pS2, cyclin D1, nuclear respiratory factor-1 (NRF-1), and progesterone receptor transcription. Expression of oncomiR miR-21 was lower in TAM-resistant LCC9 and LY2 cells compared to MCF-7 cells. Transfection with ERalpha46 altered the pharmacology of E(2) regulation of miR-21 expression from inhibition to stimulation, consistent with the hypothesis that hERalpha46 inhibits ERalpha activity. Established miR-21 targets PTEN and PDCD4 were reduced in ERalpha46-transfected, E(2)-treated MCF-7 cells. In conclusion, ERalpha46 appears to enhance endocrine responses by inhibiting selected ERalpha66 responses.

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.

<h4>Background</h4>Hepatocellular carcinoma (HCC) is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions.<h4>Methods and findings</h4>We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption). Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy.<h4>Conclusion</h4>Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

POU3F2 is a POU-Homeodomain transcription factor expressed in neurons and melanoma cells. In melanoma lesions, cells expressing high levels of POU3F2 show enhanced invasive and metastatic capacity that can in part be explained by repression of Micropthalmia-associated Transcription Factor (MITF) expression via POU3F2 binding to its promoter. To identify other POU3F2 target genes that may be involved in modulating the properties of melanoma cells, we performed ChIP-chip experiments in 501Mel melanoma cells. 2108 binding loci located in the regulatory regions of 1700 potential target genes were identified. Bioinformatic and experimental assays showed the presence of known POU3F2-binding motifs, but also many AT-rich sequences with only partial similarity to the known motifs at the occupied loci. Functional analysis indicates that POU3F2 regulates the stem cell factor (Kit ligand, Kitl) promoter via a cluster of four closely spaced binding sites located in the proximal promoter. Our results suggest that POU3F2 may regulate the properties of melanoma cells via autocrine KIT ligand signalling.

The neurodegenerative polyglutamine diseases are caused by an expansion of unstable polyglutamine repeats in various disease proteins. Although these mutant proteins are expressed ubiquitously in neuronal and non-neuronal cells, they cause selective degeneration of specific neuronal populations. Recently, increasing evidence shows that polyglutamine disease proteins also affect non-neuronal cells. However, it remains unclear how the expression of polyglutamine proteins in non-neuronal cells contributes to the course of the polyglutamine diseases. Here, we discuss recent findings about the expression of mutant polyglutamine proteins in non-neuronal cells and their influence on neurological symptoms. Understanding the contribution of non-neuronal polyglutamine proteins to disease progression will help elucidate disease mechanisms and also help in the development of new treatment options.

<h4>Objectives</h4>Irritable bowel syndrome (IBS) clusters in families, but previous studies documented family history only from patients. We have shown that patient-relative agreement on IBS status is poor. Thus, we conducted a family case-control study with direct survey of symptoms from family members to better quantitate the aggregation of IBS in families. The aims of this study were to (i) compare the prevalence of IBS in case-relatives with control-relatives, and (ii) determine whether gender, relationship, predominant symptom, and environmental risk factors affect familial aggregation.<h4>Methods</h4>Outpatients with IBS, matched controls, and their first-degree relatives completed a bowel symptom questionnaire. Percent of cases and controls with a family history were compared and odds ratios were computed using chi(2)-test; recurrence risks to relatives were computed using logistic regression and generalized estimating equations.<h4>Results</h4>Data were collected from 477 cases, 297 controls, 1,492 case-relatives, and 936 control-relatives. Probands had a median age of 50 and 78% were women. 50% of case and 27% of control families had at least another relative with IBS yielding an odds ratio of 2.75 (95% CI: 2.01-3.76, P<0.0001). When aggregation estimates were reevaluated stratifying by relative relationship or proband gender, generational and gender effects were not observed. Familial clustering by bowel habit was weakest for diarrhea, and strongest for alternating bowel habits.<h4>Conclusions</h4>IBS aggregates strongly in families. The strength of the association does vary somewhat by relationship to proband, but the lack of association in spouses supports either a possible genetic etiology or a shared household environmental exposure as an underlying cause of IBS.

Protein aggregation, arising from the failure of the cell to regulate the synthesis or degradation of aggregation-prone proteins, underlies many neurodegenerative disorders. However, the balance between the synthesis, clearance, and assembly of misfolded proteins into neurotoxic aggregates remains poorly understood. Here we study the effects of modulating this balance for the amyloid-beta (Abeta) peptide by using a small engineered binding protein (Z(Abeta3)) that binds with nanomolar affinity to Abeta, completely sequestering the aggregation-prone regions of the peptide and preventing its aggregation. Co-expression of Z(Abeta3) in the brains of Drosophila melanogaster expressing either Abeta(42) or the aggressive familial associated E22G variant of Abeta(42) abolishes their neurotoxic effects. Biochemical analysis indicates that monomer Abeta binding results in degradation of the peptide in vivo. Complementary biophysical studies emphasize the dynamic nature of Abeta aggregation and reveal that Z(Abeta3) not only inhibits the initial association of Abeta monomers into oligomers or fibrils, but also dissociates pre-formed oligomeric aggregates and, although very slowly, amyloid fibrils. Toxic effects of peptide aggregation in vivo can therefore be eliminated by sequestration of hydrophobic regions in monomeric peptides, even when these are extremely aggregation prone. Our studies also underline how a combination of in vivo and in vitro experiments provide mechanistic insight with regard to the relationship between protein aggregation and clearance and show that engineered binding proteins may provide powerful tools with which to address the physiological and pathological consequences of protein aggregation.

Peste des Petits Ruminants (PPR) is a highly contagious animal disease caused by the Peste des Petits Ruminants virus (PPRV) belonging to the genus morbillivirus and family Paramyxoviridae. The disease results in high morbidity and mortality in goats, sheep and in some small wild ruminants. The presence of large number of small ruminants reared in endemic areas makes PPR a notorious disease threatening the livelihood of poor farmers. Conventional vaccination using a live, attenuated vaccine gives adequate protection but cannot be used in case of eradication of the disease due to difficulty in differentiation of infected animals from the vaccinated ones.In the present study, we constructed two recombinant viruses using attenuated Modified Vaccinia virus Ankara virus (MVA) namely MVA-F and MVA-H expressing the full length PPRV fusion (F) and hemagglutinin (H) glycoproteins, respectively. Goats were vaccinated intramuscularly with 105 plaque forming units (PFU) each of the recombinant viruses and a live attenuated vaccine (RAKSHA PPR) and challenged 4 months later with PPRV challenge virus (10(3) goat LD(50)). All goats were completely protected from the clinical disease. This study gave an indication that mass vaccination of small ruminants with either of the above or both recombinant inexpensive virus vaccines could help in possible eradication of PPRV from endemic countries like India and subsequent seromonitoring of the disease for differentiation of infected animals from vaccinated ones.

Affective disorders represent the most common psychiatric diseases, with substantial co-morbidity existing between major depressive disorders (MDD) and anxiety disorders. The lack of truly novel acting compounds has led to non-monoaminergic based research and hypotheses in recent years. The large number of brain neuropeptides, characterized by discrete synthesis sites and multiple receptors, represent likely research candidates for novel therapeutic targets. The present review summarises the available preclinical and human evidence regarding the neuropeptide, oxytocin, and its implications in the aetiology and treatment of MDD. While the evidence is not conclusive at present additional studies are warranted to determine whether OXT may be of therapeutic benefit in subsets of MDD patients such as those with comorbid anxiety symptoms and low levels of social attachment.

Despite the excellent bioactivity of hydroxyapatite (HA) ceramics, poor mechanical strength has limited the applications of these materials primarily to coatings and other non-load-bearing areas as bone grafts. Using synthesized HA nanopowder, dense compacts with grain sizes in the nanometer to micrometer range were processed via microwave sintering between 1000 and 1150 degrees C for 20 min. Here we demonstrate that the mechanical properties, such as compressive strength, hardness and indentation fracture toughness, of HA compacts increased with a decrease in grain size. HA with 168 +/- 86 nm grain size showed the highest compressive strength of 395 +/- 42 MPa, hardness of 8.4+/-0.4 GPa and indentation fracture toughness of 1.9 +/- 0.2 MPa m(1/2). To study the in vitro biological properties, HA compacts with grain size between 168 nm and 1.16 microm were assessed for in vitro bone cell-material interactions with human osteoblast cell line. Vinculin protein expression for cell attachment and bone cell proliferation using MTT assay showed that surfaces with finer grains provided better bone cell-material interactions than coarse-grained samples. Our results indicate simultaneous improvements in mechanical and biological properties in microwave sintered HA compacts with nanoscale grain size.

Evidence of marked variability in response among people exposed to the same environmental risk implies that individual differences in genetic susceptibility might be at work. The study of such Gene-by-Environment (GxE) interactions has gained momentum. In this article, the authors review research about one of the most extensive areas of inquiry: variation in the promoter region of the serotonin transporter gene (SLC6A4; also known as 5-HTT) and its contribution to stress sensitivity. Research in this area has both advanced basic science and generated broader lessons for studying complex diseases and traits. The authors evaluate four lines of evidence about the 5-HTT stress-sensitivity hypothesis: 1) observational studies about the serotonin transporter linked polymorphic region (5-HTTLPR), stress sensitivity, and depression in humans; 2) experimental neuroscience studies about the 5-HTTLPR and biological phenotypes relevant to the human stress response; 3) studies of 5-HTT variation and stress sensitivity in nonhuman primates; and 4) studies of stress sensitivity and genetically engineered 5-HTT mutations in rodents. The authors then dispel some misconceptions and offer recommendations for GxE research. The authors discuss how GxE interaction hypotheses can be tested with large and small samples, how GxE research can be carried out before as well as after replicated gene discovery, the uses of GxE research as a tool for gene discovery, the importance of construct validation in evaluating GxE research, and the contribution of GxE research to the public understanding of genetic science.

<h4>Background</h4>Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.<h4>Methods and results</h4>Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2) receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2) receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.<h4>Conclusion</h4>We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2) receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

Conventional anticancer drug screening is typically performed in the absence of accessory cells of the tumor microenvironment, which can profoundly alter antitumor drug activity. To address this limitation, we developed the tumor cell-specific in vitro bioluminescence imaging (CS-BLI) assay. Tumor cells (for example, myeloma, leukemia and solid tumors) stably expressing luciferase are cultured with nonmalignant accessory cells (for example, stromal cells) for selective quantification of tumor cell viability, in presence versus absence of stromal cells or drug treatment. CS-BLI is high-throughput scalable and identifies stroma-induced chemoresistance in diverse malignancies, including imatinib resistance in leukemic cells. A stroma-induced signature in tumor cells correlates with adverse clinical prognosis and includes signatures for activated Akt, Ras, NF-kappaB, HIF-1alpha, myc, hTERT and IRF4; for biological aggressiveness; and for self-renewal. Unlike conventional screening, CS-BLI can also identify agents with increased activity against tumor cells interacting with stroma. One such compound, reversine, shows more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. Use of CS-BLI, therefore, enables refined screening of candidate anticancer agents to enrich preclinical pipelines with potential therapeutics that overcome stroma-mediated drug resistance and can act in a synthetic lethal manner in the context of tumor-stroma interactions.

The aim of this study was to investigate temperature and thermal dose distributions of thermobrachytherapy surface applicators (TBSAs) developed for concurrent or sequential high dose rate (HDR) brachytherapy and microwave hyperthermia treatment of chest wall recurrence and other superficial diseases. A steady-state thermodynamics model coupled with the fluid dynamics of a water bolus and electromagnetic radiation of the hyperthermia applicator is used to characterize the temperature distributions achievable with TBSAs in an elliptical phantom model of the human torso. Power deposited by 915 MHz conformal microwave array (CMA) applicators is used to assess the specific absorption rate (SAR) distributions of rectangular (500 cm(2)) and L-shaped (875 cm(2)) TBSAs. The SAR distribution in tissue and fluid flow distribution inside the dual-input dual-output (DIDO) water bolus are coupled to solve the steady-state temperature and thermal dose distributions of the rectangular TBSA (R-TBSA) for superficial tumor targets extending 10-15 mm beneath the skin surface. Thermal simulations are carried out for a range of bolus inlet temperature (T(b) = 38-43 degrees C), water flow rate (Q(b) = 2-4 L min(-1)) and tumor blood perfusion (omega(b) = 2-5 kg m(-3) s(-1)) to characterize their influence on thermal dosimetry. Steady-state SAR patterns of the R- and L-TBSA demonstrate the ability to produce conformal and localized power deposition inside the tumor target sparing surrounding normal tissues and nearby critical organs. Acceptably low variation in tissue surface cooling and surface temperature homogeneity was observed for the new DIDO bolus at a 2 L min(-1) water flow rate. Temperature depth profiles and thermal dose volume histograms indicate bolus inlet temperature (T(b)) to be the most influential factor on thermal dosimetry. A 42 degrees C water bolus was observed to be the optimal choice for superficial tumors extending 10-15 mm from the surface even under significant blood perfusion. Lower bolus temperature may be chosen to reduce the thermal enhancement ratio (TER) in the most sensitive skin where maximum radiation dose is delivered and to extend the thermal enhancement of radiation dose deeper. This computational study indicates that well-localized elevation of tumor target temperature to 40-44 degrees C can be accomplished by large surface-conforming TBSAs using appropriate selection of coupling bolus temperature.

<h4>Background</h4>In Drosophila melanogaster, dosage compensation is mediated by the action of the dosage compensation complex (DCC). How the DCC recognizes the fly X chromosome is still poorly understood. Characteristic sequence signatures at all DCC binding sites have not hitherto been found.<h4>Results</h4>In this study, we compare the known binding sites of the DCC with oligonucleotide profiles that measure the specificity of the sequences of the D. melanogaster X chromosome. We show that the X chromosome regions bound by the DCC are enriched for a particular type of short, repetitive sequences. Their distribution suggests that these sequences contribute to chromosome recognition, the generation of DCC binding sites and/or the local spreading of the complex. Comparative data indicate that the same sequences may be involved in dosage compensation in other Drosophila species.<h4>Conclusions</h4>These results offer an explanation for the wild-type binding of the DCC along the Drosophila X chromosome, contribute to delineate the forces leading to the establishment of dosage compensation and suggest new experimental approaches to understand the precise biochemical features of the dosage compensation system.

The interplay of transcription factors and epigenetic modifiers, including histone modifications, DNA methylation and microRNAs during development is essential for the acquisition of specific cell fates. Here, we review the epigenetic "programming" of stem cells into oligodendrocytes, by analyzing three sequential stages of lineage progression. The first transition from pluripotent stem cells to neural precursors is characterized by repression of pluripotency genes and restriction of the lineage potential to the neural fate. The second transition from multipotential precursors to oligodendrocyte progenitors is associated with the progressive loss of plasticity and the repression of neuronal and astrocytic genes. The last step of differentiation of oligodendrocyte progenitors into myelin-forming cells is defined by a model of derepression of myelin genes.

Mutations in the COCH (coagulation factor C homology) gene have been attributed to DFNA9 (deafness, autosomal-dominant 9), an autosomal-dominant non-syndromic hearing loss disorder. However, the mechanisms responsible for DFNA9 hearing loss remain unknown. Here, we demonstrate that mutant cochlin, the protein product of the COCH gene, forms a stable dimer that is sensitive to reducing agent. In contrast, wild-type (WT) cochlin may form only dimers transiently. Interestingly, the presence of mutant cochlin can stabilize WT cochlin in dimer conformation, providing a possible mechanism for the dominant nature of DFNA9 mutations. Furthermore, the expression of mutant cochlin eventually induces WT cochlin to form stable oligomers that are resistant to reducing agent. Finally, we show that mutant cochlin is cytotoxic in vitro and in vivo. Our study suggests a possible molecular mechanism underlying DFNA9 hearing loss and provides an in vitro model that may be used to explore protein-misfolding diseases in general.

JNK is a member of the MAPK family and has essential roles in inflammation and cell differentiation and apoptosis. In recent years, there have been accumulating data indicating a novel role for JNK in cell growth and migration. In this report, we demonstrate that JNK activity is necessary for serotonin (5-HT)-induced proliferation and migration of bovine pulmonary artery smooth muscle cells (PASMCs). Stimulation with 5-HT was found to lead to activation of JNK with a maximal activation at 10 min. Inhibition of JNK with its specific inhibitor, SP-600125, or its dominant-negative form, DN-JNK, significantly reduced 5-HT-stimulated [(3)H]thymidine incorporation and cyclin D1 expression. A similar inhibitory effect on SMC migration produced by 5-HT, as detected by a wound healing assay, was observed with inhibition of JNK. Furthermore, inhibition of 5-HT receptors (1B) and (2A), but not inhibition of the 5-HT transporter, blocked 5-HT-induced JNK activation. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY-294002 and wortmannin had little or no effect on 5-HT-induced JNK phosphorylation, but JNK inhibitor SP-600125 and DN-JNK blocked 5-HT-stimulated phosphorylation of Akt and its downstream effectors, p70S6K1 and S6, indicating that Akt is a downstream effector of JNK. Activation of Akt by 5-HT was blocked only minimally, if at all, by inhibitors of ERK and p38 MAPK, indicating a uniqueness of JNK MAPK in this activation of Akt. Coimmunoprecipitation showed binding of Akt to JNK, further supporting the interaction of JNK and Akt. Thus JNK is a critical molecule in 5-HT-induced PASMC proliferation and migration and may act at an important point for cross talk of the MAPK and PI3K pathways. Its activation by 5-HT is initiated through 5-HT (1B) and (2A) receptors, and its stimulation of SMC proliferation and migration occurs through the Akt pathway.

Using recently available mass sequencing and assembly technologies, we have been able to identify and quantify unique cell-free DNA motifs in the blood of patients with multiple sclerosis (MS). The most common MS clinical syndrome, relapsing-remitting MS (RRMS), is accompanied by a unique fingerprint of both inter- and intragenic cell-free circulating nucleic acids as specific DNA sequences that provide significant clinical sensitivity and specificity. Coding genes that are differentially represented in MS serum encode cytoskeletal proteins, brain-expressed regulators of growth, and receptors involved in nervous system signal transduction. Although coding genes distinguish RRMS and its clinical activity, several repeat sequences, such as the L1M family of LINE elements, are consistently different in all MS patients and clinical status versus the normal database. These data demonstrate that DNA motifs observed in serum are characteristic of RRMS and disease activity and are promising as a clinical tool in monitoring patient responses to treatment modalities.

<h4>Background</h4>Advanced-stage ovarian cancer patients are generally treated with platinum/taxane-based chemotherapy after primary debulking surgery. However, there is a wide range of outcomes for individual patients. Therefore, the clinicopathological factors alone are insufficient for predicting prognosis. Our aim is to identify a progression-free survival (PFS)-related molecular profile for predicting survival of patients with advanced-stage serous ovarian cancer.<h4>Methodology/principal findings</h4>Advanced-stage serous ovarian cancer tissues from 110 Japanese patients who underwent primary surgery and platinum/taxane-based chemotherapy were profiled using oligonucleotide microarrays. We selected 88 PFS-related genes by a univariate Cox model (p<0.01) and generated the prognostic index based on 88 PFS-related genes after adjustment of regression coefficients of the respective genes by ridge regression Cox model using 10-fold cross-validation. The prognostic index was independently associated with PFS time compared to other clinical factors in multivariate analysis [hazard ratio (HR), 3.72; 95% confidence interval (CI), 2.66-5.43; p<0.0001]. In an external dataset, multivariate analysis revealed that this prognostic index was significantly correlated with PFS time (HR, 1.54; 95% CI, 1.20-1.98; p = 0.0008). Furthermore, the correlation between the prognostic index and overall survival time was confirmed in the two independent external datasets (log rank test, p = 0.0010 and 0.0008).<h4>Conclusions/significance</h4>The prognostic ability of our index based on the 88-gene expression profile in ridge regression Cox hazard model was shown to be independent of other clinical factors in predicting cancer prognosis across two distinct datasets. Further study will be necessary to improve predictive accuracy of the prognostic index toward clinical application for evaluation of the risk of recurrence in patients with advanced-stage serous ovarian cancer.

There is growing evidence that gene expression profiling of peripheral blood cells is a valuable tool for assessing gene signatures related to exposure, drug-response, or disease. However, the true promise of this approach can not be estimated until the scientific community has robust baseline data describing variation in gene expression patterns in normal individuals. Using a large representative sample set of postmenopausal women (N = 286) in the Norwegian Women and Cancer (NOWAC) postgenome study, we investigated variability of whole blood gene expression in the general population. In particular, we examined changes in blood gene expression caused by technical variability, normal inter-individual differences, and exposure variables at proportions and levels relevant to real-life situations. We observe that the overall changes in gene expression are subtle, implying the need for careful analytic approaches of the data. In particular, technical variability may not be ignored and subsequent adjustments must be considered in any analysis. Many new candidate genes were identified that are differentially expressed according to inter-individual (i.e. fasting, BMI) and exposure (i.e. smoking) factors, thus establishing that these effects are mirrored in blood. By focusing on the biological implications instead of directly comparing gene lists from several related studies in the literature, our analytic approach was able to identify significant similarities and effects consistent across these reports. This establishes the feasibility of blood gene expression profiling, if they are predicated upon careful experimental design and analysis in order to minimize confounding signals, artifacts of sample preparation and processing, and inter-individual differences.

<h4>Background</h4>The distribution of C282Y and H63D variants of the HFE gene was determined in donors with evidence of phenotypical hemochromatosis. The ferritin level and the effect of a donation on iron status in the different HFE genotypes were studied.<h4>Study design and methods</h4>Forty women and 107 men with hemochromatosis were compared to HFE wild-type donors. The influence of a blood donation was assessed by the change in ferritin between two consecutive donations.<h4>Results</h4>In women and men, 85 and 59%, respectively, were C282Y homozygote. None of the women had H63D alleles. There was no significant difference in the donation history or the ferritin level between the HFE genotypes. Donation frequencies were, respectively, 3.3 and 3.7 per year for women and men. The ferritin level was significantly higher in women with hemochromatosis, while in men it was similar compared to the respective wild types. The negative influence of a donation on iron status was similar in hemochromatotic and wild-type women, while men with hemochromatosis were significantly less vulnerable to a blood donation than genetically wild-type men.<h4>Conclusion</h4>Subjects with hemochromatosis are valuable as blood donors independent of their HFE genotype. In general, the ferritin level tended to be higher in those with hemochromatosis than in wild types. The negative influence of a blood donation on iron status was less in male donors with hemochromatosis than in wild types.

A persistent mystery about the ataxias has been why mutations in genes--many of which are expressed widely in the brain--primarily cause ataxia, and not, for example, epilepsy or dementia. Why should a polyglutamine stretch in the TATA-binding protein (that is important in all cells) particularly disrupt cerebellar coordination? We propose that advances in the genetics of cerebellar ataxias suggest a rational hypothesis for how so many different genes lead to predominantly cerebellar defects. We argue that the unifying feature of many genes involved in cerebellar ataxias is their impact on the signaling protein ITPR1 (inositiol 1,4,5-triphosphate receptor type 1), that underlies coincidence detection in Purkinje cells and could play an important role in cerebellar coordination.

Diploid genomes with divergent chromosomes present special problems for assembly software as two copies of especially polymorphic regions may be mistakenly constructed, creating the appearance of a recent segmental duplication. We developed a method for identifying such false duplications and applied it to four vertebrate genomes. For each genome, we corrected mis-assemblies, improved estimates of the amount of duplicated sequence, and recovered polymorphisms between the sequenced chromosomes.

Extensive studies have led to a variety of hypotheses for the molecular basis of depression and related mood disorders, but a definite pathogenic mechanism has yet to be defined. The monoamine hypothesis, in conjunction with the efficacy of antidepressants targeting monoamine systems, has long been the central topic of depression research. While it is widely embraced that the initiation of antidepressant efficacy may involve acute changes in monoamine systems, apparently, the focus of current research is moving toward molecular mechanisms that underlie long-lasting downstream changes in the brain after chronic antidepressant treatment, thereby reaching for a detailed view of the pathophysiology of depression and related mood disorders. In this minireview, we briefly summarize major themes in current approaches to understanding mood disorders focusing on molecular views of depression and antidepressant action.

Poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with gamma-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with gamma-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate gamma-PGA. The activation of DCs by gamma-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of gamma-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with gamma-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8(+) T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of gamma-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freund's adjuvant. These results suggest that gamma-PGA NPs induce a CD8(+) T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, gamma-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.

<h4>Background</h4>Expression of the iron exporter ferroportin at the plasma membrane of macrophages is enhanced by iron loading and is decreased by hepcidin. We previously showed that ferroportin is present in specific cell surface domains suggestive of lipid rafts. Herein, we have clarified the localization of ferroportin in macrophage membranes and tested whether raft-mediated endocytosis plays a role in hepcidin activity.<h4>Design and methods</h4>Raft/detergent-resistant membranes from murine bone marrow-derived macrophages and J774a1 cells were analyzed by Western blotting. The effect of lipid raft- or clathrin-dependent endocytosis inhibitors was studied on hepcidin activity. For this purpose, after treatment, ferroportin expression was analyzed by fluorescence microscopy, Western blotting of total protein extracts or plasma membrane protein samples, and by quantitative immunofluorescence assay (In-Cell-Western).<h4>Results</h4>Macrophage ferroportin was mostly detected in detergent-resistant membranes containing raft markers (caveolin 1, flotillin 1). Interestingly, iron overload strongly increased the presence of ferroportin in the lightest raft fraction. Moreover, lipid raft breakdown by cholesterol sequestration (filipin) or depletion (methyl-beta-cyclodextrin) decreased hepcidin activity on macrophage ferroportin. Cell surface biotinylation and immunofluorescence studies indicated that the process of both hepcidin mediated endocytosis and degradation of ferroportin were affected. By contrast, the inhibition of clathrin dependent endocytosis did not interfere with hepcidin effect.<h4>Conclusions</h4>Macrophage ferroportin is present in lipid rafts which contribute to hepcidin activity. These observations reveal the existence of a new cellular pathway in hepcidin mediated degradation of ferroportin and open a new area of investigation in mammalian iron homeostasis.

Huntington disease (HD) is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis.

Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi), neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in stem cell differentiation.

<h4>Background</h4>Astrocytes exert a wide variety of functions in health and disease and respond to a wide range of signaling pathways, including members of the Janus-kinase signal transducers and activators of transcription (Jak-STAT) family. We have recently shown that STAT3 is an important regulator of astrocyte reactivity after spinal cord injury in vivo[1].<h4>Methodology/principal findings</h4>Here, we used both a conditional gene deletion strategy that targets the deletion of STAT3 selectively to astrocytes (STAT3-CKO), and a pharmacological inhibitor of JAK-2, AG490, in cultured astrocytes in vitro, to investigate potential functions and molecules influenced by STAT3 signaling in relation to mitochondrial function and oxidative stress. Our findings show that the absence of STAT3 signaling in astrocytes leads to (i) increased production of superoxide anion and other reactive oxygen species and decreased level of glutathione, (ii) decreased mitochondrial membrane potential and decreased ATP production, and (iii) decreased rate of cell proliferation. Many of the differences observed in STAT3-CKO astrocytes were distinctly altered by exposure to rotenone, suggesting a role for complex I of the mitochondrial electron transport chain. Gene expression microarray studies identified numerous changes in STAT3-CKO cells that may have contributed to the identified deficits in cell function.<h4>Conclusions/significance</h4>Taken together, these STAT3-dependent alterations in cell function and gene expression have relevance to both reactive gliosis and to the support and protection of surrounding cells in neural tissue.

The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, beta-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (>or=54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.

Endometriosis, defined as the presence of endometrial tissue outside the uterus, is a common gynecological disease with poorly understood pathogenesis. MicroRNAs are members of a class of small noncoding RNA molecules that have a critical role in posttranscriptional regulation of gene expression by repression of target mRNAs translation. We assessed differentially expressed microRNAs in ectopic endometrium compared with eutopic endometrium in 3 patients through microarray analysis. We identified 50 microRNAs differentially expressed and the differential expression of five microRNAs was validated by real-time RT-PCR in other 13 patients. We identified in silico their predicted targets, several of which match the genes that have been identified to be differentially expressed in ectopic versus eutopic endometrium in studies of gene expression. A functional analysis of the predicted targets indicates that several of these are involved in molecular pathways implicated in endometriosis, thus strengthening the hypothesis of the role of microRNAs in this pathology.

A major goal of modern neuroscience research is to understand the cellular and molecular processes that control the formation, function, and remodeling of chemical synapses. In this article, we discuss the numerous studies that implicate molecules initially discovered for their functions in axon guidance as critical regulators of synapse formation and plasticity. Insights from these studies have helped elucidate basic principles of synaptogenesis, dendritic spine formation, and structural and functional synapse plasticity. In addition, they have revealed interesting dual roles for proteins and cellular mechanisms involved in both axon guidance and synaptogenesis. Much like the dual involvement of morphogens in early cell fate induction and axon guidance, many guidance-related molecules continue to play active roles in controlling the location, number, shape, and strength of neuronal synapses during development and throughout the lifetime of the organism. This article summarizes key findings that link axon guidance molecules to specific aspects of synapse formation and plasticity and discusses the emerging relationship between the molecular and cellular mechanisms that control both axon guidance and synaptogenesis.

<h4>Background</h4>Very little is known about how intervertebral disc (IVD) is formed or maintained. Members of the TGF-beta superfamily are secreted signaling proteins that regulate many aspects of development including cellular differentiation. We recently showed that deletion of Tgfbr2 in Col2a expressing mouse tissue results in alterations in development of IVD annulus fibrosus. The results suggested TGF-beta has an important role in regulating development of the axial skeleton, however, the mechanistic basis of TGF-beta action in these specialized joints is not known. One of the hurdles to understanding development of IVD is a lack of known markers. To identify genes that are enriched in the developing mouse IVD and to begin to understand the mechanism of TGF-beta action in IVD development, we undertook a global analysis of gene expression comparing gene expression profiles in developing mouse vertebrae and IVD. We also compared expression profiles in tissues from wild type and Tgfbr2 mutant mice as well as in sclerotome cultures treated with TGF-beta or BMP4.<h4>Results</h4>Lists of IVD and vertebrae enriched genes were generated. Expression patterns for several genes were verified either through in situ hybridization or literature/database searches resulting in a list of genes that can be used as markers of IVD. Cluster analysis using genes listed under the Gene Ontology terms multicellular organism development and pattern specification indicated that mutant IVD more closely resembled vertebrae than wild type IVD. We also generated lists of genes regulated by TGF-beta or BMP4 in cultured sclerotome. As expected, treatment with BMP4 resulted in up-regulation of cartilage marker genes including Acan, Sox 5, Sox6, and Sox9. In contrast, treatment with TGF-beta1 did not regulate expression of cartilage markers but instead resulted in up-regulation of many IVD markers including Fmod and Adamtsl2.<h4>Conclusions</h4>We propose TGF-beta has two functions in IVD development: 1) to prevent chondrocyte differentiation in the presumptive IVD and 2) to promote differentiation of annulus fibrosus from sclerotome. We have identified genes that are enriched in the IVD and regulated by TGF-beta that warrant further investigation as regulators of IVD development.

<h4>Objective</h4>Recent genome-wide association studies have identified multiple novel loci associated with obesity in Europeans. We hypothesized that these genetic variants may be associated with obesity and type 2 diabetes (T2D) in Chinese.<h4>Research design and methods</h4>We examined 14 associated single-nucleotide polymorphisms at 12 loci (NEGR1, SEC16B, TMEM18, ETV5/DGKG, GNPDA2, LIN7C/BDNF, MTCH2, BCDIN3D/FAIM2, SH2B1, FTO, MC4R, and KCTD15) in 605 healthy adults, 1087 healthy adolescents and 6013 T2D patients from Hong Kong.<h4>Results</h4>The European at-risk alleles at five loci including GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 were significantly associated with increased body mass index (BMI), waist circumference (4.5 x 10(-8) < P < 0.024), and/or obesity risk (odds ratio 1.14-1.22, 2.0 x 10(-5) < P < 0.002) in our Chinese populations. The former four loci as well as LIN7C/BDNF were also modestly associated with T2D risk (odds ratio 1.09-1.22, 0.008 < P < 0.041), but the associations were lost after adjustment for BMI, suggesting their roles in T2D risk are mediated through modulation of adiposity. Joint effect analyses of the five adiposity loci revealed an increase of about 0.29 kg/m(2) in BMI with each additional copy of at-risk allele (P(trend) = 4.2 x 10(-12)).<h4>Conclusions</h4>Our findings support the important contribution of GNPDA2, BCDIN3D/FAIM2, SH2B1, FTO, and KCTD15 in the regulation of adiposity, which in turn affects T2D risk in Chinese.

<h4>Purpose</h4>Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes.<h4>Experimental design</h4>Using single nucleotide polymorphism-based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome.<h4>Results</h4>We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the "cell death" network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets.<h4>Conclusions</h4>We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma.

Accumulating evidence suggests that alcohol, hepatitis C virus infection, steatosis with obesity, and insulin resistance are accompanied by iron overload states. Phlebotomy and oral iron chelators are effective treatments for these conditions and for hemochromatosis. However, the mechanisms by which iron depletion improves clinical factors remain unclear. We examined the effect of iron depletion in a model of type 2 diabetes, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Age-matched Long-Evans Tokushima Otsuka (LETO) rats were used as controls for all experiments. Iron restriction was performed by eliminating iron in the diet from 15 wk of age or by phlebotomy. Phlebotomy was commenced at 29 wk of age by removing 4 and 3 ml of blood from the tail vein every week in OLETF and LETO rats, respectively. Rats were euthanized at 43 wk of age, and detailed analyses were performed. The plasma ferritin concentration was markedly higher in OLETF rats and decreased in iron-deficient (ID) diet and phlebotomy rats. Hemoglobin A(1c) (Hb A(1c)) was decreased significantly in OLETF rats fed the ID diet and in the phlebotomy group. Increased levels of triglycerides, glucose, free fatty acids, and total cholesterol were found in ID OLETF rats. Plasma, liver, and pancreas lipid peroxidation and hepatic superoxide production decreased in both groups. Pancreatic fibrosis and insulin levels improved in both groups of OLETF rats. Pancreatic levels of peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) ligands and hypoxia-inducible factor (HIF)-1alpha were decreased significantly in OLETF rats. These factors were normalized in both rats fed ID and phlebotomy groups of OLETF rats. In conclusion, iron depletion improved diabetic complications by inhibition of oxidative stress and TGFbeta signal pathways and the maintenance of pancreatic PPARbeta/delta and HIF-1alpha pathways.

<h4>Purpose</h4>We evaluated the cytotoxic effects of combining suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, with taxanes in human gastric cancer cell lines and assessed the pre-treatment difference of gene expression to identify genes that could potentially mediate the cytotoxic response.<h4>Methods</h4>Gastric cancer cell lines were treated with SAHA and paclitaxel or docetaxel, and the synergistic interaction between the drugs was evaluated in vitro using the combination index (CI) method. We performed significance analysis of microarray (SAM) to identify chemosensitivity-related genes in gastric cancer cell lines that were concomitantly treated with SAHA and taxane. We generated a correlation matrix between gene expression and CI values to identify genes whose expression correlated with a combined effect of taxanes and SAHA.<h4>Results</h4>Combination treatment with taxane and SAHA had a synergistic cytotoxic effect against taxane-resistant gastric cancer cells. We identified 49 chemosensitivity-related genes via SAM analysis. Among them, nine common genes (SLIT2, REEP2, EFEMP2, CDC42SE1, FSD1, POU1F1, ZNF79, ETNK1, and DOCK5) were extracted from the subsequent correlation matrix analysis.<h4>Conclusions</h4>The combination of taxane and SAHA could be efficacious for the treatment of gastric cancer. The genes that were related to the synergistic response to taxane and SAHA could serve as surrogate biomarkers to predict the therapeutic response in gastric cancer patients.

In Bilateria, many axons cross the midline of the central nervous system, forming well-defined commissures. Whereas in mammals the functions of commissures in the forebrain and in the visual system are well established, functions at other axial levels are less clearly understood. Here, we have dissected the function of several hindbrain commissures using genetic methods. By taking advantage of multiple Cre transgenic lines, we have induced site-specific deletions of the Robo3 receptor. These lines developed with the disruption of specific commissures in the sensory, motor, and sensorimotor systems, resulting in severe and permanent functional deficits. We show that mice with severely reduced commissures in rhombomeres 5 and 3 have abnormal lateral eye movements and auditory brainstem responses, respectively, whereas mice with a primarily uncrossed climbing fiber/Purkinje cell projection are strongly ataxic. Surprisingly, although rerouted axons remain ipsilateral, they still project to their appropriate neuronal targets. Moreover, some Cre;Robo3 lines represent potential models that can be used to study human syndromes, including horizontal gaze palsy with progressive scoliosis (HGPPS). To our knowledge, this study is one of the first to link defects in commissural axon guidance with specific cellular and behavioral phenotypes.

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the world's developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.

When transplanted into Xenopus oocytes, the nuclei of mammalian somatic cells are reprogrammed to express stem cell genes such as Oct4, Nanog, and Sox2. We now describe an experimental system in which the pluripotency genes Sox2 and Oct4 are repressed in retinoic acid-treated ES cells but are reprogrammed up to 100% within 24 h by injection of nuclei into the germinal vesicle (GV) of growing Xenopus oocytes. The isolation of GVs in nonaqueous medium allows the reprogramming of individual injected nuclei to be seen in real time. Analysis using fluorescence recovery after photobleaching shows that nuclear transfer is associated with an increase in linker histone mobility. A simultaneous loss of somatic H1 linker histone and incorporation of the oocyte-specific linker histone B4 precede transcriptional reprogramming. The loss of H1 is not required for gene reprogramming. We demonstrate both by antibody injection experiments and by dominant negative interference that the incorporation of B4 linker histone is required for pluripotency gene reactivation during nuclear reprogramming. We suggest that the binding of oocyte-specific B4 linker histone to chromatin is a key primary event in the reprogramming of somatic nuclei transplanted to amphibian oocytes.

Rhabdoid tumors (RT) are aggressive tumors characterized by genetic loss of SMARCB1 (SNF5, INI-1), a component of the SWI/SNF chromatin remodeling complex. No effective treatment is currently available. This study seeks to shed light on the SMARCB1-mediated pathogenesis of RT and to discover potential therapeutic targets. Global gene expression of 10 RT was compared with 12 cellular mesoblastic nephromas, 16 clear cell sarcomas of the kidney, and 15 Wilms tumors. In all, 114 top genes were differentially expressed in RT (P<0.001, fold change >2 or <0.5). Among these were downregulation of SMARCB1 and genes previously associated with SMARCB1 (ATP1B1, PTN, DOCK4, NQO1, PLOD1, PTP4A2, PTPRK); 28/114 top differentially expressed genes were involved with neural or neural crest development and were all sharply downregulated. This was confirmed by Gene Set Enrichment Analysis (GSEA). Neural and neural crest stem cell marker proteins SOX10, ID3, CD133, and Musashi were negative by immunohistochemistry, whereas Nestin was positive. Decreased expression of CDKN1A, CDKN1B, CDKN1C, CDKN2A, and CCND1 was identified, while MYC-C was upregulated. GSEA of independent gene sets associated with bivalent histone modification and polycomb group targets in embryonic stem cells showed significant negative enrichment in RT. Several differentially expressed genes were associated with tumor suppression, invasion, and metastasis, including SPP1 (osteopontin), COL18A1 (endostatin), PTPRK, and DOCK4. We conclude that RTs arise within early progenitor cells during a critical developmental window in which loss of SMARCB1 directly results in repression of neural development, loss of cyclin-dependent kinase inhibition, and trithorax/polycomb dysregulation.

Compelling evidence suggests that inflammation, cell survival, and cancer are linked, with a central role played by NF-kappaB. Recent studies implicate some TLRs in tumor development based on their ability to facilitate tumor growth; however, to our knowledge, involvement of neither TLR7 nor TLR78 has yet been demonstrated. Here we have demonstrated expression of TLR7 and TLR8, the natural receptors for single-stranded RNA, by tumor cells in human lung cancer in situ and in human lung tumor cell lines. Stimulation with TLR7 or TLR8 agonists led to activated NF-kappaB, upregulated expression of the antiapoptotic protein Bcl-2, increased tumor cell survival, and chemoresistance. Transcriptional analysis performed on human primary lung tumor cells and TLR7- or TLR8-stimulated human lung tumor cell lines revealed a gene expression signature suggestive of chronic stimulation of tumor cells by TLR ligands in situ. Together, these data emphasize that TLR signaling can directly favor tumor development and further suggest that researchers developing anticancer immunotherapy using TLR7 or TLR8 agonists as adjuvants should take into account the expression of these TLRs in lung tumor cells.

New synthetic methods for the preparation of biologically active phospholipids and lysophospholipids (LPLs) are very important in solving problems of membrane-chemistry and biochemistry. Traditionally considered just as second-messenger molecules regulating intracellular signalling pathways, LPLs have recently shown to be involved in many physiological and pathological processes such as inflammation, reproduction, angiogenesis, tumorogenesis, atherosclerosis and nervous system regulation. Elucidation of the mechanistic details involved in the enzymological, cell-biological and membrane-biophysical roles of LPLs relies obviously on the availability of structurally diverse compounds. A variety of chemical and enzymatic routes have been reported in the literature for the synthesis of LPLs: the enzymatic transformation of natural glycerophospholipids (GPLs) using regiospecific enzymes such as phospholipases A1 (PLA1), A2 (PLA2) phospholipase D (PLD) and different lipases, the coupling of enzymatic processes with chemical transformations, the complete chemical synthesis of LPLs starting from glycerol or derivatives. In this review, chemo-enzymatic procedures leading to 1- and 2-LPLs will be described.

New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.

Mammalian p62/sequestosome-1 protein binds to both LC3, the mammalian homologue of yeast Atg8, and polyubiquitinated cargo proteins destined to undergo autophagy-mediated degradation. We previously identified a cargo receptor-binding domain in Atg8 that is essential for its interaction with the cargo receptor Atg19 in selective autophagic processes in yeast. We, thus, sought to determine whether this interaction is evolutionally conserved from yeast to mammals. Using an amino acid replacement approach, we demonstrate that cells expressing mutant LC3 (LC3-K30D, LC3-K51A, or LC3-L53A) all exhibit defective lipidation of LC3, a disrupted LC3-p62 interaction, and impaired autophagic degradation of p62, suggesting that the p62-binding site of LC3 is localized within an evolutionarily conserved domain. Importantly, whereas cells expressing these LC3 mutants exhibited similar overall autophagic activity comparable to that of cells expressing wild-type LC3, autophagy-mediated clearance of the aggregation-prone mutant Huntingtin was defective in the mutant-expressing cells. Together, these results suggest that p62 directly binds to the evolutionarily conserved cargo receptor-binding domain of Atg8/LC3 and selectively mediates the clearance of mutant Huntingtin.

<h4>Background</h4>We have previously sequenced more than 500 kb of the duplicated MHC class I regions in Atlantic salmon. In the IA region we identified the loci for the MHC class I gene Sasa-UBA in addition to a soluble MHC class I molecule, Sasa-ULA. A pseudolocus for Sasa-UCA was identified in the nonclassical IB region. Both regions contained genes for antigen presentation, as wells as orthologues to other genes residing in the human MHC region.<h4>Results</h4>The genomic localisation of two MHC class I lineages (Z and S) has been resolved. 7 BACs were sequenced using a combination of standard Sanger and 454 sequencing. The new sequence data extended the IA region with 150 kb identifying the location of one Z-lineage locus, ZAA. The IB region was extended with 350 kb including three new Z-lineage loci, ZBA, ZCA and ZDA in addition to a UGA locus. An allelic version of the IB region contained a functional UDA locus in addition to the UCA pseudolocus. Additionally a BAC harbouring two MHC class I genes (UHA) was placed on linkage group 14, while a BAC containing the S-lineage locus SAA (previously known as UAA) was placed on LG10. Gene expression studies showed limited expression range for all class I genes with exception of UBA being dominantly expressed in gut, spleen and gills, and ZAA with high expression in blood.<h4>Conclusion</h4>Here we describe the genomic organization of MHC class I loci from the U-, Z-, and S-lineages in Atlantic salmon. Nine of the described class I genes are located in the extension of the duplicated IA and IB regions, while three class I genes are found on two separate linkage groups. The gene organization of the two regions indicates that the IB region is evolving at a different pace than the IA region. Expression profiling, polymorphic content, peptide binding properties and phylogenetic relationship show that Atlantic salmon has only one MHC class Ia gene (UBA), in addition to a multitude of nonclassical MHC class I genes from the U-, S- and Z-lineages.

Mineralocorticoid receptor (MR) plays a critical role in brain function. However, the regulatory mechanisms controlling neuronal MR expression that constitutes a key element of the hormonal response are currently unknown. Two alternative P1 and P2 promoters drive human MR gene transcription. To examine promoter activities and their regulation during neuronal differentiation and in mature neurons, we generated stably transfected recombinant murine embryonic stem cell (ES) lines, namely P1-GFP and P2-GFP, in which each promoter drove the expression of the reporter gene green fluorescent protein (GFP). An optimized protocol, using embryoid bodies and retinoic acid, permitted us to obtain a reproducible neuronal differentiation as revealed by the decrease in phosphatase alkaline activity, the concomitant appearance of morphological changes (neurites), and the increase in the expression of neuronal markers (nestin, beta-tubulin III, and microtubule-associated protein-2) as demonstrated by immunocytochemistry and quantitative PCR. Using these cell-based models, we showed that MR expression increased by 5-fold during neuronal differentiation, MR being preferentially if not exclusively expressed in mature neurons. Although the P2 promoter was always weaker than the P1 promoter during neuronal differentiation, their activities increased by 7- and 5-fold, respectively, and correlated with MR expression. Finally, although progesterone and dexamethasone were ineffective, aldosterone stimulated both P1 and P2 activity and MR expression, an effect that was abrogated by knockdown of MR by small interfering RNA. In conclusion, we provide evidence for a tight transcriptional control of MR expression during neuronal differentiation. Given the neuroprotective and antiapoptotic role proposed for MR, the neuronal differentiation of ES cell lines opens potential therapeutic perspectives in neurological and psychiatric diseases.

Butyrophilin (BTN) genes encode a set of related proteins. Studies in mice have shown that one of these, BTN1A1, is required for milk lipid secretion in lactation, whereas butyrophilin-like 2 is a coinhibitor of T cell activation. To understand these disparate roles of BTNs, we first compared the expression and functions of mouse Btn1a1 and Btn2a2. Btn1a1 transcripts were not restricted to lactating mammary tissue but were also found in virgin mammary tissue and, interestingly, spleen and thymus. In confirmation of this, BTN1A1 protein was detected in thymic epithelial cells. By contrast, Btn2a2 transcripts and protein were broadly expressed. Cell surface BTN2A2 protein, such as the B7 family molecule programmed death ligand 1, was upregulated upon activation of T cells. We next examined the potential of both BTN1A1 and BTN2A2 to interact with T cells. Recombinant Fc fusion proteins of murine BTN2A2 and, surprisingly BTN1A1, bound to activated T cells, suggesting the presence of one or more receptors on these cells. Immobilized BTN-Fc fusion proteins, but not MOG-Fc protein, inhibited the proliferation of CD4 and CD8 T cells activated by anti-CD3. BTN1A1 and BTN2A2 also inhibited T cell metabolism, IL-2, and IFN-gamma secretion. Inhibition of proliferation was not abrogated by exogenous IL-2 but could be overcome following costimulation with high levels of anti-CD28 Ab. These data are consistent with a coinhibitory role for mouse BTNs, including BTN1A1, the BTN expressed in the lactating mammary gland and on milk lipid droplets.

Increased iron deposition is often seen in liver explants with alpha-1-antitrypsin deficiency, but it remains unclear if this is a nonspecific effect of end-stage liver disease or if individuals with alpha-1-antitrypsin deficiency and excess iron are at increased risk for HFE mutations. To further examine this question, 45 liver explants with alpha-1-antitrypsin deficiency and 33 control livers with chronic hepatitis C were examined for histological iron accumulation, graded on a scale of 0 to 4+, and HFE mutations. Interestingly, the alpha-1-antitrypsin cirrhotic livers showed a bimodal distribution of iron accumulation, with peaks at grades 1 and 3. In contrast, hepatitis C cirrhotic livers showed a unimodal distribution with a peak at grade 2. HFE mutations in livers with alpha-1-antitrypsin deficiency were as follows: C282Y=2%, H63D=42%. H63D mutations were more frequent in alpha-1-antitrypsin deficiency cases than in controls (42 vs 27%), but was not statistically significant, P=0.17. However, there was a significant association with HFE mutations in alpha-1-antitrypsin deficiency livers with grade 3+ or 4+ iron, P=0.02. In contrast, livers with hepatitis C showed a similar frequency of HFE mutations as the general population: C282Y=15%, H63D=27%. A rare S65C mutation and a novel A271S mutation were also found in this study; the latter patient had 4+ iron in the liver and later developed heart failure with cardiac iron. In conclusion, total H63D mutations were high (42%) in cirrhotics with alpha-1-antitrypsin deficiency and there was a significant association between HFE mutations and high levels of iron accumulation.

<h4>Background</h4>The individual contribution of genes in the HLA region to the risk of developing type 1 diabetes (T1D) is confounded by the high linkage disequilibrium (LD) in this region. Using a novel approach we have combined genetic association data with information on functional protein-protein interactions to elucidate risk independent of LD and to place the genetic association into a functional context.<h4>Methodology/principal findings</h4>Genetic association data from 2300 single nucleotide polymorphisms (SNPs) in the HLA region was analysed in 2200 T1D family trios divided into six risk groups based on HLA-DRB1 genotypes. The best SNP signal in each gene was mapped to proteins in a human protein interaction network and their significance of clustering in functional network modules was evaluated. The significant network modules identified through this approach differed between the six HLA risk groups, which could be divided into two groups based on carrying the DRB1*0301 or the DRB1*0401 allele. Proteins identified in networks specific for DRB1*0301 carriers were involved in stress response and inflammation whereas in DRB1*0401 carriers the proteins were involved in antigen processing and presentation.<h4>Conclusions/significance</h4>In this study we were able to hypothesise functional differences between individuals with T1D carrying specific DRB1 alleles. The results point at candidate proteins involved in distinct cellular processes that could not only help the understanding of the pathogenesis of T1D, but also the distinction between individuals at different genetic risk for developing T1D.

Tolerizing mice polygenically predisposed to lupus-like disease (NZB/NZW F1 females) with a peptide mimicking anti-DNA IgG sequences containing MHC class I and class II T cell determinants (pConsensus, pCons) results in protection from full-blown disease attributable in part to the induction of CD4(+)CD25(+)Foxp3+ and CD8(+)Foxp3+ regulatory T cells. We compared 45 000 murine genes in total white blood cells (WBC), CD4(+) T cells, and CD8(+) T cells from splenocytes of (NZBxNZW) F1 lupus-prone mice tolerized with pCons vs untreated naïve mice and found two-fold or greater differential expression for 448 WBC, 174 CD4, and 60 CD8 genes. We identified differentially expressed genes that played roles in the immune response and apoptosis. Using real-time PCR, we validated differential expression of selected genes (IFI202B, Bcl2, Foxp3, Trp-53, CCR7 and IFNar1) in the CD8(+)T cell microarray and determined expression of selected highly upregulated genes in different immune cell subsets. We also determined Smads expression in different immune cell subsets, including CD4(+) T cells and CD8(+) T cells, to detect the effects of TGF-beta, known to be the major cytokine that accounts for the suppressive capacity of CD8(+) Treg in this system. Silencing of anti-apoptotic gene Bcl2 or interferon genes (IFI202b and IFNar1 in combination) in CD8(+) T cells from tolerized mice did not affect the expression of the other selected genes. However, silencing of Foxp3 reduced expression of Foxp3, Ifi202b and PD1-all of which are involved in the suppressive capacity of CD8(+) Treg in this model.

Epigenetic modifications of chromatin play an important role in the regulation of gene expression. KMT4/Dot1 is a conserved histone methyltransferase capable of methylating chromatin on Lys79 of histone H3 (H3K79). Here we report the identification of a multisubunit Dot1 complex (DotCom), which includes several of the mixed lineage leukemia (MLL) partners in leukemia such as ENL, AF9/MLLT3, AF17/MLLT6, and AF10/MLLT10, as well as the known Wnt pathway modifiers TRRAP, Skp1, and beta-catenin. We demonstrated that the human DotCom is indeed capable of trimethylating H3K79 and, given the association of beta-catenin, Skp1, and TRRAP, we investigated, and found, a role for Dot1 in Wnt/Wingless signaling in an in vivo model system. Knockdown of Dot1 in Drosophila results in decreased expression of a subset of Wingless target genes. Furthermore, the loss of expression for the Drosophila homologs of the Dot1-associated proteins involved in the regulation of H3K79 shows a similar reduction in expression of these Wingless targets. From yeast to human, specific trimethylation of H3K79 by Dot1 requires the monoubiquitination of histone H2B by the Rad6/Bre1 complex. Here, we demonstrate that depletion of Bre1, the E3 ligase required for H2B monoubiquitination, leads specifically to reduced bulk H3K79 trimethylation levels and a reduction in expression of many Wingless targets. Overall, our study describes for the first time the components of DotCom and links the specific regulation of H3K79 trimethylation by Dot1 and its associated factors to the Wnt/Wingless signaling pathway.

Accelerated intimal and medial calcification and sclerosis accompany the increased cardiovascular mortality of dialysis patients, but the pathomechanisms initiating microcalcifications of the media are largely unknown. In this study, we systematically investigated the ultrastructural properties of medial calcifications from patients with uremia. We collected iliac artery segments from 30 dialysis patients before kidney transplantation and studied them by radiography, microcomputed tomography, light microscopy, and transmission electron microscopy including electron energy loss spectrometry, energy dispersive spectroscopy, and electron diffraction. In addition, we performed synchrotron x-ray analyses and immunogold labeling to detect inhibitors of calcification. Von Kossa staining revealed calcification of 53% of the arteries. The diameter of these microcalcifications ranged from 20 to 500 nm, with a core-shell structure consisting of up to three layers (subshells). Many of the calcifications consisted of 2- to 10-nm nanocrystals and showed a hydroxyapatite and whitlockite crystalline structure and mineral phase. Immunogold labeling of calcification foci revealed the calcification inhibitors fetuin-A, osteopontin, and matrix gla protein. These observations suggest that uremic microcalcifications originate from nanocrystals, are chemically diverse, and intimately associate with proteinaceous inhibitors of calcification. Furthermore, considering the core-shell structure of the calcifications, apoptotic bodies or matrix vesicles may serve as a calcification nidus.

<h4>Purpose</h4>Inhibitors of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades neurotransmitters including serotonin and norepinephrine, are commonly used to treat neurological conditions including depression. Recently, we and others identified high expression of MAOA in normal basal prostatic epithelium and high-grade primary prostate cancer (PCa). In contrast, MAOA is low in normal secretory prostatic epithelium and low-grade PCa. An irreversible inhibitor of MAOA, clorgyline, induced secretory differentiation in primary cultures of normal basal epithelial cells and high-grade PCa. Furthermore, clorgyline inhibited several oncogenic pathways in PCa cells, suggesting clinical value of MAOA inhibitors as a pro-differentiation and anti-oncogenic therapy for high-risk PCa. Here, we extended our studies to a model of advanced PCa, VCaP cells, which were derived from castration-resistant metastatic PCa and express a high level of MAOA.<h4>Methods</h4>Growth of VCaP cells in the presence or absence of clorgyline was evaluated in vitro and in vivo. Gene expression changes in response to clorgyline were determined by microarray and validated by quantitative real-time polymerase chain reaction.<h4>Results</h4>Treatment with clorgyline in vitro inhibited growth and altered the transcriptional pattern of VCaP cells in a manner consistent with the pro-differentiation and anti-oncogenic effects seen in treated primary PCa cells. Src, beta-catenin, and MAPK oncogenic pathways, implicated in androgen-independent growth and metastasis, were significantly downregulated. Clorgyline treatment of mice bearing VCaP xenografts slowed tumor growth and induced transcriptome changes similar to those noted in vitro.<h4>Conclusion</h4>Our results support the possibility that anti-depressant drugs that target MAOA might find a new application in treating PCa.

The mechanisms underlying synovial joint development remain poorly understood. Here we use complete and cell-specific gene inactivation to identify the roles of the redundant chondrogenic transcription factors Sox5 and Sox6 in this process. We show that joint development aborts early in complete mutants (Sox5(-/-)6(-/-)). Gdf5 and Wnt9a expression is punctual in articular progenitor cells, but Sox9 downregulation and cell condensation in joint interzones are late. Joint cell differentiation is unsuccessful, regardless of lineage, and cavitation fails. Sox5 and Sox6 restricted expression to chondrocytes in wild-type embryos and continued Erg expression and weak Ihh expression in Sox5(-/-)6(-/-) growth plates suggest that growth plate failure contribute to this Sox5(-/-)6(-/-) joint morphogenesis block. Sox5/6 inactivation in specified joint cells and chondrocytes (Sox5(fl/fl)6(fl/fl)Col2Cre) also results in a joint morphogenesis block, whereas Sox5/6 inactivation in specified joint cells only (Sox5(fl/fl)6(fl/fl)Gdf5Cre) results in milder joint defects and normal growth plates. Sox5(fl/fl)6(fl/fl)Gdf5Cre articular chondrocytes remain undifferentiated, as shown by continued Gdf5 expression and pancartilaginous gene downregulation. Along with Prg4 downregulation, these defects likely account for joint tissue overgrowth and incomplete cavitation in adult mice. Together, these data suggest that synovial joint morphogenesis relies on essential roles for Sox5/6 in promoting both growth plate and articular chondrocyte differentiation.

<h4>Background</h4>Previous studies have suggested a probable association between the polymorphism of a microsatellite locus located in the promoter of IGF1 (Insulin-like growth factor 1) gene and the serum level of IGF1, as well as many age-related diseases. Based on these results, we hypothesized that this polymorphism may influence longevity in humans. We performed an association study in a Han Chinese population to test this hypothesis.<h4>Findings</h4>We recruited 493 elderly Han Chinese individuals (females >/= 94; males >/= 90) and 425 young individuals (controls) from Dujiangyan (Sichuan province, China). The genotype distributions and allele frequencies of the microsatellite site in the elderly and control groups were compared by chi square test.Our results suggested that there was no association between the microsatellite polymorphism and longevity in our Han Chinese population. However, there were more male persons with 18/21 genotype in elderly group than that in control group (11.11 vs. 5.45%, p = 0.011). As the difference was not significant when corrected by Bonferroni method, we speculate that the 18/21 genotype can not be functional in longevity; however, it may link with the real functional loci as there is a long haplotype block embracing the microsatellite locus.<h4>Conclusions</h4>There was no association between polymorphism of the microsatellite in promoter of IGF1 gene and longevity in our study. Future association studies containing the long haplotype block are deserved and can test our speculation of the potential linkage of 18/21 genotype and functional loci.

Protocadherins are a subfamily of the cadherin superfamily, but little is known about their functions. We identified a homozygous loss of protocadherin (PCDH) 17 in the course of a program to screen a panel of esophageal squamous cell carcinoma (ESCC) cell lines for genomic copy number aberrations. PCDH17 messenger RNA was expressed in normal esophageal tissue but not in the majority of ESCC cell lines without a homozygous deletion of this gene and restored in gene-silenced ESCC cells after treatment with 5-aza-2'-deoxycytidine. The DNA methylation status of the PCDH17 CpG island correlated inversely with the PCDH17 expression, and a putative methylation target region showed promoter activity. The methylation of the PCDH17 promoter was also associated with the silencing of gene expression in primary ESCC partly. Among primary ESCC cases, the silencing of PCDH17 protein expression was associated with a poorer differentiation status of ESCC cells and possibly with prognosis in a subset of this tumour. Restoration of PCDH17 expression in ESCC cells reduced cell proliferation and migration/invasion. These results suggest that silencing of PCDH17 expression through hypermethylation of the promoter or other mechanisms leads to loss of its tumour-suppressive activity, which may be a factor in the carcinogenesis of a subgroup of ESCCs.

Canine parvovirus (CPV) and its relative feline panleukopenia virus (FPV) bind the transferrin receptor type 1 (TfR) to infect their host cells but show differences in the interactions with the feline and canine TfRs that determine viral host range and tissue tropism. We changed apical and protease-like domain residues by introducing point mutations and adding or removing glycosylation signals, and we then examined the interactions of those mutant TfRs with the capsids. Most substitutions had little effect on virus binding and uptake. However, mutations of several sites in the apical domain of the receptor either prevented binding to the capsids or reduced the affinity of receptor binding to various degrees. Glycans within the virus binding face of the apical domain also controlled capsid binding. CPV, but not the related feline parvovirus, could use receptors containing a canine TfR-specific glycosylation to mediate efficient infection, while addition of other N-linked glycosylation sites into the virus binding face of the feline apical domain reduced or eliminated both binding and infection. Replacement of critical feline TfR residue 221 with every amino acid had effects on binding and infection which were significantly associated with the biochemical properties of the residue replaced. Receptors with reduced affinities mostly showed proportional changes in their ability to mediate infection. Testing feline TfR variants for their binding and uptake patterns in cells showed that low-affinity versions bound fewer capsids and also differed in attachment to the cell surface and filopodia, but transport to the perinuclear endosome was similar.

Iron-refractory, iron-deficiency anemia (IRIDA) is a familial disorder characterized by iron deficiency anemia unresponsive to oral iron treatment but partially responsive to intravenous iron therapy. Previously, we showed that IRIDA patients harbor loss-of-function mutations in TMPRSS6, a type II transmembrane serine protease primarily expressed by the liver. Both humans and mice with TMPRSS6 mutations show inappropriately elevated levels of the iron-regulatory hormone hepcidin, suggesting that TMPRSS6 acts to negatively regulate hepcidin expression. Here we investigate the relationship between Tmprss6 and the bone morphogenetic protein (BMP)-Smad signaling pathway, a key pathway promoting hepcidin transcription in hepatocytes. We show that livers from mice deficient for Tmprss6 have decreased iron stores and decreased Bmp6 mRNA, but markedly increased mRNA for Id1, a target gene of Bmp6 signaling. In contrast, mice deficient for both Tmprss6 and hemojuvelin (Hjv), a BMP coreceptor that augments hepcidin expression in hepatocytes, showed markedly decreased hepatic levels of hepcidin and Id1 mRNA, markedly increased hepatic Bmp6 mRNA levels, and systemic iron overload similar to mice deficient for Hjv alone. These findings suggest that down-regulation of Bmp/Smad signaling by Tmprss6 is required for regulation of hepcidin expression and maintenance of systemic iron homeostasis.

Tesofensine is a novel monoamine reuptake inhibitor that inhibits both norepinephrine, 5-HT, and dopamine (DA) reuptake function. Tesofensine is currently in clinical development for the treatment of obesity, however, the pharmacological basis for its strong effect in obesity management is not clarified. Using a rat model of diet-induced obesity (DIO), we characterized the pharmacological mechanisms underlying the appetite suppressive effect of tesofensine. DIO rats treated with tesofensine (2.0 mg/kg, s.c.) for 16 days showed significantly lower body weights than vehicle-treated DIO rats, being reflected by a marked hypophagic response. Using an automatized food intake monitoring system during a 12 h nocturnal test period, tesofensine-induced hypophagia was investigated further by studying the acute interaction of a variety of monoamine receptor antagonists with tesofensine-induced hypophagia in the DIO rat. Tesofensine (0.5-3.0 mg/kg, s.c.) induced a dose-dependent and marked decline in food intake with an ED(50) of 1.3 mg/kg. The hypophagic response of tesofensine (1.5 mg/kg, s.c.) was almost completely reversed by co-administration of prazosin (1.0 mg/kg, alpha(1) adrenoceptor antagonist) and partially antagonized by co-administration of SCH23390 (0.03 mg/kg, DA D(1) receptor antagonist). In contrast, tesofensine-induced hypophagia was not affected by RX821002 (0.3 mg/kg, alpha(2) adrenoceptor antagonist), haloperidol (0.03 mg/kg, D(2) receptor antagonist), NGB2904 (0.1 mg/kg, D(3) receptor antagonist), or ritanserin (0.03 mg/kg, 5-HT(2A/C) receptor antagonist). Hence, the mechanism underlying the suppression of feeding by tesofensine in the obese rat is dependent on the drug's ability to indirectly stimulate alpha(1) adrenoceptor and DA D(1) receptor function.

No abstract available.

<h4>Background</h4>Purine catabolism may be an unappreciated, but important component of the homeostatic response of mitochondria to oxidant stress. Accumulating evidence suggests a pivotal role of oxidative stress in schizophrenia pathology.<h4>Methodology/principal findings</h4>Using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system, we compared 6 purine metabolites simultaneously in plasma between first-episode neuroleptic-naïve patients with schizophrenia (FENNS, n = 25) and healthy controls (HC, n = 30), as well as between FENNS at baseline (BL) and 4 weeks (4w) after antipsychotic treatment. Significantly higher levels of xanthosine (Xant) and lower levels of guanine (G) were seen in both patient groups compared to HC subjects. Moreover, the ratios of G/guanosine (Gr), uric acid (UA)/Gr, and UA/Xant were significantly lower, whereas the ratio of Xant/G was significantly higher in FENNS-BL than in HC. Such changes remained in FENNS-4w with exception that the ratio of UA/Gr was normalized. All 3 groups had significant correlations between G and UA, and Xan and hypoxanthine (Hx). By contrast, correlations of UA with each of Xan and Hx, and the correlation of Xan with Gr were all quite significant for the HC but not for the FENNS. Finally, correlations of Gr with each of UA and G were significant for both HC and FENNS-BL but not for the FENNS-4w.<h4>Conclusions/significance</h4>During purine catabolism, both conversions of Gr to G and of Xant to Xan are reversible. Decreased ratios of product to precursor suggested a shift favorable to Xant production from Xan, resulting in decreased UA levels in the FENNS. Specifically, the reduced UA/Gr ratio was nearly normalized after 4 weeks of antipsychotic treatment. In addition, there are tightly correlated precursor and product relationships within purine pathways; although some of these correlations persist across disease or medication status, others appear to be lost among FENNS. Taken together, these results suggest that the potential for steady formation of antioxidant UA from purine catabolism is altered early in the course of illness.

Both acute and chronic degenerative diseases of the nervous system reduce the viability and function of neurons through changes in intracellular calcium signaling. In particular, pathological increases in the intracellular calcium concentration promote such pathogenesis. Disease involvement of numerous regulators of intracellular calcium signaling located on the plasma membrane and intracellular organelles has been documented. Diverse groups of chemical compounds targeting ion channels, G-protein coupled receptors, pumps and enzymes have been identified as potential neuroprotectants. The present review summarizes the discovery, mechanisms and biological activity of neuroprotective molecules targeting proteins that control intracellular calcium signaling to preserve or restore structure and function of the nervous system. Disease relevance, clinical applications and new technologies for the identification of such molecules are being discussed.

Celiac disease (CD) is an immune-mediated disorder triggered by the ingestion of wheat gliadin and related proteins in genetically predisposed individuals. To find a proteomic CD diagnostic signature and to gain a better understanding of pathogenetic mechanisms associated with CD, we analyzed the intestinal mucosa proteome alterations using two dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF ms) of CD patients with varying degrees of histological abnormalities defined by Marsh criteria and controls. Our results clearly evidenced the presence of two groups of patients: Group A, including controls and Marsh 0-I CD patients; and Group B, consisting of CD subjects with grade II-III Oberhuber-Marsh classification. Differentially expressed proteins were involved mainly in lipid, protein and sugar metabolism. Interestingly, in Group B, several downregulated proteins (FABP1, FABP2, APOC3, HMGCS2, ACADM and PEPCK) were implicated directly in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Moreover, Group B patients presented a deregulation of some proteins involved in apoptosis/survival pathways: phosphatidylethanolamine-binding protein 1 (PEBP1), Ras-related nuclear protein (Ran) and peroxiredoxin 4 (PRDX4). PEBP1 downregulation and RAN and PRDX4 upregulation were associated with more severe tissue damage. Likewise, IgMs were found strongly upregulated in Group B. In conclusion, our results indicate that a downregulation of proteins involved in PPAR signaling and the modulation of several cancer-related proteins are associated with the highest CD histological score according to Oberhuber-Marsh classification.

Familial adenomatous polyposis (FAP) is the second most common hereditary colorectal cancer syndrome and confers a nearly 100% lifetime risk of developing colorectal cancer. Understanding factors that facilitate and inhibit genetic testing and cancer surveillance in children who are members of families affected by FAP will better equip clinicians to clarify misunderstandings and facilitate appropriate care. The aims of this study were to examine parental attitudes and beliefs regarding endoscopic surveillance and genetic testing in minors at risk for developing FAP. This cross-sectional study includes analyses of qualitative and quantitative interview data collected from parents of children with or at risk for FAP. This report includes data from 28 parents with a total of 51 biological children between 10-17 years of age. The parents had a clinical and/or genetic diagnosis of FAP. Most commonly reported facilitators included provider recommendation (surveillance) and personalized medical management (genetic testing). Most commonly reported barriers included lack of provider recommendation (surveillance) and cost (genetic testing).

<h4>Background</h4>Hereditary hemochromatosis (HH) is an autosomal recessive disorder mainly associated with homozygosity for the C282Y and H63D mutations in the hemochromatosis (HFE) gene. The reports about the C282Y and H63D mutations and hepatocellular carninoma (HCC) were controversial. To clarify the relationship between C282Y and H63D mutations and HCC, a meta-analysis including nine studies (1102 HCC cases and 3766 controls, mainly came from European populations) was performed.<h4>Methods</h4>The association was measured using random-effect (RE) or fixed-effect (FE) odds ratios (ORs) combined with 95% confidence intervals (CIs) according to the studies' heterogeneity.<h4>Results</h4>Meta-analysis of nine studies showed that Y allele of C282Y was associated with HCC risk: RE OR reached 1.50 (95%CI: 1.05-2.14, p for heterogeneity = 0.02, I2 = 0.57). Subgroup analysis of seven studies also showed Y allele was associated with HCC risk in healthy populations: RE OR reached 1.61 (95%CI: 1.08-2.39, p for heterogeneity = 0.04, I2 = 0.55). We further did subgroup analysis in alcoholic liver cirrhosis (LC) patients of four studies (224 cases and 380 controls) and found that both the dominant model and Y allele of C282Y were associated with HCC risk (FE OR reached 4.06, 95%CI: 2.08-7.92 and 3.41, 95%CI: 1.81-6.41, respectively). There was no distinct heterogeneity among the studies (I2 = 0). Sensitivity analyses showed the results were robust in the subgroup analysis of alcoholic LC patients.<h4>Conclusions</h4>C282Y mutation was associated with HCC in European alcoholic LC patients.

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1(omega5) was purified from geranium (Pelargonium x hortorum) and shown to inhibit the proliferation of estrogen receptor alpha (ERalpha)-positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERalpha-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1(omega5) inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1(omega5) inhibited estradiol (E(2))-induced estrogen response element (ERE) reporter activity and transcription of the endogenous E(2) target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1(omega5) did not compete with E(2) for ERalpha or ERbeta binding, nor did AnAc 24:1(omega5) reduce ERalpha or ERbeta steady-state protein levels in MCF-7 cells; rather, AnAc 24:1(omega5) inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1(omega5) interaction with ERalpha ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1(omega5) interaction with the ERalpha DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1(omega5) inhibited E(2)-ERalpha interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1(omega5) inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses.

Obstructive sleep apnea may cause vascular inflammation and atherosclerosis, which has been attributed to intermittent hypoxia (IH). Recent data suggest that IH, but not sustained hypoxia (SH), activates proinflammatory genes in HeLa cells. Effects of IH and SH on the gene expression profile in human aortic endothelial cells (HAEC) have not been compared. We perfused media with alternating flow of 16% and 0% O2 (IH) or constant flow of 4% O2 (SH-4%), 8% O2 (SH-8%), or 16% O2 (control) for 8 h. Illumina gene microarrays were performed, with subsequent verification by real-time PCR. Proinflammatory cytokines in the media were measured by ELISA. Both IH and SH-4% upregulated proinflammatory genes, including heat shock protein 90-kDa B1, tumor necrosis factor superfamily member 4, and thrombospondin 1. Among all proinflammatory genes, only IL-8 mRNA showed significantly higher levels of expression (1.78-fold) during IH, compared with SH-4%, but both types of hypoxic exposure elicited striking three- to eightfold increases in IL-8 and IL-6 protein levels in the media. IH and SH-4% also upregulated antioxidant genes, including heme oxygenase-1 and nuclear factor (erythroid-derived 2)-like 2 (NRF2), whereas classical genes regulated by hypoxia-inducible factor 1 (HIF-1), such as endothelin and glucose transporter GLUT1, were not induced. SH-8% induced changes in gene expression and cytokine secretion that were similar to those of IH and SH-4%. In conclusion, short exposures to IH and SH upregulate proinflammatory and antioxidant genes in HAEC and increase secretion of proinflammatory cytokines IL-8 and IL-6 into media in similar fashions.

Dipetidyl-peptidase III is a metallopeptidase involved in a number of physiological processes and its expression has been reported to increase with the histological aggressiveness of human ovarian primary carcinomas. Because no information regarding the regulation of its expression was available, experiments were designed to clone, define and characterize the promoter region of the human dipeptidyl-peptidase III (DPP-III) gene. In this study, we cloned a 1038 bp 5'-flanking DNA fragment of the human DPP-III gene for the first time and demonstrated strong promoter activity in this region. Deletion analysis revealed that as few as 45 nucleotides proximal to the transcription start site retained approximately 40% of the activity of the full-length promoter. This promoter lacked the TATA box but contained multiple GC boxes and a single CAAT box. Similarly, two Ets-1/Elk-1-binding motifs are present in the first 25 nucleotides from the transcription start site. Binding of Ets-1/Elk-1 proteins to these motifs was visualized by electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutations of these binding sites abolished not only binding of the Ets protein, but also the intrinsic promoter activity. Increased DNA-binding activity of Ets-1/Elk-1 by v-Ha-ras also augmented the mRNA level and promoter activity of this gene. Similarly, co-transfection of DPP-III promoter-reporter constructs with Ets-1 expression vector led to a significant increase in promoter activity. From these results, we conclude that Ets-1/Elk-1 plays a critical role in transcription of the human DPP-III gene.

Skeletal bone consists of hydroxyapatite (HA) [Ca(10)(PO(4))(6)(OH)(2)] and collagen type I, both of which are osseoconductive. The goal of osseointegration of orthopedic and dental implants is the rapid achievement of a mechanically stable long-lasting fixation between bone and an implant surface. In this study, we evaluated the mechanical fixation and tissue distribution surrounding implants coated with three surfaces: plasma-sprayed HA coating, thinner coating of electrochemical-assisted deposition of HA, and an identical thin coating with a top layer of mineralized collagen. Uncoated plasma-sprayed titanium (Ti-6Al-4V) served as negative control. The electrochemical-assisted deposition was performed near physiological conditions. We used a canine experimental joint replacement model with four cylindrical implants (one of each treatment group) inserted in the humeri cancellous metaphyseal bone in a 1 mm gap. Observation time was 4 weeks. The mechanical fixation was quantified by push-out test to failure, and the peri-implant tissue formation by histomorphometric evaluation. HA coatings deposited by plasma spray technique or electrochemically, increased the mechanical fixation and bone ongrowth, but there was no statistical difference between the individual HA applications. Addition of collagen to the mineralized phase of the coating to create a more bone natural surface did not improve the osseoconductive effect of HA.

CAG-repeat length in the gene for HD is inversely correlated with age of onset (AOO). A number of statistical models elucidating the relationship between CAG length and AOO have recently been published. In the present article, we review the published formulae, summarize essential differences in participant sources, statistical methodologies, and predictive results. We argue that unrepresentative sampling and failure to use appropriate survival analysis methodology may have substantially biased much of the literature. We also explain why the survival analysis perspective is necessary if any such model is to undergo prospective validation. We use prospective diagnostic data from the PREDICT-HD longitudinal study of CAG-expanded participants to test conditional predictions derived from two survival models of AOO of HD. A prior model of the relationship of CAG and AOO originally published by Langbehn et al. yields reasonably accurate predictions, while a similar model by Gutierrez and MacDonald substantially overestimates diagnosis risk for all but the highest risk participants in this sample. The Langbehn et al. model appears accurate enough to have substantial utility in various research contexts. We also emphasize remaining caveats, many of which are relevant for any direct application to genetic counseling.

Autism is a heritable neurodevelopmental disorder with substantial genetic heterogeneity. Studies point to possible links between autism and two serotonin related genes: SLC6A4 and ITGB3 with a sex-specific genetic effect and interaction between the genes. Despite positive findings, inconsistent results have complicated interpretation. This study seeks to validate and clarify previous findings in an independent dataset taking into account sex, family-history (FH) and gene-gene effects. Family-based association analysis was performed within each gene. Gene-gene interactions were tested using extended multifactor dimensionality reduction (EMDR) and MDR-phenomics (MDR-P) using sex of affecteds and FH as covariates. No significant associations with individual SNPs were found in the datasets stratified by sex, but associations did emerge when we stratified by family history. While not significant in the overall dataset, nominally significant association was identified at RS2066713 (P = 0.006) within SLC6A4 in family-history negative (FH-) families, at RS2066713 (P = 0.038) in family-history positive (FH+) families but with the opposite risk allele as in the FH- families. For ITGB3, nominally significant association was identified at RS3809865 overall (P = 0.040) and within FH+ families (P = 0.031). However, none of the associations survived the multiple testing correction. MDR-P confirmed gene-gene effects using sex of affecteds (P = 0.023) and family history (P = 0.014, survived the multiple testing corrections) as covariates. Our results indicate the extensive heterogeneity within these two genes among families. The potential interaction between SLC6A4 and ITGB3 may be clarified using family history as an indicator of genetic architecture, illustrating the importance of covariates as markers of heterogeneity in genetic analyses.

The primary bactericidal domain of CAP37, a cationic antimicrobial protein with potent activity against Gram-negative organisms was previously shown to reside between amino acids 20 through 44 (NQGRHFCGGALIHARFVMTAASCFQ) of the native protein. In this study, we explored the efficacy of four synthetic CAP37 peptide analogs, based on this sequence, against various Candida species including fluconazole-sensitive and -resistant isolates of C. albicans. Three of the peptides demonstrated strong antifungal activity for C. albicans, including fluconazole-resistant isolates of C. albicans and were active against C. guilliermondii, C. tropicalis, C. pseudotropicalis, C. parapsilosis, and C. dubliniensis. The peptides were ineffective against C. glabrata, C. krusei, and Saccharomyces cerevisiae. For C. albicans isolates, the peptides had relatively greater activity against blastoconidia than hyphal forms, although strong antifungal activity was observed with pseudohyphal forms of the various Candida species tested. Kinetic studies demonstrated fungicidal rather than fungistatic activity. These findings indicate that synthetic peptides based on the antimicrobial domain of CAP37 also have activity against eukaryotic organisms suggesting a broader range of activity than originally demonstrated and show for the first time their potent fungicidal activity.

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self-association of disease proteins and determine whether they elicit a toxic or benign outcome.

MicroRNAs have emerged as a novel class of noncoding RNAs that regulate gene expression at the post-translational level in almost every biological event. A large body of evidence indicates that microRNAs regulate the expression of different genes that play an important role in cancer cell invasion, migration and metastasis. In this review, we briefly describe the role of various miRNAs in invasion, migration and metastasis which are essential steps during cancer progression.

To clarify the regulatory mechanism of GW112 gene expression, 5'-flanking region of the human GW112 gene was isolated and characterized in the present study. 5'-RACE analysis showed a single transcription start site, which is located 142 nucleotides upstream of the translation initiation site. Transient transfection studies with serial deletion constructs and close examination of the sequences identified a putative NF kappaB binding sequence between -442 and -430, which could be responsible for efficient expression of the GW112 gene. Indeed, GW112 gene was found to be regulated by NF kappaB signals including overexpressed p65 and I kappaB alpha, IKK inhibitor, and proteasome inhibitor. Binding of NF kappaB to its putative site was confirmed by EMSA and ChIP assays. These results suggest that NF kappaB is an essential regulatory factor for GW112 transcription. Based on this finding, we next confirmed that inhibition of GW112 expression could induce apoptosis in the presence of cytotoxic agent in gastric cancer cells. Furthermore, knocking-down or overexpression of GW112 gene in gastric cancer cells demonstrated that GW112 has an antiapoptotic property against the cytotoxic agents-induced apoptosis. Taken together, these results suggest that GW112 could be an important mediator in NF kappaB-dependent tumorigenesis of digestive tract tissues.

<h4>Unlabelled</h4>Mutations in the SLC40A1 gene, which encodes ferroportin, are associated with autosomal dominant hemochromatosis. Ferroportin is inhibited directly by hepcidin, a key iron-regulatory peptide, and functional consequences of SLC40A1 mutations account for observed phenotypic differences in patients with ferroportin disease. We describe a large pedigree with a novel SLC40A1 mutation and, through in vitro analysis, elucidate the associated molecular mechanism of iron overload. The entire coding sequence of the SLC40A1 gene was sequenced in a pedigree, presenting with autosomal dominant hyperferritinemia. The functional effects of a novel SLC40A1 mutation were studied by overexpression of wild-type and mutant ferroportin fusion proteins in human embryonic kidney cells. Iron export was studied in these cells using (59)Fe transport assays; subcellular localization of ferroportin was examined by way of confocal microscopy. A novel SLC40A1 mutation p.R489K segregated with iron overload in a family with clinical and histopathological signs of macrophage-type ferroportin disease. Human embryonic kidney cells overexpressing p.R489K ferroportin showed decreased iron export capacity when compared with wild-type ferroportin overexpressing cells. Subcellular localization studies demonstrated that p.R489K ferroportin was retained abnormally within an intracellular compartment.<h4>Conclusion</h4>We report a novel pathological SLC40A1 variant associated with abnormal cell surface expression of ferroportin due to intracellular retention of the mutant protein. These findings predict macrophage-type ferroportin disease, the phenotype observed in this kindred. Study of the molecular cell biology of ferroportin and its mutants is key to understanding the pathogenesis of this increasingly recognized form of hemochromatosis, which responds poorly to conventional therapy.

The replacement histone variant H2AX senses DNA double-strand breaks (DSBs) and recruits characteristic sets of proteins at its phosphorylated (gamma-H2AX) foci for concurrent DNA repair. We reasoned that the H2AX interaction network, or interactome, formed in the tumor-associated DNA DSB environment such as in hepatocellular carcinoma (HCC) cells, where preneoplastic lesions frequently occur, is indicative of HCC pathogenic status. By using an in vivo dual-tagging quantitative proteomic method, we identified 102 H2AX-specific interacting partners in HCC cells that stably expressed FLAG-tagged H2AX at close to the endogenous level. Using bioinformatics tools for data-dependent network analysis, we further found binary relationships among these interactors in defined pathway modules, implicating H2AX in a multifunctional role of coordinating a variety of biological pathways involved in DNA damage recognition and DNA repair, apoptosis, nucleic acid metabolism, Ca(2+)-binding signaling, cell cycle, etc. Furthermore, our observations suggest that these pathways interconnect through key pathway components or H2AX interactors. The physiological accuracy of our quantitative proteomic approach in determining H2AX-specific interactors was evaluated by both coimmunoprecipitation/ immunoblotting and confocal colocalization experiments performed on HCC cells. Due to their involvement in diverse functions, the H2AX interactors involved in different pathway modules, such as Poly(ADP-ribose) polymerase 1, 14-3-3 zeta, coflin 1, and peflin 1, were examined for their relative H2AX binding affinities in paired hepatocytes and HCC cells. Treatment with the DSB-inducing agent bleomycin enhanced binding of these proteins to H2AX, suggesting an active role of H2AX in coordinating the functional pathways of each protein in DNA damage recognition and repair.

Construction of a genomic DNA library from Pantoea agglomerans strain CU0119 and screening against the plant pathogen Erwinia amylovora yielded a new family of antibiotics, dapdiamides A-E (1-5). The structures were established through 2D-NMR experiments and mass spectrometry, as well as the synthesis of dapdiamide A (1). Transposon mutagenesis of the active cosmid allowed identification of the biosynthetic gene cluster. The dapdiamide family's promiscuous biosynthetic pathway contains two unconventional amide ligases that are predicted to couple its constituent monomers.

Histone deacetylases (HDACs) play critical roles in silencing tumor suppressor genes. HDAC inhibitors reactivate tumor suppressor genes and inhibit tumor cell growth in vitro and in vivo, and several HDAC inhibitors are currently being evaluated in clinical trials for cancer therapy. A comprehensive analysis of proteins regulated by HDAC inhibitors would enhance our ability to define and characterize their essential therapeutic targets. Here, we employed stable isotope labeling with amino acids in cell culture-based quantitative proteomics to identify acetylated proteins in human breast cancer cells. Treatment with the clinically relevant HDAC inhibitor, suberoylanilide hydroxamic acid (vorinostat), induces lysine acetylation of 61 proteins in MDA-MB-231 human breast cancer cells. Suberoylanilide hydroxamic acid not only induces lysine acetylation in chromatin-associated proteins, but also acetylates previously unrecognized nonhistone proteins, including transcriptional factors and regulators, chaperones, cell structure proteins, and glycolytic enzymes in a time-dependent manner. Knowledge of the full repertoire of acetylated proteins will provide a foundation for further defining the functions of HDACs in cancer cells.

Epigenetic remodeling is a hallmark of cancer, with the frequent acquisition of de novo DNA methylation in CpG islands. However, the functional relevance of de novo DNA methylation in cancer is less well defined. To begin to address this issue in B-cells, we used BeadArray assays to survey the methylation status of over 1,500 cancer-related CpG loci in two molecular subtypes of diffuse large B-cell lymphoma (ABC-DLBCL and GCB-DLBCL) and cognate normal B-cell populations. We identified 81 loci that showed frequent de novo DNA methylation in GCB-DLBCL and 67 loci that showed frequent de novo DNA methylation in ABC-DLBCL. These de novo methylated CpG loci included reported targets of polycomb repressive complexes (PRC) in stem cells. All candidate loci in GCB-DLBCL are proximal to genes that are poorly expressed or silent in purified normal germinal center (GC) B-cells. This is consistent with the hypothesis that de novo DNA methylation in cancer is more frequently involved in the maintenance rather than the initiation of gene silencing (de novo repression). This suggests that epigenetic switching occurs during tumorigenesis with de novo DNA methylation locking in gene silencing normally mediated by transcriptional repressors. Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis.

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into many cell types. Chondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor beta (TGFbeta). Interestingly, TGFbeta can also induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGF beta 3 upregulated SMC genes, whereas cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA, but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGF beta 3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis.

<h4>Purpose of review</h4>To determine the advances made in the genetics of scleroderma in candidate gene association studies.<h4>Recent findings</h4>Over the past 18 months, a number of candidate gene studies using large case-control series in scleroderma have been reported. The studies have identified multiple genes involved in immune regulation including BANK1, C8orf13-BLK, IL-23R, IRF5, STAT4, TBX21, and TNFSF4 as susceptibility genes for the development of SSc. Furthermore, gene-gene interaction studies suggest that IRF5, STAT4, and BANK1 as well as TBX21 and STAT4 interact with regard to scleroderma susceptibility. Many of the genetic variants associated with SSc susceptibility are shared among other autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.<h4>Summary</h4>Candidate gene association studies have substantially advanced our understanding of the pathogenesis of SSc and demonstrate that SSc is a polygenic, autoimmune disease.

APPL1 is a membrane-associated adaptor protein implicated in various cellular processes, including apoptosis, proliferation, and survival. Although there is increasing interest in the biological roles as well as the protein and membrane interactions of APPL1, a comprehensive phosphorylation profile has not been generated. In this study, we use mass spectrometry (MS) to identify 13 phosphorylated residues within APPL1. By using multiple proteases (trypsin, chymotrypsin, and Glu C) and replicate experiments of linear ion trap (LTQ) MS and LTQ-Orbitrap-MS, a combined sequence coverage of 99.6% is achieved. Four of the identified sites are located in important functional domains, suggesting a potential role in regulating APPL1. One of these sites is within the BAR domain, two cluster near the edge of the PH domain, and one is located within the PTB domain. These phosphorylation sites may control APPL1 function by regulating the ability of APPL1 domains to interact with other proteins and membranes.

In this work, we describe a novel polyamidoamine (PAMAM) dendrimer hydrogel (DH) platform with potential for tissue engineering and drug delivery. With PAMAM dendrimer G3.0 being the underlying carrier, polyethylene glycol (PEG) chains of various lengths (MW = 1500, 6000, or 12000 g mol(-1)) were coupled to the dendrimer to different extents, and the resulting PEGylated PAMAM dendrimers were further coupled with acrylate groups to yield photoreactive dendrimer macromonomers for gel formation. It was found that gelation based on photoreactive PAMAM G3.0 macromonomers was restricted by the degree of PEGylation, PEG chain length, and the distribution of acrylate groups on the dendrimer surface. Further, the architecture of the photoreactive macromonomers affects the structural stability and swelling of the resultant networks. A completely cross-linked network (DH-G3.0-12000(H)) with a high water swelling ratio was created by UV-curing of PAMAM dendrimer G3.0 coupled with 28 PEG 12000 chains in the presence of the eosin Y-based photoinitiating system. The disintegration of DH-G3.0-12000(H) was pH-insensitive. DH-G3.0-12000(H) was found to have similar cytocompatibility to un-cross-linked G3.0-12000(H) but a significantly lower cellular uptake by macrophages. With PAMAM dendrimer G3.5 being the underlying carrier, the dendrimer modified with 43 PEG 1500 chains was able to form a completely cross-linked network (DH-G3.5-1500(H)) by UV-curing in the presence of the eosin Y-based photoinitiating system. DH-G3.5-1500(H) exhibited pH-dependent disintegration. Its disintegration ratio increased with pH. PAMAM dendrimer hydrogels uniquely express the structural characteristics of both PEG hydrogel and PAMAM dendrimer and have potential for various applications in tissue engineering and drug delivery.

<h4>Background</h4>Ambient particles are associated with cardiovascular events and recently with total plasma homocysteine. High total plasma homocysteine is a risk for human health. However, the biologic mechanisms are not fully understood. One of the putative pathways is through oxidative stress. We aimed to examine whether associations of PM2.5 and black carbon with homocysteine were modified by genotypes including HFE H63D, C282Y, CAT (rs480575, rs1001179, rs2284367, and rs2300181), NQO1 (rs1800566), GSTP1 I105V, GSTM1, GSTT1 (deletion vs. nondeletion), and HMOX-1 (any short vs. both long). We attempted to replicate identified genes in an analysis of heart rate variability and in other outcomes reported in the literature.<h4>Methods</h4>Study subjects were 1000 white non-Hispanic men in the Boston area, participating in a cohort study of aging. PM2.5, black carbon, total plasma homocysteine, and other covariates were measured at several points in time between 1995 and 2006. We fit mixed models to examine effect modification of genes on associations of pollution with total plasma homocysteine.<h4>Results</h4>Interquartile range increases in PM2.5 and black carbon (7-day moving averages) were associated with 1.5% (95% confidence interval = 0.2% to 2.8%) and 2.2% (0.6% to 3.9%) increases in total plasma homocysteine, respectively. GSTT1 and HFE C282Y modified effects of black carbon on total plasma homocysteine, and HFE C282Y and CAT (rs2300181) modified effects of PM2.5 on homocysteine. Several genotypes marginally modified effects of PM2.5 and black carbon on various endpoints. All genes with significant interactions with particulate air pollution had modest main effects on total plasma homocysteine.<h4>Conclusions</h4>: Effects of PM2.5 and black carbon on various endpoints appeared to be mediated by genes related to oxidative stress pathways.

Over the past decades, hydrogels have been widely studied as biomaterials for various biomedical applications like implants, drugs and cell delivery carriers because of their high biocompatibility, high water contents and excellent permeability for nutrients and metabolites. Especially, in situ forming hydrogel systems have received much attention because of their easy application based on minimal invasive techniques. Chemical cross-linking systems fabricated using enzymatic reactions have various advantages, such as high biocompatibility and easy control of reaction rates under mild condition. In this study, we report enzyme-triggered injectable and biodegradable hydrogels composed of Tetronic-tyramine conjugates. The Tetronic-tyramine conjugates were synthesized by first reacting Tetronic with succinic anhydride and subsequent conjugation with tyramine using DCC/NHS as coupling reagents. The chemical structure of Tetronic-succinic anhydride-tyramine (Tet-SA-TA) copolymer was characterized by (1)H NMR and FTIR. The hydrogels were prepared from a Tet-SA-TA solution above 3 wt % in the presence of horseradish peroxidase (HRP) and H(2)O(2) under physiological conditions. Their mechanical property, gelation time, swelling ratio and degradation time were evaluated at different polymer, HRP, and H(2)O(2) concentrations. In addition, a cyto-compatibility study was performed using the MC3T3-E1 cell line. In the cytotoxicity test, it was clear that the Tet-SA-TA hydrogel had no apparent cytotoxicity except for the hydrogel formed with 0.25 wt % H(2)O(2) due to the cytotoxicity of residual H(2)O(2). In conclusion, the obtained results demonstrated that the Tet-SA-TA hydrogel has great potential for use as an injectable scaffold for tissue engineering and as a drug carrier for controlled drug delivery systems.

Many signaling pathways that contribute to tumorigenesis are also functional in pregnancy, although they are dysregulated in the former and tightly regulated in the latter. Transformation-related protein 53 (Trp53), which encodes p53, is a tumor suppressor gene whose mutation is strongly associated with cancer. However, its role in normal physiological processes, including female reproduction, is poorly understood. Mice that have a constitutive deletion of Trp53 exhibit widespread development of carcinogenesis at early reproductive ages, compromised spermatogenesis, and fetal exencephaly, rendering them less amenable to studying the role of p53 in reproduction. To overcome this obstacle, we generated mice that harbor a conditional deletion of uterine Trp53 and examined pregnancy outcome in females with this genotype. These mice had normal ovulation, fertilization, and implantation; however, postimplantation uterine decidual cells showed terminal differentiation and senescence-associated growth restriction with increased levels of phosphorylated Akt and p21, factors that are both known to participate in these processes in other systems. Strikingly, uterine deletion of Trp53 increased the incidence of preterm birth, a condition that was corrected by oral administration of the selective COX2 inhibitor celecoxib. We further generated evidence to suggest that deletion of uterine Trp53 induces preterm birth through a COX2/PGF synthase/PGF(2alpha) pathway. Taken together, our observations underscore what we believe to be a new critical role of uterine p53 in parturition.

Often guided by analogy with nonphospholipid products from oxidative cleavage of polyunsaturated fatty acids, we previously identified a variety of biologically active oxidatively truncated phospholipids. Previously, 4,5-epoxy-2(E)-decenal (4,5-EDE) was found to be produced by oxidative cleavage of 13-(S)-hydroperoxy-9,11-(Z,E)-octadeca-dienoic acid (13-HPODE). 4,5-EDE reacts with deoxy-adenosine (dAdo) and deoxy-guanosine (dGuo) to form mutagenic etheno derivatives. We hypothesized that a functionally similar and potentially mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid (OETA), would be generated from 9-HPODE through an analogous fragmentation. We expected that an ester of 2-lysophosphatidylcoline (PC), OETA-PC, would be produced by oxidative cleavage of 9-HPODE-PC in biological membranes. An efficient, unambiguous total synthesis of trans-OETA-PC was first executed to provide a standard that could facilitate the identification of this phospholipid epoxyalkenal that was shown to be produced during oxidation of the linoleic acid ester of 2-lysoPC. Finally, trans-OETA-PC was detected in a lipid extract from rat retina. The identity of the naturally occurring oxidatively truncated phospholipid was further confirmed by derivatization with methoxylamine that produced characteristic mono and bis adducts. The average amount of trans-OETA-PC in rat retina, 0.33 pmol, is relatively low as compared to other oxidatively truncated PCs, for example, the 4-hydroxy-7-oxohept-5-enoic acid PC ester (2.5 pmol) or the 4-keto-7-oxohept-5-enoic acid PC ester (1.7 pmol), derived from the docosahexaenoic acid ester of 2-lysoPC. This, most likely, is because docosahexaenoate PCs are particularly abundant in the retina as compared to the linoleate PC ester precursor of OETA-PC. As predicted by analogy with 4,5-EDE, OETA-PC reacts with dAdo and dGuo, as well as with DNA, to form mutagenic etheno adducts.

This report describes the synthesis and biological characterization of novel granisetron derivatives that are antagonists of the human serotonin (5-HT(3)A) receptor. Some of these substituted granisetron derivatives showed low nanomolar binding affinity and allowed the identification of positions on the granisetron core that might be used as attachment points for biophysical tags. A BODIPY fluorophore was appended to one such position and specifically bound to 5-HT(3)A receptors in mammalian cells.

Parkinson's disease (PD) is characterized by dopaminergic neurodegeneration in the nigrostriatal region of the brain; however, the neurodegeneration extends well beyond dopaminergic neurons. To gain a better understanding of the molecular changes relevant to PD, we applied two-dimensional LC-MS/MS to comparatively analyze the proteome changes in four brain regions (striatum, cerebellum, cortex, and the rest of brain) using a MPTP-induced PD mouse model with the objective to identify potential nigrostriatal-specific and other region-specific protein abundance changes. The combined analyses resulted in the identification of 4,895 nonredundant proteins with at least two unique peptides per protein. The relative abundance changes in each analyzed brain region were estimated based on the spectral count information. A total of 518 proteins were observed with substantial MPTP-induced abundance changes across different brain regions. A total of 270 of these proteins were observed with specific changes occurring either only in the striatum and/or in the rest of the brain region that contains substantia nigra, suggesting that these proteins are associated with the underlying nigrostriatal pathways. Many of the proteins that exhibit changes were associated with dopamine signaling, mitochondrial dysfunction, the ubiquitin system, calcium signaling, the oxidative stress response, and apoptosis. A set of proteins with either consistent change across all brain regions or with changes specific to the cortex and cerebellum regions were also detected. Ubiquitin specific protease (USP9X), a deubiquination enzyme involved in the protection of proteins from degradation and promotion of the TGF-beta pathway, exhibited altered abundance in all brain regions. Western blot validation showed similar spatial changes, suggesting that USP9X is potentially associated with neurodegeneration. Together, this study for the first time presents an overall picture of proteome changes underlying both nigrostriatal pathways and other brain regions potentially involved in MPTP-induced neurodegeneration. The observed molecular changes provide a valuable reference resource for future hypothesis-driven functional studies of PD.

Schizophrenia is a complex genetic disorder manifesting combined environmental and genetic causation. Recently, genome-wide association experiments yielded remarkable new experimental evidence that is leading to a better understanding of the genetic models and the biological risk factors involved in schizophrenia. These studies have discovered uncommon copy number variations (mainly deletions) and common single nucleotide polymorphisms with alleles associated with schizophrenia. The aggregate data provide support for polygenic inheritance and for genetic overlap of schizophrenia with autism and with bipolar disorder. It is anticipated that the application of a myriad of tools from systems biology, in combination with biological functional experiments, will lead to a delineation of biological pathways involved in the pathophysiology of schizophrenia, and eventually to new therapies.

Although twin studies demonstrate that ADHD is a highly heritable condition, molecular genetic studies suggest that the genetic architecture of ADHD is complex. The handful of genome-wide linkage and association scans that have been conducted thus far show divergent findings and are, therefore, not conclusive. Similarly, many of the candidate genes reviewed here (ie, DBH, MAOA, SLC6A2, TPH-2, SLC6A4, CHRNA4, GRIN2A) are theoretically compelling from neurobiological systems perspective but available data are sparse and inconsistent. However, candidate gene studies of ADHD have produced substantial evidence implicating several genes in the etiology of the disorder, with meta-analyses supportive of a role of the genes coding for DRD4, DRD5, SLC6A3, SNAP-25, and HTR1B in the etiology of ADHD.

Existing psychotropic medications for the treatment of mental illnesses, including antidepressants, mood stabilizers, and antipsychotics, are clinically suboptimal. They are effective in only a subset of patients or produce partial responses, and they are often associated with debilitating side effects that discourage adherence. There is growing enthusiasm in the promise of pharmacogenetics to personalize the use of these treatments to maximize their efficacy and tolerability; however, there is still a long way to go before this promise becomes a reality. This article reviews the progress that has been made in research toward understanding how genetic factors influence psychotropic drug responses and the challenges that lie ahead in translating the research findings into clinical practices that yield tangible benefits for patients with mental illnesses.

Catalytic graphitization for (14)C-accelerator mass spectrometry ((14)C-AMS) produced various forms of elemental carbon. Our high-throughput Zn reduction method (C/Fe = 1:5, 500 degrees C, 3 h) produced the AMS target of graphite-coated iron powder (GCIP), a mix of nongraphitic carbon and Fe(3)C. Crystallinity of the AMS targets of GCIP (nongraphitic carbon) was increased to turbostratic carbon by raising the C/Fe ratio from 1:5 to 1:1 and the graphitization temperature from 500 to 585 degrees C. The AMS target of GCIP containing turbostratic carbon had a large isotopic fractionation and a low AMS ion current. The AMS target of GCIP containing turbostratic carbon also yielded less accurate/precise (14)C-AMS measurements because of the lower graphitization yield and lower thermal conductivity that were caused by the higher C/Fe ratio of 1:1. On the other hand, the AMS target of GCIP containing nongraphitic carbon had higher graphitization yield and better thermal conductivity over the AMS target of GCIP containing turbostratic carbon due to optimal surface area provided by the iron powder. Finally, graphitization yield and thermal conductivity were stronger determinants (over graphite crystallinity) for accurate/precise/high-throughput biological, biomedical, and environmental (14)C-AMS applications such as absorption, distribution, metabolism, elimination (ADME), and physiologically based pharmacokinetics (PBPK) of nutrients, drugs, phytochemicals, and environmental chemicals.

Recombination, together with mutation, generates the raw material of evolution, is essential for reproduction and lies at the heart of all genetic analysis. Recent advances in our ability to construct genome-scale, high-resolution recombination maps and new molecular techniques for analysing recombination products have substantially furthered our understanding of this important biological phenomenon in humans and mice: from describing the properties of recombination hot spots in male and female meiosis to the recombination landscape along chromosomes. This progress has been accompanied by the identification of trans-acting systems that regulate the location and relative activity of individual hot spots.

Transport of proteins to and from cilia is crucial for normal cell function and survival, and interruption of transport has been implicated in degenerative and neoplastic diseases. It has been hypothesized that the ciliary axoneme and structures adjacent to and including the basal bodies of cilia impose selective barriers to the movement of proteins into and out of the cilium. To examine this hypothesis, using confocal and multiphoton microscopy we determined the mobility of the highly soluble photoactivatable green fluorescent protein (PAGFP) in the connecting cilium (CC) of live Xenopus retinal rod photoreceptors, and in the contiguous subcellular compartments bridged by the CC, the inner segment (IS) and the outer segment (OS). The estimated axial diffusion coefficients are D(CC) = 2.8 +/- 0.3, D(IS) = 5.2 +/- 0.6, and D(OS) = 0.079 +/- 0.009 microm(2) s(-1). The results establish that the CC does not pose a major barrier to protein diffusion within the rod cell. However, the results also reveal that axial diffusion in each of the rod's compartments is substantially retarded relative to aqueous solution: the axial diffusion of PAGFP was retarded approximately 18-, 32- and 1,000-fold in the IS, CC, and OS, respectively, with approximately 20-fold of the reduction in the OS attributable to tortuosity imposed by the lamellar disc membranes. Previous investigation of PAGFP diffusion in passed, spherical Chinese hamster ovary cells yielded D(CHO) = 20 microm(2) s(-1), and estimating cytoplasmic viscosity as D(aq)/D(CHO) = 4.5, the residual 3- to 10-fold reduction in PAGFP diffusion is ascribed to sub-optical resolution structures in the IS, CC, and OS compartments.

<h4>Objective</h4>The genetic contributions to the multifactorial disorder osteoarthritis (OA) have been increasingly recognized. The goal of the current study was to use OA-related biomarkers of severity and disease burden as quantitative traits to identify genetic susceptibility loci for OA.<h4>Methods</h4>In a large multigenerational extended family (n = 350), we measured 5 OA-related biomarkers: hyaluronan (HA), cartilage oligomeric matrix protein (COMP), N-propeptide of type IIA collagen (PIIANP), C-propeptide of type II procollagen (CPII), and type II collagen neoepitope (C2C). Single-nucleotide polymorphism markers (n = 6,090) covering the whole genome were genotyped using the Illumina HumanLinkage-12 BeadChip. Variance components analysis, as implemented in the Sequential Oligogenic Linkage Analysis Routines, was used to estimate heritabilities of the quantitative traits and to calculate 2-point and multipoint logarithm of odds (LOD) scores using a polygenic model.<h4>Results</h4>After adjusting for age and sex, we found that 4 of the 5 biomarkers exhibited significant heritability (PIIANP 0.57, HA 0.49, COMP 0.43, C2C 0.30; P < or = 0.01 for all). Fourteen of the 19 loci that had multipoint LOD scores of >1.5 were near to or overlapped with previously reported OA susceptibility loci. Four of these loci were identified by more than 1 biomarker. The maximum multipoint LOD scores for the heritable quantitative biomarker traits were 4.3 for PIIANP (chromosome 8p23.2), 3.2 for COMP (chromosome 8q11.1), 2.0 for HA (chromosome 6q16.3), and 2.0 for C2C (chromosome 5q31.2).<h4>Conclusion</h4>Herein, we report the first evidence of genetic susceptibility loci identified by OA-related biomarkers in an extended family. Our results demonstrate that serum concentrations of PIIANP, HA, COMP, and C2C have substantial heritable components, and using these biomarkers, several genetic loci potentially contributing to the genetic diversity of OA were identified.

<h4>Background</h4>Gastric cancer is one of the most common malignancies worldwide and the second most common cause of cancer related death. Gene copy number alterations play an important role in the development of gastric cancer and a change in gene copy number is one of the main mechanisms for a cancer cell to control the expression of potential oncogenes and tumor suppressor genes.<h4>Methods</h4>To highlight genes of potential biological and clinical relevance in gastric cancer, we carried out a systematic array-based survey of gene expression and copy number levels in primary gastric tumors and gastric cancer cell lines and validated the results using an affinity capture based transcript analysis (TRAC assay) and real-time qRT-PCR.<h4>Results</h4>Integrated microarray analysis revealed altogether 256 genes that were located in recurrent regions of gains or losses and had at least a 2-fold copy number- associated change in their gene expression. The expression levels of 13 of these genes, ALPK2, ASAP1, CEACAM5, CYP3A4, ENAH, ERBB2, HHIPL2, LTB4R, MMP9, PERLD1, PNMT, PTPRA, and OSMR, were validated in a total of 118 gastric samples using either the qRT-PCR or TRAC assay. All of these 13 genes were differentially expressed between cancerous samples and nonmalignant tissues (p < 0.05) and the association between copy number and gene expression changes was validated for nine (69.2%) of these genes (p < 0.05).<h4>Conclusion</h4>In conclusion, integrated gene expression and copy number microarray analysis highlighted genes that may be critically important for gastric carcinogenesis. TRAC and qRT-PCR analyses validated the microarray results and therefore the role of these genes as potential biomarkers for gastric cancer.

A 21-year-old man who was diagnosed with Diamond-Blackfan anemia at 2 years of age came to our hospital with the hope of undergoing bone marrow transplantation (BMT). He had been red cell transfusion-dependent for about 8 years; at presentation he had transfusion-induced hemochromatosis, a subsequent low left ventricular ejection fraction (LVEF) of 43%, and diabetes mellitus requiring insulin therapy. He received unrelated BMT with reduced intensity conditioning and sufficient GVHD prophylaxis. Regardless of the cardiac dysfunction, he had an uneventful course during pre- and post-grafting periods, and is currently doing well with an improved LVEF (55%), although he is still insulin dependent.

<h4>Background</h4>Iron overload observed in chronic hepatitis C (CHC) has been suggested to be one of the negative prognostic factors influencing liver disease progression and failure of treatment with recombinant interferon in monotherapy or in combination with ribavirin. The aim of this study was to assess occurrence of iron overload in relation to polymorphism of the HFE and the influence of both these factors on efficacy of antiviral treatment with pegylated interferon and ribavirin in patients with CHC.<h4>Material/methods</h4>Liver function tests, serum indices of iron metabolism, and HFE mutations were assayed in 152 patients with CHC from Poland. Histopathological examination of the liver biopsy specimen was performed in 138 patients. Sixty-one patients were treated with pegylated interferon alfa-2 and ribavirin. The comparative analysis was performed in 2 groups of patients: those with and those without elevated serum indices of iron metabolism.<h4>Results</h4>Increased biochemical iron metabolism parameters correlated with older age, higher ALT activity, more advanced liver fibrosis and treatment failure. Iron deposits in liver specimens were not accompanied by exacerbation of necro-inflammatory activity and advanced fibrosis. Elevated biochemical values of iron metabolism parameters and presence of hepatic iron deposits correlated positively with C282Y mutations. The lack of sustained viral response after treatment with pegylated interferon and ribavirin was observed more frequently in carriers of HFE mutations.<h4>Conclusions</h4>Iron overload was frequently detected in patients with CHC, and was associated only with C282Y alleles. Biochemical markers of iron overload and HFE gene mutations were negative prognostic factors of antiviral treatment.

<h4>Background</h4>Budd-Chiari syndrome (BCS) generally implies thrombosis of the hepatic veins and/or the intrahepatic or suprahepatic inferior vena cava. Treatment depends on the underlying cause, the anatomic location, the extent of the thrombotic process and the functional capacity of the liver. It can be divided into medical treatment including anticoagulation and thrombolysis, radiological procedures such as angioplasty and transjugular intrahepatic porto-systemic shunt (TIPS) and surgical interventions including orthotopic liver transplantation (OLT). Controlled trials or reports on larger cohorts are limited due to rare disease frequency. The aim of this study was to report our single centre long term results of patients with BCS receiving one of three treatment options i.e. medication only, TIPS or OLT on an individually based decision of our local expert group.<h4>Methods</h4>20 patients with acute, subacute or chronic BCS were treated between 1988 and 2008. Clinical records were analysed with respect to underlying disease, therapeutic interventions, complications and overall outcome.<h4>Results</h4>16 women and 4 men with a mean age of 34 +/- 12 years (range: 14-60 years) at time of diagnosis were included. Myeloproliferative disorders or a plasmatic coagulopathy were identified as underlying disease in 13 patients, in the other patients the cause of BCS remained unclear. 12 patients presented with an acute BCS, 8 with a subacute or chronic disease. 13 patients underwent TIPS, 4 patients OLT as initial therapy, 2 patients required only symptomatic therapy, and one patient died from liver failure before any specific treatment could be initiated. Eleven of 13 TIPS patients required 2.5 +/- 2.4 revisions (range: 0-8). One patient died from his underlying hematologic disease. The residual 12 patients still have stable liver function not requiring OLT. All 4 patients who underwent OLT as initial treatment, required re-OLT due to thrombembolic complications of the graft. Survival in the TIPS group was 92.3% and in the OLT group 75% during a median follow-up of 4 and 11.5 years, respectively.<h4>Conclusion</h4>Our results confirm the role of TIPS in the management of patients with acute, subacute and chronic BCS. The limited number of patients with OLT does not allow to draw a meaningful conclusion. However, the underlying disease may generate major complications, a reason why OLT should be limited to patients who cannot be managed by TIPS.

<h4>Background</h4>Acute exposure to ambient air pollution has been associated with acute changes in cardiac outcomes, often within hours of exposure.<h4>Objectives</h4>We examined the effects of air pollutants on heart-rate-corrected QT interval (QTc), an electrocardiographic marker of ventricular repolarization, and whether these associations were modified by participant characteristics and genetic polymorphisms related to oxidative stress.<h4>Methods</h4>We studied repeated measurements of QTc on 580 men from the Veterans Affairs Normative Aging Study (NAS) using mixed-effects models with random intercepts. We fitted a quadratic constrained distributed lag model to estimate the cumulative effect on QTc of ambient air pollutants including fine particulate matter <or= 2.5 microm in aerodynamic diameter (PM2.5), ozone (O3), black carbon (BC), nitrogen dioxide (NO2), carbon monoxide (CO), and sulfur dioxide (SO2) concentrations during the 10 hr before the visit. We genotyped polymorphisms related to oxidative stress and analyzed pollution-susceptibility score interactions using the genetic susceptibility score (GSS) method.<h4>Results</h4>Ambient traffic pollutant concentrations were related to longer QTc. An interquartile range (IQR) change in BC cumulative during the 10 hr before the visit was associated with increased QTc [1.89 msec change; 95% confidence interval (CI), -0.16 to 3.93]. We found a similar association with QTc for an IQR change in 1-hr BC that occurred 4 hr before the visit (2.54 msec change; 95% CI, 0.28-4.80). We found increased QTc for IQR changes in NO2 and CO, but the change was statistically insignificant. In contrast, we found no association between QTc and PM2.5, SO2, and O3. The association between QTc and BC was stronger among participants who were obese, who had diabetes, who were nonsmokers, or who had higher GSSs.<h4>Conclusions</h4>Traffic-related pollutants may increase QTc among persons with diabetes, persons who are obese, and nonsmoking elderly individuals; the number of genetic variants related to oxidative stress increases this effect.

<h4>Objective</h4>Type 2 diabetes is heterogeneous in its clinical features, pathogenesis, and predisposing or causal genetic factors. This report examines what is known and what needs to be learned about the potential to individualize glycemic therapies in type 2 diabetes, based on phenotypes and genotypes.<h4>Participants</h4>A 29-member international working group with expertise in diabetes epidemiology, physiology, genetics, clinical trials, and clinical care participated in formal presentations and discussions at a conference on April 16-17, 2009. A writing group subsequently prepared this summary and recommendations. The conference was coendorsed by The Endocrine Society and the American Diabetes Association and was supported by an unrestricted educational grant from Novo Nordisk.<h4>Evidence</h4>Participants reviewed and discussed published literature, plus their own unpublished data.<h4>Consensus process</h4>The summary and recommendations were supported unanimously by the writing group as representing the majority or unanimous opinions of the working group.<h4>Conclusions</h4>Recent advances in genetics, such as the identification of Kir6.2 mutations and the responsible genes for several forms of maturity onset diabetes of the young (MODY), have established precedents linking specifically effective therapies to defined diabetes subtypes. The recent increase in identified polygenic factors related to type 2 diabetes and our understanding of the pathogenesis of diabetes provide potential opportunities to individualize therapy. To further this process, we recommend expanded analysis of existing data sources and the development of new basic and clinical research studies, including a greater focus on identifying type 2 diabetes subtypes, their response to different therapies, and quantitation of cost-effectiveness.

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

The condensin complex is a key determinant of higher-ordered chromosome structure. We show here that the complex is also important for the correct alignment of chromosomes on the meiosis I spindle. Unlike during mitosis and meiosis II, when sister chromatids attach to microtubules emanating from opposite spindle poles (biorientation), accurate meiosis I chromosome segregation requires that sister chromatids attach to microtubules emanating from the same spindle pole (co-orientation). The monopolin complex, consisting of Lrs4, Csm1, and the meiosis-specific component Mam1, brings about meiosis I co-orientation. We find that in the absence of functional condensin complexes, a fraction of sister kinetochores biorient on the meiosis I spindle and association of the monopolin complex subunit Mam1 with kinetochores is decreased. Our studies uncover a new locus-specific effect of the condensin complex.

Endoreplication, also known as endoreduplication, is a phyogenetically widespread modified version of the cell cycle in which DNA replication is not followed by cell division. The SIAMESE (SIM) gene of Arabidopsis thaliana encodes the founding member of a novel class of plant-specific cyclin-dependent kinase (CDK) inhibitors and is a key regulator of endoreplication during the development of trichomes (shoot epidermal hairs). Here, we have identified mutations in the CCS52A1 gene as genetic modifiers of the multicellular trichome phenotype of sim mutants. Loss-of-function ccs52A1 mutations dramatically enhance the multicellularity of sim mutants trichomes in double mutants, whereas overexpression of CCS52A1 completely suppresses the sim mutant phenotype. CCS52A1 encodes a CDH1/FZR-like protein, a class of proteins that function as activators of the anaphase-promoting complex. Unicellular ccs52A1 trichomes become multicellular upon overexpression of B-type cyclin, consistent with repression of the accumulation of mitotic cyclins in the developing trichome by CCS52A1. As these M-phase-specific cyclins are known to accumulate in sim mutant trichomes, our data suggest that CCS52A1 and SIM cooperate in repressing accumulation of mitotic cyclins to establish the trichome endocycle. Comparison with endoreplication pathways in Drosophila and mammals indicates that while these organisms all use similar components to initiate endoreplication, the components are deployed differently in each organism.

<h4>Background</h4>We compare the genotype distribution for the serotonin transporter polymorphism (5-HTTLPR) in a sample of older Taiwanese adults with samples of various racial and ethnic groups collected in other studies. We also explore interactions among sex, stressors, and 5-HTTLPR genotype on depressive symptoms in our sample.<h4>Methods</h4>Using a nationally representative sample of 984 Taiwanese aged 53 and older, we model depressive symptoms as a function of 5-HTTLPR genotype and two classes of stressors: lifetime trauma and recent major life events. We test two- and three-way interactions among stressors, 5-HTTLPR, and sex.<h4>Results</h4>This sample exhibits higher frequency of S/S and lower frequency of L/L genotype than Western samples, but the distribution is comparable to those in East Asian populations. Nearly 9% carry an allele (XL) that has rarely been reported in the literature. Although the gene-environment (GxE) interaction with recent major life events is not significant, our results suggest that trauma has a worse effect on depressive symptoms for those with S/S or S/L genotype than for those who do not carry the S allele (P<0.05). We find no evidence that this GxE interaction varies by sex.<h4>Conclusions</h4>Previous studies of this GxE interaction have been inconclusive, perhaps because interactions between genotype and stressful events are more prominent under extreme stressors. Our findings underscore the need to move beyond a bi-allelic parameterization of the 5-HTTLPR polymorphism and raise questions about why East Asian populations exhibit low rates of depression despite a high frequency of the S allele.

<h4>Background</h4>Alcohol dependence is a complex disease, and although linkage and candidate gene studies have identified several genes associated with the risk for alcoholism, these explain only a portion of the risk.<h4>Methods</h4>We carried out a genome-wide association study (GWAS) on a case-control sample drawn from the families in the Collaborative Study on the Genetics of Alcoholism. The cases all met diagnostic criteria for alcohol dependence according to the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition; controls all consumed alcohol but were not dependent on alcohol or illicit drugs. To prioritize among the strongest candidates, we genotyped most of the top 199 single nucleotide polymorphisms (SNPs) (p < or = 2.1 x 10(-4)) in a sample of alcohol-dependent families and performed pedigree-based association analysis. We also examined whether the genes harboring the top SNPs were expressed in human brain or were differentially expressed in the presence of ethanol in lymphoblastoid cells.<h4>Results</h4>Although no single SNP met genome-wide criteria for significance, there were several clusters of SNPs that provided mutual support. Combining evidence from the case-control study, the follow-up in families, and gene expression provided strongest support for the association of a cluster of genes on chromosome 11 (SLC22A18, PHLDA2, NAP1L4, SNORA54, CARS, and OSBPL5) with alcohol dependence. Several SNPs nominated as candidates in earlier GWAS studies replicated in ours, including CPE, DNASE2B, SLC10A2, ARL6IP5, ID4, GATA4, SYNE1, and ADCY3.<h4>Conclusions</h4>We have identified several promising associations that warrant further examination in independent samples.

The study of haemoglobin switching has represented a focus in haematology due in large part to the clinical relevance of the fetal to adult haemoglobin switch for developing targeted approaches to ameliorate the severity of the beta-haemoglobinopathies. Additionally, the process by which this switch occurs represents an important paradigm for developmental gene regulation. In this review, we provide an overview of both the embryonic primitive to definitive switch in haemoglobin expression, as well as the fetal to adult switch that is unique to humans and old world monkeys. We discuss the nature of these switches and models of their regulation. The factors that have been suggested to regulate this process are then discussed. With the increased understanding and discovery of molecular regulators of haemoglobin switching, such as BCL11A, new avenues of research may lead ultimately to novel therapeutic, mechanism-based approaches to fetal haemoglobin reactivation in patients.

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP-ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Combinatorial interactions among transcription factors are critical to directing tissue-specific gene expression. To build a global atlas of these combinations, we have screened for physical interactions among the majority of human and mouse DNA-binding transcription factors (TFs). The complete networks contain 762 human and 877 mouse interactions. Analysis of the networks reveals that highly connected TFs are broadly expressed across tissues, and that roughly half of the measured interactions are conserved between mouse and human. The data highlight the importance of TF combinations for determining cell fate, and they lead to the identification of a SMAD3/FLI1 complex expressed during development of immunity. The availability of large TF combinatorial networks in both human and mouse will provide many opportunities to study gene regulation, tissue differentiation, and mammalian evolution.

No abstract available.

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island "shores" and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations-the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.

The presence of intracellular ubiquitylated inclusions in neurodegenerative disorders and the role of the ubiquitin/proteasome system (UPS) in degrading abnormal hazardous proteins have given rise to the hypothesis that UPS-impairment underlies neurodegenerative processes. However, this remains controversial for polyglutamine disorders such as Huntington disease (HD). Whereas studies in cellular models have provided evidence in favor of UPS-impairment attributable to expression of the N-terminal fragment of mutant huntingtin (N-mutHtt), similar studies on mouse models failed to do so. Furthermore, we have recently shown that the increase in polyubiquitin conjugates reported in the brain of N-mutHtt mice occurs in the absence of a general UPS-impairment. In the present study we aim to clarify the potential of N-mutHtt to impair UPS function in vivo as well as the mechanisms by which neurons may adapt after prolonged exposure to N-mutHtt in genetic models. By combining UPS reporter mice with an inducible mouse model of HD, we demonstrate for the first time polyglutamine-induced global UPS-impairment in vivo. UPS-impairment occurred transiently after acute N-mutHtt expression and restoration correlated with appearance of inclusion bodies (IBs). Consistently, UPS recovery did not take place when IB formation was prevented through administration of N-mutHtt aggregation-inhibitors in both cellular and animal models. Finally, no UPS-impairment was detected in old mice constitutively expressing N-mutHtt despite the age-associated decrease in brain proteasome activity. Therefore, our data reconcile previous contradictory reports by showing that N-mutHtt can indeed impair UPS function in vivo and that N-mutHtt aggregation leads to long lasting restoration of UPS function.

The finding of lipid accumulation in the liver, so-called hepatic steatosis or non-alcoholic fatty liver disease, is a common condition frequently found in healthy subjects. Its prevalence, in fact, has been estimated by magnetic resonance studies to be about 35% in the general population and 75% in obese persons. Nevertheless, its presence generates liver damage only in a small percentage of subjects not affected by other liver diseases. It should be defined as a "co-factor" capable of affecting severity and progression, and also therapeutic perspectives, of liver diseases to which it is associated. Herein we will evaluate the impact of hepatic steatosis and obesity on the most common liver diseases: chronic viral hepatitis C and B, and alcoholic liver disease.

<h4>Aim</h4>To study the relation between hepatitis C virus (HCV) genotype 4 and microalbuminuria and renal impairment in relation to hepatic histology, and viremia in the absence of cryoglobulinemia, and to examine the effect of treatment on microalbuminuria.<h4>Methods</h4>Three hundred subjects, including 233 HCV genotype-4 infected patients, were tested for cryoglobulinemia, microalbuminuria, albumin creatinine ratio (ACR), urea, creatinine, and estimated glomerular filtration rate (eGFR). The parameters were measured again in the HCV patients after 48 wk of treatment with pegylated interferon and ribavirin.<h4>Results</h4>Significantly higher levels of microalbuminuria were detected in HCV-positive patients compared to HCV-negative controls (median 9.5 vs 5.9, respectively, Kruskal-Wallis P = 0.017). Log microalbuminuria was significantly correlated with hepatic inflammation (r = 0.13, P = 0.036) and fibrosis (r = 0.12, P = 0.061), but not with viral load (r = -0.03, P = 0.610), or alanine transaminase (r = -0.03, P = 0.617). Diabetes mellitus neither significantly moderated (chi(2) = 0.13, P = 0.720), nor mediated (Sobel test P = 0.49) the HCV effect. HCV status was significantly associated with log microalbuminuria (chi(2) = 4.97, P = 0.026), adjusting for age, gender, diabetes, cryoglobulinemia, urea and creatinine. A positive HCV status was not significantly associated with low eGFR (< 60 mL/min every 1.73 m(2)) [odds ratio (OR): 0.5, 95% confidence interval (CI): 0.2-1.4], nor with high ACR (OR: 1.7, 95% CI: 0.7-4.1). End-of-treatment response (ETR) was achieved in 51.9% of patients. Individuals with ETR had significantly lower microalbuminuria post-treatment (chi(2) = 8.19, P = 0.004).<h4>Conclusion</h4>HCV affected the development of microalbuminuria independent of diabetes or cryoglobulinemia. Combination therapy of pegylated interferon-ribavirin had a positive effect in reducing microalbuminuria.

To investigate the role of microRNAs in regulating oligodendrocyte (OL) differentiation and myelination, we utilized transgenic mice in which microRNA processing was disrupted in OL precursor cells (OPCs) and OLs by targeted deletion of Dicer1. We found that inhibition of OPC-OL miRNA processing disrupts normal CNS myelination and that OPCs lacking mature miRNAs fail to differentiate normally in vitro. We identified three miRNAs (miR-219, miR-138, and miR-338) that are induced 10-100x during OL differentiation; the most strongly induced of these, miR-219, is necessary and sufficient to promote OL differentiation, and partially rescues OL differentiation defects caused by total miRNA loss. miR-219 directly represses the expression of PDGFRalpha, Sox6, FoxJ3, and ZFP238 proteins, all of which normally help to promote OPC proliferation. Together, these findings show that miR-219 plays a critical role in coupling differentiation to proliferation arrest in the OL lineage, enabling the rapid transition from proliferating OPCs to myelinating OLs.

MicroRNAs (miRNAs) regulate various biological processes, but evidence for miRNAs that control the differentiation program of specific neural cell types has been elusive. To determine the role of miRNAs in the formation of myelinating oligodendrocytes, we selectively deleted a miRNA-processing enzyme, Dicer1, in oligodendrocyte lineage cells. Mice lacking Dicer1 display severe myelinating deficits despite an expansion of the oligodendrocyte progenitor pool. To search for miRNAs responsible for the induction of oligodendrocyte maturation, we identified miR-219 and miR-338 as oligodendrocyte-specific miRNAs in spinal cord. Overexpression of these miRNAs is sufficient to promote oligodendrocyte differentiation. Additionally, blockage of these miRNA activities in oligodendrocyte precursor culture and knockdown of miR-219 in zebrafish inhibit oligodendrocyte maturation. miR-219 and miR-338 function in part by directly repressing negative regulators of oligodendrocyte differentiation, including transcription factors Sox6 and Hes5. These findings illustrate that miRNAs are important regulators of oligodendrocyte differentiation, providing new targets for myelin repair.

Endoplasmic reticulum-associated degradation (ERAD) is a system by which proteins accumulated in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol and degraded by the ubiquitin-proteasome pathway. HRD1 is expressed in brain neurons and acts as an ERAD ubiquitin ligase. Amyloid precursor protein (APP) is processed into amyloid-beta peptides (Abetas) that form plaque deposits in the brains of Alzheimer's disease (AD) patients. We found significantly decreased HRD1 protein levels in the cerebral cortex of AD patients. HRD1 colocalized with APP in brain neurons and interacted with APP through the proline-rich region of HRD1. HRD1 promoted APP ubiquitination and degradation, resulting in decreased generation of Abeta. Furthermore, suppression of HRD1 expression induced APP accumulation that led to increased production of Abeta associated with ER stress. Immunohistochemical analysis revealed that suppression of HRD1 expression inhibited APP aggresome formation, resulting in apoptosis. In addition, we found that the ATF6- and XBP1-induced upregulation of ERAD led to APP degradation and reduced Abeta production. These results suggest that the breakdown of HRD1-mediated ERAD causes Abeta generation and ER stress, possibly linked to AD.

Huntington's disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine stretch in the protein huntingtin (Htt). HD neurons are dysfunctional at multiple levels and have increased susceptibility to stress and apoptotic stimuli. We have discovered that synthesis of the ganglioside GM1 is reduced in fibroblasts from HD patients and in cell and animal models of HD, and that decreased GM1 levels contribute to heighten HD cell susceptibility to apoptosis. The apoptotic susceptibility is recapitulated through inhibition of ganglioside synthesis in wild-type striatal cells, suggesting that decreased GM1 levels might be one of the key events leading to HD pathogenesis and progression. Administration of GM1 restores ganglioside levels in HD cells and promotes activation of AKT and phosphorylation of mutant Htt, leading to decreased mutant Htt toxicity and increased survival of HD cells. Our data identify GM1 as a potential treatment for HD.

Localization of mRNAs to postsynaptic sites and their subsequent translation is thought to contribute to synapse-specific plasticity. However, the direct visualization of dendritic RNA transport in living neurons remains a major challenge. Here, we analyze the transport of Alexa-labeled RNAs microinjected into mature hippocampal neurons. We show that microinjected MAP2 and CaMKIIalpha RNAs form particles that localize into dendrites as their endogenous counterparts. In contrast, nonlocalizing RNAs or truncated CaMKIIalpha, lacking the dendritic targeting element, remain in the cell body. Furthermore, our microinjection approach allowed us to identify a novel dendritically localized RNA, Septin7. Time-lapse videomicroscopy of neurons injected with CaMKIIalpha and Septin7 RNAs demonstrates fast directional movement along the dendrites of hippocampal neurons, with similar kinetics to Staufen1 ribonucleoprotein particles (RNPs). Coinjection and simultaneous visualization of two RNAs, as well as double detection of the corresponding endogenous RNAs, reveal that neuronal transcripts are differentially sorted in dendritic RNPs.

This article reviews the evidence that ties the development of hepatocellular carcinoma (HCC) to the natural immune pro-inflammatory response to chronic liver disease, with a focus on the role of Toll-like receptor (TLR) signaling as the mechanism of liver stem cell/progenitor transformation to HCC. Two exemplary models of this phenomenon are reviewed in detail. One model applies chronic ethanol/lipopolysaccharide feeding to the activated TLR4 signaling pathway. The other applies chronic feeding of a carcinogenic drug, in which TLR2 and 4 signaling pathways are activated. In the drug-induced model, two major methyl donors, S-adenosylmethionine and betaine, prevent the upregulation of the TLR signaling pathways and abrogate the stem cell/progenitor proliferation response when fed with the carcinogenic drug. This observation supports a nutritional approach to liver cancer prevention and treatment. The observation that upregulation of the TLR signaling pathways leads to liver tumor formation gives evidence to the popular concept that the chronic pro-inflammatory response is an important mechanism of liver oncogenesis. It provides a nutritional approach, which could prevent HCC from developing in many chronic liver diseases.

Motor neurons are functionally related, but represent a diverse collection of cells that show strict preferences for specific axon pathways during embryonic development. In this article, we describe the ligands and receptors that guide motor axons as they extend toward their peripheral muscle targets. Motor neurons share similar guidance molecules with many other neuronal types, thus one challenge in the field of axon guidance has been to understand how the vast complexity of brain connections can be established with a relatively small number of factors. In the context of motor guidance, we highlight some of the temporal and spatial mechanisms used to optimize the fidelity of pathfinding and increase the functional diversity of the signaling proteins.

<h4>Background</h4>Interferon-alpha (IFN-alpha) treatment frequently induces depression, potentially leading to early dose reductions or a shorter duration of treatment, which can adversely affect outcomes, including quality of life.<h4>Objective</h4>Defining relevant risk factors for IFN-alpha-induced depression is essential in order to identify prophylactic treatment strategies.<h4>Method</h4>The authors examined whether a functional polymorphism (5-HTTLPR) in the gene encoding the serotonin transporter moderates IFN-alpha-induced depressive symptoms in 1,015 patients with chronic hepatitis C (CHC) receiving pegylated IFN-alpha and ribavirin. Depressive symptoms were assessed at baseline, 12 weeks, and 20 weeks of treatment.<h4>Results</h4>Depression symptoms increased during antiviral treatment; 5-HTTLPR genotype moderated IFN-alpha-induced depression symptoms in both non-Hispanic Caucasians and Hispanic patients, although the opposite risk allele was associated with depression in the two populations.<h4>Conclusion</h4>5-HTTLPR may moderate risk for the development of depressive symptoms during IFN-alpha therapy for CHC in a population-specific manner.

<h4>Aim</h4>To establish a quantitative method to measure the amount of lipids.<h4>Methods</h4>The livers of 53 male Wistar rats (225 g) with different degrees of hepatic steatosis were studied. This model of hepatic steatosis was based on a high carbohydrate, fat-free modified diet. Biopsies were classified into four grades depending on fat accumulation, using the Kleiner and Brunt classification. Total fat was studied by the Soxtec method (Soxtec 2050 Auto Fat Extraction System), and agreement between both assays was assessed by calculating the kappa coefficient.<h4>Results</h4>According to the histological classification, 38% of rats presented grade 0, 21% grade 1, 22% grade 2 and 20% grade 3. The amount of fat per 100 g tissue was 2.60 +/- 0.64 g for grade 0, 3.87 +/- 1.59 g for grade 1, 5.82 +/- 1.37 g for grade 2 and 8.68 +/- 2.30 g for grade 3. Statistically significant differences were found between the mean values for each of the histological grades (P < 0.05). The correlation for the quantification of fat in the liver between both assays was moderate (kappa = 0.60).<h4>Conclusion</h4>The biochemical quantification of fat in liver tissue by the Soxtec method was correlated with the histological classification, although the agreement between the two tests was only moderate.

Apoptosis of neurons in the maturing neocortex has been recorded in a wide variety of mammals, but very little is known about its effects on cortical differentiation. Recent research has implicated the RhoA GTPase subfamily in the control of apoptosis in the developing nervous system and in other tissue types. Rho GTPases are important components of the signaling pathways linking extracellular signals to the cytoskeleton. To investigate the role of the RhoA GTPase subfamily in neocortical apoptosis and differentiation, we have engineered a mouse line in which a dominant-negative RhoA mutant (N19-RhoA) is expressed from the Mapt locus, such that all neurons of the developing nervous system are expressing the N19-RhoA inhibitor. Postnatal expression of N19-RhoA led to no major changes in neocortical anatomy. Six layers of the neocortex developed and barrels (whisker-related neural modules) formed in layer IV. However, the density and absolute number of neurons in the somatosensory cortex increased by 12-26% compared with wild-type littermates. This was not explained by a change in the migration of neurons during the formation of cortical layers but rather by a large decrease in the amount of neuronal apoptosis at postnatal day 5, the developmental maximum of cortical apoptosis. In addition, overexpression of RhoA in cortical neurons was seen to cause high levels of apoptosis. These results demonstrate that RhoA-subfamily members play a major role in developmental apoptosis in postnatal neocortex of the mouse but that decreased apoptosis does not alter cortical cytoarchitecture and patterning.

Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.

<h4>Study objective</h4>To test the hypothesis that a 44-base-pair insertion/deletion polymorphism in the 5' regulatory region of the serotonin transporter gene (5-HTTLPR) is associated with primary insomnia.<h4>Design</h4>Association study.<h4>Setting</h4>Sleep laboratory at the Central Institute of Mental Health, Mannheim, Germany.<h4>Patients</h4>157 patients with primary insomnia and 827 healthy controls.<h4>Interventions</h4>N/A.<h4>Measurement and results</h4>We found the short (s-) allele of the 5-HTTLPR to be significantly more frequent in patients suffering from insomnia than in control individuals (47.1% vs. 39.9%: OR = 1.34).<h4>Conclusions</h4>This finding contributes to the understanding of the pathophysiology of primary insomnia and suggests a biological basis between the prevalent comorbidity of primary insomnia and other psychiatric disorders.

The fibroblast growth factor receptor tyrosine kinases (FGFR1, 2, 3, and 4) represent promising therapeutic targets in a number of cancers. We have developed the first potent and selective irreversible inhibitor of FGFR1, 2, 3, and 4, which we named FIIN-1 that forms a covalent bond with cysteine 486 located in the P loop of the FGFR1 ATP binding site. We demonstrated that the inhibitor potently inhibits Tel-FGFR1-transformed Ba/F3 cells (EC(50) = 14 nM) as well as numerous FGFR-dependent cancer cell lines. A biotin-derivatized version of the inhibitor, FIIN-1-biotin, was shown to covalently label FGFR1 at Cys486. FIIN-1 is a useful probe of FGFR-dependent cellular phenomena and may provide a starting point of the development of therapeutically relevant irreversible inhibitors of wild-type and drug-resistant forms of FGFR kinases.

The Genetics of Kidneys in Diabetes (GoKinD) study was initiated to facilitate research aimed at identifying genes involved in diabetic nephropathy (DN) in type 1 diabetes. In this review, we present an overview of this study and the various reports that have used its collection. At the forefront of these efforts is the recent genome-wide association scan implemented on the GoKinD collection. We highlight the results from our analysis of these data and describe compelling evidence from animal models that further support the potential role of associated loci in the susceptibility of DN. To enhance our analysis of genetic associations in GoKinD, using genome-wide imputation, we expanded our analysis of this collection to include genotype data from more than 2.4 million common single nucleotide polymorphisms. We illustrate the added utility of this enhanced dataset through the comprehensive fine-mapping of candidate genomic regions previously linked with DN and the targeted investigation of genes involved in candidate pathways implicated in its pathogenesis. Collectively, genome-wide association and genome-wide imputation data from the GoKinD collection will serve as a springboard for future investigations into the genetic basis of DN in type 1 diabetes.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by a loss of tolerance to self-antigens and the production of high titers of serum autoantibodies. Lupus nephritis can affect up to 74% of SLE patients, particularly those of Hispanic and African ancestries, and remains a major cause of morbidity and mortality. A genetic etiology in SLE is now well substantiated. Thanks to extensive collaborations, extraordinary progress has been made in the past few years and the number of confirmed genes predisposing to SLE has catapulted to approximately 30. Studies of other forms of genetic variation, such as copy number variants and epigenetic alterations, are emerging and promise to revolutionize our knowledge about disease mechanisms. However, to date little progress has been made on the identification of genetic factors specific to lupus nephritis. On the near horizon, two large-scale efforts, a collaborative meta-analysis of lupus nephritis based on all genome-wide association data in Caucasians and parallel scans in four other ethnicities, are poised to make fundamental discoveries in the genetics of lupus nephritis. Collectively, these findings will show that a broad array of pathways underlines the genetic heterogeneity of SLE and lupus nephritis, and provide potential avenues for the development of novel therapies.

PURPOSE: The DBA/2J (D2) mouse carries mutations in two of its genes, Tyrp1 and Gpnmb. These alterations result in the development of an immune response in the iris, leading to iris atrophy and pigment dispersion. The development of elevated intraocular pressure (IOP) in this model of glaucoma is considered to be a significant factor leading to the death of retinal ganglion cells (RGCs). Changes in gene expression in the retina have already been correlated with the appearance of elevated IOP in the D2 mouse. The purpose of the present study was to determine if any changes in gene expression occur prior to the development of IOP. METHODS: The IOP was measured monthly using a rebound tonometer in D2 and age-matched C57/BL6 (B6) mice (normal controls). D2 animals with normal IOP at 2 and 4 M were used. In addition, mice at the age of 6-7 M were included to look for any trends in gene expression that might develop during the progression of the disease. Separate RNA samples were prepared from each of three individual retinas for each age, and gene expression profiles were determined with the aid of mouse oligonucleotide arrays (Agilent). A subset of genes was examined with the aid of real-time PCR. Immunocytochemistry was used to visualize changes in the retina for some of the gene-products. RESULTS: Four hundred and thirteen oligonucleotide probes were differentially expressed in the retinas of 4 M versus 2 M old D2 mice. The most significantly up-regulated genes (181) were associated with immune responses including interferon signaling, the complement system and the antigen presentation pathway, whereas the down-regulated genes (232) were linked to pathways related to cell death and known neurological diseases/disorders. These particular changes were not revealed in the age-matched B6 mice. By 6 M, when IOP started to increase in many of the D2 mice, more robust changes of these same genes were observed. Changes in the levels of selected genes, representative of different functions/pathways, were validated with RT-PCR, and changes in glial responses were visualized in the retina with immunocytochemistry. CONCLUSIONS: The results showed that the expression of genes related to the immune response and acute stress were altered independently of the development of elevated IOP, and indicated early involvement of the immune system in the onset of the disease. The later development of elevated IOP, observed in this animal model, was coincident with continued changes in expression of genes observed at earlier time points. Further studies are warranted to identify the roles of specific genes identified here with respect to the death of the RGCs.

Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD(140Q/140Q)), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD(140Q/140Q) mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.

The innate immune response is the first line of host defense against infections. This system employs a number of different types of cells, which in turn activate different sets of genes. Microarray studies of human and mouse cells infected with various pathogens identified hundreds of differentially expressed genes. However, combining these datasets to identify common and unique response patterns remained a challenge. We developed methods based on probabilistic graphical models to combine expression experiments across species, cells, and pathogens. Our method analyzes homologous genes in different species concurrently overcoming problems related to noise and orthology assignments. Using our method, we identified both core immune response genes and genes that are activated in macrophages in both human and mouse but not in dendritic cells, and vice versa. Our results shed light on immune response mechanisms and on the differences between various types of cells that are used to fight infecting bacteria. For supporting website, see www.cs.cmu.edu/-lyongu/pub/immune/.

Hierarchical functional data are widely seen in complex studies where sub-units are nested within units, which in turn are nested within treatment groups. We propose a general framework of functional mixed effects model for such data: within unit and within sub-unit variations are modeled through two separate sets of principal components; the sub-unit level functions are allowed to be correlated. Penalized splines are used to model both the mean functions and the principal components functions, where roughness penalties are used to regularize the spline fit. An EM algorithm is developed to fit the model, while the specific covariance structure of the model is utilized for computational efficiency to avoid storage and inversion of large matrices. Our dimension reduction with principal components provides an effective solution to the difficult tasks of modeling the covariance kernel of a random function and modeling the correlation between functions. The proposed methodology is illustrated using simulations and an empirical data set from a colon carcinogenesis study. Supplemental materials are available online.

Insulinomas are rare neuroendocrine tumors of pancreatic islet cells that retain the ability to produce and secrete insulin. In contrast to normally differentiated β-cells, insulinoma cells continue to secrete insulin and proinsulin at low blood glucose. This deregulated insulin secretion manifests clinically as fasting hypoglycemia. The molecular pathways that characterize normal insulin secretion and β-cell growth are reviewed and contrasted to the biology of insulinomas. The second half of this review summarizes the clinical approach to the disorder. The diagnosis of insulinoma is established by demonstrating inappropriately high insulin levels with coincident hypoglycemia at the time of a supervised fast. Localization of insulinomas is challenging owing to their small size but should be attempted to maximize the chance for successful surgical resection and avoid risks associated with reoperation. In the majority of cases, successful surgical resection leads to lifelong cure.

<h4>Objective</h4>This study was performed to investigate the impact of HAART versus no HAART and nucleoside free versus nucleoside containing HAART on the efficacy and safety of pegylated interferon and ribavirin therapy for the treatment of chronic HCV infection in HIV/HCV co-infected patients. In addition a control group of HCV mono-infected patients undergoing anti-HCV therapy was evaluated.<h4>Methods</h4>Multicenter, partially randomized, controlled clinical trial. HIV-negative and -positive patients with chronic HCV infection were treated with pegylated interferon alfa-2a and ribavirin (800 - 1200 mg/day) for 24 - 48 weeks in one of four treatment arms: HIV-negative (A), HIV-positive without HAART (B) and HIV-positive on HAART (C). Patients within arm C were randomized to receive open label either a nucleoside containing (C1) or a nucleoside free HAART (C2).<h4>Results</h4>168 patients were available for analysis. By intent-to-treat analysis similar sustained virological response rates (SVR, negative HCV-RNA 24 weeks after the end of therapy) were observed comparing HIV-negative and -positive patients (54% vs. 54%, p = 1.000). Among HIV-positive patients SVR rates were similar between patients off and on HAART (57% vs. 52%, p = 0.708). Higher SVR rates were observed in patients on a nucleoside free HAART compared to patients on a nucleoside containing HAART, though confounding could not be ruled out and in the intent-to-treat analysis the difference was not statistically significant (64% vs. 46%, p = 0.209).<h4>Conclusions</h4>Similar response rates for HCV therapy can be achieved in HIV-positive and -negative patients. Patients on nucleoside free HAART reached at least equal rates of sustained virological response compared to patients on standard HAART.

The association between polymorphisms in genes COMT, HFE that takes part in oxidative stress regulation, and chromosome aberration frequency in lymphocytes was assessed in 278 female residents of radiation polluted regions of Central Russia: Bryansk (322 kBk/m2) and Tula Districts (137Cs - 171 kBk/m2). The C187G, G845A genotyping of HFE and G1947A (H/L) of COMT was done by means of polymerase chain reaction-restriction fragment length polymorphism. Studied population was divided into 3 subgroups by level of chromosome aberrations per cell (0-2, 3-4, >5). There was shown statistically significant difference in distribution of COMTand HFE genotypes between the groups. The high frequency of chromosome aberrations (> or = 5%) was associated with homozygotes of the high activity COMT G/G and HFE CC. Heterozygotes for G1947A COMT and C187G HFE reveal negative association with the high frequency of chromosome aberrations and correspond to "resistance factors".

The F-BAR domain is emerging as an important player in membrane remodeling pathways. F-BAR domain proteins couple membrane remodeling with actin dynamics associated with endocytic pathways and filopodium formation. Here, we provide a comprehensive analysis of F-BAR domain proteins in terms of their evolutionary relationships and protein function. F-BAR domain containing proteins can be categorized into five subfamilies based on their phylogeny which is consistent with the additional protein domains they possess, for example, RhoGAP domains, Cdc42 binding sites, SH3 domains and tyrosine kinase domains. We derive a protein-protein interaction network suggesting that dynamin1/2, N-WASP, Huntingtin, intersectin and Cdc42 are central nodes influencing F-BAR domain protein function.

Cerebellar ataxias are a group of disabling neurological disorders. Patients exhibit a cerebellar syndrome and can also present with extra-cerebellar deficits, namely pigmentary retinopathy, extrapyramidal movement disorders, pyramidal signs, cortical symptoms (seizures, cognitive impairment/behavioural symptoms), and peripheral neuropathy. Recently, deficits in cognitive operations have been unraveled. Cerebellar ataxias are heterogeneous both at the phenotypic and genotypic point of view. Therapeutical trials performed during these last 4 decades have failed in most cases, in particular because drugs were not targeting a deleterious pathway, but were given to counteract putative defects in neurotransmission. The identification of the causative mutations of many hereditary ataxias, the development of relevant animal models and the recent identifications of the molecular mechanisms underlying ataxias are impacting on the development of new drugs. We provide an overview of the pharmacological treatments currently used in the clinical practice and we discuss the drugs under development.

The incidence of hepatocellular carcinoma is increasing worldwide as well as the associated risk factors, some of which include exposure to aflatoxin B1, Hepatitis B (HBV) virus and hepatitis C (HCV) virus. Mutation of tumour suppressor gene p53 at codon 249(ser) at exon 7 has been found to contribute significantly to replication of damaged DNA and subsequent tumour progression. The x gene of HBV (HBx) is the most common open reading frame integrated into the host genome in hepatocellular carcinoma and the integrated HBx is frequently mutated in hepatocellular carcinoma. Mutant HBx proteins still retain their ability to bind to p53 thereby attenuating DNA repair and p53-mediated apoptosis.

Colorectal cancer (CCR) is one of the most frequent cancers in developed countries. It poses a major public health problem and there is renewed interest in understanding the basic principles of the molecular biology of colorectal cancer. It has been established that sporadic CCRs can arise from at least two different carcinogenic pathways. The traditional pathway, also called the suppressor or chromosomal instability pathway, follows the Fearon and Vogelstein model and shows mutation in classical oncogenes and tumour suppressor genes, such as K-ras, adenomatous polyposis coli, deleted in colorectal cancer, or p53. Alterations in the Wnt pathway are also very common in this type of tumour. The second main colorectal carcinogenesis pathway is the mutator pathway. This pathway is present in nearly 15% of all cases of sporadic colorectal cancer. It is characterized by the presence of mutations in the microsatellite sequences caused by a defect in the DNA mismatch repair genes, mostly in hMLH1 or hMSH2. These two pathways have clear molecular differences, which will be reviewed in this article, but they also present distinct histopathological features. More strikingly, their clinical behaviours are completely different, having the "mutator" tumours a better outcome than the "suppressor" tumours.

A 56-year-old male visited our hospital for evaluation of an occipital mass. Contrast computed tomography showed hypervascular enhancement with osteolytic change in the skull and a huge enhanced mass in the liver. Magnetic resonance imaging showed bone metastasis in the thoracic vertebrae. Assays for hepatitis B surface antigen and hepatitis B core antibody were positive and his liver condition was Child-Pugh grade A. Our diagnosis was hepatocellular carcinoma (HCC) with skull and vertebrae metastases on chronic hepatitis B. He was treated with radiation therapy for bone metastases and transcatheter arterial chemoembolization for HCC. But he developed acute respiratory failure because of aspiration pneumonia, congestion and oedema with haemorrhage of the lungs and died. Dissection showed HCC with multiple bone metastases. The liver tumor was categorized as well-differentiated HCC, Edmondson classification I, trabecular type and pseudoglandular type. In the liver mild infiltration of lymphocytes was seen in Glisson's capsules which were significantly enlarged with well preserved limiting plates. Piecemeal necrosis was not obvious. No fibrosis was noted. An 8 cm × 7 cm × 3 cm metastatic lesion had formed in the left occipitotemporal part of the cranial bone. The lesion was osteolytic and showed invasion into the dura mater. Neither the subdural cavity nor the brain showed involvement from the metastatic tumor. However, skull metastasis from HCC is very rare and it affects the patient's prognosis and the quality of life. Therefore, it is very important to make an early diagnosis and carry out proper management of skull metastasis from HCC.

<h4>Aim</h4>To identify new markers of hepatocellular carcinoma (HCC) using a proteomic analysis.<h4>Methods</h4>Patients with liver cirrhosis of the three most frequent etiologies: hepatitis C virus, hepatitis B virus and alcoholic liver disease, were included in the study. The samples were analysed by 2D-electrophoresis in order to determine the differential protein expression. The proteins were separated according to the charge in immobilized pH 3-10 gradient strips and then by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins of interest were excised, digested with trypsin and the resulting peptides were separated and identified.<h4>Results</h4>Three differentially expressed apolipoproteins (Apo) were identified based on the protein profile using proteomic techniques: Apo-A1, Apo-A4 and Apo-E. Apo-A4 levels were significantly lower in HCC than in non-HCC patients regardless of etiology (P < 0.01). Multivariate logistic regression showed that Apo-A4 and Apo-A1 were the only independent factors related to HCC diagnosis (P < 0.05). The receiver operating characteristic (ROC) curve including both Apo-A4 and Apo-A1 showed an area under the ROC of 0.944 (P < 0.001), a sensitivity of 0.89 and a specificity of 0.81 for diagnosis of HCC.<h4>Conclusion</h4>Apo-A4 and Apo-A1 may be used clinically as biomarkers of HCC with a high sensibility and specificity. These findings may provide additional insights into the mechanism of HCC development and progression.