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Despite identification of the gene for huntingtin (Htt) as causal in Huntington's Disease (HD), explication of HD symptoms and selective damage to the corpus striatum has been elusive. The small G protein Rhes Ras homolog enriched in striatum, highly localized to the striatum, binds selectively to mutant Htt (mHtt) and enhances sumoylation of mHtt. Sumoylation disaggregates mHtt and augments its cytotoxicity. Thus, it appears likely that Rhes-mHtt interaction accounts in substantial part for the selective striatal neurotoxicity of HD with associated extrapyramidal symptomatology. Rhes also binds and activates mTOR, enhancing its influence on protein synthesis, and may be the principal determinant of striatal mTOR activation. In HD, sequestration of Rhes by mHtt may decrease its access to mTOR. The attendant loss of protein translational stimulation may explain the pronounced striatal atrophy of HD. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

A hallmark of many neurodegenerative diseases is accumulation of misfolded proteins within neurons, leading to cellular dysfunction and cell death. Although several mechanisms have been proposed to link protein misfolding to cellular toxicity, the connection remains enigmatic. Here, we report a cell death pathway involving protein disulfide isomerase (PDI), a protein chaperone that catalyzes isomerization, reduction and oxidation of disulfides. Through a small molecule screening approach, we discovered five structurally distinct compounds that prevent apoptosis induced by mutant huntingtin protein. Using modified Huisgen cycloaddition chemistry, we then identified PDI as the molecular target of these small molecules. Expression of polyglutamine-expanded huntingtin exon 1 in PC12 cells caused PDI to accumulate at mitochondrial-associated ER membranes and trigger apoptotic cell death via mitochondrial outer-membrane permeabilization. Inhibiting PDI in rat brain cells suppressed the toxicity of mutant huntingtin exon 1 and Aβ peptides processed from the amyloid precursor protein. This pro-apoptotic function of PDI represents a new mechanism linking protein misfolding and apoptotic cell death.

The ENCODE project is an international consortium with a goal of cataloguing all the functional elements in the human genome. The ENCODE Data Coordination Center (DCC) at the University of California, Santa Cruz serves as the central repository for ENCODE data. In this role, the DCC offers a collection of high-throughput, genome-wide data generated with technologies such as ChIP-Seq, RNA-Seq, DNA digestion and others. This data helps illuminate transcription factor-binding sites, histone marks, chromatin accessibility, DNA methylation, RNA expression, RNA binding and other cell-state indicators. It includes sequences with quality scores, alignments, signals calculated from the alignments, and in most cases, element or peak calls calculated from the signal data. Each data set is available for visualization and download via the UCSC Genome Browser (http://genome.ucsc.edu/). ENCODE data can also be retrieved using a metadata system that captures the experimental parameters of each assay. The ENCODE web portal at UCSC (http://encodeproject.org/) provides information about the ENCODE data and links for access.

Colorectal cancer is a complex disease resulting from somatic genetic and epigenetic alterations, including locus-specific CpG island methylation and global DNA or LINE-1 hypomethylation. Global molecular characteristics such as microsatellite instability (MSI), CpG island methylator phenotype (CIMP), global DNA hypomethylation, and chromosomal instability cause alterations of gene function on a genome-wide scale. Activation of oncogenes including KRAS, BRAF and PIK3CA affects intracellular signalling pathways and has been associated with CIMP and MSI. Traditional epidemiology research has investigated various factors in relation to an overall risk of colon and/or rectal cancer. However, colorectal cancers comprise a heterogeneous group of diseases with different sets of genetic and epigenetic alterations. To better understand how a particular exposure influences the carcinogenic and pathologic process, somatic molecular changes and tumour biomarkers have been studied in relation to the exposure of interest. Moreover, an investigation of interactive effects of tumour molecular changes and the exposures of interest on tumour behaviour (prognosis or clinical outcome) can lead to a better understanding of tumour molecular changes, which may be prognostic or predictive tissue biomarkers. These new research efforts represent 'molecular pathologic epidemiology', which is a multidisciplinary field of investigations of the inter-relationship between exogenous and endogenous (eg, genetic) factors, tumoural molecular signatures and tumour progression. Furthermore, integrating genome-wide association studies (GWAS) with molecular pathological investigation is a promising area (GWAS-MPE approach). Examining the relationship between susceptibility alleles identified by GWAS and specific molecular alterations can help elucidate the function of these alleles and provide insights into whether susceptibility alleles are truly causal. Although there are challenges, molecular pathological epidemiology has unique strengths, and can provide insights into the pathogenic process and help optimise personalised prevention and therapy. In this review, we overview this relatively new field of research and discuss measures to overcome challenges and move this field forward.

The inheritance of DNA methylation patterns is a popular theory to explain the influence of parental genetic and environmental factors on the phenotype of their offspring but few studies have examined this relationship in humans. Using 120 paired maternal-umbilical cord blood samples randomly selected from a prospective birth cohort in Bangladesh, we quantified DNA methylation by pyrosequencing seven CpG positions in the promoter region of p16, four CpG positions in the promoter region of p53, LINE-1 and Alu. Positive correlations were observed between maternal and umbilical cord blood at p16, LINE-1, and Alu but not p53. Multiple linear regression models observed a significant association between maternal and umbilical cord blood at LINE-1 and Alu (LINE-1: β = 0.63, p<0.0001; Alu: β = 0.28, p = 0.009). After adjusting for multiple comparisons, maternal methylation of p16 at position 4 significantly predicted methylation at the same position in umbilical cord blood (β = 0.43, p = <0.0001). These models explained 48%, 5% and 16% of the observed variability in umbilical cord %5mC for LINE-1, Alu and p16 at position 4, respectively. These results suggest that DNA methylation in maternal blood was correlated with her offspring at LINE-1, Alu, and p16 but not p53. Additional studies are needed to confirm whether these observed associations were due to the inheritance of epigenetic events or the shared environment between mother and fetus. Future studies should also use a multi-generational family-based design that would quantify both maternal and paternal contributions to DNA methylation in offspring across more than one generation.

Gene network information has been used to improve gene selection in microarray-based studies by selecting marker genes based both on their expression and the coordinate expression of genes within their gene network under a given condition. Here we propose a new network-embedded gene selection model. In this model, we first address the limitations of microarray data. Microarray data, although widely used for gene selection, measures only mRNA abundance, which does not always reflect the ultimate gene phenotype, since it does not account for post-transcriptional effects. To overcome this important (critical in certain cases) but ignored-in-almost-all-existing-studies limitation, we design a new strategy to integrate together microarray data with the information of microRNA, the major post-transcriptional regulatory factor. We also handle the challenges led by gene collaboration mechanism. To incorporate the biological facts that genes without direct interactions may work closely due to signal transduction and that two genes may be functionally connected through multi paths, we adopt the concept of diffusion distance. This concept permits us to simulate biological signal propagation and therefore to estimate the collaboration probability for all gene pairs, directly or indirectly-connected, according to multi paths connecting them. We demonstrate, using type 2 diabetes (DM2) as an example, that the proposed strategies can enhance the identification of functional gene partners, which is the key issue in a network-embedded gene selection model. More importantly, we show that our gene selection model outperforms related ones. Genes selected by our model 1) have improved classification capability; 2) agree with biological evidence of DM2-association; and 3) are involved in many well-known DM2-associated pathways.

<h4>Background</h4>For years, the genetics of metastatic colorectal cancer (CRC) have been studied using a variety of techniques. However, most of the approaches employed so far have a relatively limited resolution which hampers detailed characterization of the common recurrent chromosomal breakpoints as well as the identification of small regions carrying genetic changes and the genes involved in them.<h4>Methodology/principal findings</h4>Here we applied 500K SNP arrays to map the most common chromosomal lesions present at diagnosis in a series of 23 primary tumours from sporadic CRC patients who had developed liver metastasis. Overall our results confirm that the genetic profile of metastatic CRC is defined by imbalanced gains of chromosomes 7, 8q, 11q, 13q, 20q and X together with losses of the 1p, 8p, 17p and 18q chromosome regions. In addition, SNP-array studies allowed the identification of small (<1.3 Mb) and extensive/large (>1.5 Mb) altered DNA sequences, many of which contain cancer genes known to be involved in CRC and the metastatic process. Detailed characterization of the breakpoint regions for the altered chromosomes showed four recurrent breakpoints at chromosomes 1p12, 8p12, 17p11.2 and 20p12.1; interestingly, the most frequently observed recurrent chromosomal breakpoint was localized at 17p11.2 and systematically targeted the FAM27L gene, whose role in CRC deserves further investigations.<h4>Conclusions/significance</h4>In summary, in the present study we provide a detailed map of the genetic abnormalities of primary tumours from metastatic CRC patients, which confirm and extend on previous observations as regards the identification of genes potentially involved in development of CRC and the metastatic process.

Neogenin is a type I transmembrane glycoprotein with a large ectodomain containing tandem immunoglobulin-like and fibronectin type III (FNIII) domains. Closely related to the tumor suppressor gene DCC, neogenin functions in critical biological processes through binding to various ligands, including netrin, repulsive guidance molecules, and the iron regulatory protein hemojuvelin. We previously reported that neogenin binds to hemojuvelin through its membrane-proximal fifth and sixth FNIII domains (FN5-6), with domain 6 (FN6) contributing the majority of critical binding interactions. Here we present the crystal structure of FN5-6, the hemojuvelin-binding fragment of human neogenin, at 1.8Å. The two FNIII domains are orientated nearly linearly, a domain arrangement most similar to that of a tandem FNIII-containing fragment within the cytoplasmic tail of the β4 integrin. By mapping surface-exposed residues that differ between neogenin FN5-6 and the comparable domains from DCC, which does not bind hemojuvelin, we identified a potential hemojuvelin-binding site on neogenin FN6. Neogenin FN5, which does not bind hemojuvelin in isolation, exhibits a highly electropositive surface, which may be involved in interactions with negatively-charged polysaccharides or phospholipids in the membrane bilayer. The neogenin FN5-6 structure can be used to facilitate a molecular understanding of neogenin's interaction with hemojuvelin to regulate iron homeostasis and with hemojuvelin-related repulsive guidance molecules to mediate axon guidance.

<h4>Aims</h4>a public health strategy to promote early diagnosis of hemochromatosis gene (HFE)-related hemochromatosis (HFE-HH) largely depends on people's acceptance of available screening tests. The present study aimed at evaluating patient awareness of HFE-HH and their acceptance of DNA testing in western Romania.<h4>Results</h4>a total of 221 participants were randomly recruited from the ambulatory unit of the Emergency County Hospital in Timisoara, Romania. They received brief information on HFE-HH and were assessed for the signs and symptoms of hemochromatosis. HFE genotyping was offered to all of them. Only two cases (0.9%) had previous knowledge of HFE-HH. Twenty-one cases (9.5%) underwent genetic testing. Characteristics associated with test acceptance were age <45 years, male gender, and educational attainment. Acceptance was associated with a desire to know if they had HFE-HH (85.7%). The most prevalent refusal reason was a desire for more information (41%).<h4>Conclusions</h4>larger educational programs are required to increase people's awareness about HFE-HH in western Romania. Nevertheless, within health care settings, the importance of disease detection and patient's educational background appear to be essential for achieving high rates of participation in the genetic test.

<h4>Background</h4>Rearrangement of the mixed-lineage leukemia gene (MLL) is found in 80% of infant acute lymphoblastic leukemia (ALL) and is associated with poor prognosis and resistance to glucocorticoids (GCs). We have recently observed that GC resistance in T-ALL cell lines is associated with a proliferative metabolism and reduced expression of MLL. In this study we have further explored the relationship between MLL status and GC sensitivity.<h4>Results</h4>Negative correlation of MLL expression with GC resistance in 15 T-ALL cell lines was confirmed by quantitative RT-PCR. The absence of MLL-rearrangements suggested that this relationship represented expression of wild-type MLL. Analysis of MLL expression patterns revealed a negative relationship with cellular metabolism, proliferation and anti-apoptotic transcriptional networks. In silico analysis of published data demonstrated that reduced levels of MLL mRNA are associated with relapse and prednisolone resistance in T-ALL patients and adverse clinical outcome in children with MLL-rearranged ALL. RNAi knockdown of MLL expression in T-ALL cell lines significantly increased resistance to dexamethasone and gamma irradiation indicating an important role for wild-type MLL in the control of cellular apoptosis.<h4>Conclusions</h4>The data suggests that reduced expression of wild-type MLL can contribute to GC resistance in ALL patients both with and without MLL-translocations.

The multifaceted potentialities of expanded pyridiniums (EPs), based on one pyridinium core bearing a 4-pyridyl or 4-pyridylium as the N-pyridinio group, are established at both experimental and theoretical levels. Two classes of head-to-tail (htt) EPs were designed, and their first representative elements were synthesized and fully characterized. The branched (B) family is made up of 2,6-diphenyl-4-aryl-1,4'-bipyridin-1-ium (or 1,1'-diium) species, denoted 1B and 2B for monocationic EPs (with aryl = phenyl and biphenyl, respectively) and 1B(Me) and 2B(Me) for related quaternarized dicationic species. The series of fused (F) analogues comprises 9-aryl-benzo[c]benzo[1,2]quinolizino[3,4,5,6-ija][1,6]naphthyridin-15-ium species, denoted 1F and 2F, and their 2,15-diium derivatives referred to as 1F(Me) and 2F(Me). Electrochemistry (in MeCN vs SCE) reveals that branched EPs undergo a single reversible bielectronic reduction at ca. -0.92 V for 1B/2B and -0.59 V for 1B(Me)/2B(Me), whereas pericondensed species show two reversible monoelectronic reductions at ca. -0.83 and -1.59 V for 1F/2F and ca. -0.42 and -1.07 V for 1F(Me)/2F(Me). Regarding electronic absorption features, all htt-EP chromophores show absorptivity in the range of ca. 1-4 × 10(4) M(-1) cm(-1), with red-edge absorptions extending toward 450 and 500 nm (in MeCN) for 2B(Me) and 2F(Me), respectively. These lowest-energy pi-pi* transitions are ascribed to intramolecular charge transfer between the electron-releasing biphenyl group and the htt-bipyridinium electron-withdrawing subsystems. EPs display room-temperature photoemission quantum yields ranging from 10% to 50%, with the exception of 1B, and branched luminophores are characterized by larger Stokes shifts (8000-10 000 cm(-1)) than fused ones. Lastly, a method to predict the efficiency of photobiscyclization of branched EPs into fused ones, based on the analysis of computed difference maps in total electron density for singlet excited states, is proposed.

The use of glucocorticoids in the treatment of rheumatoid arthritis has been widely employed, but, owing to their systemic side-effects and also their susceptibility to the first pass metabolism, their use is being discouraged. To circumvent this, triamcinolone (TA) were encapsulated in chitosan microspheres with glutaraldehyde as the cross-linking agent to achieve a prolonged drug release. The percentage of drug loading, encapsulation efficiency, and surface morphology by Scanning electron microscopy (SEM), Phase transition by Differential scanning colorimetry (DSC), as well as Fourier transform infrared spectroscopy (FTIR) studies was carried out to characterize the chitosan microspheres. In-vitro and in-vivo release studies revealed that microspheres were able to control the release of TA with a uniform release pattern up to a period of 36 days and thereafter an extended release up to 63 days. The clinical parameters were investigated for changes in paw volume, hematological parameters like Erythrocyte sedimentation rate (ESR), Paced cell volume (PCV), Total leucocyte count (TLC), Hb, and Differential cell count (DCC) in Fruend's complete adjuvant induced arthritic rats. Histopathological findings as well as radiology (X-ray) further confirmed the effectiveness of TA encapsulated microspheres in mitigating the rat arthritic model.

Identifying the genes underlying quantitative trait loci (QTL) for disease is difficult, mainly because of the low resolution of the approach and the complex genetics involved. However, recent advances in bioinformatics and the availability of genetic resources now make it possible to narrow the genetic intervals, test candidate genes, and define pathways affected by these QTL. In this study, we mapped three significant QTL and one suggestive QTL for an increased albumin-to-creatinine ratio on chromosomes (Chrs) 1, 4, 15, and 17, respectively, in a cross between the inbred MRL/MpJ and SM/J strains of mice. By combining data from several sources and by utilizing gene expression data, we identified Tlr12 as a likely candidate for the Chr 4 QTL. Through the mapping of 33,881 transcripts measured by microarray on kidney RNA from each of the 173 male F2 animals, we identified several downstream pathways associated with these QTL, including the glycan degradation, leukocyte migration, and antigen-presenting pathways. We demonstrate that by combining data from multiple sources, we can identify not only genes that are likely to be causal candidates for QTL but also the pathways through which these genes act to alter phenotypes. This combined approach provides valuable insights into the causes and consequences of renal disease.

<h4>Rationale</h4>MicroRNAs (miRs) are expanding our understanding of cardiac disease and have the potential to transform cardiovascular therapeutics. One miR can target hundreds of individual mRNAs, but existing methodologies are not sufficient to accurately and comprehensively identify these mRNA targets in vivo.<h4>Objective</h4>To develop methods permitting identification of in vivo miR targets in an unbiased manner, using massively parallel sequencing of mouse cardiac transcriptomes in combination with sequencing of mRNA associated with mouse cardiac RNA-induced silencing complexes (RISCs).<h4>Methods and results</h4>We optimized techniques for expression profiling small amounts of RNA without introducing amplification bias and applied this to anti-Argonaute 2 immunoprecipitated RISCs (RISC-Seq) from mouse hearts. By comparing RNA-sequencing results of cardiac RISC and transcriptome from the same individual hearts, we defined 1645 mRNAs consistently targeted to mouse cardiac RISCs. We used this approach in hearts overexpressing miRs from Myh6 promoter-driven precursors (programmed RISC-Seq) to identify 209 in vivo targets of miR-133a and 81 in vivo targets of miR-499. Consistent with the fact that miR-133a and miR-499 have widely differing "seed" sequences and belong to different miR families, only 6 targets were common to miR-133a- and miR-499-programmed hearts.<h4>Conclusions</h4>RISC-sequencing is a highly sensitive method for general RISC profiling and individual miR target identification in biological context and is applicable to any tissue and any disease state.

<h4>Background</h4>Prostasomes are exosome-like vesicles that are secreted by the prostate and incorporated into semen during ejaculation. Human prostasomes are proposed to function in regulation of sperm function, immunosuppression, and prostate cancer progression. Previously, we identified galectin-3 on the surface of prostasomes. Galectin-3 is a β-galactoside binding protein involved in immunomodulation, cell interactions, and cancer progression, including prostate cancer. Functional characterization of galectin-3 in a given biological environment includes identification of its target glycoprotein ligands.<h4>Methods</h4>Candidate galectin-3 ligands in prostasomes were identified by tandem mass spectrometry of proteins that co-purified with galectin-3 during lactose affinity chromatography. Immunochemical and biochemical methods were used to investigate the association of Mac-2 binding protein (M2BP) with prostasomes.<h4>Results</h4>Proteins identified by tandem mass spectrometry included M2BP, CD26/dipeptidyl peptidase IV, prolactin-inducible protein (PIP), olfactomedin-4 (OLFM4), and semenogelins I and II (SgI and SgII). M2BP is a known galectin-3 ligand that was not previously described in prostasomes. M2BP protein bands were detected in the testis, epididymis, vas deferens, prostate, seminal vesicle, and sperm extracts. In seminal plasma, M2BP was identified in the soluble fraction and in purified prostasomes. Surface biotinylation and immunofluorescence studies indicated that M2BP is present on the prostasome surface and on sperm, respectively.<h4>Conclusions</h4>M2BP, CD26, PIP, OLFM4, and SgI and SgII are candidate glycoprotein ligands for galectin-3 in prostasomes. Given their overlap in functional significance with prostasomes and galectin-3, the identification of these glycoproteins as galectin-3 ligands in prostasomes lays the groundwork for future studies of prostasomes in reproduction and prostate cancer.

<h4>Background</h4>The peroxiredoxins (PRDXs) are emerging as regulators of antioxidant defense, apoptosis, and therapy resistance in cancer. Because their significance in prostate cancer (PCa) is unclear, we investigated their expression and clinical associations in PCa.<h4>Methods</h4>Transcript expression of PRDX1-6 in PCa was evaluated in cancer gene microarray datasets, whereas protein expression was evaluated by immunoblotting in prostate cell lines, and by immunohistochemistry (IHC) in prostate tissue microarrays (TMAs) containing tumor (n = 80) and control (n = 17) tissues. PRDX3 was also analyzed in TMAs containing PCa tissues from African-American and Caucasian patients (n = 150 per group). PRDX expression was correlated with patients' clinicopathologic characteristics.<h4>Results</h4>Analysis of PRDX expression in cancer microarray datasets revealed consistent upregulation (tumor vs. normal) of PRDX3 and 4. All PRDXs exhibited elevated protein expression in PCa cell lines, compared with non-tumor cells. IHC revealed significant overexpression of PRDX3 and 4 in PCa, associated with age, increased prostate specific antigen (PSA), tumor stage, or Gleason score. High PRDX3 staining was associated with early age and elevated Gleason score at time of radical prostatectomy in African-American but not in Caucasian patients with PCa. PSA recurrence free survival in patients with low PRDX3 tumor expression was significantly longer in Caucasians compared to African-Americans, but no difference was detected for high expression.<h4>Conclusions</h4>PRDXs exhibit differential expression in prostate tumors, with PRDX3 and 4 consistently upregulated. Their role in PCa development, and their potential as biological determinants of PCa health disparities and novel therapeutic targets, deserve further investigation.

Huntington's disease (HD) is a genetic neurodegenerative disease caused by abnormal CAG expansion. MicroRNAs (miRNAs) are short RNA molecules regulating gene expression, and are implicated in a variety of diseases including HD. However, the profiles and regulation of miRNAs in HD are not fully understood. Here, we analyzed the miRNA expression and miRNA regulators in two transgenic models of HD, YAC128 and R6/2 mice, and in a 3-nitropropionic acid (3NP)-induced striatal degeneration rat model. After characterizing the phenotypes by behavioral tests and histological analyses, we profiled striatal miRNAs using a miRNA microarray and we measured the key molecules involved in miRNA biogenesis and function. YAC128 mice showed upregulation-dominant miRNA expressions at 5 months and downregulation-dominant expressions at 12 months. Concomitantly, the expressions of Drosha-DGCR8, Exportin-5, and Dcp1 were increased at 5months, and the expression of Dicer was decreased at 12 months. In 10-week-old R6/2 mice, downregulation was dominant in the miRNA expressions and the level of Drosha decreased concomitantly. Nine miRNAs (miR-22, miR-29c, miR-128, miR-132, miR-138, miR-218, miR-222, miR-344, and miR-674*) were commonly down-regulated in both the 12-month-old YAC128 and 10-week-old R6/2 mice. Meanwhile, 3NP rats showed dynamic changes in the miRNA profiles during disease development and a few miRNAs with altered expression. Our results show that transgenic HD mice have abnormal miRNA biogenesis. This information should aid in future studies on therapeutic application of miRNAs in HD.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first exon of the gene Huntingtin (Htt). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells (NSCs) from HD-iPS cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained with β-III tubulin, calbindin and GABA but not DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs (from iPS or H9 NSCs). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD mechanisms and drug screening.

Carbazole 1,9a-dioxygenase (CARDO) is the initial dioxygenase in the carbazole-degradation pathway of Novosphingobium sp. KA1. The CARDO from KA1 consists of a terminal oxygenase (Oxy), a putidaredoxin-type ferredoxin and a ferredoxin reductase. The Oxy from Novosphingobium sp. KA1 was crystallized at 277 K using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. Diffraction data were collected to a resolution of 2.1 Å. The crystals belonged to the monoclinic space group P2(1). Self-rotation function analysis suggested that the asymmetric unit contained two Oxy trimers; the Matthews coefficient and solvent content were calculated to be 5.9 Å(3) Da(-1) and 79.1%, respectively.

In vivo iron load must be monitored to prevent complications from iron overload diseases such as hemochromatosis or transfusion-dependent anemias. While liver biopsy is the gold standard for determining in vivo iron load, MRI offers a noninvasive approach. MR phantoms have been reported that estimate iron concentration in the liver and mimic relaxation characteristics of in vivo deposits of hemosiderin. None of these phantoms take into account the size distribution of hemosiderin, which varies from patient to patient based on iron load. We synthesized stable and reproducible microsphere-ferritin conjugates (ferribeads) of different sizes that are easily characterized for several parameters that are necessary for modeling such as iron content and bead fraction. T(1) s and T(2) s were measured on a 1.41-T low-resolution NMR spectrometer and followed a size-dependent trend. Ferribeads imaged at 4.7 and 14.1 T showed that signal intensities are dependent on the distribution of ferritin around the bead rather than the iron concentration alone. These particles can be used to study the effects of particle size, ferritin distribution, and bead fraction on proton relaxation and may be of use in mimicking hemosiderin in a phantom for estimating iron concentration.

Mathematics ability and disability is as heritable as other cognitive abilities and disabilities, however its genetic etiology has received relatively little attention. In our recent genome-wide association study of mathematical ability in 10-year-old children, 10 SNP associations were nominated from scans of pooled DNA and validated in an individually genotyped sample. In this paper, we use a 'SNP set' composite of these 10 SNPs to investigate gene-environment (GE) interaction, examining whether the association between the 10-SNP set and mathematical ability differs as a function of ten environmental measures in the home and school in a sample of 1888 children with complete data. We found two significant GE interactions for environmental measures in the home and the school both in the direction of the diathesis-stress type of GE interaction: The 10-SNP set was more strongly associated with mathematical ability in chaotic homes and when parents are negative.

Testis is the one and only location of spermatogenesis and sexual hormone production. Spermatogenesis is a complicated physiological process regulated by many genes specifically and differentially expressed in the testis. In this study, Transmembrane Protein 225 (TMEM225), which is specifically expressed in rat testis, has been identified. TMEM225 was cloned from the testis cDNA library and was mapped to chromosome 8q22 by browsing the University of California Santa Cruz genomic database. It contains an open reading frame with a length of 696 bp, encoding a protein with four putative transmembrane helices. TMEM225 mRNA expression was evaluated by reverse transcription-polymerase chain reaction and in situ hybridization. In addition, the subcellular location of TMEM225 was evaluated. The results obtained highlighted age related specific expression of TMEM225 in testis, specifically during the adult period after age of 13 months. In situ hybridization analysis indicated that TMEM225 mRNA was mainly expressed in spermatocyte cells and round spermatids. Green fluorescence protein localization analysis showed that rat TMEM225 mainly surrounded the nuclear membrane, with a minority distribution in the cytoplasm, and the distribution of TMEM225 was affected by the deletion of N-terminal transmembrane domain. As the expression phase is not related to the first wave of spermatozoon development, our data presented here suggest that TMEM225 may play an important role in sperm degeneration but not in spermatogenesis.

Immediately after spinal cord injury (SCI), a devastating paralysis results from the loss of brain stem and cortical innervation of spinal neurons that control movement, including a loss of serotonergic (5-HT) innervation of motoneurons. Over time, motoneurons recover from denervation and function autonomously, exhibiting large persistent calcium currents (Ca PICs) that both help with functional recovery and contribute to uncontrolled muscle spasms. Here we systematically evaluated which 5-HT receptor subtypes influence PICs and spasms after injury. Spasms were quantified by recording the long-lasting reflexes (LLRs) on ventral roots in response to dorsal root stimulation, in the chronic spinal rat, in vitro. Ca PICs were quantified by intracellular recording in synaptically isolated motoneurons. Application of agonists selective to 5-HT(2B) and 5-HT(2C) receptors (including BW723C86) significantly increased the LLRs and associated Ca PICs, whereas application of agonists to 5-HT(1), 5-HT(2A), 5-HT(3), or 5-HT(4/5/6/7) receptors (e.g., 8-OH-DPAT) did not. The 5-HT(2) receptor agonist-induced increases in LLRs were dose dependent, with doses for 50% effects (EC(50)) highly correlated with published doses for agonist receptor binding (K(i)) at 5-HT(2B) and 5-HT(2C) receptors. Application of selective antagonists to 5-HT(2B) (e.g., RS127445) and 5-HT(2C) (SB242084) receptors inhibited the agonist-induced increase in LLR. However, antagonists that are known to specifically be neutral antagonists at 5-HT(2B/C) receptors (e.g., RS127445) had no effect when given by themselves, indicating that these receptors were not activated by residual 5-HT in the spinal cord. In contrast, inverse agonists (such as SB206553) that block constitutive activity at 5-HT(2B) or 5-HT(2C) receptors markedly reduced the LLRs, indicating the presence of constitutive activity in these receptors. 5-HT(2B) or 5-HT(2C) receptors were confirmed to be on motoneurons by immunolabeling. In summary, 5-HT(2B) and 5-HT(2C) receptors on motoneurons become constitutively active after injury and ultimately contribute to recovery of motoneuron function and emergence of spasms.

As the genomic basis for Down syndrome (DS), human trisomy 21 is the most common genetic cause of intellectual disability in children and young people. The genomic regions on human chromosome 21 (Hsa21) are syntenic to three regions in the mouse genome, located on mouse chromosome 10 (Mmu10), Mmu16, and Mmu17. Recently, we have developed three new mouse models using chromosome engineering carrying the genotypes of Dp(10)1Yey/+, Dp(16)1Yey/+, or Dp(17)1Yey/+, which harbor a duplication spanning the entire Hsa21 syntenic region on Mmu10, Mmu16, or Mmu17, respectively. In this study, we analyzed the hippocampal long-term potentiation (LTP) and cognitive behaviors of these models. Our results show that, while the genotype of Dp(17)1Yey/+ results in abnormal hippocampal LTP, the genotype of Dp(16)1Yey/+ leads to both abnormal hippocampal LTP and impaired learning/memory. Therefore, these mutant mice can serve as powerful tools for further understanding the mechanism underlying cognitively relevant phenotypes associated with DS, particularly the impacts of different syntenic regions on these phenotypes.

Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways, i.e., macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In this review, we focus on CMA, a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. CMA participates in quality control to maintain normal cell functions by clearing "old" proteins and provides energy to cells under nutritional stress. Deregulation of CMA has recently been shown to underlie some diseases, especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor. Therefore, targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.

Blastomere fate and embryonic genome activation (EGA) during human embryonic development are unsolved areas of high scientific and clinical interest. Forty-nine blastomeres from 5- to 8-cell human embryos have been investigated following an efficient single-cell cDNA amplification protocol to provide a template for high-density microarray analysis. The previously described markers, characteristic of Inner Cell Mass (ICM) (n = 120), stemness (n = 190) and Trophectoderm (TE) (n = 45), were analyzed, and a housekeeping pattern of 46 genes was established. All the human blastomeres from the 5- to 8-cell stage embryo displayed a common gene expression pattern corresponding to ICM markers (e.g., DDX3, FOXD3, LEFTY1, MYC, NANOG, POU5F1), stemness (e.g., POU5F1, DNMT3B, GABRB3, SOX2, ZFP42, TERT), and TE markers (e.g., GATA6, EOMES, CDX2, LHCGR). The EGA profile was also investigated between the 5-6- and 8-cell stage embryos, and compared to the blastocyst stage. Known genes (n = 92) such as depleted maternal transcripts (e.g., CCNA1, CCNB1, DPPA2) and embryo-specific activation (e.g., POU5F1, CDH1, DPPA4), as well as novel genes, were confirmed. In summary, the global single-cell cDNA amplification microarray analysis of the 5- to 8-cell stage human embryos reveals that blastomere fate is not committed to ICM or TE. Finally, new EGA features in human embryogenesis are presented.

<h4>Background</h4>The involvement of iron in anthracycline cardiotoxicity is supported by extensive experimental data, and by the preventive efficacy of dexrazoxane, an iron chelator. However, no clinical evidence of anthracycline-induced cardiac iron accumulation is available and the influence of previous iron overload or of genetic factors in human-induced heart disease is largely unknown. Our aim was to test the hypothesis that anthracyclines increase iron heart concentration and that HFE genotype modulates this iron deposit.<h4>Methods</h4>We retrospectively evaluated cardiac events, cardiac iron and HFE genotype in 97 consecutive necropsies from patients with solid and hematological neoplasms. Heart and liver iron concentration was determined by atomic absorption spectroscopy. HFE gene mutations (C282Y and H63D) linked to hereditary hemochromatosis were analyzed by Fluorescence Resonance Energy Transfer (FRET) genotyping.<h4>Results</h4>Heart iron concentration was increased in cases treated with a cumulative doxorubicin dose greater than 200mg/m(2) (490 vs 240 μg/g; p=0.01), independently of liver iron load or transfusion history. HFE mutated haplotypes 282C/63D (p=0.049) and 282Y/63H (p=0.027) were associated to higher cardiac iron deposits. The haplotype C282Y-Y/H63D-H interacted with anthracyclines for increasing cardiac iron load. In a multivariate linear regression analysis both HFE genotypes and anthracyclines contributed to heart iron concentration (R(2)=0.284).<h4>Conclusions</h4>Our data support the occurrence of an HFE-modulated heart iron accumulation in individuals treated with anthracyclines, independently of systemic iron load. If prospectively confirmed, iron-related parameters might be useful as predictive factors for anthracycline cardiotoxicity.

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)-dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.

<h4>Background</h4>High-density lipoprotein (HDL) protects the artery wall by removing cholesterol from lipid-laden macrophages. However, recent evidence suggests that HDL might also inhibit atherogenesis by combating inflammation.<h4>Methods and results</h4>To identify potential antiinflammatory mechanisms, we challenged macrophages with lipopolysaccharide, an inflammatory microbial ligand for Toll-like receptor 4. HDL inhibited the expression of 30 (277 of 911) of the genes normally induced by lipopolysaccharide, microarray analysis revealed. One of its major targets was the type I interferon response pathway, a family of potent viral immunoregulators controlled by Toll-like receptor 4 and the TRAM/TRIF signaling pathway. Unexpectedly, the ability of HDL to inhibit gene expression was independent of macrophage cholesterol stores. Immunofluorescent studies suggested that HDL promoted TRAM translocation to intracellular compartments, which impaired subsequent signaling by Toll-like receptor 4 and TRIF. To examine the potential in vivo relevance of the pathway, we used mice deficient in apolipoprotein A-I, the major protein of HDL. After infection with Salmonella typhimurium, a Gram-negative bacterium that expresses lipopolysaccharide, apolipoprotein A-I-deficient mice had 6-fold higher plasma levels of interferon-β, a key regulator of the type I interferon response, than did wild-type mice.<h4>Conclusions</h4>HDL inhibits a subset of lipopolysaccharide-stimulated macrophage genes that regulate the type I interferon response, and its action is independent of sterol metabolism. These findings raise the possibility that regulation of macrophage genes by HDL might link innate immunity and cardioprotection.

Novel 8-substituted base and sugar-modified analogues of the Ca(2+) mobilizing second messenger cyclic adenosine 5'-diphosphate ribose (cADPR) were synthesized using a chemoenzymatic approach and evaluated for activity in sea urchin egg homogenate (SUH) and in Jurkat T-lymphocytes; conformational analysis investigated by (1)H NMR spectroscopy revealed that a C2'endo/syn conformation of the "southern" ribose is crucial for agonist or antagonist activity at the SUH-, but not at the T cell-cADPR receptor.

The synthetic retinoid 13-cis-retinoic acid (13-cis-RA), prescribed for the treatment of severe nodular acne, has been linked to an increased incidence of depression. Chronic treatment studies in rodents have shown that 13-cis-RA induces an increase in depression-related behaviours and a functional uncoupling of the hippocampus and dorsal raphe nucleus (DRN). Changes in the number of serotoninergic neurons in the DRN have been reported in depressed human patients. Given that retinoids have apoptotic effects, we hypothesized that a decrease in the number of serotoninergic neurons in the DRN or median raphe nucleus (MRN) would lead to decreased serotoninergic tone and in turn to the behavioural changes seen with 13-cis-RA administration. Here, we used immunolabelling and unbiased stereological methods to estimate the number of serotonin (5-hydroxytryptamine, 5-HT) neurons in the MRN and DRN of vehicle control and 13-cis-RA-treated adult mice. In the MRN, the number of 5-HT immunolabelled cells was 1815±194 in control, compared with 1954±111 in 13-cis-RA treated tissues. The number of 5-HT immunolabelled cells was much higher in the DRN, with 7148±377 cells in the control, compared with 7578±424 in the 13-cis-RA treated group. Further analysis of the DRN revealed that there were no changes in the number of 5-HT neurons within distinct subregions of the DRN. Similarly, changes in the density of serotoninergic neurons or in the volume of the MRN or DRN were not observed in 13-cis-RA treated animals. These data show that apoptotic actions of 13-cis-RA do not occur in vivo at drug concentrations that induce changes in depression-related behaviour and functional uncoupling of the DRN and hippocampus. The potential pro-depressant behavioural and molecular effects associated with chronic administration of 13-cis-RA may result from changes in serotoninergic activity rather than changes in the number of serotoninergic neurons.

Defects in cellular energy metabolism represent an early feature in a variety of human neurodegenerative diseases. Recent studies have shown that targeting energy metabolism can protect against neuronal cell death in such diseases. Here, we show that meclizine, a clinically used drug that we have recently shown to silence oxidative metabolism, suppresses apoptotic cell death in a murine cellular model of polyglutamine (polyQ) toxicity. We further show that this protective effect extends to neuronal dystrophy and cell death in Caenorhabditis elegans and Drosophila melanogaster models of polyQ toxicity. Meclizine's mechanism of action is not attributable to its anti-histaminergic or anti-muscarinic activity, but rather, strongly correlates with its ability to suppress mitochondrial respiration. Since meclizine is an approved drug that crosses the blood-brain barrier, it may hold therapeutic potential in the treatment of polyQ toxicity disorders, such as Huntington's disease.

The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.

<h4>Objective</h4>Psychiatric disorders such as depression, anxiety and alcohol dependence are associated with serotonin metabolism. We assessed the methylation level of the serotonin transporter (5-HTT) promoter region in control and alcohol dependent patients.<h4>Methods</h4>Twenty seven male patients who met the Diagnostic and Statistical Manual of Mental Disorder IV (DSM-IV) criteria for alcohol dependence were compared with fifteen controls. Polymerase chain reaction (PCR) assays of bisulfate-modified DNA were designed to amplify a part of the CpG island in the 5HTT gene. Pyrosequencing was performed and the methylation level at seven CpG island sites was measured.<h4>Results</h4>We found no differences in the methylation patterns of the serotonin transporter linked promoter region (5-HTTLPR) between alcohol-dependent and control subjects.<h4>Conclusion</h4>Our negative finding may be because 5-HTT epigenetic variation may not affect the expression for 5-HTT or there may be other methylation site critical for its expression. To find out more conclusive result, repeating the study in more methylation sites with a larger number of samples in a well-controlled setting is needed.

Nanoparticles are the new entities that can greatly limit the various side effects of systemic chemotherapy, and that coupled with a targeting moiety enables site-specific delivery of drugs. Folate receptors are overexpressed in retinoblastoma cells, thus these can specifically uptake the drug-loaded nanoparticles, thereby increasing the cytotoxicity at the tumor site. In our work, doxorubicin-loaded chitosan nanoparticles was prepared and then conjugated to folic acid. The conjugation efficiency was characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. Thereafter, the efficacy of FA-conjugated DOX-CNPs on retinoblastoma cells (Y-79) was analyzed by MTT assay which demonstrated superior cytotoxic effects as compared to unconjugated DOX-CNPs and native DOX. This may be due to enhanced intracellular uptake of DOX-CNPs-FA (30%) than that of DOX-CNPs (13.24%) and native DOX (5.01%), resulting from the high affinity of FA for folate receptors. Finally, the mechanism of doxorubicin-mediated apoptosis in retinoblastoma cell line (Y-79) was investigated which demonstrated that the mitochondrial pathway is activated and that the FA-conjugated DOX-CNPs are most effective and causes enhanced release of cytochrome <i>c</i> as well as the activation of downstream caspases to assist in apoptosis. Thus, the FA-targeted NPs were proved to possess sustainable, controlled, and targeted delivery of anticancer drugs with DOX as a model drug, which may provide a drug delivery system of precise control and targeting effect for the treatment of retinoblastoma.

A 19-year-old male with perinatally acquired HIV infection and AIDS (clinical stage 3) presented with a 9.1 × 5.0 cm hepatic mass. The resected mass was determined to be a hepatocellular carcinoma (HCC) without metastasis. The patient did not have active hepatitis B or C coinfection, as revealed by polymerase chain reaction (PCR), nor other risk factors for development of cirrhosis or HCC, and comprises only the second explicitly stated case of an HIV-positive individual developing HCC in the absence of concomitant hepatitis virus infection or other risk factors. This case illustrates the fact that as survival of perinatally infected individuals increases in the highly active antiretroviral therapy (HAART) era, new associations between HIV infection and other disease processes may be uncovered.

Genetic factors contribute to the development of digital osteoarthritis, whose heritability has been estimated at 48 to 65%. Among the manifestations of digital osteoarthritis, only Heberden's nodes are transmitted by Mendelian inheritance, as a dominant trait in women and a recessive trait in men. The other forms of digital osteoarthritis are multifactorial, with a major gene and a residual multifactorial component that probably interacts with environmental factors. Hindrances to molecular studies include the absence to date of a universally accepted definition of the phenotype and the late onset of the manifestations. Genetic association studies of selected class I and II HLA genes produced conflicting results. The T303M polymorphism of the MATN3 gene, which was initially described as associated with hand osteoarthritis, may be more closely linked to trapeziometacarpal osteoarthritis than to digital osteoarthritis. Genome-wide scans have identified numerous loci linked to digital osteoarthritis. Replication has been achieved for some of these loci, most notably those located at 2p, 2q, 3p, 4q, and 7p. A recently published genome-wide association study showed that an A2BP1 gene polymorphism was significantly associated with hand osteoarthritis. Many candidate-gene studies found associations with AGC1, ASPN, ENPP1, HFE, KL, VDR, IL-1 cluster, and IL-6, although the results were not consistently reproducible. In one study, women with hand osteoarthritis had significant telomere shortening. Telomere shortening has also been reported in other age-related conditions.

A number of studies have demonstrated that the common polymorphism in the serotonin transporter gene (5-HTT-LPR) moderates the increased risk for major depression seen in persons exposed to early adverse experiences. Several mouse models of this interaction have been recently established to investigate the increased vulnerability of individuals carrying the 5-HTT-LPR S allele to both early and adult life stressful events. Identifying the immediate effects of an adverse early environment on genetically susceptible individuals is critical to develop effective prevention of its long-term negative consequences of such an interaction. For this purpose we investigated molecular and neurochemical effects promoted by variable amount of maternal care in the brain of developing (postnatal day 10) wild type and heterozygous serotonin transporter knockout mice. Pups experiencing low level of maternal care showed increased levels of brain-derived neurotrophic factor (BDNF) messenger RNA within the hippocampus and primary somato-sensory cortex, and increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor binding in hippocampus in comparison with pups experiencing high level of maternal care. Strikingly, only heterozygous serotonin transporter knockout pups experiencing high maternal care showed increased hippocampal levels of serotonin and norepinephrine and decreased serotonin turnover compared to wild-type littermates. These findings support the hypothesis that maternal care affects the development of the hippocampus and primary somato-sensory cortex of individuals characterized by genetic variants of the serotonin transporter.

<h4>Objective</h4>These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells.<h4>Materials and methods</h4>DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis.<h4>Results</h4>Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor.<h4>Conclusions</h4>DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.

Healthy Living Partnerships to Prevent Diabetes (HELP PD) is a randomized controlled trial designed to translate the Diabetes Prevention Program (DPP) lifestyle intervention into a community setting using community health workers engaged through an existing Diabetes Care Center (DCC). Overweight and obese (BMI 25-40 kg/m²) individuals with pre-diabetes (fasting blood glucose 95-125 mg/dl) with no medical contraindications to participate in a lifestyle intervention were recruited for participation in this study. Standard recruitment strategies were employed, including mass mailing, direct provider referral, and community events. Participant recruitment and randomization for this trial began in 2007 and was concluded in 2009. 1818 screenings were conducted; of these, 326 (17.9%) qualified and 301 (16.6%) participants were randomized over a 21 month period. 23.8% of potential participants were excluded during the initial telephone screening, primarily for BMI and recent history of CVD. The majority of participants (220, 73.1%) reported mass mailing as their primary source of information about the study. Mass mailing was more effective with participants who identified themselves as white when compared to African-Americans. The cost of recruitment per randomized participant was $816, which includes direct costs and staff effort. 41% of the randomized participants were male and approximately 27% reported a race or ethnicity other than white. In comparison to the DPP study cohort, the HELP PD population is older, more educated and predominately white. These differences, reflecting in part the community in which HELP PD was conducted, may have implications for retention and adherence in the lifestyle intervention group.

Daily, systemic injections of a positive AMPA-type glutamate receptor modulator (ampakine) have been shown to reduce synaptic plasticity defects in rodent models of aging and early-stage Huntington's disease (HD). Here we report that long-term ampakine treatment markedly slows the progression of striatal neuropathology and locomotor dysfunction in the R6/2 HD mouse model. Remarkably, these effects were produced by an ampakine, CX929, with a short half-life. Injected once daily for 4-7 weeks, the compound increased protein levels of brain-derived neurotrophic factor (BDNF) in the neocortex and striatum of R6/2 but not wild-type mice. Moreover, ampakine treatments prevented the decrease in total striatal area, blocked the loss of striatal DARPP-32 immunoreactivity and reduced by 36% the size of intra-nuclear huntingtin aggregates in R6/2 striatum. The CX929 treatments also markedly improved motor performance of R6/2 mice on several measures (rotarod, vertical pole descent) but did not influence body weight or lifespan. These findings describe a minimally invasive, pharmacologically plausible strategy for treatment of HD and, potentially, other neuropathological diseases.

Homoharringtonine (HHT) is a plant alkaloid that inhibits the elongation phase of translation that is currently in clinical trials. Because the intrinsically short-lived antiapoptotic protein myeloid cell leukemia-1 (Mcl-1) has been reported to support the survival of chronic lymphocytic leukemia (CLL) cells, we hypothesized that inhibition of protein synthesis by HHT would decrease Mcl-1 expression and induce apoptosis in CLL. In primary CLL cells, HHT induced significant apoptosis independent of the prognostic characteristics of the patients. This was associated with inhibition of translation and decreased Mcl-1 levels in CLL cells. Mcl-1 reduction was evident as early as 2 hours and continued to decrease in the next 6-8 hours, whereas cell death started in 2 hours and continued to increase for 24 hours. Reduction of the Mcl-1 level was due to translation inhibition and proteasome degradation rather than to transcription inhibition or caspase cleavage. HHT and the transcription inhibitor SNS-032 induced synergistic cell killing. Although stromal cells induced Mcl-1 expression and protected CLL cells from the toxicity of fludarabine, this induction was reversed by HHT, which overcame stromal cell-mediated protection. Thus, these results provide a rationale for clinical development of HHT in CLL as single agent or in combinations.

Longstanding results with calorie and growth factor restriction plus recent results with the first interventional drug suggest that retarding the pace of aging to improve the quality of life of older people is at hand. The biological system targeted by these approaches is the target of rapamycin (TOR), which is central for cellular responses to a variety of stimuli including stressors, growth factors, and nutrients and energy states. That the life-extending response to reducing its activity is highly conserved from yeast to mammals is consistent with the evolution of aging as a strategy to preserve reproductive potential of young cells and animals.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

<h4>Background</h4>According to mRNA microarray, proteomics and other studies, biological abnormalities of eutopic endometrium (EM) are involved in the pathogenesis of endometriosis, but the relationship between mRNA and protein expression in EM is not clear. We tested for the first time the hypothesis that EM TRIzol extraction allows proteomic Surface Enhanced Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry (SELDI-TOF MS) analysis and that these proteomic data can be related to mRNA (microarray) data obtained from the same EM sample from women with and without endometriosis.<h4>Methods</h4>Proteomic analysis was performed using SELDI-TOF-MS of TRIzol-extracted EM obtained during secretory phase from patients without endometriosis (n = 6), patients with minimal-mild (n = 5) and with moderate-severe endometriosis (n = 5), classified according to the system of the American Society of Reproductive Medicine. Proteomic data were compared to mRNA microarray data obtained from the same EM samples.<h4>Results</h4>In our SELDI-TOF MS study 32 peaks were differentially expressed in endometrium of all women with endometriosis (stages I-IV) compared with all controls during the secretory phase. Comparison of proteomic results with those from microarray revealed no corresponding genes/proteins.<h4>Conclusion</h4>TRIzol treatment of secretory phase EM allows combined proteomic and mRNA microarray analysis of the same sample, but comparison between proteomic and microarray data was not evident, probably due to post-translational modifications.

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.

<h4>Background and purpose</h4>Atherosclerosis is a complex subclinical cardiovascular disorder with a substantial genetic component. This study sought to identify genetic loci influencing carotid plaque in 2 independent samples.<h4>Methods</h4>B-mode ultrasound was performed to determine the presence and area of carotid plaque. Variance components analysis was used to test for linkage using 383 autosomal microsatellite markers in 1308 subjects from 100 Dominican families. Multiple linear and logistic regression models were used to investigate the association between plaque traits and 18,904 single nucleotide polymorphisms under the 1-logarithm of odds unit down regions of linkage peaks in an independent community-based data set (N = 941, 41% Dominicans) from the Northern Manhattan Study.<h4>Results</h4>After adjustment for age, hypertension, diabetes mellitus, cigarette pack-years, body mass index, and waist-to-hip ratio, significant heritability was detected for plaque presence (h² = 0.50 ± 0.14, P < 0.0001) and plaque area (h²=0.17 ± 0.04, P < 0.0001). Quantitative and dichotomous trait linkage analyses obtained similar results and identified 4 regions with multipoint logarithm of odds scores ≥ 2.00 on 7q36, 11p15, 14q32, and 15q23. In the association analysis of the 4 linkage peaks, several single nucleotide polymorphisms in or near SOX6, FSD2, AP3S2, EFTUD1, and MYOD1 were associated with carotid plaque traits with a nominal P ≤ 0.0005 in the Northern Manhattan Study data set and with a P ≤ 0.01 in Northern Manhattan Study Dominican subset.<h4>Conclusions</h4>Carotid plaque has considerable heritability and may be influenced by loci on chromosomes 11p15, 14q32, and 15q23. The SOX6 gene within the bone morphogenic protein pathway could be a candidate for carotid plaque. Larger independent studies are needed to validate these findings.

Increased values of multiple adiposity-related anthropometric traits are important risk factors for many common complex diseases. We performed a genome-wide association (GWA) study for four quantitative traits related to body size and adiposity (BMI, weight, waist circumference, and height) in a cohort of 1,792 adult Filipino women from the Cebu Longitudinal Health and Nutrition Survey (CLHNS). This is the first GWA study of anthropometric traits in Filipinos, a population experiencing a rapid transition into a more obesogenic environment. In addition to identifying suggestive evidence of additional single-nucleotide polymorphism (SNP) association signals (P < 10(-5)), we replicated (P < 0.05, same direction of additive effect) associations previously reported in European populations of both BMI and weight with MC4R and FTO, of BMI with BDNF, and of height with EFEMP1, ZBTB38, and NPPC, but none with waist circumference. We also replicated loci reported in Japanese or Korean populations as associated with BMI (OTOL1) and height (HIST1H1PS2, C14orf145, GPC5). A difference in local linkage disequilibrium (LD) between European and Asian populations suggests a narrowed association region for BDNF, while still including a proposed functional nonsynonymous amino acid substitution variant (rs6265, Val66Met). Finally, we observed significant evidence (P < 0.0042) for age-by-genotype interactions influencing BMI for rs17782313 (MC4R) and rs9939609 (FTO), and for a study year-by-genotype interaction for rs4923461 (BDNF). Our results show that several genetic risk factors are associated with anthropometric traits in Filipinos and provide further insight into the effects of BDNF, FTO, and MC4R on BMI.

In human mitochondria, polyadenylation of mRNA, undertaken by the nuclear-encoded mitochondrial poly(A) RNA polymerase, is essential for maintaining mitochondrial gene expression. Our molecular investigation of an autosomal-recessive spastic ataxia with optic atrophy, present among the Old Order Amish, identified a mutation of MTPAP associated with the disease phenotype. When subjected to poly(A) tail-length assays, mitochondrial mRNAs from affected individuals were shown to have severely truncated poly(A) tails. Although defective mitochondrial DNA maintenance underlies a well-described group of clinical disorders, our findings reveal a defect of mitochondrial mRNA maturation associated with human disease and imply that this disease mechanism should be considered in other complex neurodegenerative disorders.

Association studies of over 1 million SNPs capturing most of the human genome common variation became possible thanks to the information provided by the HapMap International project and the development of high-throughput genotyping technologies at accessible prices. Genome-wide scans analyzing thousands of individuals have now identified most if not all of the major genes involved in susceptibility for several systemic autoimmune diseases. In particular, results for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc) are reviewed here. While most genes are shared between diseases, few seem to be unique reflecting that we still are long before knowing all genes, their interactions with other genes and the environment and their impact on biological functions.

<h4>Background & aims</h4>The use of low-molecular-weight heparins (LMWH) in patients with advanced liver diseases is frequently avoided because of the enhanced risk of bleeding complications. However, many patients with impaired liver function are at a high risk of thrombosis or have an indication for therapeutic anticoagulation. Therefore, the aim of this study was to evaluate the pharmacokinetics of LMWH in patients with cirrhosis.<h4>Methods</h4>Eighty-four consecutive patients with cirrhosis and a clinical indication for prophylactic or therapeutic anticoagulation were included. The LMWH doses were chosen according to current guidelines. Antifactor Xa activity (anti-Xa) was assessed on two consecutive days, 4 h after drug administration. The severity of liver disease was quantified using Child-Turcotte-Pugh score, the MELD score and clinical features and was correlated with the anti-Xa value and the occurrence of complications.<h4>Results</h4>Antifactor Xa activity was negatively correlated with the severity of the liver disease, and a positive correlation was observed between antithrombin-III (AT) levels and anti-Xa value. AT itself was negatively correlated with the severity of liver disease. Seven patients had an episode of variceal bleeding. No patient died during the observation interval and no thromboembolic events occurred.<h4>Conclusion</h4>Prophylactic use of LMWH in patients with cirrhosis appears to be safe. A decreased anti-Xa value in cirrhotic patients and a negative correlation with liver function challenge the unconditional use of anti-Xa assays in LMWH monitoring in cirrhotic patients and reveals a potential limitation of anti-Xa analysis in these patients. Low levels of AT, because of reduced hepatic synthesis, are the most likely cause of this phenomenon.

<h4>Background</h4>Since human brain tissue is often unavailable for transcriptional profiling studies, blood expression data is frequently used as a substitute. The underlying hypothesis in such studies is that genes expressed in brain tissue leave a transcriptional footprint in blood. We tested this hypothesis by relating three human brain expression data sets (from cortex, cerebellum and caudate nucleus) to two large human blood expression data sets (comprised of 1463 individuals).<h4>Results</h4>We found mean expression levels were weakly correlated between the brain and blood data (r range: [0.24,0.32]). Further, we tested whether co-expression relationships were preserved between the three brain regions and blood. Only a handful of brain co-expression modules showed strong evidence of preservation and these modules could be combined into a single large blood module. We also identified highly connected intramodular "hub" genes inside preserved modules. These preserved intramodular hub genes had the following properties: first, their expression levels tended to be significantly more heritable than those from non-preserved intramodular hub genes (p < 10⁻⁹⁰); second, they had highly significant positive correlations with the following cluster of differentiation genes: CD58, CD47, CD48, CD53 and CD164; third, a significant number of them were known to be involved in infection mechanisms, post-transcriptional and post-translational modification and other basic processes.<h4>Conclusions</h4>Overall, we find transcriptome organization is poorly preserved between brain and blood. However, the subset of preserved co-expression relationships characterized here may aid future efforts to identify blood biomarkers for neurological and neuropsychiatric diseases when brain tissue samples are unavailable.

<h4>Background</h4>Various hemostatic markers are associated with the risk of cardiovascular disease; however, limited information exists on their relationship with the occurrence and prognosis of atrial fibrillation (AF).<h4>Objectives</h4>To assess whether hemostatic markers are associated with the incidence and prognosis of AF.<h4>Methods</h4>We studied 14,858 men and women in the Atherosclerosis Risk in Communities cohort, aged 45-64 and free of AF at baseline (1987-1989). Fibrinogen, von Willebrand factor (vWf), factor VII activity (VIIc), factor VIII activity (VIIIc), protein C, antithrombin III (ATIII), and activated partial thromboplastin time (aPTT) were measured in blood samples at baseline. AF and other cardiovascular outcomes through 2005 were determined following standardized protocols.<h4>Results</h4>During a median follow-up of 16.8 years, 1209 cases of AF were identified. In multivariable Cox models, the hazard ratios (HR) and 95% confidence intervals (CI) of incident AF associated with a 1-standard deviation (SD) increase in each marker were 1.13 (1.07-1.20) for fibrinogen, 1.17 (1.11-1.23) for vWf, 1.17 (1.11-1.24) for factor VIIIc, 0.93 (0.88-1.00) for factor VIIc, 0.98 (0.92-1.04) for protein C, 1.00 (0.94-1.06) for aPTT and 1.00 (0.95-1.06) for ATIII. Greater factor VIIIc, fibrinogen and vWf were consistently associated with a higher risk of cardiovascular outcomes and mortality in those with and without incident AF, while greater protein C was associated with a lower risk of ischemic stroke.<h4>Conclusion</h4>Several hemostatic markers are associated with the incidence of AF independently of other cardiovascular risk factors. Their role in the risk stratification of AF patients should be further studied.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic tumor resulting from the malignant transformation of immature T-cell progenitors. Originally associated with a dismal prognosis, the outcome of T-ALL patients has improved remarkably over the last two decades as a result of the introduction of intensified chemotherapy protocols. However, these treatments are associated with significant acute and long-term toxicities, and the treatment of patients presenting with primary resistant disease or those relapsing after a transient response remains challenging. T-ALL is a genetically heterogeneous disease in which numerous chromosomal and genetic alterations cooperate to promote the aberrant proliferation and survival of leukemic lymphoblasts. However, the identification of activating mutations in the NOTCH1 gene in over 50% of T-ALL cases has come to define aberrant NOTCH signaling as a central player in this disease. Therefore, the NOTCH pathway represents an important potential therapeutic target. In this review, we will update our current understanding of the molecular basis of T-ALL, with a particular focus on the role of the NOTCH1 oncogene and the development of anti-NOTCH1 targeted therapies for the treatment of this disease.

Oxidative stress causes damage to multiple cellular components such as DNA, proteins, and lipids, and is implicated in various human diseases including cancer, neurodegeneration, inflammatory diseases, and aging. In response to oxidative attack, cells have developed an antioxidant defense system to maintain cellular redox homeostasis and to protect cells from damage. The thiol-containing small molecules (e.g. glutathione), reactive oxygen species-inactivating enzymes (e.g. glutathione peroxidase), and phase 2 detoxifying enzymes (e.g. NAD(P)H: quinine oxidoreductase 1 and glutathione-S-transferases) are members of this antioxidant system. NF-E2-related factor 2 (Nrf2) is a CNC-bZIP transcription factor which regulates the basal and inducible expression of a wide array of antioxidant genes. Following dissociation from the cytosolic protein Keap1, a scaffolding protein which binds Nrf2 and Cul3 ubiquitin ligase for proteasome degradation, Nrf2 rapidly accumulates in the nucleus and transactivates the antioxidant response element in the promoter region of many antioxidant genes. The critical role of Nrf2 has been demonstrated by various animal studies showing that mice with a targeted disruption of the nrf2 gene are prone to develop lesions in response to environmental toxicants/carcinogens, drugs, and inflammatory insults. In this review, we discuss the role of the Nrf2 system, with particular focus on Nrf2-controlled target genes and the potential pleiotropic effects of Nrf2 activation of indirect antioxidants.

<h4>Objective</h4>To provide an overview of the genetic epidemiology of substance use and misuse in adolescents.<h4>Method</h4>A selective review of genetically informative research strategies, their limitations, and key findings examining issues related to the heritability of substance use and substance use disorders in children and adolescents is presented.<h4>Results</h4>Adoption, twin, and extended-family designs have established there is a strong heritable component to liability to nicotine, alcohol, and illicit drug dependence in adults. However, shared environmental influences are relatively stronger in youth samples and at earlier stages of substance involvement (e.g., use). There is considerable overlap in the genetic influences associated with the abuse/dependence across drug classes, and shared genetic influences contribute to the commonly observed associations between substance-use disorders and externalizing and, to a lesser extent, internalizing psychopathology. Rapid technologic advances have made the identification of specific gene variants that influence risks for substance-use disorders feasible, and linkage and association (including genomewide association studies) have identified promising candidate genes implicated in the development of substance-use disorders.<h4>Conclusions</h4>Studies using genetically informative research designs, including those that examine aggregate genetic factors and those examining specific gene variants, individually and in interaction with environmental influences, offer promising avenues not only for delineating genetic effects on substance-use disorders but also for understanding the unfolding of risk across development and the interaction between environmental and genetic factors in the etiology of these disorders.

Modulation of serotonin transporter (5-HTT) function causes changes in affective behavior, both in humans and rodents. Stressful life events likewise affect emotional behavior. In humans, a low-expressing genetic 5-htt variant, the s allele of the 5-htt linked promoter region, has been associated with increased risk for depression only where there was a history of stressful life events. To investigate this gene by environment interaction in mice, we compared the effects of inescapable shocks on the behavior of wild-type (5-htt+/+), heterozygote (5-htt+/-) and serotonin transporter deficient (5-htt-/-) mice. Inescapable shocks induce behavioral changes including a shock escape deficit, in a subsequent test when escape is possible. Confirming a gene by environment interaction, we found that stress increases escape latencies in a gene-dose dependent manner (5-htt-/->5-htt+/->5-htt +/+), where as there were no differences among the genotypes in the unstressed condition. The vulnerability to increased escape latency could not be accounted for by enhanced fear learning, as 5-htt-/- mice did not show heightened fear conditioning. The interaction of 5-htt genotype and stress appeared to produce a selective behavioral vulnerability, because no interaction of 5-htt genotype and stress was observed in other measures of anxiety and depression-linked behavior, including the open field, novelty suppressed feeding, and forced swim tests. We replicated prior findings that the 5-htt-/- displays heightened anxiety and depression-like behavior at baseline (unstressed condition). In conclusion, our data offer the possibility for future investigation of the neural basis underlying 5-htt genotype-by-stress interaction shown here.

Investigation of biochemical cues in isolation or in combinations in cell culture systems is crucial for unraveling the mechanisms that govern neural development and repair. The most widely used experimental paradigms that elicit axon guidance in vitro utilize as the source of the gradient a pulsatile pipette, transfected cells, or a loaded gel, producing time-varying gradients of poor reproducibility which are not well suited for studying slow-growing mammalian cells. Although microfluidic device design have allowed for generating stable, complex gradients of diffusible molecules, the flow-induced shear forces in a microchannel has made it impossible to maintain viable mammalian neuronal cultures for sufficiently long times. In this paper, we describe axonal responses of mouse cortical neurons in a "neuron-benign" gradient-generator device based on an open chamber that can establish highly stable gradients of diffusible molecules for at least 6 h with negligible shear stress, and also allows the neurons to thrive for at least 2 weeks. Except for the period when the gradient is on, the cells in the gradient are under the same conditions as the cells on the control surfaces, which ensure a consistent set of micro-environmental variables. The gradient stability and uniformity over the cell culture surface achieved by the device, together with our software platform for acquiring, post-processing and quantitatively analyzing the large number of images allowed us to extract valuable information even from small datasets. We report a directed response of primary mammalian neurons (from E14 embryonic mice cortex) to a diffusible gradient of netrin in vitro. We infer from our studies that a large majority (∼73%) of the neurons that extend axons during the gradient application grow towards the netrin source, and our data analysis also indicates that netrin acts as a growth factor for this same population of neurons.

<h4>Background</h4>Most cases of ovarian cancer are epithelial in origin and diagnosed at advanced stage when the cancer is widely disseminated in the peritoneal cavity. The objective of this study was to establish an immunocompetent syngeneic mouse model of disseminated epithelial ovarian cancer (EOC) to facilitate laboratory-based studies of ovarian tumor biology and preclinical therapeutic strategies.<h4>Methods</h4>Individual lines of TgMISIIR-TAg transgenic mice were phenotypically characterized and backcrossed to inbred C57BL/6 mice. In addition to a previously described line of EOC-prone mice, two lines (TgMISIIR-TAg-Low) were isolated that express the oncogenic transgene, but have little or no susceptibility to tumor development. Independent murine ovarian carcinoma (MOVCAR) cell lines were established from the ascites of tumor-bearing C57BL/6 TgMISIIR-TAg transgenic mice, characterized and tested for engraftment in the following recipient mice: 1) severe immunocompromised immunodeficient (SCID), 2) wild type C57BL/6, 3) oophorectomized tumor-prone C57BL/6 TgMISIIR-TAg transgenic and 4) non-tumor prone C57BL/6 TgMISIIR-TAg-Low transgenic. Lastly, MOVCAR cells transduced with a luciferase reporter were implanted in TgMISIIR-TAg-Low mice and in vivo tumor growth monitored by non-invasive optical imaging.<h4>Results</h4>Engraftment of MOVCAR cells by i.p. injection resulted in the development of disseminated peritoneal carcinomatosis in SCID, but not wild type C57BL/6 mice. Oophorectomized tumor-prone TgMISIIR-TAg mice developed peritoneal carcinomas with high frequency, rendering them unsuitable as allograft recipients. Orthotopic or pseudo-orthotopic implantation of MOVCAR cells in TgMISIIR-TAg-Low mice resulted in the development of disseminated peritoneal tumors, frequently accompanied by the production of malignant ascites. Tumors arising in the engrafted mice bore histopathological resemblance to human high-grade serous EOC and exhibited a similar pattern of peritoneal disease spread.<h4>Conclusions</h4>A syngeneic mouse model of human EOC was created by pseudo-orthotopic and orthotopic implantation of MOVCAR cells in a susceptible inbred transgenic host. This immunocompetent syngeneic mouse model presents a flexible system that can be used to study the consequences of altered gene expression (e.g., by ectopic expression or RNA interference strategies) in an established MOVCAR tumor cell line within the ovarian tumor microenvironment and for the development and analysis of preclinical therapeutic agents including EOC vaccines and immunotherapeutic agents.

<h4>Background</h4>RAE-1 is a ligand of the activating receptor NKG2D expressed by NK cells, NKT, γδT and some CD8(+)T lymphocytes. RAE-1 is overexpressed in tumor cell lines and its expression is induced after viral infection and genotoxic stress. We have recently demonstrated that RAE-1 is expressed in the adult subventricular zone (SVZ) from C57BL/6 mice. RAE-1 is also expressed in vitro by neural stem/progenitor cells (NSPCs) and plays a non-immune role in cell proliferation. The C57BL/6 mouse genome contains two rae-1 genes, rae-1δ and rae-1ε encoding two different proteins. The goals of this study are first to characterize the in vivo and in vitro expression of each gene and secondly to elucidate the mechanisms underlying their respective expression, which are far from known.<h4>Principal findings</h4>We observed that Rae-1δ and Rae-1ε transcripts are differentially expressed according to tissues, pathological conditions and cell lines. Embryonic tissue and the adult SVZ mainly expressed Rae-1δ transcripts. The NSPCs derived from the SVZ also mainly expressed RAE-1δ. The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε. On the contrary, cell lines expressed either similar levels of RAE-1δ and RAE-1ε proteins or only RAE-1ε. Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression. We indeed identified two different promoter regions for each gene: one mainly involved in the control of rae-1δ gene expression and the other in the control of rae-1ε expression.<h4>Conclusions/significance</h4>RAE-1δ and RAE-1ε differ with respect to their function and the control of their expression. Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

<h4>Background</h4>Prostate cancer is initially dependent on androgens for survival and growth, making hormonal therapy the cornerstone treatment for late-stage tumors. However, despite initial remission, the cancer will inevitably recur. The present study was designed to investigate how androgen-dependent prostate cancer cells eventually survive and resume growth under androgen-deprived and antiandrogen supplemented conditions. As model system, we used the androgen-responsive PC346C cell line and its therapy-resistant sublines: PC346DCC, PC346Flu1 and PC346Flu2.<h4>Methodology/principal findings</h4>Microarray technology was used to analyze differences in gene expression between the androgen-responsive and therapy-resistant PC346 cell lines. Microarray analysis revealed 487 transcripts differentially-expressed between the androgen-responsive and the therapy-resistant cell lines. Most of these genes were common to all three therapy-resistant sublines and only a minority (∼5%) was androgen-regulated. Pathway analysis revealed enrichment in functions involving cellular movement, cell growth and cell death, as well as association with cancer and reproductive system disease. PC346DCC expressed residual levels of androgen receptor (AR) and showed significant down-regulation of androgen-regulated genes (p-value = 10(-7)). Up-regulation of VAV3 and TWIST1 oncogenes and repression of the DKK3 tumor-suppressor was observed in PC346DCC, suggesting a potential AR bypass mechanism. Subsequent validation of these three genes in patient samples confirmed that expression was deregulated during prostate cancer progression.<h4>Conclusions/significance</h4>Therapy-resistant growth may result from adaptations in the AR pathway, but androgen-independence may also be achieved by alternative survival mechanisms. Here we identified TWIST1, VAV3 and DKK3 as potential players in the bypassing of the AR pathway, making them good candidates as biomarkers and novel therapeutical targets.

Growing axons encounter numerous developmental signals to which they must promptly respond in order to properly form complex neural circuitry. In the axons, these signals are often transduced into a local increase or decrease in protein levels. Contrary to the traditional view that the cell bodies are the exclusive source of axonal proteins, it is becoming increasingly clear not only that de novo protein synthesis takes place in axons, but also that it is required for the axons to respond to certain signals. Here we review the current knowledge of local mRNA translation in developing neurons with a special focus on protein synthesis occurring in axons and growth cones.

<h4>Objective</h4>Hereditary hemochromatosis has an autosomal recessive inheritance. The C282Y homozygosity is the most common genetic mutation in persons of Northern European descent. A screen of two multiethnic populations in Northern Norway was performed to investigate whether the prevalence of hereditary hemochromatosis was consistent with previous results in Northern Europe.<h4>Material and methods</h4>Participants in two population-based studies in Northern Norway were analyzed for serum ferritin (s-ferritin) and transferrin saturation. Participants with s-ferritin or transferrin saturation above the reference limits in two separate blood samples were tested for three different HFE mutations, namely C282Y, H63D and S65.<h4>Results</h4>The estimated prevalence of the C282Y/C282Y mutation in the two municipalities studied was lower than in comparable studies in Norway. The prevalence was the lowest in the Sør-Varanger population (men 0.19% and women 0.22%), which also had the highest proportion of individuals with Sami and Kven affiliation. In Tromsø, the prevalence was consistent with previous results in Norway.<h4>Conclusions</h4>The prevalence of hereditary hemochromatosis is lower in multiethnic populations in Northern Norway than in previous studies from other parts of Norway.

<h4>Background</h4>Numerous studies reported genomic alterations in colorectal human tumors but few focused on rectal tumors with the specification of preoperative-treated or untreated tumors. The goals of this study were to list chromosome allelic imbalances and correlate their frequency with tumor progression and to identify potential molecular markers of progression in rectal chromosomally unstable tumors without preoperative treatment.<h4>Methods</h4>Genomic alterations of 57 rectal tumors assessed by allelotyping targeting 33 chromosomal loci, were clusterised and compared to those of 151 left colon tumors.<h4>Results</h4>Clustering separated the rectal tumors without preoperative treatment into three subtypes according to the allelic imbalance frequency and genomic alteration associations. The tumors without preoperative treatment displayed a significantly higher allelic imbalance frequency (54%) than the tumors with preoperative treatment (33%), suggesting that treatment could target highly altered tumor clones. Interestingly, the survival analysis identified three potential prognostic molecular survival markers, D1S197, D5S430, and D14S65, for tumors without preoperative treatment.<h4>Conclusion</h4>Based on the genomic status of 33 chromosomal loci, we observed that rectal tumors without preoperative treatment segregate according to the global allelic imbalance frequency but without correlation to the tumor progression. Moreover, the detailed associations of alterations in rectal tumors are different from those described in colon tumors suggesting that rectal and left tumors should be considered as separate entities. Finally, potential prognostic genomic molecular markers for survival are proposed which status could specify the clinical course of the tumors.

As a central regulator of iron metabolism, hepcidin inhibits dietary iron absorption and macrophage iron recycling. Its expression is regulated by multiple factors including iron availability and erythropoietic activity. To investigate the role of transferrin (Tf) in the regulation of hepcidin expression by these factors in vivo, we employed the hypotransferrinemic (hpx) mouse. These Tf-deficient mice have severe microcytic anemia, tissue iron overload, and hepcidin deficiency. To determine the relationship of Tf levels and erythropoiesis to hepcidin expression, we subjected hpx mutant and control mice to a number of experimental manipulations. Treatment of hpx mice with Tf injections corrected their anemia and restored hepcidin expression. To investigate the effect of erythropoiesis on hepcidin expression, we suppressed erythropoiesis with blood transfusions or myeloablation with chemotherapeutic drugs. Transfusion of hpx animals with wild-type red blood cells led to increased hepcidin expression, while hepcidin expression in myeloablated hpx mice increased only if Tf was administered postablation. These results suggest that hepcidin expression in hpx mice is regulated both by Tf-restricted erythropoiesis and by Tf through a mechanism independent of its role in erythropoiesis.

Epidemiological studies have correlated arsenic exposure with cancer, skin diseases, and adverse developmental outcomes such as spontaneous abortions, neonatal mortality, low birth weight, and delays in the use of musculature. The current study used C2C12 mouse myoblast cells to examine whether low concentrations of arsenic could alter their differentiation into myotubes, indicating that arsenic can act as a developmental toxicant. Myoblast cells were exposed to 20 nM sodium arsenite, allowed to differentiate into myotubes, and expression of the muscle-specific transcription factor myogenin, along with the expression of tropomyosin, suppressor of cytokine signaling 3 (Socs3), prostaglandin I2 synthesis (Ptgis), and myocyte enhancer 2 (Mef2), was investigated using QPCR and immunofluorescence. Exposing C2C12 cells to 20 nM sodium arsenite delayed the differentiation process, as evidenced by a significant reduction in the number of multinucleated myotubes, a decrease in myogenin mRNA expression, and a decrease in the total number of nuclei expressing myogenin protein. The expression of mRNA involved in myotube formation, such as Ptgis and Mef2 mRNA, was also significantly reduced by 1.6-fold and 4-fold during differentiation. This was confirmed by immunofluorescence for Mef2, which showed a 2.6-fold reduction in nuclear translocation. Changes in methylation patterns in the promoter region of myogenin (-473 to +90) were examined by methylation-specific PCR and bisulfite genomic sequencing. Hypermethylated CpGs were found at -236 and -126 bp, whereas hypomethylated CpGs were found at -207 bp in arsenic-exposed cells. This study indicates that 20 nM sodium arsenite can alter myoblast differentiation by reducing the expression of the transcription factors myogenin and Mef2c, which is likely due to changes in promoter methylation patterns. The delay in muscle differentiation may lead to developmental abnormalities.

We designed to determine whether pretreatment of allografts with methoxypolyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and an anti-OX40L monoclonal antibody (McAb) can relieve acute graft-versus-host disease in allogeneic bone marrow transplantation recipients. Responder splenocytes from C57BL/6 donor mice were incubated with stimulator splenocytes from BALB/c recipient mice for 7 days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA modification. Donor BM cells plus mixed culture T cells were then transplanted into myeloablatively irradiated BALB/c mice. The signs of GVHD were less evident in mice of groups B (mPEG-SPA modification group), C (anti-OX40L McAb pretreated group) and D (dual-treated group), with average survival durations all longer than those in group A (non-treated BMT group) (P < 0.05). The survival rates on day 60 post-BMT in groups B, C and D were 50, 41.7 and 66.7%, respectively. After BMT, serum IL-4 and IL-10 levels elevated in groups B, C (P < 0.05) and even more significantly increased in group D (P < 0.01), while serum IFN-gamma levels decreased in these three groups (P < 0.01). In conclusion, the combination of mPEG-SPA and anti-OX40L McAb can block T cell-activated antigens, co-stimulatory pathways and induce the immune shift of Th cells toward Th2 cells; their effects in ameliorating GVHD are synergistic.

The capacity to act as an electron donor and acceptor makes iron an essential cofactor of many vital processes. Its balance in the body has to be tightly regulated since its excess can be harmful by favouring oxidative damage, while its deficiency can impair fundamental activities like erythropoiesis. In the brain, an accumulation of iron or an increase in its availability has been associated with the development and/or progression of different degenerative processes, including Parkinson's disease, while iron paucity seems to be associated with cognitive deficits, motor dysfunction, and restless legs syndrome. In the search of DNA sequence variations affecting the individual predisposition to develop movement disorders, we scanned by DHPLC the exons and intronic boundary regions of ceruloplasmin, iron regulatory protein 2, hemopexin, hepcidin and hemojuvelin genes in cohorts of subjects affected by Parkinson's disease and idiopathic neurodegeneration with brain iron accumulation (NBIA). Both novel and known sequence variations were identified in most of the genes, but none of them seemed to be significantly associated to the movement diseases of interest.

<h4>Background</h4>During 2008, Singapore experienced its largest ever outbreak of hand, foot and mouth disease (HFMD), resulting in 29686 cases, including four cases of encephalitis and one fatality.<h4>Methods</h4>A total of 51 clinical specimens from 43 patients with suspected HFMD at the National University Hospital, Singapore were collected for virus isolation and identification by reverse transcription polymerase chain reaction (RT-PCR) and sequencing.<h4>Results</h4>Enteroviruses were identified in 34 samples (66.7%), with 11 samples (21.6%) being positive for enterovirus 71 (EV71). Other non-EV71 enteroviruses (including coxsackievirus A4, A6, A10, and A16) were identified in 23 samples (45.1%). The most prevalent virus serotypes were CA6, CA10, and EV71. CA6 and CA10 accounted for 35.3% of all HFMD cases, which may explain the high transmissibility and low fatality that characterized this unprecedented epidemic associated with relatively mild disease. Phylogenetic analyses of 10 circulating EV71 strains indicated that they belonged to two subgenogroups, i.e., B5 (80%) and C2 (20%). The VP1 sequences of the 2008 EV71 strains also exhibited continuous mutations during the outbreak, reflecting the relatively high mutation rate of the EV71 capsid protein, which may have implications for future vaccine development.<h4>Conclusions</h4>A safe and effective vaccine against EV71 is certainly warranted in view of its potential neurovirulence and its role in HFMD epidemics of recurring frequency with resultant fatalities in Asia, as well as other parts of the world.

Palate, lung and nasal epithelial clone (PLUNC) proteins are structural homologues to the innate defence molecules LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). PLUNCs make up the largest portion of the wider BPI/LBP/PLUNC-like protein family and are amongst the most rapidly evolving mammalian genes. In this study we systematically identified and characterised BPI/LBP/PLUNC-like protein-encoding genes in the chicken genome. We identified eleven complete genes (and a pseudogene). Five of them are clustered on a >50 kb locus on chromosome 20, immediately adjacent to BPI. In addition to BPI, we have identified presumptive orthologues LPLUNCs 2, 3, 4 and 6, and BPIL-2. We find no evidence for the existence of single domain containing proteins in birds. Strikingly our analysis also suggests that there is no LBP orthologue in chicken. This observation may in part account for the relative resistance to LPS toxicity observed in birds. Our results indicate significant differences between the avian and mammalian repertoires of BPI/LBP/PLUNC-like genes at the genomic and transcriptional levels and provide a framework for further functional analyses of this gene family in chickens.

<h4>Background</h4>Carotid intima-medial thickness (IMT) is a surrogate marker of subclinical atherosclerosis. This study aimed to investigate the impacts of genetic variants on IMT and future development of ischemic stroke in a cohort, followed by an independent replication study.<h4>Methods</h4>B-mode carotid ultrasound was performed among 3330 healthy adults in the CVDFACT cohort study, and the genetic effects of atherosclerosis-related genes including connexin37 (GJA4), C-reactive protein (CRP), paraoxonase (PON1), adiponectin (ACDC), angiotensin-converting enzyme (ACE), beta-adrenergic receptor (ADRB1, ADRB2), antithrombin III (SERPINC1), and kinesin family member 6 (KIF6) were evaluated by a multivariate regression model, adjusting for traditional vascular risk factors. Study subjects were prospectively followed for the development of ischemic stroke to assess the prognostic impacts of these genetic variants. An independent case-control study was performed to replicate the genetic association from the cohort study.<h4>Results</h4>The T allele of connexin37 C1019T polymorphism significantly affected IMT (β=0.014, p=0.013) after adjusting for traditional risk factors. During an average follow-up period of 10.7 years, 80 patients with ischemic stroke (2.4%) were identified. The connexin37 1019T allele was significantly associated with an increased rate of ischemic stroke under an additive model, with hazard ratios (HR) of 2.83 (95% confidence interval, 1.2-6.66) and 1.69 (95% confidence interval, 1.06-2.71), comparing TT and CT genotype with CC, respectively. After Cox analysis, age (HR, 1.78 every 10 years), diabetes mellitus (HR, 2.63), hypertension (HR, 2.08), and the T allele of C1019T polymorphism of GJA4 (HR, 1.69) were identified as independent predictors of ischemic stroke. The relationship between T allele of C1019T polymorphism of GJA4 gene and ischemic stroke was also confirmed by an independent association study.<h4>Conclusion</h4>Connexin 37 genetic variants significantly affect carotid IMT and contribute to future development of ischemic stroke.

During the past 15 years, an impressive amount of genetic information has become available in the research field of psychiatry, particularly as it relates to attention-deficit/hyperactivity disorder (ADHD). However, the classical clinical approach to ADHD has minimally affected and not significantly been improved by this genetic revolution. It is difficult to predict how long it will take for genetic findings to alter the way clinicians treat patients with ADHD. New medications or treatment protocols may take years to become routine clinical practice. However, when taken together, recent successes in genomics, pharmacogenomics, and genetic epidemiology have the potential (1) to prevent comorbid consequences of ADHD, (2) to individualize therapies for patients with ADHD, and (3) to define new epidemiological policies to aid with the impact of ADHD on society. Here, we present an overview of how genetic research may affect and improve the quality of life of patients with ADHD: as an example, we use the discovery of LPHN3, a new gene in which variants have recently been shown to be associated with ADHD.

The identification of secreted proteins that are differentially expressed between non-neoplastic and esophageal squamous cell carcinoma (ESCC) cells can provide potential biomarkers of ESCC. We used a SILAC-based quantitative proteomic approach to compare the secretome of ESCC cells with that of non-neoplastic esophageal squamous epithelial cells. Proteins were resolved by SDS-PAGE, and tandem mass spectrometry analysis (LC-MS/MS) of in-gel trypsin-digested peptides was carried out on a high-accuracy qTOF mass spectrometer. In total, we identified 441 proteins in the combined secretomes, including 120 proteins with > 2-fold upregulation in the ESCC secretome vs. that of non-neoplastic esophageal squamous epithelial cells. In this study, several potential protein biomarkers previously known to be increased in ESCC including matrix metalloproteinase 1, transferrin receptor, and transforming growth factor beta-induced 68 kDa were identified as overexpressed in the ESCC-derived secretome. In addition, we identified several novel proteins that have not been previously reported to be associated with ESCC. Among the novel candidate proteins identified, protein disulfide isomerase family a member 3 (PDIA3), GDP dissociation inhibitor 2 (GDI2), and lectin galactoside binding soluble 3 binding protein (LGALS3BP) were further validated by immunoblot analysis and immunohistochemical labeling using tissue microarrays. This tissue microarray analysis showed overexpression of protein disulfide isomerase family a member 3, GDP dissociation inhibitor 2, and lectin galactoside binding soluble 3 binding protein in 93%, 93% and 87% of 137 ESCC cases, respectively. Hence, we conclude that these potential biomarkers are excellent candidates for further evaluation to test their role and efficacy in the early detection of ESCC.

Infections are a devastating complication of titanium alloy orthopedic implants. Current therapy includes antibiotic-impregnated bone cement and antibiotic-containing coatings. We hypothesized that daptomycin, a Gram-positive peptide antibiotic, could prevent bacterial colonization on titanium alloy surfaces if covalently bonded via a flexible, hydrophilic spacer. We designed and synthesized a series of daptomycin conjugates for bonding to the surface of 1.0 cm² Ti6Al4V foils through bisphosphonate groups, reaching a maximum yield of 180 pmol/cm². Daptomycin-bonded foils killed 53 ± 5% of a high challenge dose of 3 × 10⁵ cfu Staphylococcus aureus ATCC 29213.

<h4>Objective</h4>The aim of this study was to investigate the effects of therapeutic hypothermia (TH) on coagulopathy and cerebral microcirculation disorder after cardiopulmonary resuscitation (CPR) in rabbits.<h4>Methods</h4>Cardiac ventricular fibrillation was induced by alternating current in 24 New Zealand rabbits, and hypothermia was induced by surface cooling or normothermia (NT) was maintained for 12 hours after the return of spontaneous circulation (ROSC). Several physiologic indexes were measured before CPR and at 4, 8, and 12 hours after ROSC. The microcirculation flow in the cerebral cortex was measured with a PERIMED Multichannel Laser Doppler system (Perimid, Sweden), and glomerular fibrin deposition was determined by microscopy.<h4>Results</h4>Compared with the NT group, the prothrombin time, activated partial thromboplastin time, and international normalized ratio in the TH group were increased; there were no differences in anti-thrombin-III, protein C, and d-dimer indexes. The microcirculation flow in the cerebral cortex before CPR and after ROSC at 4, 8, and 12 hours was 401.60 ± 11.76, 258.86 ± 34.58, 317.59 ± 23.36, and 371.98 ± 5.79 mL/min, respectively, in the NT group, and 398.18 ± 12.91, 336.19 ± 19.27, 347.76 ± 13.80, and 383.78 ± 3.29 mL/min, respectively, in the TH group. There were apparent disparities at each checkpoint after ROSC in these 2 groups (4 hours: P = .001; 8 hours: P = .011; 12 hours: P = .009). The Pearson correlation test showed that the microcirculation flow in the cerebral cortex was positively correlated with activated partial thromboplastin time after ROSC (4 hours: r = 0.503, P = .033; 8 hours: r = 0.565, P = .035; 12 hours: r = 0.774, P = .009), but not with other coagulation parameters.<h4>Conclusions</h4>Therapeutic hypothermia might cause coagulant dysfunction but concomitantly improves the microcirculation flow in the cerebral cortex, which might be an effect of TH that results in cerebral protection.

There is currently no known natural animal model that fully complements type 2 diabetes in humans. Criteria for a true natural animal model include the presence of a fasting hyperglycemia, evidence of insulin resistance, and pathologies matching that reported in humans. To investigate the bottlenose dolphin (Tursiops truncatus) as a comparative model for type 2 diabetes in humans, hourly plasma and urine chemistry changes, including glucose, were analyzed among five healthy, adult dolphins for 24 h following ingestion of 2.5-3.5 kg of mackerel or 2-3 L of 10% dextrose in ionosol. Fasting and 2 h post-prandial insulin levels were also determined among five adult dolphins to assess the presence of hyperinsulinemia. Finally, a case-control study compared insulin and glucagon levels among dolphins with and without iron overload, a condition associated with insulin resistance in humans. Both protein and dextrose meals caused significant increases in plasma glucose during the 0-5 h post-prandial period; dolphins fed dextrose demonstrated a sustained hyperglycemia lasting 5-10 h. Fasting plasma insulin levels among healthy dolphins mimicked those found in humans with some insulin resistance. Dolphins with hemochromatosis had higher post-prandial plasma insulin levels compared to controls. We conclude that bottlenose dolphins can demonstrate metabolic responses consistent with type 2 diabetes, specifically sustained hyperglycemia and hyperinsulinemia. Understanding more about how and why dolphins have a diabetes-like metabolism may provide new research avenues for diabetes in humans.

Huntington's disease (HD) is an inherited, progressive and ultimately fatal neurodegenerative disorder that is characterized by psychiatric, cognitive and motor symptoms. Among the pathways implicated in HD are those involving mitogen-activated protein kinase signaling and particularly the Ras-extracellular signal-regulated kinase (ERK) cascade. Studies in both cells and animal models suggest that ERK activation might provide a novel therapeutic target for the treatment of HD but compounds that specifically activate ERK are few. To test the hypothesis that pharmaceutical activation of ERK might be protective for HD, a polyphenol, fisetin, which was previously shown to activate the Ras-ERK cascade, was tested in three different models of HD: PC12 cells expressing mutant Httex1 under the control of an inducible promoter, Drosophila expressing mutant Httex1 and the R6/2 mouse model of HD. The results indicate that fisetin can reduce the impact of mutant huntingtin in each of these disease models. Prompted by this observation, we determined that the related polyphenol, resveratrol, also activates ERK and is protective in HD models. Notably, although more than a dozen small molecule inhibitors of ERK activation are in clinical trials, very few small molecule activators of ERK signaling are reported. Thus, fisetin, resveratrol and related compounds might be useful for the treatment of HD by virtue of their unique ability to activate ERK.

<h4>Background</h4>The bone morphogenetic protein BMP6 regulates hepcidin production by the liver. However, it is not yet known whether BMP6 derives from the liver itself or from other sources such as the small intestine, as has been recently suggested. This study was aimed at investigating the source of BMP6 further.<h4>Design and methods</h4>We used three different strains of mice (C57BL/6, DBA/2, and 129/Sv) with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene. We examined Bmp6 expression at both the mRNA (by quantitative PCR) and protein (by immunohistochemistry and Western blotting analyses) levels.<h4>Results</h4>We showed that iron overload induces Bmp6 mRNA expression in the liver but not in the duodenum of these mice. Bmp6 is also detected by immunohistochemistry in liver tissue sections of mice with iron overload induced either by an iron-enriched diet or by inactivation of the Hfe gene, but not in liver tissue sections from iron-loaded Bmp6-deficient mice. Bmp6 in the duodenum was below immunodetection threshold, thus confirming quantitative PCR data. Lack of specificity of available antibodies together with slight heterogeneity between 129 substrains may account for the differences with previously published data.<h4>Conclusions</h4>Our data strongly support the importance of liver BMP6 for regulation of iron metabolism. Indeed, they demonstrate that intestinal Bmp6 expression is modulated by iron neither at the mRNA nor at the protein level.

Uveitis is a major and common cause of visual disability. Recent studies have shown that Th17 cells are implicated in the pathogenesis of this serious intraocular disorder. Activated T cells express an inducible costimulatory molecule called OX40, and OX40 in turn promotes the activation and proliferation of these lymphocytes. Nevertheless, it is unclear whether OX40 plays a vital role in enhancing the effector function of Th17 cells as well as the severity of uveitis. In this study, we demonstrated an increase of OX40 transcription in ovalbumin-induced uveitis, whereas anti-OX40L antibody substantially inhibited the antigen-specific ocular inflammation. Next, results from flow cytometry showed that activated Th17 cells expressed OX40, and OX40-activating antibody significantly augmented the production of Th17 cytokines in vitro. To validate the impact of OX40 in vivo, we stimulated ovalbumin-specific T cells with the OX40-activating antibody. Compared to donor cells without OX40 activation, adoptive transfer of OX40-stimulated lymphocytes elicited more severe ocular inflammation. Furthermore, an interleukin-17-neutralizing antibody attenuated OX40-mediated uveitis. In conclusion, our findings suggest that activation of OX40 augments Th17 cell function and thereby contributes to ocular inflammation. This study thus enhances our knowledge of costimulatory molecule-mediated immunopathological mechanisms of uveitis and suggests a future therapeutic strategy to treat uveitis by the targeting of OX40.

Porphyria cutanea tarda (PCT) is a cutaneous porphyria with sporadic (type 1) and familial (type 2) subtypes, both resulting from decreased hepatic uroporphyrinogen decarboxylase (UROD) activity. Environmental and genetic factors are involved in the development of PCT, and genetic variants in the cytochrome P450 (CYP ) genes, CYP1A1 and CYP1A2, have been implicated. We investigated the association between PCT and variants in CYP1A1, CYP1A2 and CYP2E1, and the glutathione-S-transferase (GST ) genes, GSTM1 and GSTT1. PCT diagnosis was based on urinary or plasma porphyrin profiles. Patients were classified as type 1 or 2 PCT based on UROD mutation analysis. The CYP1A2*1F promoter A allele frequency was significantly higher (P < 0.022) and the A/A genotype frequency marginally higher in PCT patients overall (P < 0.057), with the A/A genotype significantly more common in type 1 PCT (P < 0.043). The presence of the wild-type GSTM1 allele also was associated significantly with PCT (P < 0.019). Neither hemochromatosis (HFE) mutations, tobacco smoking, hepatitis C and HIV infection, ethanol consumption, nor estrogen use were associated with these allelic variants. Age at onset was significantly lower in type 2 PCT patients (P < 0.001), as observed previously. Thus, positive associations between PCT and the CYP1A2*1F promoter A allele and A/A genotype and the wild-type GSTM1 allele indicates that these functional hepatic biotransformation enzymes are risk factors for the development of this disease.

Although heritability of human body weight is assumed to be high, only a small fraction of the variance can as yet be attributed to molecular genetic factors. Single monogenic forms of obesity have been identified. Functionally relevant coding mutations in the melanocortin-4 receptor gene occur in 1-6% of extremely obese children and adolescents and thus represent the most common major gene effect. Genome-wide association studies (GWAS) had previously identified 14 obesity loci with genome-wide significant (p < 5 x 10-8) associations. Many of the respective genes are expressed in the central nervous system. The GIANT (Genetic Investigation of ANtropometric Traits) Consortium has now performed a meta-analysis of GWAS data based on 123,865 individuals of European ancestry followed by confirmatory analyses for the 42 best independent loci in up to 125,931 independent individuals (Speliotes et al: Association analyses of 249,796 individuals reveal eighteen new loci associated with body mass index. Nature Genetics; epub October 2010 [1]). Apart from confirming the 14 known loci, 18 novel BMI-associated loci (p < 5 x 10-8) were identified. Several of the new loci point to genes involved in key hypothalamic pathways of energy balance. The identified variants mostly have small to very small effect sizes; only 1-2% of the BMI variance is explained. Currently, a consensus explanation for this 'missing heritability' in complex diseases has not yet emerged.

<h4>Purpose</h4>The pathophysiological events that occur in advanced glaucoma are not well characterized. The principal purpose of this study is to characterize the gene expression changes that occur in advanced glaucoma.<h4>Methods</h4>Retinal RNA was obtained from canine eyes with advanced glaucoma as well as from healthy eyes. Global gene expression patterns were determined using oligonucleotide microarrays and confirmed by real-time PCR. The presence of tumor necrosis factor (TNF) and its receptors was evaluated by immunolabeling. Finally, we evaluated the presence of serum autoantibodies directed against retinal epitopes using western blot analyses.<h4>Results</h4>We identified over 500 genes with statistically significant changes in expression level in the glaucomatous retina. Decreased expression levels were detected for large number of functional groups, including synapse and synaptic transmission, cell adhesion, and calcium metabolism. Many of the molecules with decreased expression levels have been previously shown to be components of retinal ganglion cells. Genes with elevated expression in glaucoma are largely associated with inflammation, such as antigen presentation, protein degradation, and innate immunity. In contrast, expression of many other pro-inflammatory genes, such as interferons or interleukins, was not detected at abnormal levels.<h4>Conclusions</h4>This study characterizes the molecular events that occur in the canine retina with advanced glaucoma. Our data suggest that in the dog this stage of the disease is accompanied by pronounced retinal neuroinflammation.

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene and characterized by chronic anemia caused by ineffective erythropoiesis, and accompanied by a variety of serious secondary complications such as extramedullary hematopoiesis, splenomegaly, and iron overload. In the past few years, numerous studies have shown that such secondary disease conditions have a genetic basis caused by the abnormal expression of genes with a role in controlling erythropoiesis and iron metabolism. In this article, the most recent discoveries related to the mechanism(s) responsible for anemia/ineffective erythropoiesis and iron overload are discussed in detail. Particular attention is paid to the pathway(s) controlling the expression of hepcidin, which is the main regulator of iron metabolism, and the Epo/EpoR/Jak2/Stat5 signaling pathway, which regulates erythropoiesis. Better understanding of how these pathways function and are altered in β-thalassemia has revealed several possibilities for development of new therapeutic approaches to treat of the complications of this disease.

The effect of temperature on isolation and characterization of hydroxyapatite (HAp) from tuna bone was evaluated at different temperatures ranging from 200 °C to 1200 °C. The calcined bones were characterized by thermo gravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDX) and cytotoxicity assay. The FTIR and TGA results revealed the presence of inorganic and organic matrices in raw bone and a preserved carbonated group in the derived HAp. The XRD results of the derived HAp were coherent with the Joint Committee on Powder Diffraction Standards (JCPDS-09-0432/1996) data. In addition, FE-SEM results revealed the formation of nanostructured HAp (80-300 nm) at 600 °C and crystal agglomeration was observed with an increase in temperature. The calcium to phosphorous weight ratio was determined by EDX results of treated bones. Derived HAp with various crystal sizes had no cytotoxicity on the MG 63 cell line. Based on the analysis, we conclude that varying the isolation temperature between 600-900 °C has tremendous impact on the production of HAp from Thunnus obesus with required properties.

<h4>Background</h4>In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate.<h4>Results</h4>In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms.<h4>Conclusions</h4>Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.

The prevalence of obesity in children and adults in the United States has increased dramatically over the past decade. Genomic copy number variations (CNVs) have been strongly implicated in subjects with extreme obesity and coexisting developmental delay. To complement these previous studies, we addressed CNVs in common childhood obesity by examining children with a BMI in the upper 5(th) percentile but excluding any subject greater than three standard deviations from the mean in order to reduce severe cases in the cohort. We performed a whole-genome CNV survey of our cohort of 1080 defined European American (EA) childhood obesity cases and 2500 lean controls (< 50(th) percentile BMI) who were genotyped with 550,000 SNP markers. Positive findings were evaluated in an independent African American (AA) cohort of 1479 childhood obesity cases and 1575 lean controls. We identified 17 CNV loci that were unique to at least three EA cases and were both previously unreported in the public domain and validated via quantitative PCR. Eight of these loci (47.1%) also replicated exclusively in AA cases (six deletions and two duplications). Replicated deletion loci consisted of EDIL3, S1PR5, FOXP2, TBCA, ABCB5, and ZPLD1, whereas replicated duplication loci consisted of KIF2B and ARL15. We also observed evidence for a deletion at the EPHA6-UNQ6114 locus when the AA cohort was investigated as a discovery set. Although these variants may be individually rare, our results indicate that CNVs contribute to the genetic susceptibility of common childhood obesity in subjects of both European and African ancestry.

Positron emission tomography (PET) is used in drug development to assist dose selection and to establish the relationship between blood and tissue pharmacokinetics (PKs). We present a new biomathematical approach that allows prediction of repeat-dose (RD) brain target occupancy (TO) using occupancy data obtained after administration of a single dose (SD). A PET study incorporating a sequential adaptive design was conducted in 10 healthy male adults who underwent 4 PET scans with [(11)C]DASB ([(11)C]N,N-dimethyl-2-(2-amino-4-cyanophenylthio) benzylamine): 1 at baseline, 2 after 20 mg SD of the 5-hydroxytryptamine transporter (5-HTT) inhibitor duloxetine, and 1 after 4 days daily administration of 20 mg duloxetine. An adaptive design was used to select optimal times after SD for measurement of occupancy. Both direct and indirect PK/TO models were fitted to the SD data to characterise the model parameters and then applied to a predicted RD duloxetine plasma time course to predict the 5-HTT occupancy after RD. Repeat-dose prediction from the indirect model (OC(50)=2.62±0.93 ng/mL) was significantly better (P<0.05) than that from the direct model (OC(50)=2.29±1.11 ng/mL). This approach increases the value of SD occupancy studies that are performed as part of first time in human drug development programmes by providing an estimate of the dose required to achieve the desired TO at RD.

<h4>Background</h4>Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.<h4>Method</h4>Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.<h4>Results</h4>CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.<h4>Conclusions</h4>Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Dosage compensation is a strategy to deal with the imbalance of sex chromosomal gene products relative to autosomes and also between the sexes. The mechanisms that ensure dosage compensation for X-chromosome activity have been extensively studied in mammals, worms, and flies. Although each entails very different mechanisms to equalize the dose of X-linked genes between the sexes, they all involve the co-ordinate regulation of hundreds of genes specifically on the sex chromosomes and not the autosomes. In addition to chromatin modifications and changes in higher order chromatin structure, nuclear organization is emerging as an important component of these chromosome-wide processes and in the specific targeting of dosage compensation complexes to the sex chromosomes. Preferential localization within the nucleus and 3D organization are thought to contribute to the differential treatment of two identical homologs within the same nucleus, as well as to the chromosome-wide spread and stable maintenance of heterochromatin.

Diabetes insipidus is a rare disorder in pregnant women, predating pregnancy or appearing for the first time during gestation. In pregnancy it usually affects women with HELLP syndrome or acute fatty liver of pregnancy and results from the reduced hepatic degradation of placental vasopressinase leading to its increased activity. Although infiltrative diseases have been found to cause diabetes insipidus in non-pregnant population, very few studies showed that these disorders may manifest for the first time during gestation. We describe here the case of transient diabetes insipidus in two subsequent pregnancies of a female with hemochromatosis. The first symptoms of this disease appeared for the first time at the beginning of the third trimester of her second pregnancy, and diagnosis was established on the basis of typical clinical presentation, confirmed by a water deprivation test. Diabetes insipidus resulted from the increased activity of vasopressinase, caused by hemochromatosis-induced liver dysfunction, the presence of which was confirmed between the pregnancies by liver biopsy and identification of the HFE gene mutation. Subsequent desferrioxamine treatment resulted in a less severe clinical course of diabetes insipidus in the last patient's pregnancy. In both pregnancies, the patient was successfully treated with oral desmopressin, which is resistant to degradation by placental vasopressinase. Although unrecognized pituitary disorders may pose a serious health problem to the mother and fetus, hemochromatosis-induced diabetes insipidus, as the case of our patient demonstrates, if effectively diagnosed and treated, cannot be regarded as a contraindication for pregnancy.

<h4>Background</h4>Both anxiety and elevated heart rate (HR) have been implicated in the development of hypertension. The HyperGen cohort, consisting of siblings with severe and mild hypertension, an age-matched random sample of persons from the same base populations, and unmedicated adult offspring of the hypertensive siblings (N = 1,002 men and 987 women), was analyzed for an association of the angiotenisinogen AGTM235T genotype (TT, MT, MM) with an endophenotype, heart rate (HR) in high and low anxious groups.<h4>Methodology</h4>The interaction of AGTM genotype with anxiety, which has been independently associated with hypertension, was investigated adjusting for age, hypertension status, smoking, alcohol consumption, beta blocker medication, body mass index, physical activity and hours of television viewing (sedentary life style).<h4>Principal findings</h4>Although there was no main effect of genotype on HR in men or women, high anxious men with the TT genotype had high HR, whereas high anxious men with the MM genotype had low HR. In women, HR was inversely associated with anxiety but there was no interaction with genotype.<h4>Conclusion/significance</h4>The results suggest that high anxiety in men with the TT genotype may increase risk for hypertension whereas the MM genotype may be protective in high anxious men. This type of gene x environment interaction may be one reason why genome wide association studies sometimes fail to replicate. The locus may be important only in combination with certain environmental factors.

After spermatogenesis, testicular spermatozoa are not able to fertilize an oocyte, they must undergo sequential maturational processes. Part of these essential processes occurs during the transit of the spermatozoa through the male reproductive tract. Since the sperm become silent in terms of translation and transcription at the testicular level, all the maturational changes that take place on them are dependent on the interaction of spermatozoa with epididymal and accessory gland fluids. During the last decades, reproductive biotechnologies applied to bovine species have advanced significantly. The knowledge of the bull reproductive physiology is really important for the improvement of these techniques and the development of new ones. This paper focuses on the importance of the sperm interaction with the male reproductive fluids to acquire the fertilizing ability, with special attention to the role of the membranous vesicles present in those fluids and the recent mechanisms of protein acquisition during sperm maturation.

Despite the advances in the understanding of neural and genetic foundations of violence, the investigation of the biological bases of a mental disorder is rarely included in psychiatric evaluation of mental insanity. Here we report on a case in which cognitive neuroscience and behavioral genetics methods were applied to a psychiatric forensic evaluation conducted on a young woman, J.F., tried for a violent and impulsive murder. The defendant had a history of multidrug and alcohol abuse and non-forensic clinical evaluation concluded for a diagnosis of borderline personality disorder. We analyzed the defendant's brain structure in order to underlie possible brain structural abnormalities associated with pathological impulsivity. Voxel-based morphometry indexed a reduced gray matter volume in the left prefrontal cortex, in a region specifically associated with response inhibition. Furthermore, J.F.'s DNA was genotyped in order to identify genetic polymorphisms associated with various forms of violence and impulsive behavior. Five polymorphisms that are known to be associated with impulsivity, violence, and other severe psychiatric illnesses were identified in J.F.'s DNA. Taken together, these data provided evidence for the biological correlates of a mental disorder characterized by high impulsivity and aggressive tendencies. Our claim is that the use of neuroscience and behavioral genetics do not change the rationale underlying the determination of criminal liability, which must be based on a causal link between the mental disorder and the crime. Rather, their use is crucial in providing objective data on the biological bases of a defendant's mental disorder.

In this study, we applied electrical polarization technique to increase adsorption and control protein release from biphasic calcium phosphate (BCP). Three different biphasic calcium phosphate (BCP) composites, with hydroxyapatite (HAp) and β-tricalcium phosphate (β-TCP), were processed and electrically polarized. Our study showed that stored charge was increased in the composites with the increase in HAp percentage. Adsorption of bovine serum albumin (BSA), as a model protein, on the poled as well as unpoled surfaces of the composites was studied. The highest amount of BSA adsorption was obtained on positively poled surfaces of each composite. Adsorption isotherm study suggested a multilayer adsorption of BSA on the BCP composites. The effect of electrical polarization on BSA release kinetics from positively charged BCP surfaces was studied. A gradual increase in percent BSA release from positively charged BCP surfaces with decreasing stored charge was observed. Our study showed that the BCP based composites have the potential to be used as a drug or growth factor delivery vehicle.

Apatite-binding peptides discovered by phage display provide an alternative design method for creating functional biomaterials for bone and tooth tissue repair. A limitation of this approach is the absence of display peptide phosphorylation--a post-translational modification important to mineral-binding proteins. To refine the material specificity of a recently identified apatite-binding peptide, and to determine critical design parameters (net charge, charge distribution, amino acid sequence and composition) controlling peptide affinity for mineral, we investigated the effects of phosphorylation and sequence scrambling on peptide adsorption to four different apatites (bone-like mineral, and three types of apatite containing initially 0, 5.6 and 10.5% carbonate). Phosphorylation of the VTKHLNQISQSY peptide (VTK peptide) led to a 10-fold increase in peptide adsorption (compared to nonphosphorylated peptide) to bone-like mineral, and a 2-fold increase in adsorption to the carbonated apatite, but there was no effect of phosphorylation on peptide affinity to pure hydroxyapatite (without carbonate). Sequence scrambling of the nonphosphorylated VTK peptide enhanced its specificity for the bone-like mineral, but scrambled phosphorylated VTK peptide (pVTK) did not significantly alter mineral-binding suggesting that despite the importance of sequence order and/or charge distribution to mineral-binding, the enhanced binding after phosphorylation exceeds any further enhancement by altered sequence order. Osteoblast culture mineralization was dose-dependently inhibited by pVTK and to a significantly lesser extent by scrambled pVTK, while the nonphosphorylated and scrambled forms had no effect, indicating that inhibition of osteoblast mineralization is dependent on both peptide sequence and charge. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy upon phosphorylation that was unaffected by scrambling. In conclusion, phosphorylation of serine residues increases peptide specificity for bone-like mineral, whose adsorption is determined primarily by sequence composition and net charge as opposed to sequence order. However, sequence order in addition to net charge modulates the mineralization of osteoblast cultures. The ability of such peptides to inhibit mineralization has potential utility in the management of pathologic calcification.

Huntington's disease (HD) is a fatal progressive neurodegenerative disorder with autosomal dominant inheritance. In humans mutated huntingtin (htt) induces a preferential loss of medium spiny neurons (MSN) of the striatum and causes motor, cognitive and emotional deficits. One of the proposed cellular mechanism underlying medium spiny neurons degeneration is excitotoxic pathways mediated by glutamate receptors. The hypothesis proposed is restoration of medium spiny neurons in Huntington's disease using neural progenitor cell implantation and attenuation of glutamate mediated excitotoxicity using a partial glutamate antagonist - Memantine. Memantine can block the NMDA receptors and will prevent excess calcium influx into the neurons decreases the vulnerability of medium spiny neurons to glutamate mediated excitotoxicity. Neural progenitor cell implantation can enhance endogenous neurogenesis process replacing the degenerated medium spiny neurons in the striatum. This has immense significance in the management of Huntington's disease.

Autophagy is a highly conserved intracellular pathway involved in the elimination of proteins and organelles by lysosomes. Known originally as an adaptive response to nutrient deprivation in mitotic cells, autophagy is now recognized as an arbiter of neuronal survival and death decisions in neurodegenerative diseases. Studies using postmortem human tissue, genetic and toxin-induced animal and cellular models indicate that many of the etiological factors associated with neurodegenerative disorders can perturb the autophagy process. Emerging data support the view that dysregulation of autophagy might play a critical role in the pathogenesis of neurodegenerative disorders. In this review, we highlight the pathophysiological roles of autophagy and its potential therapeutic implications in debilitating neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's, Parkinson's and Huntington's diseases.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. In this study, we investigated the role of the POU domain-containing transcription factor Brn2 in keratinocyte differentiation. Immunohistochemical analysis showed that Brn2 is expressed primarily in the upper granular layer. Consistent with its epidermal localization, Brn2 expression was highly induced at 14 days after calcium treatment of cultured normal human epidermal keratinocytes. When Brn2 was overexpressed by adenoviral transduction, Brn2 led to increased expression of the differentiation-related genes involucrin, filaggrin, and loricrin in addition to inhibition of their proliferation. Chromatin immunoprecipitation demonstrated that Brn2 bound to the promoter regions of these differentiation-related genes. We injected the purified Brn2 adenovirus into rat skin, which led to a thickened epidermis with increased amounts of differentiation related markers. The histopathologic features of adenovirus-Brn2 injected skin tissues looked similar to the features of lichen planus, a human skin disease showing chronic inflammation and well-differentiated epidermal changes. Moreover, Brn2 is shown to be expressed in almost all cell nuclei of the thickened epidermis of lichen planus, and Brn2 also attracts T lymphocytes. Our results demonstrate that Brn2 is probably a transcriptional factor playing an important role in keratinocyte differentiation and probably also in the pathogenesis of lichen planus lesions.

<h4>Background</h4>To explore the gender difference of ALT elevation and its association with high hemoglobin levels.<h4>Methods</h4>A cross-sectional study of 3547 adolescents (2005 females, mean age of 16.5?.3 years) who were negative for hepatitis B surface antigen received health checkups in 2006. Body mass index (BMI), levels of hemoglobin, ALT and cholesterol were measured. ALT >42 U/L was defined as elevated ALT. Elevated ALT levels were detected in 112 of the 3547 participants (3.3%), more prevalent in males than in females (5.4% vs. 1.4%, p<0.001). Hemoglobin levels had a significant linear correlation with ALT levels in both genders. Abnormal ALT started to occur if hemoglobin >11 g/dl in females or >13.5 g/dl in males, but the cumulative cases of elevated ALT increased more quickly in males. Proportion of elevated ALT increased as either the BMI or hemoglobin level rise, more apparent in male adolescents. Logistic regression modeling showed odds ratio (95% confidence interval) were 24.7 (15.0-40.6) for BMI ≥27 kg/m(2); 5.5 (2.9-10.4) for BMI 24-27 kg/m(2); 2.7 (1.3-5.5) for Q5 (top 20th percentile) hemoglobin level; and 2.6 (1.6-4.1) for male gender. Further separately fitting the logistic models for two genders, the significance of Q5 hemoglobin level only appeared in the males.<h4>Conclusions</h4>High hemoglobin level is a significant risk factor of ALT elevation after control hepatitis B, obesity and gender. Males have greater risk of abnormal liver function which may be associated with higher hemoglobin levels.

Mcl-1 inhibits apoptosis in well-differentiated cells by sequestering BAD, BID, and BAX and other apoptotic molecules. pAKT blocks apoptotsis by facilitating the interaction of BAD with BCL-XL. Expression of pAKT and Mcl-1 have been described in colon cancer, however, the relationship between pAKT and Mcl-1 has not. Mcl-1 and pAKT immunohistochemistry was performed using colorectal cancer tissue microarray (TMA). The Holm step-down method was used to adjust for multiple testing. Mcl-1 and pAKT scores, stage, and grade were compared using Spearman's correlation coefficient. Metastasis and no metastasis groups were compared using the Wilcoxon rank sum test. Mcl-1 and pAKT scores were compared for normal colorectal mucosa (NR), adenoma (AD), and colorectal cancer (CRC) cohorts. The mean (SD) pAKT expression in NR (14) was 2.0 (1.4), in AD (8) was 3.0 (1.7), and in CRC (101) was 5.6 (2.4). These differences were statistically significant. For Mcl-1 the mean (SD) expression was 4.1 (1.7) in NR, 3.2 (1.2) in AD, and 3.3 (2.6) in CRC. Mcl-1 and pAKT scores were directly correlated during various stages of colon car-cinogenesis (p = 0.04). Mcl-1 showed direct correlation with tumor grade (p = 0.001) and tumor stage (p = 0.02) and with presence of metastasis (p = 0.008). We report the correlation of Mcl-1 protein expression with higher grade and stage in colorectal cancer. Mcl-1 correlated also with pAKT expression. We also report the up regulation of pAKT during the transition from NR to CRC.

A Thiazole Orange conjugated with folate derivative was synthesized in two steps. Firstly, folate was coupled with 1-(3-aminopropyl)-4-methylquinolinium bromide to afford folate-methylquinolinium bromide, which then reacted with benzothiazolium to obtain the title folate-conjugated compound. The compound was evaluated by ¹H-NMR MS, TG/DTA and fluorescence spectroscopic methods. The title compound could selectively target folate receptor expressing tumors according to the in vivo fluorescence imaging preliminarily performed on nude mice with breast tumors.

Disruptive behavior disorders (DBDs) are reflected by a great variety of symptoms ranging from impulsive-hot-tempered quarrels to purposeful and goal-directed acts of cruelty. A growing body of data indicates that there are neurobiological factors that increase the risk for developing DBDs. In this review, we give a broad overview of recent studies investigating physiological, neural, genetic factors, and specific neurotransmitter systems. We also discuss the impact of psychosocial risk and consider the effects of gene-environment interactions. Due to the heterogeneity of DBDs, it is concluded that specific subtypes of disruptive behavior should be considered both in terms their biological basis and in regard to specific treatment needs.

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.

<h4>Background</h4>Variation in the effects of genetic variants on physiological traits over time or with age may alter the trajectories of these traits. However, few studies have investigated this possibility for variants associated with type 2 diabetes or obesity, and these show little consensus. We aimed to characterise the possible longitudinal associations of common diabetes-susceptibility variants in the KCNJ11, PPARG, TCF7L2, IGF2BP2, CDKAL1, SLC30A8 and HHEX gene loci, with fasting glucose level; and of an obesity-associated variant in the FTO gene, with body mass index (BMI).<h4>Methods</h4>The study analysed data from the Busselton Health Study (n = 4,554). Cross-sectional association analyses included family data and used the total association test. Longitudinal association analyses of unrelated participant data (n = 2,864) used linear mixed-effects models.<h4>Results</h4>In cross-sectional analyses, we observed associations of the T allele at the IGF2BP2 single nucleotide polymorphism (SNP) rs4402960 with raised fasting glucose (p = 0.045), and the A allele at the FTO SNP rs9939609 with raised BMI (p = 0.003). Longitudinal analyses showed no significant associations between SNPs and changes in fasting glucose or BMI in the same individuals, either over mean follow-up times of 18.7 and 21.8 years respectively, or with age during adulthood.<h4>Conclusions</h4>There was no indication that the effects of common type 2 diabetes variants on fasting glucose varied with age during adulthood or over time.

Smith-Magenis syndrome (SMS) is a complex syndrome involving intellectual disabilities, sleep disturbance, behavioural problems, and a variety of craniofacial, skeletal, and visceral anomalies. While the majority of SMS cases harbor an ~3.5 Mb common deletion on 17p11.2 that encompasses the retinoic acid induced-1 (RAI1) gene, some patients carry small intragenic deletions or point mutations in RAI1. We present data on two cases of Smith-Magenis syndrome with mutation of RAI1. Both cases are phenotypically consistent with SMS and RAI1 mutation but also have other anomalies not previously reported in SMS, including spontaneous pneumothoraces. These cases also illustrate variability in the SMS phenotype not previously shown for RAI1 mutation cases, including hearing loss, absence of self-abusive behaviours, and mild global delays. Sequencing of RAI1 revealed mutation of the same heptameric C-tract (CCCCCCC) in exon 3 in both cases (c.3103delC one case and and c.3103insC in the other), resulting in frameshift mutations. Of the seven reported frameshift mutations occurring in poly C-tracts in RAI1, four cases (~57%) occur at this heptameric C-tract. Collectively, these results indicate that this heptameric C-tract is a preferential hotspot for single nucleotide insertion/deletions (SNindels) and therefore, should be considered a primary target for analysis in patients suspected for mutations in RAI1. We expect that as more patients are sequenced for mutations in RAI1, the incidence of frameshift mutations in this hotspot will become more evident.

In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas-247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)-by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.

<h4>Objective</h4>The purpose of this study is to present an unusual and rarely described case of occupational hand arthropathy involving the metacarpophalangeal (MCP) joints.<h4>Clinical features</h4>A 62-year-old male truck driver (of 35 years) presented to a chiropractic clinic with pain and stiffness along the third metacarpal and MCP joint of the left hand. Examination revealed severe pain and limited flexion at the third MCP joint. Bilateral radiographs demonstrated severe osteoarthritis (OA) of this joint in the left (nondominant) hand and mild-to-moderate (asymptomatic) OA in the same joint on the right. Results of laboratory blood tests were unremarkable for metabolic, inflammatory, or infectious joint disease.<h4>Intervention and outcome</h4>The patient was diagnosed with bilateral, third MCP joint OA associated with manual labor. He was treated unsuccessfully with a short course of low-level laser therapy, MCP joint mobilization, and hand-stretching exercises. After 3½ years, the patient continues to work despite ongoing and worsening symptoms. Three serial left hand radiographs are presented, highlighting the progressive nature of this arthropathy.<h4>Conclusion</h4>The differential diagnosis in patients presenting with manual labor MCP joint OA should include hemochromatosis and calcium pyrophosphate dihydrate crystal deposition disease. Because of the increased risk of serious systemic disease, it is imperative that these latter 2 disorders are ruled out before the former is diagnosed.

Much evidence suggests that variation in expression of the 5-hydroxytryptamine (5-HT) transporter (5-HTT) is linked to risk of psychiatric illness, but the neurobiological basis of this association is uncertain. In this study, we investigated the impact of variation in 5-HTT expression on subsecond fluctuations in extracellular 5-HT concentrations ([5-HT](o) ). Stimulus-evoked [5-HT](o) was detected using fast-scan cyclic voltammetry at carbon-fibre microelectrodes in the substantia nigra in brain slices from 5-HTT knockout (KO) and 5-HTT over-expressing (OE) mice. Compared with wild-type (WT) controls, evoked [5-HT](o) was greater in KO and less in OE mice. In WT controls, evoked [5-HT](o) was frequency-sensitive; however, in both KO and OE mice, evoked [5-HT](o) showed a striking loss of frequency sensitivity. The latter was observed in WT mice after application of a 5-HTT blocker. These data show that while variation in 5-HTT expression modified the peak magnitude of [5-HT](o) evoked by any given stimulus in a gene dose dependent manner, there was a non-linear relationship between 5-HTT expression and frequency sensitivity. Overall, the findings suggest that variation in 5-HTT expression has a marked effect on frequency sensitivity which is a fundamental property of normal 5-HT transmission.

Cidofovir (HPMPC), a broad spectrum antiviral agent, cannot be administered orally due to ionization of its phosphonic acid group at physiological pH. One prodrug approach involves conversion to the cyclic form (cHPMPC, 1) and esterification by the side chain hydroxyl group of a peptidomimetic serine. Transport studies in a rat model have shown enhanced levels of total cidofovir species in the plasma after oral dosing with L-Val-L-Ser-OMe cHPMPC, 2a. To explore the possibility that 2a and its three L/D stereoisomers 2b-d undergo active transport mediated by the peptide-specific intestinal transporter PEPT1, we performed radiotracer uptake and electrophysiology experiments applying the two-electrode voltage clamp technique in Xenopus laevis oocytes overexpressing human PEPT1 (hPEPT1, SLC15A1). 2a-d did not induce inward currents, indicating that they are not transported, but the stereoisomers with an L-configuration at the N-terminal valine (2a and 2b) potently inhibited transport of the hPEPT1 substrate glycylsarcosine (Gly-Sar). A "reversed" dipeptide conjugate, L-Ser-L-Ala-OiPr cHPMPC (4), also did not exhibit detectable transport, but completely abolished the Gly-Sar signal, suggesting that affinity of the transporter for these prodrugs is not impaired by a proximate linkage to the drug in the N-terminal amino acid of the dipeptide. Single amino acid conjugates of cHPMPC (3a and 3b) or cHPMPA (5, 6a and 6b) were not transported and only weakly inhibited Gly-Sar transport. The known hPEPT1 prodrug substrate valacyclovir (7) and its L-Val-L-Val dipeptide analogue (8) were used to verify coupled transport by the oocyte model. The results indicate that the previously observed enhanced oral bioavailability of 2a relative to the parent drug is unlikely to be due to active transport by hPEPT1. Syntheses of the novel compounds 2b-d and 3-6 are described, including a convenient solid-phase method to prepare 5, 6a and 6b.

During its earliest stages, the avian embryo is approximately planar. Through a complex series of folds, this flat geometry is transformed into the intricate three-dimensional structure of the developing organism. Formation of the head fold (HF) is the first step in this cascading sequence of out-of-plane tissue folds. The HF establishes the anterior extent of the embryo and initiates heart, foregut and brain development. Here, we use a combination of computational modeling and experiments to determine the physical forces that drive HF formation. Using chick embryos cultured ex ovo, we measured: (1) changes in tissue morphology in living embryos using optical coherence tomography (OCT); (2) morphogenetic strains (deformations) through the tracking of tissue labels; and (3) regional tissue stresses using changes in the geometry of circular wounds punched through the blastoderm. To determine the physical mechanisms that generate the HF, we created a three-dimensional computational model of the early embryo, consisting of pseudoelastic plates representing the blastoderm and vitelline membrane. Based on previous experimental findings, we simulated the following morphogenetic mechanisms: (1) convergent extension in the neural plate (NP); (2) cell wedging along the anterior NP border; and (3) autonomous in-plane deformations outside the NP. Our numerical predictions agree relatively well with the observed morphology, as well as with our measured stress and strain distributions. The model also predicts the abnormal tissue geometries produced when development is mechanically perturbed. Taken together, the results suggest that the proposed morphogenetic mechanisms provide the main tissue-level forces that drive HF formation.

The molecular pathways that are activated and contribute to physiological remodeling of skeletal muscle in response to endurance exercise have not been fully characterized. We previously reported that ∼800 gene transcripts are regulated following 6 wk of supervised endurance training in young sedentary males, referred to as the training-responsive transcriptome (TRT) (Timmons JA et al. J Appl Physiol 108: 1487-1496, 2010). Here we utilized this database together with data on biological variation in muscle adaptation to aerobic endurance training in both humans and a novel out-bred rodent model to study the potential regulatory molecules that coordinate this complex network of genes. We identified three DNA sequences representing RUNX1, SOX9, and PAX3 transcription factor binding sites as overrepresented in the TRT. In turn, miRNA profiling indicated that several miRNAs targeting RUNX1, SOX9, and PAX3 were downregulated by endurance training. The TRT was then examined by contrasting subjects who demonstrated the least vs. the greatest improvement in aerobic capacity (low vs. high responders), and at least 100 of the 800 TRT genes were differentially regulated, thus suggesting regulation of these genes may be important for improving aerobic capacity. In high responders, proangiogenic and tissue developmental networks emerged as key candidates for coordinating tissue aerobic adaptation. Beyond RNA-level validation there were several DNA variants that associated with maximal aerobic capacity (Vo(₂max)) trainability in the HERITAGE Family Study but these did not pass conservative Bonferroni adjustment. In addition, in a rat model selected across 10 generations for high aerobic training responsiveness, we found that both the TRT and a homologous subset of the human high responder genes were regulated to a greater degree in high responder rodent skeletal muscle. This analysis provides a comprehensive map of the transcriptomic features important for aerobic exercise-induced improvements in maximal oxygen consumption.

Mitochondrial defects that affect cellular energy metabolism have long been implicated in the etiology of Huntington's disease (HD). Indeed, several studies have found defects in the mitochondrial functions of the central nervous system and peripheral tissues of HD patients. In this study, we investigated the in vivo oxidative metabolism of exercising muscle in HD patients. Ventilatory and cardiometabolic parameters and plasma lactate concentrations were monitored during incremental cardiopulmonary exercise in twenty-five HD subjects and twenty-five healthy subjects. The total exercise capacity was normal in HD subjects but notably the HD patients and presymptomatic mutation carriers had a lower anaerobic threshold than the control subjects. The low anaerobic threshold of HD patients was associated with an increase in the concentration of plasma lactate. We also analyzed in vitro muscular cell cultures and found that HD cells produce more lactate than the cells of healthy subjects. Finally, we analyzed skeletal muscle samples by electron microscopy and we observed striking mitochondrial structural abnormalities in two out of seven HD subjects. Our findings confirm mitochondrial abnormalities in HD patients' skeletal muscle and suggest that the mitochondrial dysfunction is reflected functionally in a low anaerobic threshold and an increased lactate synthesis during intense physical exercise.

Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.

<h4>Background</h4>Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation.<h4>Methodology/principal findings</h4>We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo.<h4>Conclusions/significance</h4>Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.

The ubiquitin-interacting motif (UIM) is a short peptide with dual function of binding ubiquitin (Ub) and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12) both in free and Ub-bound forms. The solution structure of free AT3-UIM12 consists of two α-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates.

Most genome-wide association (GWA) studies have focused on populations of European ancestry with limited assessment of the influence of the sequence variants on populations of other ethnicities. To determine whether markers that we have recently shown to associate with Bone Mineral Density (BMD) in Europeans also associate with BMD in East-Asians we analysed 50 markers from 23 genomic loci in samples from Korea (n = 1,397) and two Chinese Hong Kong sample sets (n = 3,869 and n = 785). Through this effort we identified fourteen loci that associated with BMD in East-Asian samples using a false discovery rate (FDR) of 0.05; 1p36 (ZBTB40, P = 4.3×10(-9)), 1p31 (GPR177, P = 0.00012), 3p22 (CTNNB1, P = 0.00013), 4q22 (MEPE, P = 0.0026), 5q14 (MEF2C, P = 1.3×10(-5)), 6q25 (ESR1, P = 0.0011), 7p14 (STARD3NL, P = 0.00025), 7q21 (FLJ42280, P = 0.00017), 8q24 (TNFRSF11B, P = 3.4×10(-5)), 11p15 (SOX6, P = 0.00033), 11q13 (LRP5, P = 0.0033), 13q14 (TNFSF11, P = 7.5×10(-5)), 16q24 (FOXL1, P = 0.0010) and 17q21 (SOST, P = 0.015). Our study marks an early effort towards the challenge of cataloguing bone density variants shared by many ethnicities by testing BMD variants that have been established in Europeans, in East-Asians.

Raf-1 kinase inhibitor protein (RKIP) plays a pivotal role in modulating multiple signaling networks. Here we report backbone and side chain resonance assignments of uniformly (15)N, (13)C labeled human RKIP.

<h4>Background</h4>Systematic evaluation of psychosocial distress in oncology outpatients is an important issue. We assessed feasibility and benefit of standardized routine screening using the Distress Thermometer (DT) and Problem List (PL) in all patients of our community-based hematooncology group practice.<h4>Patients and methods</h4>One thousand four hundred forty-six patients were screened between July 2008 and September 2008. Five hundred randomly selected patients were sent a feedback form.<h4>Results</h4>The average distress level was 4.7, with 37% indicating a distress level >5. Patients with nonmalignant diseases (81% autoimmune diseases or hereditary hemochromatosis) showed the highest distress level of 5.2. Most distressed were patients who just learned about relapse or metastases (6.4), patients receiving best supportive care (5.4) and patients receiving adjuvant antihormonal therapy (5.4). Ninety-seven percent of patients appreciated to speak to their doctor about their distress. Fifty-six percent felt better than usual after this consultation.<h4>Conclusion</h4>DT and PL are feasible instruments to measure distress in hematooncology outpatients receiving routine care. DT and PL are able to improve doctor-patient communication and thus should be implemented in routine patient care. The study shows that distress is distributed differently between individuals, disease groups and treatment phases.

Human anal cancers are associated with high-risk human papillomaviruses (HPV) that cause other anogenital cancers and head and neck cancers. As with other cancers, HPV16 is the most common high-risk HPV in anal cancers. We describe the generation and characterization of a mouse model for human anal cancer. This model makes use of K14E6 and K14E7 transgenic mice in which the HPV16 E6 and E7 genes are directed in their expression to stratified squamous epithelia. HPV16 E6 and E7 possess oncogenic properties including, but not limited to, their capacity to inactivate the cellular tumor suppressors p53 and pRb, respectively. Both E6 and E7 were found to be functionally expressed in the anal epithelia of K14E6/K14E7 transgenic mice. To assess the susceptibility of these mice to anal cancer, mice were treated topically with dimethylbenz[a]anthracene (DMBA), a chemical carcinogen that is known to induce squamous cell carcinomas in other sites. Nearly 50% of DMBA-treated HPV16 E6/E7 transgenic mice showed overt signs of tumors, whereas none of the like-treated nontransgenic mice showed tumors. Histopathologic analyses confirmed that the HPV16 transgenic mice were increased in their susceptibility to anal cancers and precancerous lesions. Biomarker analyses demonstrated that these mouse anal cancers exhibit properties that are similar to those observed in HPV-positive precursors to human anal cancer. This is the first mouse model for investigating the contributions of viral and cellular factors in anal carcinogenesis, and should provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer.

Muscle fibres have different properties with respect to force, contraction speed, endurance, oxidative/glycolytic capacity etc. Although adult muscle fibres are normally post-mitotic with little turnover of cells, the physiological properties of the pre-existing fibres can be changed in the adult animal upon changes in usage such as after exercise. The signal to change is mainly conveyed by alterations in the patterns of nerve-evoked electrical activity, and is to a large extent due to switches in the expression of genes. Thus, an excitation-transcription coupling must exist. It is suggested that changes in nerve-evoked muscle activity lead to a variety of activity correlates such as increases in free intracellular Ca(2+) levels caused by influx across the cell membrane and/or release from the sarcoplasmatic reticulum, concentrations of metabolites such as lipids and ADP, hypoxia and mechanical stress. Such correlates are detected by sensors such as protein kinase C (PKC), calmodulin, AMP-activated kinase (AMPK), peroxisome proliferator-activated receptor δ (PPARδ), and oxygen dependent prolyl hydroxylases that trigger intracellular signaling cascades. These complex cascades involve several transcription factors such as nuclear factor of activated T-cells (NFAT), myocyte enhancer factor 2 (MEF2), myogenic differentiation factor (myoD), myogenin, PPARδ, and sine oculis homeobox 1/eyes absent 1 (Six1/Eya1). These factors might act indirectly by inducing gene products that act back on the cascade, or as ultimate transcription factors binding to and transactivating/repressing genes for the fast and slow isoforms of various contractile proteins and of metabolic enzymes. The determination of size and force is even more complex as this involves not only intracellular signaling within the muscle fibres, but also muscle stem cells called satellite cells. Intercellular signaling substances such as myostatin and insulin-like growth factor 1 (IGF-1) seem to act in a paracrine fashion. Induction of hypertrophy is accompanied by the satellite cells fusing to myofibres and thereby increasing the capacity for protein synthesis. These extra nuclei seem to remain part of the fibre even during subsequent atrophy as a form of muscle memory facilitating retraining. In addition to changes in myonuclear number during hypertrophy, changes in muscle fibre size seem to be caused by alterations in transcription, translation (per nucleus) and protein degradation.

<h4>Background</h4>The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS)-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC) death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6) plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis.<h4>Results</h4>We have shown that RGCs exposed to hypoxia (1%) or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM), revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6.<h4>Conclusion</h4>Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder that usually presents in adulthood with characteristic motor and cognitive features and with variable and diverse psychiatric disturbances. Following the discovery of the causative defect in the HTT gene in 1993, great advances in understanding the pathogenesis of HD have been made, yet no effective disease-modifying therapy has been identified. In this new era of HD research, we have seen the emergence of a number of large clinical trials, the systematic search for novel biomarkers and the recent initiation of the first pre-manifest HD clinical studies. In this review, we seek to provide an overview of the clinical and genetic features of HD together with a summary of clinical research at this time.

Bones provide mechanical and protective function, while also serving as housing for marrow and a site for regulation of calcium ion homeostasis. The properties of bones do not remain constant with age; rather, they change throughout life, in some cases improving in function, but in others, function deteriorates. Here we review the modifications in the mechanical function and shape of bones, the bone cells, the matrix they produce, and the mineral that is deposited on this matrix, while presenting recent theories about the factors leading to these changes.

Recent development of proteomic array technology, including protein profiling coupling ProteinChip array with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS), provides a potentially powerful tool for discovery of new biomarkers by comparison of its profiles according to patient phenotypes. We used this approach to identify the host factors associated with treatment response in patients with chronic hepatitis C (CHC) receiving a 48-wk course of pegylated interferon (PEG-IFN) alpha 2b plus ribavirin (RBV). Protein profiles of pretreatment serum samples from 32 patients with genotype 1b and high viral load were conducted by SELDI-TOF/MS by using the three different ProteinChip arrays (CM10, Q10, IMAC30). Proteins showed significantly different peak intensities between sustained virological responders (SVRs), and non-SVRs were identified by chromatography, SDS-PAGE, TOF/MS and tandem mass spectrometry (MS/MS) assay. Eleven peak intensities were significantly different between SVRs and non-SVRs. The three SVR-increased peaks could be identified as two apolipoprotein (Apo) fragments and albumin and, among the eight non-SVR-increased proteins, four peaks identified as two iron-related and two fibrogenesis-related protein fragments, respectively. Multivariate analysis showed that the serum ferritin and three peak intensity values (Apo A1, hemopexin and transferrin) were independent variables associated with SVRs, and the area under the receiver operating characteristic (ROC) curves for SVR prediction by using the Apo A1/hemopexin and hemopexin/transferrin were 0.964 and 0.936. In conclusion, pretreatment serum protein profiling by SELDI-TOF/MS is variable for identification of response-related host factors, which are useful for treatment efficacy prediction in CHC receiving PEG-IFN plus RBV. Our data also may help us understand the mechanism for treatment resistance and development of more effective antiviral therapy targeted toward the modulation of lipogenesis or iron homeostasis in CHC patients.

Huntington disease (HD) is a dominantly inherited progressive neurological disease characterized by chorea, an involuntary brief movement that tends to flow between body regions. HD is typically diagnosed based on clinical findings in the setting of a family history and may be confirmed with genetic testing. Predictive testing is available to those at risk, but only experienced clinicians should perform the counseling and testing. Multiple areas of the brain degenerate mainly involving the neurotransmitters dopamine, glutamate, and γ-aminobutyric acid. Although pharmacotherapies theoretically target these neurotransmitters, few well-conducted trials for symptomatic or neuroprotective interventions yielded positive results. Tetrabenazine (TBZ) is a dopamine-depleting agent that may be one of the more effective agents for reducing chorea, although it has a risk of potentially serious adverse effects. Some newer antipsychotic agents, such as olanzapine and aripiprazole, may have adequate efficacy with a more favorable adverse-effect profile than older antipsychotic agents for treating chorea and psychosis. This review will address the epidemiology and diagnosis of HD as background for understanding potential pharmacological treatment options. Because TBZ is the only US Food and Drug Administration-approved medication in the United States for HD, the focus of this review will be on its pharmacology, efficacy, safety, and practical uses. There are no current treatments to change the course of HD, but education and symptomatic therapies can be effective tools for clinicians to use with patients and families affected by HD.

At a first glance, ribosome biogenesis and chromatin remodeling are quite different processes, but they share a common problem involving interactions between charged nucleic acids and small basic proteins that may result in unwanted intracellular aggregations. The multifunctional nuclear acidic chaperone NPM1 (B23/nucleophosmin) is active in several stages of ribosome biogenesis, chromatin remodeling, and mitosis as well as in DNA repair, replication and transcription. In addition, NPM1 plays an important role in the Myc-ARF-p53 pathway as well as in SUMO regulation. However, the relative importance of NPM1 in these processes remains unclear. Provided herein is an update on the expanding list of the diverse activities and interacting partners of NPM1. Mechanisms of NPM1 nuclear export functions of NPM1 in the nucleolus and at the mitotic spindle are discussed in relation to tumor development. It is argued that the suggested function of NPM1 as a histone chaperone could explain several, but not all, of the effects observed in cells following changes in NPM1 expression. A future challenge is to understand how NPM1 is activated, recruited, and controlled to carry out its functions.

Surgical repair of a discontinuity in traumatized or degenerated soft tissues is traditionally accomplished using sutures. A current trend is to reinforce this primary repair with surgical grafts, meshes, or patches secured with perforating mechanical devices (i.e., sutures, staples, or tacks). These fixation methods frequently lead to chronic pain and mesh detachment. We developed a series of biodegradable adhesive polymers that are synthetic mimics of mussel adhesive proteins (MAPs), composed of 3,4-dihydroxyphenylalanine (DOPA)-derivatives, polyethylene glycol (PEG), and polycaprolactone (PCL). These polymers can be cast into films, and their mechanical properties, extent of swelling, and degradation rate can be tailored through the composition of the polymers as well as blending with additives. When coated onto a biologic mesh used for hernia repair, these adhesive constructs demonstrated adhesive strengths significantly higher than fibrin glue. With further development, a precoated bioadhesive mesh may represent a new surgical option for soft tissue repair.

The promise of personalised medicine is now a clinical reality, with colorectal cancer genetics at the forefront of this next major advance in clinical medicine. This is no more evident than in the recent advances in testing of colorectal cancers for specific molecular alterations in order to guide treatment with the monoclonal antibody therapies cetuximab and panitumumab, which target the epidermal growth factor receptor. In this review, genetic mechanisms of colorectal cancer and how these alterations relate to emerging biomarkers for early detection and risk stratification (diagnostic markers), prognosis (prognostic markers) and the prediction of treatment responses (predictive markers) are examined.

The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease β-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.

The possible mechanism of casein aggregation and micelle buildup was studied in a new approach by letting α-casein adsorb from low concentration (0.1 mg·ml(-1)) solutions onto the charged surfaces of polyelectrolyte films. It was found that α-casein could adsorb onto both positively and negatively charged surfaces. However, only when its negative phosphoseryl clusters remained free, i.e. when it adsorbed onto a negative surface, could calcium phosphate (CaP) nanoclusters bind to the casein molecules. Once the CaP clusters were in place, step-by-step building of multilayered casein architectures became possible. The presence of CaP was essential; neither Ca(2+) nor phosphate could alone facilitate casein aggregation. Thus, it seems that CaP is the organizing motive in the casein micelle formation. Atomic force microscopy revealed that even a single adsorbed casein layer was composed of very small (in the range of tens of nanometers) spherical forms. The stiffness of the adsorbed casein layer largely increased in the presence of CaP. On this basis, we can imagine that casein micelles emerge according to the following scheme. The amphipathic casein monomers aggregate into oligomers via hydrophobic interactions even in the absence of CaP. Full scale, CaP-carrying micelles could materialize by interlocking these casein oligomers with CaP nanoclusters. Such a mechanism would not contradict former experimental results and could offer a synthesis between the submicelle and the block copolymer models of casein micelles.

The genomic revolution has identified therapeutic targets for a plethora of diseases, creating a need to develop robust technologies for combination drug therapy. In the present work, we describe a self-assembled polymeric nanoparticle (NP) platform to target and control precisely the codelivery of drugs with varying physicochemical properties to cancer cells. As proof of concept, we codelivered cisplatin and docetaxel (Dtxl) to prostate cancer cells with synergistic cytotoxicity. A polylactide (PLA) derivative with pendant hydroxyl groups was prepared and conjugated to a platinum(IV) [Pt(IV)] prodrug, c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)COOH)(OH)Cl(2)] [PLA-Pt(IV)]. A blend of PLA-Pt(IV) functionalized polymer and carboxyl-terminated poly(D,L-lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer in the presence or absence of Dtxl, was converted, in microfluidic channels, to NPs with a diameter of ∼100 nm. This process resulted in excellent encapsulation efficiency (EE) and high loading of both hydrophilic platinum prodrug and hydrophobic Dtxl with reproducible EEs and loadings. The surface of the NPs was derivatized with the A10 aptamer, which binds to the prostate-specific membrane antigen (PSMA) on prostate cancer cells. These NPs undergo controlled release of both drugs over a period of 48-72 h. Targeted NPs were internalized by the PSMA-expressing LNCaP cells via endocytosis, and formation of cisplatin 1,2-d(GpG) intrastrand cross-links on nuclear DNA was verified. In vitro toxicities demonstrated superiority of the targeted dual-drug combination NPs over NPs with single drug or nontargeted NPs. This work reveals the potential of a single, programmable nanoparticle to blend and deliver a combination of drugs for cancer treatment.

Sleep and wakefulness are regulated primarily by inhibitory interactions between the hypothalamus and brainstem. The expression of the states of rapid eye movement (REM) sleep and non-REM (NREM) sleep also are correlated with the activity of groups of REM-off and REM-on neurons in the dorsal brainstem. However, the contribution of ventral brainstem nuclei to sleep regulation has been little characterized to date. Here we examined sleep and wakefulness in mice deficient in a homeobox transcription factor, Goosecoid-like (Gscl), which is one of the genes deleted in DiGeorge syndrome or 22q11 deletion syndrome. The expression of Gscl is restricted to the interpeduncular nucleus (IP) in the ventral region of the midbrain-hindbrain transition. The IP has reciprocal connections with several cell groups implicated in sleep/wakefulness regulation. Although Gscl(-/-) mice have apparently normal anatomy and connections of the IP, they exhibited a reduced total time spent in REM sleep and fewer REM sleep episodes. In addition, Gscl(-/-) mice showed reduced theta power during REM sleep and increased arousability during REM sleep. Gscl(-/-) mice also lacked the expression of DiGeorge syndrome critical region 14 (Dgcr14) in the IP. These results indicate that the absence of Gscl and Dgcr14 in the IP results in altered regulation of REM sleep.

The production of neurons from neural progenitor cells, the growth of axons and dendrites and the formation and reorganization of synapses are examples of neuroplasticity. These processes are regulated by cell-autonomous and intercellular (paracrine and endocrine) programs that mediate responses of neural cells to environmental input. Mitochondria are highly mobile and move within and between subcellular compartments involved in neuroplasticity (synaptic terminals, dendrites, cell body and the axon). By generating energy (ATP and NAD(+)), and regulating subcellular Ca(2+) and redox homoeostasis, mitochondria may play important roles in controlling fundamental processes in neuroplasticity, including neural differentiation, neurite outgrowth, neurotransmitter release and dendritic remodelling. Particularly intriguing is emerging data suggesting that mitochondria emit molecular signals (e.g. reactive oxygen species, proteins and lipid mediators) that can act locally or travel to distant targets including the nucleus. Disturbances in mitochondrial functions and signalling may play roles in impaired neuroplasticity and neuronal degeneration in Alzheimer's disease, Parkinson's disease, psychiatric disorders and stroke.

Natural antisense transcripts represent a class of regulatory RNA molecules, which are characterized by their complementary sequence to another RNA transcript. Extensive sequencing efforts suggest that natural antisense transcripts are prevalent throughout the mammalian genome; however, their biological significance has not been well defined. We performed a loss-of-function RNA interference (RNAi) screen, which targeted 797 evolutionary conserved antisense transcripts, and found evidence for a regulatory role for a number of natural antisense transcripts. Specifically, we found that natural antisense transcripts for CCPG1 and RAPGEF3 may functionally disrupt signaling pathways and corresponding biological phenotypes, such as cell viability, either independently or in parallel with the corresponding sense transcript. Our results show that the large-scale siRNA screen can be applied to evaluate natural antisense transcript modulation of fundamental cellular events.

<h4>Background</h4>Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers.<h4>Methodology/principal findings</h4>A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC₅₀ concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation.<h4>Conclusions/significance</h4>Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling.

<h4>Objective</h4>The gene coding for the 5-HT transporter (5-HTT) is considered as a candidate gene for bipolar disorder, either as a "vulnerability" or as a "modifying the phenotype" gene. Psychotic symptoms occur at least once in one bipolar patient out of two, the relevant risk factors being insufficiently understood. The gene × environment interaction approach offers the opportunity to disentangle the latter, including childhood sexual abuse and cannabis abuse.<h4>Methods</h4>We investigated the 5-HTTLPR of the 5-HTT gene (G) and the presence of childhood sexual abuse and cannabis comorbidity (E) in 137 bipolar patients with (versus without) lifetime psychotic symptoms.<h4>Results</h4>The short allele and cannabis abuse were significantly more frequent among patients with psychotic symptoms than in those without (p=0.01 and p=0.004, respectively), while childhood sexual abuse was not. Complex interactions were found between presence of the short allele, cannabis abuse or dependence and childhood sexual abuse.<h4>Conclusions</h4>The short allele of the 5-HTTLPR polymorphism of the 5-HTT gene was a risk factor for psychotic symptoms in bipolar disorder in the present sample, directly but also indirectly, through the presence of cannabis abuse or dependence, as an exacerbating factor heightening psychotic symptoms.

Clear cell meningiomas (CCM) can be difficult to distinguish from metastatic clear cell renal cell carcinomas by standard light microscopy. Distinction is important in deciding patient management and establishing prognosis. The purpose of this study is to evaluate the use of immunomarkers CA9, CD10, and RCC in differentiating between CCM and clear cell renal cell carcinoma. The study retrospectively reviewed the clinicopathologic features of 18 patients with CCM (9 females, 9 males; age range at the time of surgery 16 to 86 y) including immunostaining results with antibodies to CA9, CD10, and RCC. Immunostaining results were compared with those found in 26 cases of clear cell renal cell carcinoma. The most common sites of origin for the CCM included the meninges overlying the frontal lobe (n=7), cavernous sinus (n=3), and cerebellopontine angle/posterior fossa (n=2). All tumors had at least a 10% clear cell component (mean 41%). All tumors showed a sheet-like growth pattern. Other commonly observed morphologic features included increased cellularity (n=12), nucleolation (n=8), small cell change (n=6), microcalcifications (n=5), and necrosis (n=5). A mean of 1.9 mitotic figures per 10 high-power fields and a mean Ki-67 labeling index of 12.1% were observed. Seven tumors (38.9%) showed CA9 immunoreactivity, 5 tumors (27.8%) CD10 staining, and 0 cases showed RCC staining. Immunostaining results observed in the clear cell renal cell carcinoma group included 93.8% CA9 staining (15/16 cases evaluated), 100% CD10 staining (15/15 cases), and 36.4% RCC staining (4/11 cases). Follow-up was available in 16 CCM patients (mean follow-up of 58.9 mo); 10 patients (62.5%) developed at least 1 recurrence requiring surgical intervention. In conclusion, meningiomas with at least a 10% clear cell component tend to behave in a more aggressive fashion with increased risk of recurrence. Immunohistochemical staining with antibodies to CA9, CD10, and RCC are potentially useful in differentiating CCM from metastatic renal cell carcinoma. In the majority of cases in which immunostaining was observed in meningiomas, staining was focal (involving <5% of neoplastic cells) in comparison with CA9 and CA10 immunostaining in renal cell carcinomas in which more than 50% of tumor cells stained the majority of cases.

This study reports the prevalence of child (CSA) and adult (ASA) sexual abuse among 535 African American HIV serodiscordant couples from four major United State cities, and its relationship to personal and couple related vulnerabilities and HIV risk factors. As part of a randomized, clinical trial, CSA and ASA histories were obtained through face-to-face interviews. Results indicate that HIV positive women were significantly more likely to report one kind of abuse (32.32%), either before or since age 18 or both (32.6%). HIV-positive men (34.9%) were significantly more likely to report CSA than HIV-negative men (22.0%). Overall, 72% of couples reported that one or both had CSA histories. These findings underscore the heightened emotional vulnerability, and STI and HIV transmission risk taking practices, associated with sexual abuse. Sexual abuse histories among couples should be assessed to better understand how these histories may contribute to couples dynamics and risk-taking practices.

This paper reports baseline behavioral and biological data collected from a cohort of 535 African American HIV serodiscordant couples enrolled in the Eban study across four urban metro areas. Data were collected on (1) the prevalence of risky sexual behaviors that occur within a couple and with concurrent sexual partners, (2) the STD prevalence for each member of the couple and (3) the correlates of STDs in the male partner as well as in the female partner. Presentation of the sociodemographic characterization and HIV risk behavior profiles of African American HIV serodiscordant couples represents an important initial description of a hidden, vulnerable population. Future research should be conducted with diverse samples of African American couples (i.e., younger couples, non-stable couples) to explore other potential correlates of STD prevalence.

<h4>Objective</h4>We previously observed the association of the co-occurrence of the HLA-DRB1 shared epitope (SE) and RANKL single-nucleotide polymorphisms (SNPs) with younger age at the onset of rheumatoid arthritis (RA) in 182 rheumatoid factor (RF)-positive European American patients with early-onset RA. The aim of this study was to fine-map the 48-kb RANKL region in the extended cohort of 210 European American RF-positive patients with early RA, to seek replication of RA-associated SNPs in additional RA cohorts of 501 European Americans and 298 African Americans, and to explore the functional consequences of RA-associated SNPs.<h4>Methods</h4>SNP genotyping was conducted using pyrosequencing or TaqMan polymerase chain reaction (PCR) assays. Associations of rs7984870 with RANKL expression in plasma, peripheral blood mononuclear cells, and isolated T cells were quantified using enzyme-linked immunosorbent assay and reverse transcription-PCR. Site-directed mutagenesis of rs7984870 within the 2-kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.<h4>Results</h4>A single promoter SNP, rs7984870, was consistently significantly associated with earlier age at the onset of RA in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody) RA cohorts but not in seronegative RA patients. The C risk allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive patients with RA, significantly elevated RANKL messenger RNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to the transcription factor SOX5.<h4>Conclusion</h4>The RANKL promoter allele that increased transcription levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to a younger age at the onset of RA in seropositive European American and African American patients.

Generation of reactive oxygen species (ROS) is a common denominator in many conditions leading to cell death in the cochlea, yet little is known of the cochlea's endogenous mechanisms involved in preventing oxidative stress and its consequences in the cochlea. We have recently described a corticotropin-releasing factor (CRF) signaling system in the inner ear involved in susceptibility to noise-induced hearing loss. We use biochemical and proteomics assays to define further the role of CRF signaling in the response of cochlear cells to aminoglycoside exposure. We demonstrate that activity via the CRF(2) class of receptors protects against aminoglycoside-induced ROS production and activation of cell death pathways. This study suggests for the first time a role for CRF signaling in protecting the cochlea against oxidative stress, and our proteomics data suggest novel mechanisms beyond induction of free radical scavengers that are involved in its protective mechanisms.

<h4>Background</h4>Conflicting results have been reported in studies evaluating the relationship between serum markers of iron overload, liver iron deposits, and HFE mutations (C282Y and H63D) in chronic hepatitis C patients, and also their impact on the response to therapy in these patients.<h4>Aim</h4>To evaluate the role of HFE mutations in the severity of liver disease and in the response to therapy in chronic hepatitis C.<h4>Methods</h4>Two hundred and sixty-four hepatitis C patients treated with standard interferon and ribavirin were divided into two groups according to type of antiviral response: sustained virological response (SVR) and nonresponse or relapse. We evaluated the relationship between HFE mutation and the type of antiviral response, clinical data, biochemical tests, liver histopathology, virological data, and HFE mutations.<h4>Results</h4>Of the 264 patients, 88 (32.1%) had SVR whereas 67.9% had nonresponse or relapse. Liver iron deposits were observed in 49.2% of the patients. The factors associated with SVR were hepatitis C virus genotype 2 or 3, transferrin saturation value of 45% or less, and detection of the H63D mutation. HFE mutation was more frequent in patients with iron deposits, but without association with serum iron biochemistry or severity of liver disease. Steatosis was more frequent in patients with liver iron deposits. CONCLUSION THE: H63D mutation was an independent factor associated with SVR in chronic hepatitis C patients, as also were hepatitis C virus genotype 2 or 3 and transferrin saturation value of 45% or less. Moreover, the H63D mutation was associated with liver iron deposits.

Current bone tissue engineering strategies aim to grow a tissue similar to native bone by combining cells and biologically active molecules with a scaffold material. In this study, a macroporous scaffold made from the seaweed-derived polymer alginate was synthesized and mineralized for cell-based bone tissue engineering applications. Nucleation of a bone-like hydroxyapatite mineral was achieved by incubating the scaffold in modified simulated body fluids (mSBF) for 4 weeks. Analysis using scanning electron microscopy and energy dispersive x-ray analysis indicated growth of a continuous layer of mineral primarily composed of calcium and phosphorous. X-ray diffraction analysis showed peaks associated with hydroxyapatite, the major inorganic constituent of human bone tissue. In addition to the mineral characterization, the ability to control nucleation on the surface, into the bulk of the material, or on the inner pore surfaces of scaffolds was demonstrated. Finally, human MSCs attached and proliferated on the mineralized scaffolds and cell attachment improved when seeding cells on mineral coated alginate scaffolds. This novel alginate- HAP composite material could be used in bone tissue engineering as a scaffold material to deliver cells, and perhaps also biologically active molecules.

The division of the neocortex into functional areas (the cortical map) differs little between individuals, although brain lesions in development can lead to substantial re-organization of regional identity. We are studying how the cortical map is established in the human brain as a first step towards understanding this plasticity. Previous work on rodent development has identified certain transcription factors (e.g. Pax6, Emx2) expressed in gradients across the neocortex that appear to control regional expression of cell adhesion molecules and organization of area-specific thalamocortical afferent projections. Although mechanisms may be shared, the human neocortex is composed of different and more complex local area identities. Using Affymetrix gene chips of human foetal brain tissue from 8 to 12.5 post-conceptional weeks [PCW, equivalent to Carnegie stage (CS) 23, to Foetal stage (F) 4], human material obtained from the MRC-Wellcome Trust Human Developmental Biology Resource (http://www.hdbr.org), we have identified a number of genes that exhibit gradients along the anterior-posterior axis of the neocortex. Gene probe sets that were found to be upregulated posteriorally compared to anteriorally, included EMX2, COUPTFI and FGF receptor 3, and those upregulated anteriorally included cell adhesion molecules such as cadherins and protocadherins, as well as potential motor cortex markers and frontal markers (e.g. CNTNAP2, PCDH17, ROBO1, and CTIP2). Confirmation of graded expression for a subset of these genes was carried out using real-time PCR. Furthermore, we have established a dissociation cell culture model utilizing tissue dissected from anteriorally or posteriorally derived developing human neocortex that exhibits similar gradients of expression of these genes for at least 72 h in culture.

Three independent risk factors (immature gestation, absence of antenatal glucocorticoid exposure, and presence of the rs2817399(A) allele of the gene TFAP2B) are associated with patent ductus arteriosus (PDAs) that fail to close during prostaglandin inhibition. We hypothesized that these three factors may affect a common set of genes that increase the risk of persistent PDA after birth. We studied baboon ductus from term, preterm, and glucocorticoid-treated preterm fetuses and found that both immature gestation and absence of antenatal glucocorticoid exposure decreased RNA expression of calcium- and potassium-channel genes involved in oxygen-induced constriction, and phosphodiesterase genes (that modulate cAMP/cGMP signaling). Ductus obtained from second trimester human pregnancies were genotyped for TFAP2B polymorphisms. When present, the rs2817399(A) allele also was associated with decreased expression of calcium- and potassium-channel genes. In contrast, alleles of two other TFAP2B polymorphisms, rs2817419(G) and rs2635727(T), which are not related to the incidence of PDA after birth, had no effect on RNA expression. In conclusion, three calcium- and potassium-channel genes (CACNA1G/ alpha1G, CACNB 2/CaL-beta2, and KCNA2/ Kv1.2) were similarly affected by each of the PDA risk factors. We speculate that these channels may play a significant role in closing the preterm ductus during prostaglandin inhibition and may be potential targets for future pharmacologic manipulations.

The TFAP2C transcription factor is involved in mammary development, differentiation, and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERalpha) and ERBB2 (Her2) in breast carcinomas. However, the role of TFAP2C in different breast cancer phenotypes has not been examined in detail. To develop a more complete characterization of TFAP2C target genes, ChIP-seq with anti-TFAP2C antibody and expression arrays with TFAP2C knock down were analyzed in MCF-7 breast carcinoma cells. Genomic sequences common to the ChIP-seq data set defined the consensus sequence for TFAP2C chromatin binding as the nine base sequence SCCTSRGGS (S = G/C, r = A/G), which closely matches the previously defined optimal in vitro binding site. Comparing expression arrays before and after knock down of TFAP2C with ChIP-seq data demonstrated a conservative estimate that 8% of genes altered by TFAP2C expression are primary target genes and includes genes that are both induced and repressed by TFAP2C. A set of 447 primary target genes of TFAP2C was identified, which included ESR1 (ERalpha), FREM2, RET, FOXA1, WWOX, GREB1, MYC, and members of the retinoic acid response pathway. The identification of ESR1, WWOX, GREB1, and FOXA1 as primary targets confirmed the role of TFAP2C in hormone response. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer and its target genes are different than for the Her2 breast cancer phenotype.

Chromatin-modifying complexes typically contain signature domains that either have catalytic activity or recognize and bind to specific histone modifications such as acetylation, methylation, and phosphorylation. Despite tremendous progress in this area, much remains to be learned in particular about the mechanistic functions of less well characterized signature domains. One such module is the evolutionary conserved YEATS domain, found in a variety of chromatin-modifying and transcription complexes from yeast to human. Three yeast proteins contain a YEATS domain, including Yaf9, a subunit of both the histone variant H2A.Z deposition complex SWR1-C and the histone acetyltransferase complex NuA4. The three-dimensional structure of the YEATS domain from Yaf9 was solved recently, revealing the existence of three distinct structural regions. One region is characterized by a shallow groove that might constitute a potential acetyl-lysine binding pocket, raising questions about potential protein interaction partners of the Yaf9 YEATS domain.

<h4>Unlabelled</h4>Hereditary hemochromatosis (HH) is a common inherited iron overload disorder. The vast majority of patients carry the missense Cys282Tyr mutation of the HFE gene. Hepcidin, the central regulator of iron homeostasis, is deficient in HH, leading to unchecked iron absorption and subsequent iron overload. The bone morphogenic protein (BMP)/small mothers against decapentaplegic (Smad) signaling cascade is central to the regulation of hepcidin. Recent data from HH mice models indicate that this pathway may be defective in the absence of the HFE protein. Hepatic BMP/Smad signaling has not been characterized in a human HFE-HH cohort to date. Hepatic expression of BMP/Smad-related genes was examined in 20 HFE-HH males with significant iron overload, and compared to seven male HFE wild-type controls using quantitative real-time reverse transcription polymerase chain reaction. Hepatic expression of BMP6 was appropriately elevated in HFE-HH compared to controls (P = 0.02), likely related to iron overload. Despite this, no increased expression of the BMP target genes hepcidin and Id1 was observed, and diminished phosphorylation of Smad1/Smad5/Smad8 protein relative to iron burden was found upon immunohistochemical analysis, suggesting that impaired BMP signaling occurs in HFE-HH. Furthermore, Smad6 and Smad7, inhibitors of BMP signaling, were up-regulated in HFE-HH compared to controls (P = 0.001 and P = 0.018, respectively).<h4>Conclusion</h4>New data arising from this study suggest that impaired BMP signaling underlies the hepcidin deficiency of HFE-HH. Moreover, the inhibitory Smads, Smad6, and Smad7 are identified as potential disruptors of this signal and, hence, contributors to the pathogenesis of this disease.

Self-renewal in the intestinal epithelia is fueled by a population of undifferentiated intestinal stem cells (ISCs) that give rise to daughter or progenitor cells, which can subsequently differentiate into the mature cell types required for normal gut function. The cellular signals that regulate self-renewal are poorly understood and the factors that mediate the transition from a stem cell to a progenitor cell in the gut are unknown. Recent studies have suggested that ISCs are located either at the crypt base interspersed between the Paneth cells (eg, Lgr-5+ve cells) or at or near position 4 within the intestinal crypt (eg, DCAMKL-1 or Bmi-1+ve cells). This raises the possibility that distinct stem cell regions exist in the crypts and that ISC's state of activation will determine how the self-renewal is regulated in the intestinal tract.

The non-white blood cell mesenchymal elements of the intestinal lamina propria are the myofibroblasts, fibroblasts, pericytes, stromal stem cells, muscularis mucosae, and the smooth muscle of the villus core associated with the lymphatic lacteal. We review the functional anatomy of these mesenchymal cells, what is known about their origin in the embryo and their replacement in adults, their putative role in intestinal mucosal morphogenesis, and the intestinal stem cell niche, and we consider new information about myofibroblasts as nonprofessional immune cells. Although our knowledge of the function of mesenchymal cells in intestinal disease is rudimentary, we briefly consider here their roles in cancer and intestinal inflammation.

<h4>Unlabelled</h4>Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate oxidase (NOX) is required for liver fibrosis. This study investigates the role of NOX in ROS production and the differential contribution of NOX from bone marrow (BM)-derived and non-BM-derived liver cells. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline-deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47phox knockout [KO]), a component of NOX. The p47phox KO chimeric mice were generated by the combination of liposomal clodronate injection, irradiation, and BM transplantation of p47phox KO BM into WT recipients and vice versa. Upon BDL, chimeric mice with p47phox KO BM-derived cells, including Kupffer cells, and WT endogenous liver cells showed a ∼25% reduction of fibrosis, whereas chimeric mice with WT BM-derived cells and p47phox KO endogenous liver cells, including hepatic stellate cells, showed a ∼60% reduction of fibrosis. In addition, p47phox KO compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury, but a ∼50% reduction in fibrosis. Cultured WT and p47phox KO hepatocytes treated with free fatty acids had a similar increase in lipid accumulation. Free fatty acids promoted a 1.5-fold increase in ROS production both in p47phox KO and in WT hepatocytes.<h4>Conclusion</h4>NOX in both BM-derived and non-BM-derived cells contributes to liver fibrosis. NOX does not play a role in experimental steatosis and the generation of ROS in hepatocytes, but exerts a key role in fibrosis.

Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ∼60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 h. Hydrazones formed with the PMCA cross-linker showed no difference in release rates at pH 7.4 and 5.0.

Regulated secretion of neurotransmitters and neurohumoral factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoral factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 proteins in the soluble fraction and 384 membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease.

Plasma biomarkers studies are based on the differential expression of proteins between different treatment groups or between diseased and control populations. Most mass spectrometry-based methods of protein quantitation, however, are based on the detection and quantitation of peptides, not intact proteins. For peptide-based protein quantitation to be accurate, the digestion protocols used in proteomic analyses must be both efficient and reproducible. There have been very few studies, however, where plasma denaturation/digestion protocols have been compared using absolute quantitation methods. In this paper, 14 combinations of heat, solvent [acetonitrile, methanol, trifluoroethanol], chaotropic agents [guanidine hydrochloride, urea], and surfactants [sodium dodecyl sulfate (SDS) and sodium deoxycholate (DOC)] were compared with respect to their effectiveness in improving subsequent tryptic digestion. These digestion protocols were evaluated by quantitating the production of proteotypic tryptic peptides from 45 moderate- to high-abundance plasma proteins, using tandem mass spectrometry in multiple reaction monitoring mode, with a mixture of stable-isotope labeled analogues of these proteotypic peptides as internal standards. When the digestion efficiencies of these 14 methods were compared, we found that both of the surfactants (SDS and DOC) produced an increase in the overall yield of tryptic peptides from these 45 proteins, when compared to the more commonly used urea protocol. SDS, however, can be a serious interference for subsequent mass spectrometry. DOC, on the other hand, can be easily removed from the samples by acid precipitation. Examining the results of a reproducibility study, done with 5 replicate digestions, DOC and SDS with a 9 h digestion time produced the highest average digestion efficiencies (∼80%), with the highest average reproducibility (<5% error, defined as the relative deviation from the mean value). However, because of potential interferences resulting from the use of SDS, we recommend DOC with a 9 h digestion procedure as the optimum protocol.

The complexity of cell and tissue proteomes presents one of the most significant technical challenges in proteomic biomarker discovery. Multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based shotgun proteomics can be coupled with selective enrichment of cysteinyl peptides (Cys-peptides) to reduce sample complexity and increase proteome coverage. Here we evaluated the impact of Cys-peptide enrichment on global proteomic inventories. We employed a new cleavable thiol-reactive biotinylating probe, N-(2-(2-(2-(2-(3-(1-hydroxy-2-oxo-2-phenylethyl)phenoxy)acetamido)ethoxy)-ethoxy)ethyl)-5-(2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (IBB), to capture Cys-peptides after digestion. Treatment of tryptic digests with the IBB reagent followed by streptavidin capture and mild alkaline hydrolysis releases a highly purified population of Cys-peptides with a residual S-carboxymethyl tag. Isoelectric focusing (IEF) followed by LC-MS/MS of Cys-peptides significantly expanded proteome coverage in Saccharomyces cerevisiae (yeast) and in human colon carcinoma RKO cells. IBB-based fractionation enhanced detection of Cys-proteins in direct proportion to their cysteine content. The degree of enrichment typically was 2-8-fold but ranged up to almost 20-fold for a few proteins. Published copy number annotation for the yeast proteome enabled benchmarking of MS/MS spectral count data to yeast protein abundance and revealed selective enrichment of cysteine-rich, lower abundance proteins. Spectral count data further established this relationship in RKO cells. Enhanced detection of low abundance proteins was due to the chemoselectivity of Cys-peptide capture, rather than simplification of the peptide mixture through fractionation.

Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas disease, and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinylsulfone-based cysteine protease inhibitors, a series of α-ketoheterocycles were developed as reversible inhibitors of a recombinant L. mexicana cysteine protease, CPB2.8. Three isoxazoles and especially one oxadiazole compound are potent reversible inhibitors of CPB2.8; however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease.

We report on a patient presenting with cognitive delay, prenatal and postnatal growth deficiency, microcephaly, ptosis of eyelids, high and broad nasal root, and camptodactyly. Analysis of a dense whole genome single-nucleotide polymorphism (SNP) array showed a de novo 3.35 Mb deletion on 2p15-p16.1. In order to study the parental origin of the deletion we analyzed selected SNPs in the deleted area in the proband and her parents showing Mendelian incompatibilities suggesting a de novo deletion on the chromosome of paternal origin. Based on the five cases described previously in the literature, we have narrowed the critical region responsible for the 2p15-p16.1 microdeletion syndrome phenotype. The critical region does not include the VRK2 gene that had been speculated to have a role in cortical dysplasia. However, the association of the VRK2 gene with cortical dysplasia remains to be determined, as MRI imaging of the brain and gene content of the 2p15-16 deletion becomes established in more patients.

Curcumin (diferuloylmethane) is the main active ingredient of turmeric, a traditional herbal medicine and food of south Asia. Curcumin has been found to have a wide range of biological activities, including antioxidant, anti-inflammatory, chemopreventive and chemotherapeutic activities. Curcumin is currently being tested in clinical trials for treatment of various types of cancers, including multiple myeloma, pancreatic cancer and colon cancer. Although no toxicity associated with curcumin (even at very high doses) has been observed, the effects of curcumin in other solid tumors have been modest, primarily due to poor water solubility and poor bioavailability in tissues remote from the gastrointestinal tract. Therefore, there is a need for the discovery of curcumin analogs with better water solubility or greater bioavailability for the treatment of solid tumors such as prostate cancer. In this study, curcumin acetates and amino acid conjugates of curcumin were studied in terms of their proteasome inhibitory and antiproliferative effects against several human cancer cell lines. It was found that the water soluble amino acid conjugates of curcumin showed a potent antiproliferative effect and are potent proteasome inhibitors. Docking studies of the curcumin amino acid conjugates for proteasome inhibition were carried out to explain their biological activities. It is suggested that they may serve as the water soluble analogs of curcumin.

No abstract available.

Huntington disease (HD) is a progressive neurodegenerative disease caused by an expansion of a polyglutamine sequence in mutant huntingtin (mhtt) that produces abnormal folding and aggregation that results in the formation of nuclear and cytoplasmic neuronal inclusion bodies. Although the precise role of mhtt aggregates in the pathogenesis is unclear, attempts to reduce accumulated mhtt protein have ameliorated the phenotype in multiple cellular and in vivo HD models. Here, we provide critical results on intracranial delivery of a single-chain Fv intrabody, C4, which targets the first 17 amino acids of the htt protein, a region of httExon1 that is increasingly being recognized as pivotal. To assess long-term efficacy and safety issues, we used adenoassociated viral vectors (AAV2/1) to deliver intrabody genes to the striatum of inbred B6.HDR6/1 mice. Treatment initiation at various stages of the disease showed that early treatment preserved the largest number of cells without nuclear aggregates and that the accumulation of aggregated material could be delayed by several months. Even when intrabody treatment was not initiated until the clinical disease stage, significant, albeit smaller, effects were seen. These data indicate that neuronal intrabodies against critical N-terminal epitopes can be safely and effectively delivered using AAV2/1 to delay the aggregation phenotype during a sustained period in this HD model, even when delivery is initiated after disease onset.

Prostaglandin (PG) actions on disc metabolism are unclear even though certain PGs are highly expressed by disc cells under inflammatory conditions and nonsteroidal anti-inflammatory drugs (NSAIDs) are frequently used to block PG production to treat back pain. Hence this study aimed to (1) quantify gene expression of arachidonic acid cascade components responsible for PG synthesis and (2) examine the effects of key PGs on disc matrix homeostasis. Microarray analysis revealed that inflammatory stress increases expression of synthases and receptors for prostaglandin E2 (PGE(2)) and prostaglandin F2α (PGF(2α)), resulting in elevated PGE(2) and PGF(2α) production in conditioned media of disc cells. PGE(2) diminished disc cell proteoglycan synthesis, in a dose-dependent manner. Semiquantitative RT-PCR revealed differential effects of PGE(2) and PGF(2α) on disc cell expression of key matrix structural genes, aggrecan, versican, collagens type I and II. PGE(2) and PGF(2α) also decreased message for the anabolic factor, IGF-1. PGE(2) decreased mRNA expression for the anti-catabolic factor TIMP-1 while PGF(2α) increased mRNAs for catabolic factors MMP-1 and MMP-3. Thus, PGE(2) and PGF(2α) may have an overall negative impact on disc matrix homeostasis, and the use of NSAIDs may impact disc metabolism as well as treat back pain.

Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

Phosphatidylethanolamine binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), has been considered as a suppressor of metastasis and a prognostic marker in prostate cancer, breast cancer, gastrointestinal stromal tumors, melanoma, and epithelial ovarian cancer. In this report, recombinant PEBP1 was successfully expressed in an Escherichia coli system. A panel of monoclonal antibodies (mAbs) against PEBP1 with high specificity and affinity was generated and characterized using ELISA, western blot analysis, immunofluorescent staining and immunohistochemical staining. PEBP1 expression in normal 293 cells and a few pancreatic cancer cell lines was detected with mAb 7F12 in western blot analysis. To screen for a pair of mAbs with optimal binding affinity to soluble PEBP1, ForteBio's Octet system was used. Sandwich ELISA with mAb pair 4F10 and 8E2 showed a linear correlation between absorbance and PEBP1 protein concentration over a range of 7 to 100 ng/ml. MAb 4A11 detected a high level expression of PEBP1 in normal pancreatic tissue, and cancer adjacent normal pancreatic tissue in a pancreatic tissue microarray (TMA) comprising 80 human tissue cores. Pancreatic cancer tissues show a no or very weak staining intensity of PEBP1. In 69 valid cases, PEBP1 expression was significantly lower in tumor than in normal pancreas (p=8.40E-14) and adjacent normal tissue (p=8.46E-17). PEBP1 expression in pancreatic cancer was not associated with pTMN stage, differentiation grade and pathologic diagnosis. In conclusion, our results suggest that PEBP1 overexpresses in normal pancreas but significantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10, 7F12, and 8E2 are potential clinical diagnostic agents for pancreatic cancer.

KMUP-1 inhibits monocrotaline (MCT)-induced pulmonary artery (PA) proliferation by targeting serotonin (5-HT) receptors, inactivating RhoA and reducing phosphorylation of AKT/ERK. In MCT-treated rats, KMUP-1 f (5 mg/kg p.o.; 1mg/kg i.p.x 21 days) decreased proliferation (PCNA-positive) cells and 5-HTT-expression in lung and 5-HT levels in plasma. In isolated PA, KMUP-1 and simvastatin (0.1-100 μM) inhibited 5-HT (10 μM)-induced PA constriction. l-NAME-pretreatment reduced KMUP-1-induced relaxation. In pulmonary arterial smooth muscle cells (PASMCs), KMUP-1 (1-100 μM) and simvastatin (10 μM) inhibited 5-HT-induced cell migration and proliferation and KMUP-1 (1-100 μM) inhibited 5-HT-induced Ca²+ influx. Similar to Y27632, KMUP-1 (1-100 μM) inhibited 5-HT-induced RhoA/ROCK expression, while KMUP-1, Y27632 and simvastatin at 10 μM inhibited 5-HT-induced 5-HTT expression and KMUP-1 inhibited 5-HT-induced phosphorylation of AKT and ERK1/2 in PASMCs. In human pulmonary arterial endothelial cell (HPAEC), KMUP-1 (1-100 μM) increased the expression of eNOS and 5-HT(2B) and also at 10 μM augmented eNOS expression and production of nitric oxide (NO) in 5-HT-treated HPAEC. In radioligand binding, the IC₅₀/K(i) values of KMUP-1 for 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors were 0.34/0.0971, 0.04/0.0254, and 0.408/0.214 μM respectively. In conclusion, KMUP-1 inhibits MCT-induced PA proliferation by binding to 5-HT(2A), 5-HT(2B) and 5-HT(2C) receptors, increasing endothelial eNOS/5-HT(2B) receptor expression and NO release and inhibiting 5-HTT/RhoA/ROCK expression and AKT/ERK phosphorylation. KMUP-1 is suggested to be useful in the treatment of 5-HT-induced pulmonary artery proliferation.

<h4>Purpose</h4>The objective of this study was to develop a novel folate receptor-targeted drug delivery system for retinoblastoma cells using doxorubicin (DOX) as a model drug.<h4>Methods</h4>Biodegradable DOX-loaded poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) micelles (DOXM) were prepared with various solvents (dimethylsulfoxide, acetone, and dimethylformamide). The effects of solvents on entrapment efficiency, particle size, and polydispersity were examined. The effects of thermosensitive gel structure on the release of DOX from the DOXM were also studied. Qualitative and quantitative uptake studies of DOX and DOXM were carried out in Y-79 cell line. Cytotoxicity studies of DOXM were performed on Y-79 cells.<h4>Results</h4>Based on size, polydispersity, and entrapment efficiency, dimethylformamide was found to be the most suitable solvent for the preparation of DOXM. Dispersion of DOXM in PLGA-PEG-PLGA gel sustained drug release for a period of 2 weeks. Uptake of DOX was ∼4 times higher with DOXM than DOX in Y-79 cells overexpressing folate receptors. This was further confirmed from the quantitative uptake studies. DOXM exhibited higher cytotoxicity in Y-79 cells when compared with pure DOX.<h4>Conclusion</h4>These polymeric micellar systems suspended in thermosensitive gels may provide sustained and targeted delivery of anticancer agents to retinoblastoma cells following intravitreal administration.

Patients with various hematologic malignancies, including acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML), diffuse histiocytic lymphoma, and granulocytic sarcoma, have sometimes been shown to carry the PICALM-MLLT10 fusion gene (alias CALM-AF10) by various cytogenetic methodologies. Cases with the PICALM-MLLT10 fusion gene can involve a diagnostic dilemma for the following reasons: (1) the fusion gene occurs very rarely, (2) the cases do not have a distinct myeloid or lymphoid morphology and cells often appear immature, (3) cases usually have a mixed T-cell and myeloid phenotype, and (4) cases often have a mixed clinical presentation (e.g., mediastinal mass in a patient with AML). A 27-year-old woman was diagnosed with AML with the PICALM-MLLT10 fusion gene. The patient was treated on an AML regimen and achieved a complete remission. Although the reported treatment of these patients varies greatly, outcome remains very poor in the vast majority. Furthermore, central nervous system involvement at diagnosis and relapse are reported in pediatric populations. Routine acute leukemia fluorescence in situ hybridization panels do not include a probe for the PICALM-MLLT10 fusion gene, and therefore diagnosis can be made only when karyotyping is available; that delay can result in initial misdiagnosis and mistreatment. The case report and literature review here (including discussion of the poor prognosis and of management, including CNS prophylaxis) are intended to raise awareness and to inform about PICALM-MLLT10 in acute leukemia.

The neonatal Fc receptor (FcRn), also known as the Brambell receptor and encoded by Fcgrt, is a MHC class I like molecule that functions to protect IgG and albumin from catabolism, mediates transport of IgG across epithelial cells, and is involved in antigen presentation by professional antigen presenting cells. Its function is evident in early life in the transport of IgG from mother to fetus and neonate for passive immunity and later in the development of adaptive immunity and other functions throughout life. The unique ability of this receptor to prolong the half-life of IgG and albumin has guided engineering of novel therapeutics. Here, we aim to summarize the basic understanding of FcRn biology, its functions in various organs, and the therapeutic design of antibody- and albumin-based therapeutics in light of their interactions with FcRn.

The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.

Affective startle probe methodology was used to examine the effects of nicotine administration and deprivation on emotional processes among individuals carrying at least one s allele versus those with the l/l genotype of the 5-Hydroxytryptamine (Serotonin) Transporter Linked Polymorphic Region, 5-HTTLPR in the promoter region of the serotonin transporter gene [solute ligand carrier family 6 member A4 (SLC6A4) or SERT]. Smokers (n=84) completed four laboratory sessions crossing deprivation (12-h deprived vs. non-deprived) with nicotine spray (nicotine vs. placebo). Participants viewed affective pictures (positive, negative, neutral) while acoustic startle probes were administered. We found that smokers with the l/l genotype showed significantly greater suppression of the startle response when provided with nicotine vs. placebo than those with the s/s or s/l genotypes. The results suggest that l/l smokers, who may have higher levels of the serotonin transporter and more rapid synaptic serotonin clearance, experience substantial reduction in activation of the defensive system when exposed to nicotine.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world's deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects. Targets of particular interest include genes that regulate development. However, although the Ae. aegypti genome project uncovered homologs of many known developmental regulatory genes, little is known of the genetic regulation of development in Ae. aegypti or other vector mosquitoes. This article provides an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Methods for culturing, collecting and fixing developing tissues, analyzing gene and protein expression, and knocking down genes are permitting detailed analyses of the functions of developmental regulatory genes and the selective inhibition of such genes during Ae. aegypti development. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

<h4>Background</h4>Beta tricalcium phosphate is commonly used in metaphyseal defects but its use in posterolateral spinal fusion remains controversial. There are very few published animal studies in which use of beta tricalcium phosphate has been evaluated in the posterolateral lumbar arthrodesis model. Hence we conducted a study to evaluate the potential of composite graft of beta tricalcium phosphate and bone marrow aspirate in comparison to autologous bone graft, when used for posterolateral spinal fusion.<h4>Materials and methods</h4>Single level posterolateral lumbar fusion was performed in 40 adult male Indian rabbits, which were assigned randomly into one of the four groups based on graft materials implanted; a) 3 gm beta tricalcium phosphate plus 3 ml bone marrow aspirate (Group I); b) 3 ml bone marrow aspirate alone (Group II); c) 3 gm beta tricalcium phosphate (Group III) and d) 3 gm autologous bone graft (Group IV). Each group had 10 rabbits. Half of the rabbits were sacrificed by injecting Phenobarbitone intraperitoneally after eight weeks and the remaining after 24 weeks, and were evaluated for fusion by X-rays, computed tomography (CT) scans, manual palpation test and histology.<h4>Results</h4>Beta tricalcium phosphate used with bone marrow aspirate produced best results when compared to other groups (P =.0001). When beta tricalcium phosphate was used alone, fusion rates were better as compared to fusion achieved with autologous iliac crest bone graft though statistically not significant (P =0.07). Autologous bone graft showed signs of new bone formation. However, the rate of new bone formation was comparatively slow.<h4>Conclusion</h4>Composite graft of beta tricalcium phosphate and bone marrow aspirate can be used as an alternative to autologous iliac crest bone graft.

The [NiFe]-hydrogenase model complex NiFe(pdt)(dppe)(CO)(3) (1) (pdt = 1,3-propanedithiolate) has been efficiently synthesized and found to be robust. This neutral complex sustains protonation to give the first nickel-iron hydride [1H]BF(4). One CO ligand in [1H]BF(4) is readily substituted by organophosphorus ligands to afford the substituted derivatives [HNiFe(pdt)(dppe)(PR(3))(CO)(2)]BF(4), where PR(3) = P(OPh)(3) ([2H]BF(4)); PPh(3) ([3H]BF(4)); PPh(2)Py ([4H]BF(4), where Py = 2-pyridyl). Variable temperature NMR measurements show that the neutral and protonated derivatives are dynamic on the NMR time scale, which partially symmetrizes the phosphine complex. The proposed stereodynamics involve twisting of the Ni(dppe) center, not rotation at the Fe(CO)(2)(PR(3)) center. In MeCN solution, 3, which can be prepared by deprotonation of [3H]BF(4) with NaOMe, is about 10(4) stronger base than is 1. X-ray crystallographic analysis of [3H]BF(4) revealed a highly unsymmetrical bridging hydride, the Fe-H bond being 0.40 Å shorter than the Ni-H distance. Complexes [2H]BF(4), [3H]BF(4), and [4H]BF(4) undergo reductions near -1.46 V vs Fc(0/+). For [2H]BF(4), this reduction process is reversible, and we assign it as a one-electron process. In the presence of trifluoroacetic acid, proton reduction catalysis coincides with this reductive event. The dependence of i(c)/i(p) on the concentration of the acid indicates that H(2) evolution entails protonation of a reduced hydride. For [2H](+), [3H](+), and [4H](+), the acid-independent rate constants are 50-75 s(-1). For [2H](+) and [3H](+), the overpotentials for H(2) evolution are estimated to be 430 mV, whereas the overpotential for the N-protonated pyridinium complex [4H(2)](2+) is estimated to be 260 mV. The mechanism of H(2) evolution is proposed to follow an ECEC sequence, where E and C correspond to one-electron reductions and protonations, respectively. On the basis of their values for its pK(a) and redox potentials, the room temperature values of ΔG(H•) and ΔG(H-) are estimated as respectively as 57 and 79 kcal/mol for [1H](+).

<h4>Background</h4>Studies have demonstrated that flexor tendon repair strength fails to increase in the first three weeks following suturing of the tendon, a finding that correlates closely with the timing of many clinical failures. The application of growth factors holds promise for improving the tendon-repair response and obviating failure in the initial three weeks.<h4>Methods</h4>The effects of basic fibroblast growth factor on flexor tendon healing were evaluated with use of a canine model. Operative repair followed by the sustained delivery of basic fibroblast growth factor, at two different doses, was compared with operative repair alone. Histological, biochemical, and biomechanical methods were used to evaluate the tendons twenty-one days after repair.<h4>Results</h4>Vascularity, cellularity, and adhesion formation were increased in the tendons that received basic fibroblast growth factor as compared with the tendons that received operative repair alone. DNA concentration was increased in the tendons that received 1000 ng of basic fibroblast growth factor (mean and standard deviation, 5.7 ± 0.7 μg/mg) as compared with the tendons that received 500 ng of basic fibroblast growth factor (3.8 ± 0.7 μg/mg) and the matched control tendons that received operative repair alone (4.5 ± 0.9 μg/mg). Tendons that were treated with basic fibroblast growth factor had a lower ratio of type-I collagen to type-III collagen, indicating increased scar formation compared with that seen in tendons that received operative repair alone (3.0 ± 1.6 in the group that received 500-ng basic fibroblast growth factor compared with 4.3 ± 1.0 in the paired control group that received operative repair alone, and 3.4 ± 0.6 in the group that received 1000-ng basic fibroblast growth factor compared with 4.5 ± 1.9 in the paired control group that received operative repair alone). Consistent with the increases in adhesion formation that were seen in tendons treated with basic fibroblast growth factor, the range of motion was reduced in the group that received the higher dose of basic fibroblast growth factor than it was in the paired control group that received operative repair alone (16.6° ± 9.4° in the group that received 500 ng basic fibroblast growth factor, 13.4° ± 6.1° in the paired control group that received operative repair alone, and 29.2° ± 5.8° in the normal group [i.e., the group of corresponding, uninjured tendons from the contralateral forelimb]; and 15.0° ± 3.8° in the group that received 1000 ng basic fibroblast growth factor, 19.3° ± 5.5° in the paired control group that received operative repair alone, and 29.0° ± 8.8° in the normal group). There were no significant differences in tendon excursion or tensile mechanical properties between the groups that were treated with basic fibroblast growth factor and the groups that received operative repair alone.<h4>Conclusions</h4>Although basic fibroblast growth factor accelerated the cell-proliferation phase of tendon healing, it also promoted neovascularization and inflammation in the earliest stages following the suturing of the tendon. Despite a substantial biologic response, the administration of basic fibroblast growth factor failed to produce improvements in either the mechanical or functional properties of the repair. Rather, increased cellular activity resulted in peritendinous scar formation and diminished range of motion.

Changes in blood parameters, especially those related to blood coagulations, were examined on gestational days (GDs) 6, 18 and 28 in Japanese White rabbits. As compared with the non-pregnant group, the following changes were observed in the pregnant group. For blood coagulation-related parameters, fibrinogen, platelets and antithrombin time III (ATIII) was significantly increased on GD 18 and/or GD 28, and activated partial thromboplastin time (APTT) was significantly prolonged on GDs 18 and 28. For the other hematological and blood chemical parameters, hemoglobin and hematocrit values were significantly decreased on GDs 18 and 28. Reticulocyte counts significantly and prominently increased on GD 18 and then decreased to significantly lower levels than those in the non-pregnant group on GD 28. Eosinophils and basophil showed significant decreases on GD 18 and/or GD 28. The serum progesterone concentration reached its highest level on GD 18. Total protein (TP), albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), cholesterol, calcium, glucose and blood urea nitrogen were significantly decreased on GD 18 and/or GD 28. These results were almost similar to those in the pregnant New Zealand White rabbits (Mizoguchi et al., 2010).

Induced pluripotent Huntington's disease monkey stem cells (rHD-iPSCs) were established by the overexpression of rhesus macaque transcription factors (Oct4, Sox2, and Klf4) in transgenic Huntington's monkey skin fibroblasts. The rHD-iPSCs were pluripotent and capable of differentiating into neuronal cell types in vitro and developed teratoma in immune compromised mice. We also demonstrated the upregulation of endogenous Oct4 and Sox2 after successful reprogramming to pluripotency in rHD-iPSCs, which was not expressed in skin fibroblasts. rHD-iPSCs also developed cellular features comparable to Huntington's disease (HD), including the accumulation of mutant huntingtin (htt) aggregate and the formation of intranuclear inclusions (NIs) paralleling neural differentiation in vitro. Induced pluripotent stem cells from transgenic HD monkeys open a new era of nonhuman primate modeling of human diseases. rHD-iPSCs that develop key HD cellular features and parallel neural differentiation can be a powerful platform for investigating the developmental impact on HD pathogenesis and developing new therapies, which can be evaluated in HD monkeys from whom the rHD-iPSCs were derived.

<h4>Aim</h4>To assess the values of liver stiffness (LS) in patients with hepatitis B virus (HBV) chronic hepatitis and to compare them with those in patients with hepatitis C virus (HCV) chronic hepatitis.<h4>Methods</h4>The study included 140 patients with HBV chronic hepatitis, and 317 patients with HCV chronic hepatitis, in which LS was measured (FibroScan®-Echosens®) and liver biopsy was performed in the same session (assessed according to the Metavir score).<h4>Results</h4>According to the Metavir score of the 140 HBV patients: one had F0, 32 had F1, 67 had F2, 33 had F3 and 7 had F4. Of the 317 HCV patients: 5 had F0, 34 had F1, 146 had F2, 93 had F3 and 39 had F4. For the same severity of fibrosis, the mean values of LS in HBV patients were similar to those in HCV patients: F1, 6.5 ± 1.9 kPa vs 5.8 ± 2.1 kPa (P = 0.0889); F2, 7.1 ± 2 kPa vs 6.9 ± 2.5 kPa (P = 0.3369); F3, 9.1 ± 3.6 kPa vs 9.9 ± 5 kPa (P = 0.7038); F4, 19.8 ± 8.6 kPa vs 17.3 ± 6.1 kPa (P = 0.6574). A significant direct correlation between LS measurements and fibrosis was found in HCV patients (Spearman's r = 0.578, P < 0.0001), as well as in HBV patients (r = 0.408, P < 0.0001). The correlation was more significant in HCV than in HBV patients (Fisher's Z-test, Z = 2.210, P = 0.0271).<h4>Conclusion</h4>In our group, the mean values of LS in patients with chronic B hepatitis were similar to those in patients with chronic HCV hepatitis, for the same stage of fibrosis. Also, LS was correlated with the severity of fibrosis both in HBV and HCV chronic hepatitis patients.

Peroxiredoxin 6 (Prdx6) exerts its protective role through peroxidase activity against H₂O₂ and phospholipid hydroperoxides. We hypothesized that targeted disruption of Prdx6 would lead to enhanced susceptibility to cigarette smoke (CS)-mediated lung inflammation and/or emphysema in mouse lung. Prdx6 null (Prdx6⁻/⁻mice exposed to acute CS showed no significant increase of inflammatory cell influx or any alterations in lung levels of proinflammatory cytokines compared to wild-type (WT) mice. Lung levels of antioxidant enzymes were significantly increased in acute CS-exposed Prdx6⁻/⁻ compared to WT mice. Overexpressing (Prdx6⁻/⁻) mice exposed to acute CS showed significant decrease in lung antioxidant enzymes associated with increased inflammatory response compared to CS-exposed WT mice or air-exposed Prdx6⁻/⁻ mice. However, chronic 6 months of CS exposure resulted in increased lung inflammatory response, mean linear intercept (Lm), and alteration in lung mechanical properties in Prdx6⁻/⁻ when compared to WT mice exposed to CS. These data show that targeted disruption of Prdx6 does not lead to increased lung inflammatory response but is associated with increased antioxidants, suggesting a critical role of lung Prdx6 and several compensatory mechanisms during acute CS-induced adaptive response, whereas this protection is lost in chronic CS exposure leading to emphysema.

The genetic components in systemic lupus erythematosus (SLE) have long been established, however, it has been unclear for many years whether the same genetic risk factors for SLE are shared across different ethnic groups. Over the past few years, a number of genetic and genomic studies have been conducted in Asian populations to address this question. These studies have demonstrated that genetic heterogeneity does exist in SLE across different ethnic groups. With these studies, it has been established that a number of genes associated with SLE in Caucasians are also risk factors in Asians: HLA class II genes, STAT4, BANK1, BLK, IRF5, TNFSF4, ITGAM, etc., while there are also novel genetic risk factors identified by these studies in Asians, for instance, the ETS1 and WDFY4 in Chinese. For the genomic studies, the interferon signature has been confirmed as a major lupus molecular phenotype in Asians the same as in Caucasians; microRNA expression profiling and its novel role in regulating the interferon pathway has been first revealed in Asians. Further understanding of the function of lupus disease genes and delineating the key molecular pathway(s) will enhance the development of novel therapeutic targets and biomarkers for individualized clinical management for lupus patients.

When evaluating clinical characteristics and outcomes in patients on hemodialysis, the prevalence and severity of comorbidity may change over time. Knowing whether updated assessments of comorbidity enhance predictive power will assist the design of future studies. We conducted a secondary data analysis of 1846 prevalent hemodialysis patients from 15 US clinical centers enrolled in the HEMO study. Our primary explanatory variable was the Index of Coexistent Diseases score, which aggregates comorbidities, as a time-constant and time-varying covariate. Our outcomes of interest were all-cause mortality, time to first hospitalization, and total hospitalizations. We used Cox proportional hazards regression. Accounting for an updated comorbidity assessment over time yielded a more robust association with mortality than accounting for baseline comorbidity alone. The variation explained by time-varying comorbidity assessments on time to death was greater than age, baseline serum albumin, diabetes, or any other covariates. There was a less pronounced advantage of updated comorbidity assessments on determining time to hospitalization. Updated assessments of comorbidity significantly strengthen the ability to predict death in patients on hemodialysis. Future studies in dialysis should invest the necessary resources to include repeated assessments of comorbidity.

<h4>Background</h4>Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited.<h4>Methodology/principal findings</h4>To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21(Cip1) (CDKN1A) and p15(Ink4b) (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling.<h4>Conclusions/significance</h4>This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Characterizing the ionic distribution around chromatin is important for understanding the electrostatic forces governing chromatin structure and function. Here we develop an electrostatic model to handle multivalent ions and compute the ionic distribution around a mesoscale chromatin model as a function of conformation, number of nucleosome cores, and ionic strength and species using Poisson-Boltzmann theory. This approach enables us to visualize and measure the complex patterns of counterion condensation around chromatin by examining ionic densities, free energies, shielding charges, and correlations of shielding charges around the nucleosome core and various oligonucleosome conformations. We show that: counterions, especially divalent cations, predominantly condense around the nucleosomal and linker DNA, unburied regions of histone tails, and exposed chromatin surfaces; ionic screening is sensitively influenced by local and global conformations, with a wide ranging net nucleosome core screening charge (56-100e); and screening charge correlations reveal conformational flexibility and interactions among chromatin subunits, especially between the histone tails and parental nucleosome cores. These results provide complementary and detailed views of ionic effects on chromatin structure for modest computational resources. The electrostatic model developed here is applicable to other coarse-grained macromolecular complexes.

No abstract available.

Mutations in TNNT2, encoding cardiac troponin T, commonly shows early onset, aggressive dilated cardiomyopathy (DCM). This observation may influence the decision of whether to undertake clinical genetic testing for TNNT2 in later onset DCM. Further, the trigger for late onset DCM remains enigmatic. A 70-year-old woman, previously healthy with a left ventricular ejection fraction of 50%-55% at age 69, presented with DCM of unknown cause and a 4-month history progressive heart failure requiring cardiac transplantation. Clinical genetic testing revealed a novel TNNT2 R139H mutation but no relevant variants in 18 other DCM genes. Her explanted heart showed partial fatty replacement in the right ventricle. Sequencing for five arrhythmogenic right ventricular dysplasia genes was negative. Functional studies in porcine cardiac skinned fibers reconstituted with the mutant R139H troponin T protein showed decreased Ca(2+) sensitivity at pH 7, characteristic of DCM. Because fatty infiltration may acidify the myocellular environment, maximal force development examined at pH 6.5 was diminished, suggesting a possible environmental trigger. We conclude that the TNNT2 R139H mutation was likely to be disease causing. Further, later age of onset may not be relevant to exclude genetic testing for TNNT2 mutations.

Resilience has been conceptualized as a dynamic developmental process encompassing the attainment of positive adaptation within the context of significant threat, severe adversity, or trauma. Until the past decade, the empirical study of resilience predominantly focused on behavioral and psychosocial correlates of, and contributors to, the phenomenon and did not examine neurobiological or genetic correlates of and contributors to resilience. Technological advances in molecular genetics and neuroimaging, and in measuring other biological aspects of behavior, have made it more feasible to begin to conduct research on pathways to resilient functioning from a multilevel perspective. Child maltreatment constitutes a profound immersion in severe stress that challenges and frequently impairs development across diverse domains of biological and psychological functioning. Research on the determinants of resilience in maltreated children is presented as an illustration of empirical work that is moving from single-level to multilevel investigations of competent functioning in the face of adversity and trauma. These include studies of personality, neural, neuroendocrine, and molecular genetic contributors to resilient adaptation. Analogous to neural plasticity that takes place in response to brain injury, it is conjectured that it may be possible to conceptualize resilience as the ability of individuals to recover functioning after exposure to extreme stress. Multilevel randomized control prevention and intervention trials have substantial potential for facilitating the promotion of resilient functioning in diverse high-risk populations that have experienced significant adversity. Determining the multiple levels at which change is engendered through randomized control trials will provide insight into the mechanisms of change, the extent to which neural plasticity may be promoted, and the interrelations between biological and psychological processes in the development of maladaptation, psychopathology, and resilience.

YAC transgenic mice expressing poly(Q)-expanded full-length huntingtin (mhtt) recapitulate many behavioral and neuropathological features of Huntington disease (HD). We have previously observed a reduction in phosphorylation of mhtt at S421 in the presence of the mutation for HD. In addition, phosphorylation of normal S421-htt is reduced after excitotoxic stimulation of NMDA receptors (NMDARs). To test whether NMDAR stimulation contributes to reduced pS421-htt levels in HD, we determined phosphorylation of htt at Ser421 after NMDA-induced excitotoxicity in neurons from YAC128 mice. Here, we report that the total level of pS421-htt is reduced in YAC128 primary neurons after excitotoxic NMDAR stimulation. Similarly, the total level of pS421-htt is reduced in YAC128 transgenic mice after quinolinic acid injection into the striatum. In contrast, loss of phosphorylation of pS421-htt is prevented in YAC mice that never develop clinical or neuropathological features of HD [the caspase 6-resistant YAC128 transgene (C6R)]. To gain insight into the mechanisms underlying these findings, we determined that the Ser/Thr protein phosphatases PP1 and PP2A dephosphorylate pS421-htt in situ and after excitotoxic stimulation of NMDARs in neurons. Furthermore, increasing the phosphorylation of htt at S421 by blocking PP1 and PP2A activity protects YAC128 striatal neurons from NMDA-induced cell death. These results, together with the observed modulation of pS421-htt levels by dopamine, the reduced expression of PP1 inhibitor Darpp-32 in the striatum of YAC128 mice, and the reduced phosphorylation of PP1 substrate CreB, point to altered regulation of phosphatase activity in HD and highlight enhancing phosphorylation of htt at S421 as a therapeutic target.

Selective serotonin reuptake inhibitors (SSRIs) represent one of the most common treatment options in major depression and anxiety disorders. By blocking the serotonin transporter, SSRIs modulate serotonergic neurotransmission as well as the function of autoreceptors and heteroreceptors. However, treatment-induced changes on a network level primarily remain unknown. Thus, we evaluated the association between serotonin-1A (5-HT1A) autoreceptors and heteroreceptors before and after SSRIs. Twenty-one patients with anxiety disorders underwent positron emission tomography using [carbonyl-11C]WAY-100635 before and after 12 weeks of escitalopram treatment; 15 of them completed the study protocol. Additionally, 36 drug-naive healthy controls were measured once. The 5-HT1A receptor binding potential (BPND) was quantified for the dorsal raphe nucleus (DRN) using a region-of-interest approach and for the entire brain by calculating parametric maps. Voxel-wise linear regression was applied between DRN autoreceptor and whole-brain heteroreceptor 5-HT1A BPND. Consistent with previous observations, healthy subjects showed widespread positive correlations of 5-HT1A BPND between autoreceptors and heteroreceptors. Comparing patients before versus after escitalopram treatment revealed enhanced associations of autoreceptor-to-heteroreceptor 5-HT1A BPND within the amygdala and hippocampus (R2=0.21-0.28 vs 0.49-0.81; p<0.05-0.001). In contrast, no significant SSRI-induced changes were found for correlations of heteroreceptor-to-heteroreceptor 5-HT1A BPND between several limbic regions. This interregional approach suggests a treatment-induced reinforcement of the association of 5-HT1A binding between autoreceptors and heteroreceptors specifically in areas involved in anxiety disorders. These findings provide complementary information about treatment effects on a network level and confirm the central role of the DRN as a prime regulatory area.

The 1000 Genomes Project aims to provide a deep characterization of human genome sequence variation as a foundation for investigating the relationship between genotype and phenotype. Here we present results of the pilot phase of the project, designed to develop and compare different strategies for genome-wide sequencing with high-throughput platforms. We undertook three projects: low-coverage whole-genome sequencing of 179 individuals from four populations; high-coverage sequencing of two mother-father-child trios; and exon-targeted sequencing of 697 individuals from seven populations. We describe the location, allele frequency and local haplotype structure of approximately 15 million single nucleotide polymorphisms, 1 million short insertions and deletions, and 20,000 structural variants, most of which were previously undescribed. We show that, because we have catalogued the vast majority of common variation, over 95% of the currently accessible variants found in any individual are present in this data set. On average, each person is found to carry approximately 250 to 300 loss-of-function variants in annotated genes and 50 to 100 variants previously implicated in inherited disorders. We demonstrate how these results can be used to inform association and functional studies. From the two trios, we directly estimate the rate of de novo germline base substitution mutations to be approximately 10(-8) per base pair per generation. We explore the data with regard to signatures of natural selection, and identify a marked reduction of genetic variation in the neighbourhood of genes, due to selection at linked sites. These methods and public data will support the next phase of human genetic research.

Autophagy is an essential intracellular process that mediates degradation of intracellular proteins and organelles in lysosomes. Autophagy was initially identified for its role as alternative source of energy when nutrients are scarce but, in recent years, a previously unknown role for this degradative pathway in the cellular response to stress has gained considerable attention. In this review, we focus on the novel findings linking autophagic function with metabolic stress resulting either from proteins or lipids. Proper autophagic activity is required in the cellular defense against proteotoxicity arising in the cytosol and also in the endoplasmic reticulum, where a vast amount of proteins are synthesized and folded. In addition, autophagy contributes to mobilization of intracellular lipid stores and may be central to lipid metabolism in certain cellular conditions. In this review, we focus on the interrelation between autophagy and different types of metabolic stress, specifically the stress resulting from the presence of misbehaving proteins within the cytosol or in the endoplasmic reticulum and the stress following a lipogenic challenge. We also comment on the consequences that chronic exposure to these metabolic stressors could have on autophagic function and on how this effect may underlie the basis of some common metabolic disorders.

Once protein-coding, the X-inactivation center (Xic) is now dominated by large noncoding RNAs (ncRNA). X chromosome inactivation (XCI) equalizes gene expression between mammalian males and females by inactivating one X in female cells. XCI requires Xist, an ncRNA that coats the X and recruits Polycomb proteins. How Xist is controlled remains unclear but likely involves negative and positive regulators. For the active X, the antisense Tsix RNA is an established Xist repressor. For the inactive X, here, we identify Xic-encoded Jpx as an Xist activator. Jpx is developmentally regulated and accumulates during XCI. Deleting Jpx blocks XCI and is female lethal. Posttranscriptional Jpx knockdown recapitulates the knockout, and supplying Jpx in trans rescues lethality. Thus, Jpx is trans-acting and functions as ncRNA. Furthermore, ΔJpx is rescued by truncating Tsix, indicating an antagonistic relationship between the ncRNAs. We conclude that Xist is controlled by two RNA-based switches: Tsix for Xa and Jpx for Xi.

<h4>Background</h4>Hyalinizing trabecular tumor (HTT) is a rare thyroid neoplasm, which shares some histologic features with thyroid papillary carcinoma (TPC). Clinically, it is frequently misdiagnosed as papillary carcinoma, even for some experienced pathologists. The aim of this study was to investigate whether HTT is variant of TPC or HTT is an independent entity of thyroid neoplasm.<h4>Methods</h4>The expression of CK19, galectin-3, HBME-1 and MIB-1 was detected by immunohistochemical staining in 12 cases of hyalinizing trabecular tumor and 20 cases of thyroid papillary carcinoma.<h4>Results</h4>Two of the 12 HTT samples were positive or focally positive for CK19. Four of the 12 samples of HTT presented positive to galectin-3; 3 were stained strongly and the other one was focally positive. None of the 12 samples of HTT was positive for HBME-1. Five in 12 HTT samples were stained in nucleus for MIB-1. Almost all the 20 cases of thyroid papillary carcinoma were intensely stained for CK19, galectin-3 and HBME-1. Fifteen in 20 cases of thyroid papillary carcinoma showed nuclear staining for MIB-1.<h4>Conclusions</h4>HTT is an independent thyroid neoplasm, not a variant of TPC. This study could help in the differential diagnosis of HTT from TPC. CK19, galectin-3 and HBME-1 are adequate to identify HTT and TPC, but MIB-1 does not play an important role in discrimination between HTT and TPC.

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.

Cervical cancer (CC) as a single diagnostic entity exhibits differences in clinical behavior and poor outcomes in response to therapy in advanced tumors. Although infection of high-risk human papillomavirus is recognized as an important initiating event in cervical tumorigenesis, stratification of CC into subclasses for progression and response to treatment remains elusive. Existing knowledge of genetic, epigenetic and transcriptional alterations is inadequate in addressing the issues of diagnosis, progression and response to treatment. Recent technological advances in high-throughput genomics and the application of integrative approaches have greatly accelerated gene discovery, facilitating the identification of molecular targets. In this article, we discuss the results obtained by preliminary integrative analysis of DNA copy number increases and gene expression, utilizing the two most common copy number-gained regions of 5p and 20q in identifying gene targets in CC. These analyses provide insights into the roles of genes such as RNASEN, POLS and SKP2 on 5p, KIF3B, RALY and E2F1 at 20q11.2 and CSE1L, ZNF313 and B4GALT5 at 20q13.13. Future integrative applications using additional datasets, such as mutations, DNA methylation and clinical outcomes, will raise the promise of accomplishing the identification of biological pathways and molecular targets for therapies for patients with CC.

Two recurring problems with stem/neural progenitor cell (NPC) transplantation therapies for spinal cord injury (SCI) are poor cell survival and uncontrolled cell differentiation. The current study evaluated the viability and differentiation of embryonic stem cell-derived neural progenitor cells (ESNPCs) transplanted within fibrin scaffolds containing growth factors (GFs) and a heparin-binding delivery system (HBDS) to enhance cell survival and direct differentiation into neurons. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4-/4+ retinoic acid (RA) induction protocol that resulted in a population of cells that was 70% nestin positive NPCs. The ESNPCs were transplanted directly into a rat subacute dorsal hemisection lesion SCI model. ESNPCs were either encapsulated in a fibrin scaffold; encapsulated in fibrin containing the HBDS, neurotrophin-3 (NT-3) and platelet derived growth factor (PDGF-AA); or encapsulated in fibrin scaffolds with NT-3 and PDGF-AA without the HBDS. We report that the combination of GFs and fibrin scaffold (without HBDS) enhanced the total number of ESNPCs present in the treated spinal cords and increased the number of ESNPC-derived NeuN positive neurons 8 weeks after transplantation. All experimental groups treated with ESNPCs exhibited an increase in behavioral function 4 weeks after transplantation. In a subset of animals, the ESNPCs over-proliferated as evidenced by SSEA-1 positive/Ki67 positive ESCs found at 4 and 8 weeks. These results demonstrate the potential of tissue-engineered fibrin scaffolds to enhance the survival of NPCs and highlight the need to purify cell populations used in therapies for SCI.

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

Gastric cancer is the most common malignancy of the gastrointestinal tract in East Asian populations and the second most frequent cause of cancer-related mortality in the world. While previous studies have investigated the genetic factors involved in gastric carcinogenesis, there still exist relatively few studies that have investigated the genetic traits associated with the risk of gastric precancerous conditions. In this paper we will review the biology and genetic polymorphisms involved in the genesis of gastric precancerous conditions reported to date and discuss the future prospects of this field of study. The associations of gastric precancerous conditions with polymorphisms in the cytotoxin-associated gene A-related genes (e.g. PTPN11 G/A at intron 3, rs2301756), those in the genes involved in host immunity against Helicobacter pylori (H. pylori) infection (e.g. TLR4 +3725G/C, rs11536889) or polymorphisms of the genes essential for the development/ differentiation of the gastric epithelial cells (e.g. RUNX3 T/A polymorphism at intron 3, rs760805) have been reported to date. Genetic epidemiological studies of the associations between H. pylori-induced gastric precancerous conditions and other gene polymorphisms in these pathways as well as polymorphisms of the genes involved in other pathways like oxidative DNA damage repair pathways would provide useful evidence for the individualized prevention of these H. pylori-induced gastric precancerous conditions.

Nonalcoholic fatty liver disease (NAFLD), a manifestation of metabolic syndrome, includes a wide range of clinical entities from simple fatty liver, a benign condition, to nonalcoholic steatohepatitis (NASH), a condition which can progress to cirrhosis, hepatocellular carcinoma and hepatic failure. The diagnosis of NASH requires no history of previous or current significant alcohol consumption and no evidence of other chronic liver diseases. Ethanol intake levels of 20 g daily (or 140 g weekly) are endorsed as the acceptable threshold to define nonalcoholic patients. Liver biopsy is the current gold standard for the diagnosis of NASH and provides prognostic information. Histopathological diagnosis of NASH is based on the following 3 features: (1) hepatic macrovesicular steatosis; (2) lobular inflammation; and (3) ballooning degeneration of hepatocytes. It is impractical to biopsy every patient with suspected NAFLD. Although highly accurate and affordable noninvasive screening tools can differentiate NASH from NAFLD, no imaging studies or laboratory tests are able to precisely diagnose NASH. There is no universal agreement regarding the indications for liver biopsy in NAFLD patients. In Japan, liver biopsies are considered in patients with suspected NAFLD based on several criteria including low platelet counts, elevated fibrosis markers, increasing age and other deciding parameters. Further studies are needed to establish a suitable scoring system that can distinguish steatohepatitis from simple steatosis.

Focal nodular hyperplasia (FNH) is the second most common benign lesion of the liver. It is a solitary lesion and usually does not enlarge. We present the magnetic resonance imaging findings of multiple progressive FNH lesions in a patient with hemosiderosis using Gadolinium-EOB-DTPA (Eovist) as a hepatobiliary contrast agent. The possible mechanisms underlying the occurrence and progression of FNH lesions and the potential value of Eovist in characterizing the lesions were discussed.

Comprehensive management of dental caries should involve the management of disease as well as the lesion. Current decision making process in cariology is influenced by numerous factors such as the size/ depth/ activity of the carious lesion and age/ the caries risk status of the patient. Treatment decisions should involve planning the non-operative/ preventive treatment for non-cavitated or early cavitated lesions and also formulating operative treatment for cavitated lesions. Apart from these two responsibilities, a clinician should also be knowledgeable enough to decide when not to interfere in the caries dynamics and how frequently to recall the patient for follow-ups. The non-operative treatment prescriptions vary in dose, intensity and mode of delivery according to the caries risk status. Minimal invasion and maximal conservation of tooth structure has become the essence of current operative treatments. This part of the series elaborates on the paradigm shift in the management of dental caries.

The development of personality traits united by the notion of conscientiousness, should promote, along with reduction of anxiety, the physical and mental health. In order to detect the sources of conscientiousness and neuroticism formation, we evaluated associations between polymorphic markers of 5-HTT, 5-HTR2A, and GABRA6 genes and relevant scores of TCI questionnaire in a group of 369 volunteers. Associations of markers VNTR and LPR of 5-HTT gene and marker T1521C of GABRA6 gene with "self-directedness" and the effects of 5-HTR2A gene marker T102C and its interactions with the GABRA6 gene on the "harm avoidance" were detected.

Specific changes in gene expression during cancer initiation should enable discovery of biomarkers for risk assessment, early detection and targets for chemoprevention. It has been previously demonstrated that altered mRNA and proteome signatures of morphologically normal cells bearing a single inherited "hit" in a tumor suppressor gene parallel many changes observed in the corresponding sporadic cancer. Here, we report on the global gene expression profile of morphologically normal, cultured primary breast epithelial and stromal cells from Li-Fraumeni syndrome (LFS) TP53 mutation carriers. Our analyses identified multiple changes in gene expression in both morphologically normal breast epithelial and stromal cells associated with TP53 haploinsufficiency, as well as interlocking pathways. Notably, a dysregulated p53 signaling pathway was readily detectable. Pharmacological intervention with the p53 rescue compounds CP-31398 and PRIMA-1 provided further evidence in support of the central role of p53 in affecting these changes in LFS cells and treatment for this cancer. Because loss of signaling mediated by TP53 is associated with the development and survival of many human tumors, identification of gene expression profiles in morphologically normal cells that carry "one-hit" p53 mutations may reveal novel biomarkers, enabling the discovery of potential targets for chemoprevention of sporadic tumors as well.

The hypothesis that mimicry between a self and a microbial peptide antigen is strictly related to autoimmune pathology remains a debated concept in autoimmunity research. Clear evidence for a causal link between molecular mimicry and autoimmunity is still lacking. In recent studies we have demonstrated that viruses and bacteria share amino acid sequences with the human proteome at such a high extent that the molecular mimicry hypothesis becomes questionable as a causal factor in autoimmunity. Expanding upon our analysis, here we detail the bacterial peptide overlapping to the human proteome at the penta-, hexa-, hepta- and octapeptide levels by exact peptide matching analysis and demonstrate that there does not exist a single human protein that does not harbor a bacterial pentapeptide or hexapeptide motif. This finding suggests that molecular mimicry between a self and a microbial peptide antigen cannot be assumed as a basis for autoimmune pathologies. Moreover, the data are discussed in relation to the microbial immune escape phenomenon and the possible vaccine-related autoimmune effects.

<h4>Unlabelled</h4>Hereditary hemochromatosis is a genetic disturbance of iron metabolism resulting in iron overload in several organs and their functional failure. Early diagnosis is necessary to start a simple and effective therapy: bleeding. It is the most frequent genetic disease in some populations. Our objectives were 1. An estimate of the expectancy of the disease in the population of our geographic region, and 2. To diagnose the disease by applying the established methods and estimate the efficiency of our diagnosis.<h4>Methods</h4>1.To estimate the expectancy, we genotyped 200 persons for the most frequent mutations causing the disease: HFE-C282Y and HFE-H63D by PCR-RFLP. 2. To diagnose the disease phenotypically we determined plasma iron level, ferritin level and transferrin saturation index in 549 patients previously diagnosed as chronic hepatitis or cirrhosis and genotyped those with hemochromatosis phenotype.<h4>Results</h4>1. We found allelic frequencies of 1.75% and 13.25% for the HFE-C282Y and H63D mutant alleles respectively. From these frequencies we calculated that a severe case caused by a C282Y/C282Y homozygote can arise in 816 people and a mild case caused by a C282Y/H63D compound heterozygote can arise in 100 people 2. Among 549 patients we found 10 to have the phenotype of hemochromatosis and 3 out of the 10 were found to carry mutations: two in the HFE gene (one homozygous C282Y and one compound heterozygous C282Y/H63D) and one in the hemojuvelin (HJV) gene (a G320V).

Platelets play a major role in the metastatic dissemination of tumor cells in vivo . Recent evidence reveals megakaryocyte-derived platelet pre-mRNA is spliced to mRNA and then translated into functional proteins in response to external stimulation. Employing a human lung cancer model, we hypothesized a subset of megakaryocyte/platelet genes exists that are significantly over or underexpressed in metastasis compared with noncancer. Microarray analysis employing platelet mRNA followed by unsupervised hierarchical clustering revealed an expression profile that includes decreased expression of 197 of the 200 platelet genes with the most altered expression (p < 1.0 × 10(-4)). Among the 608 splicing events identified between the metastasis and negative control groups, 33 highly variable genes were identified with between 3 and 13 splicing events each. In conclusion, this preliminary study reveals a platelet-based gene expression signature that differentiates metastatic lung cancer from negative controls on the basis of decreased expression of 197 of the 200 genes with the most altered expression levels. Further study may yield a prognostic tool for future metastasis among subsets of early stage lung cancer patients.

<h4>Background</h4>The atmosphere of valley of Kashmir is ideal for fresh and dry fruit production. Millions of tons of pesticides, insecticides and fungicides (chemicals like chlorpyriphos, mancozeb, captan, dimethoate, phosalone, etc.) are being used by the orchard farmers to spray the plants, fruits and the leaves every year. The increasing trend in the incidence of primary malignant brain tumors in orchard farmers of Kashmir is alarming.<h4>Aim</h4>To determine the relationship between the patients of primary malignant brain tumors and their occupation.<h4>Materials and methods</h4>Retrospectively case files along with death certificates of 432 patients of primary malignant brain tumors and 457 controls (non-tumor neurologic diseases), admitted for treatment simultaneously over a period of 4 years from January 2005 to December 2008, to the Department of Neurosurgery, Sher-I-Kashmir Institute of Medical Sciences (SKIMS), Kashmir, were studied. Follow-up and family contact was established. The serum cholinesterase activity was measured by kinetic/DGKC calorimetric method and ethylenediaminetetraacetic acid (EDTA) samples were sent to the laboratory. The results are expressed in U/l which is U/l×1000. The laboratory at SKIMS, Srinagar, and Dr Lal PathLabs at New Delhi used a reference range for serum cholinesterase as 3167-6333 U/l.<h4>Results</h4>Analysis revealed that 90.04% (389 out of 432) patients were orchard-farm workers, orchard residents and orchard playing children exposed to the high levels of multiple types of neurotoxic and carcinogenic (chlorpyriphos, dimethoate, mancozeb and captan) chemicals for more than 10-20 years. About 31.9% (124 out of 389) of these from both sexes were younger than 40 years beginning exposure at an early age and had higher (<6334 U/l) serum cholinesterase (SCE) levels. The 9.96% (43 out of 432) patients were not exposed to pesticides. On the other hand, only 119 patients out of 457 controls had recorded history of pesticide exposure and 338 were unrelated to pesticides. Out of 389 patients, 71.7% (279 out of 389) were males and 28.3% (110 out of 389) including 7 members of three families, 6 were females and 1 male.<h4>Conclusion</h4>All orchard-related 389 patients had high grade tumors as compared to the non-pesticide tumors. Mortality in pesticide exposed tumors was 12%. Higher levels of SCE were found in 31.9% (124 out of 389) patients and decreased levels in only 45.3% (176 out of 389) orchard-related patients. The significantcase/control odds ratio (OR) of 0.28, hospital control SCE OR of 1.1 and family control SCE OR of 1.5, points the finger of suspicion toward the link between pesticides and brain cancer.

A database of Prostate Cancer Proteomics has been created by using the results of a proteomic study of human prostate carcinoma and benign hyperplasia tissues, and of some human-cultured cell lines (PCP, http://ef.inbi.ras.ru). PCP consists of 7 interrelated modules, each containing four levels of proteomic and biomedical data on the proteins in corresponding tissues or cells. The first data level, onto which each module is based, is a 2DE proteomic reference map where proteins separated by 2D electrophoresis, and subsequently identified by mass-spectrometry, are marked. The results of proteomic experiments form the second data level. The third level contains protein data from published articles and existing databases. The fourth level is formed with direct Internet links to the information on corresponding proteins in the NCBI and UniProt databases. PCP contains data on 359 proteins in total, including 17 potential biomarkers of prostate cancer, particularly AGR2, annexins, S100 proteins, PRO2675, and PRO2044. The database will be useful in a wide range of applications, including studies of molecular mechanisms of the aetiology and pathogenesis of prostate diseases, finding new diagnostic markers, etc.

MicroRNAs (miRNAs) are short RNA molecules which bind to target mRNAs, resulting in translational repression and gene silencing and are found in all eukaryotic cells. Approximately 2200 miRNA genes have been reported to exist in the mammalian genome, from which over 1000 belong to the human genome. Many major cellular functions such as development, differentiation, growth, and metabolism are known to be regulated by miRNAs. Proximity to other genes in the genome and their locations in introns of coding genes, noncoding genes and exons have been reported to have a major influence on the level of gene expressions in eukaryotic cells. miRNAs are well conserved in eukaryotic system and are believed to be an essential and evolutionary ancient component of gene regulatory networks. Therefore, in recent years miRNAs have been studied as a likely candidate for involvement in most biologic processes and have been implicated in many human diseases.

No abstract available.