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Since aberrant wound healing and chronic inflammation can promote malignant transformation, we determined whether dietary bioactive fish oil (FO)-derived n-3 polyunsaturated fatty acids (n-3 PUFA) modulate stem cell kinetics in a colitis-wounding model. Lgr5-LacZ and Lgr5-EGFP-IRES-creER(T2) mice were fed diets enriched with n-3 PUFA vs n-6 PUFA (control) and exposed to dextran sodium sulfate (DSS) for 5days in order to induce crypt damage and colitis throughout the colon. Stem cell number, cell proliferation, apoptosis, expression of stem cell (Lgr5, Sox9, Bmi1, Hopx, mTert, Ascl2, and DCAMKL-1) and inflammation (STAT3) markers were quantified. DSS treatment resulted in the ablation of Lgr5(+) stem cells in the distal colon, concurrent with the loss of distal crypt structure and proliferating cells. Lgr5, Ascl2 and Hopx mRNA expression levels were decreased in damaged colonic mucosa. Lgr5(+) stem cells reappeared at day 5 of DSS recovery, with normal levels attained by day 6 of recovery. There was no effect of diet on the recovery of stem cells. FO fed animals exhibited higher levels of phospho-STAT3 at all time points, consistent with a higher wounding by DSS in FO feeding. n-3 PUFA-fed mice exhibited a reduction in stem cell associated factors, Ascl2, Axin2 and EphB3. These results indicate that rapidly cycling Lgr5(+) stem cells residing at position 1 in the colon epithelium are highly susceptible to DSS-induced damage and that dietary cues can impact stem cell regulatory networks.

We have shown previously and confirmed in this study that the phospholipase A(2) (PLA(2)) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluates the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, strengthened ANS binding, and a lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ΔG°(D) was 3.3 ± 0.3 kcal mol(-1) for Prdx6 and 1.7 ± 0.7 kcal mol(-1) for pPrdx6, suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (50:25:15:10 DPPC:egg PC:cholesterol:PG molar ratio) were evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with a higher affinity (K(d) = 5.6 ± 1.2 μM) than Prdx6 (K(d) = 24.9 ± 4.5 μM). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (ΔH = -31.49 ± 0.22 kcal mol(-1)). Correlating our conformational studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA(2) activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA(2) enzymatic activity.

The organization of eukaryotic chromosomes into transcriptionally active euchromatin and repressed heterochromatin requires mechanisms that establish, maintain and distinguish these canonical chromatin domains. Post-translational modifications are fundamental in these processes. Monoubiquitylation of histones was discovered more than three decades ago, but its precise function has been enigmatic until recently. It is now appreciated that the spectrum of chromatin ubiquitylation is not restricted to monoubiquitylation of histones, but includes degradatory ubiquitylation of histones, histone-modifying enzymes and non-histone chromatin factors. These occur in a spatially and temporally controlled manner. In this review, we summarize our understanding of these mechanisms with a particular emphasis on how ubiquitylation shapes the physical landscape of chromatin.

Plexin transmembrane receptors and their semaphorin ligands, as well as their co-receptors (Neuropilin, Integrin, VEGFR2, ErbB2, and Met kinase) are emerging as key regulatory proteins in a wide variety of developmental, regenerative, but also pathological processes. The diverse arenas of plexin function are surveyed, including roles in the nervous, cardiovascular, bone and skeletal, and immune systems. Such different settings require considerable specificity among the plexin and semaphorin family members which in turn are accompanied by a variety of cell signaling networks. Underlying the latter are the mechanistic details of the interactions and catalytic events at the molecular level. Very recently, dramatic progress has been made in solving the structures of plexins and of their complexes with associated proteins. This molecular level information is now suggesting detailed mechanisms for the function of both the extracellular as well as the intracellular plexin regions. Specifically, several groups have solved structures for extracellular domains for plexin-A2, -B1, and -C1, many in complex with semaphorin ligands. On the intracellular side, the role of small Rho GTPases has been of particular interest. These directly associate with plexin and stimulate a GTPase activating (GAP) function in the plexin catalytic domain to downregulate Ras GTPases. Structures for the Rho GTPase binding domains have been presented for several plexins, some with Rnd1 bound. The entire intracellular domain structure of plexin-A1, -A3, and -B1 have also been solved alone and in complex with Rac1. However, key aspects of the interplay between GTPases and plexins remain far from clear. The structural information is helping the plexin field to focus on key questions at the protein structural, cellular, as well as organism level that collaboratoria of investigations are likely to answer.

<h4>Rationale</h4>MicroRNA-499 and other members of the myomiR family regulate myosin isoforms in pressure-overload hypertrophy. miR-499 expression varies in human disease, but results of mouse cardiac miR-499 overexpression are inconsistent, either protecting against ischemic damage or aggravating cardiomyopathy after pressure overload. Likewise, there is disagreement over direct and indirect cardiac mRNAs targeted in vivo by miR-499.<h4>Objective</h4>To define the associations between regulated miR-499 level in clinical and experimental heart disease and modulation of its predicted mRNA targets and to determine the consequences of increased cardiac miR-499 on direct mRNA targeting, indirect mRNA modulation, and on myocardial protein content and posttranslational modification.<h4>Methods and results</h4>miR-499 levels were increased in failing and hypertrophied human hearts and associated with decreased levels of predicted target mRNAs. Likewise, miR-499 is increased in Gq-mediated murine cardiomyopathy. Forced cardiomyocyte expression of miR-499 at levels comparable to human cardiomyopathy induced progressive murine heart failure and exacerbated cardiac remodeling after pressure overloading. Genome-wide RNA-induced silencing complex and RNA sequencing identified 67 direct, and numerous indirect, cardiac mRNA targets, including Akt and MAPKs. Myocardial proteomics identified alterations in protein phosphorylation linked to the miR-499 cardiomyopathy phenotype, including of heat shock protein 90 and protein serine/threonine phosphatase 1-α.<h4>Conclusions</h4>miR-499 is increased in human and murine cardiac hypertrophy and cardiomyopathy, is sufficient to cause murine heart failure, and accelerates maladaptation to pressure overloading. The deleterious effects of miR-499 reflect the cumulative consequences of direct and indirect mRNA regulation, modulation of cardiac kinase and phosphatase pathways, and higher-order effects on posttranslational modification of myocardial proteins.

MacroH2A is a histone H2A variant that is typically found in heterochromatic regions of the genome. A positively charged linker that connects the histone-fold with the macro-domain was suggested to have DNA-binding properties, and has been shown to promote oligomerization of chromatin fibers. Here we examine the influence of this basic linker on DNA of mononucleosomes. We find that the macro-linker reduces accessibility to extranucleosomal DNA, and appears to increase compaction of the nucleosome. These properties arise from interactions between the H1-like basic linker region and DNA around the entry/exit site, which increases protection of nucleosomal DNA from exonuclease III digestion by ∼10 bp. By stabilizing the wrapping of DNA around the histone core, this basic linker of macroH2A may alter the distribution of nucleosome-associated factors, and potentially contribute to the more compacted nature of heterochromatin.

The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces.

Over 50 genetic variants have been statistically associated with the development of SLE (or lupus). Each genetic association is a key component of a pathway to lupus pathogenesis, the majority of which requires further mechanistic studies to understand the functional changes to cellular physiology. Whereas their use in clinical practice has yet to be established, these genes guide efforts to develop more specific therapeutic approaches. The BCR signaling pathways are rich in lupus susceptibility genes and may well provide novel opportunities for the understanding and clinical treatment of this complex disease.

Polyphenols as "sensitizers" together with cytotoxic drugs as "inducers" cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer.

Proteasomes are the major enzyme complexes for non-lysosomal protein degradation in eukaryotic cells. Mammals express two sets of catalytic subunits: the constitutive subunits β1, β2 and β5 and the immunosubunits LMP2 (β1i), MECL-1 (β2i) and LMP7 (β5i). The LMP7-propeptide (proLMP7) is required for optimal maturation of LMP2/MECL-1-containing precursors to mature immunoproteasomes, but can also mediate efficient integration into mixed proteasomes containing β1 and β2. In contrast, the β5-propeptide (proβ5) has been suggested to promote preferential integration into β1/β2-containing precursors, consequently favouring the formation of constitutive proteasomes. Here, we show that proβ5 predominantly promotes integration into LMP2/MECL-1-containing precursors in IFNγ-stimulated, LMP7-deficient cells and infected LMP7-deficient mice. This demonstrates that proβ5 does not direct preferential integration into β1/β2-containing precursors, but instead promotes the formation of mixed LMP2/MECL-1/β5 proteasomes under inflammatory conditions. Moreover, the propeptides substantially differ in their capacity to promote proteasome maturation, with proLMP7 showing a significantly higher chaperone activity as compared to proβ5. Increased efficiency of proteasome maturation mediated by proLMP7 is required for optimal MHC class I cell surface expression and is equally important as the catalytic activity of immunoproteasomes. Intriguingly, induction of LMP7 by infection not only results in rapid exchange of constitutive by immunosubunits, as previously suggested, but also increases the total proteasome abundance within the infected tissue. Hence our data identify a novel LMP7-dependend mechanism to enhance the activity of the proteasome system in infection, which is based on the high chaperone activity of proLMP7 and relies on accelerated maturation of active proteasome complexes.

Diffusion coefficients of huntingtin (Htt) fragments and SOD1 mutants expressed in cells were measured using fluorescence correlation spectroscopy. The diffusion mobilities of both non-pathological Htt fragments (25 polyQs) and pathological Htt fragments (103 polyQs) were much slower than expected for monomers suggesting that they oligomerize. The mobility of these fragments was unaffected by duration of expression or by over-expression of Hsp70 and Hsp40. However in cells with HttQ103 inclusions, diffusion measurements showed that the residual cytosolic HttQ103 was monomeric. These results suggest that both non-pathological and pathological Htt fragments form soluble oligomers in the cytosol with the properties of the oligomers determining whether they cause pathology. SOD1 with point mutations (A4V, G37R, and G85R) also had slower diffusional mobility than the wild-type protein whose mobility was consistent with that of a dimer. However, the decrease in mobility of the different SOD1 mutants did not correlate with their known pathology. Therefore, while soluble oligomers always seem to be present under conditions where cell pathology occurs, the presence of the oligomers, in itself, does not determine the extent of neuropathology.

The purpose of this study was to develop two novel drug delivery systems based on biodegradable docetaxel-lipid-based-nanosuspensions. The first one was poly(ethylene glycol)- modified docetaxel-lipid-based-nanosuspensions (pLNS). It was developed to increase the cycle time of the drug within the body and enhance the accumulation of the drug at the tumor site. The second one was targeted docetaxel-lipid-based-nanosuspensions (tLNS) using folate as the target ligand. The tLNS could target the tumor cells that overexpressed folate receptor (FR). The morphology, particle size, and zeta potential of pLNS and tLNS were characterized, respectively. The in vitro cytotoxicity evaluation of Duopafei(®), pLNS, and tLNS were performed in human hepatocellular liver carcinoma HepG2 (FR-) and B16 (FR+) cells, respectively. The in vivo antitumor efficacy and pharmacokinetics, as well as the drug tissue distribution, were evaluated in Kunming mice bearing B16 cells. The particle size of pLNS was 204.2 ± 6.18 nm and tLNS had a mean particle size of 220.6 ± 9.54 nm. Cytotoxicity of tLNS against B16 (FR+) cell lines was superior to pLNS (P < 0.05), while there was no significant difference in the half maximum inhibitory concentration values for HepG2 (FR-) cells between pLNS and tLNS. The results of the in vivo antitumor efficacy evaluation showed that tLNS exhibited higher antitumor efficacy by reducing tumor volume (P < 0.01) compared with Duopafei and pLNS, respectively. The results of the in vivo biodistribution study indicate that the better antitumor efficacy of tLNS was attributed to the increased accumulation of the drug in the tumor.

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome's ability to unfold its substrate, and polyglutamine repeats such as found in Huntington's disease reduce the processivity of the proteasome in a length-dependent manner.

<h4>Unlabelled</h4>What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC.<h4>Objective</h4>• To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed.<h4>Materials and methods</h4>• To identify more differentially expressed genes with a lower false discovery rate from a previously published microarray data set, an integrative hypothesis-testing statistical approach was applied. • For validation experiments, expression and phosphorylation levels of select proteins were evaluated by western blotting.<h4>Results</h4>• Integration analysis of this transcriptomics data set with our own quantitative proteomics data set identified 10 genes that are potentially regulated by APF in vivo from 4140 differentially expressed genes identified with a false discovery rate of 1%. • Of these, five (i.e. JUP, MAPKSP1, GSPT1, PTGS2/COX-2 and XPOT) were found to be prominent after network modelling of the common genes identified in the proteomics and microarray studies. • This molecular signature reflects the biological processes of cell adhesion, cell proliferation and inflammation, which is consistent with the known physiological effects of APF. • Lastly, we found the mammalian target of rapamycin pathway was down-regulated in response to APF.<h4>Conclusion</h4>• This unbiased integration analysis of in vitro quantitative proteomics data with in vivo quantitative transcriptomics data led to the identification of potential downstream mediators of the APF signal transduction pathway.

<h4>Background</h4>Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve as therapeutic targets, CHDI Foundation has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner.<h4>Methods</h4>To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies.<h4>Results</h4>Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling). For follow-up studies, we provide a regularly updated compendium of molecular mechanism, that are associated with HD, at http://hdtt.sysbiolab.eu Additionally, we derived a candidate set of 24 novel genetic modifiers, including histone deacetylase 3 (HDAC3), metabotropic glutamate receptor 1 (GRM1), CDK5 regulatory subunit 2 (CDK5R2), and coactivator 1ß of the peroxisome proliferator-activated receptor gamma (PPARGC1B).<h4>Conclusions</h4>The results of our study give us an intriguing picture of the molecular complexity of HD. Our analyses can be seen as a first step towards a comprehensive list of biological processes, molecular functions, and pathways involved in HD, and may provide a basis for the development of more holistic disease models and new therapeutics.

Transposable elements (TEs) are abundant in the human genome, and some are capable of generating new insertions through RNA intermediates. In cancer, the disruption of cellular mechanisms that normally suppress TE activity may facilitate mutagenic retrotranspositions. We performed single-nucleotide resolution analysis of TE insertions in 43 high-coverage whole-genome sequencing data sets from five cancer types. We identified 194 high-confidence somatic TE insertions, as well as thousands of polymorphic TE insertions in matched normal genomes. Somatic insertions were present in epithelial tumors but not in blood or brain cancers. Somatic L1 insertions tend to occur in genes that are commonly mutated in cancer, disrupt the expression of the target genes, and are biased toward regions of cancer-specific DNA hypomethylation, highlighting their potential impact in tumorigenesis.

Huntington's disease (HD) is caused by a CAG expansion in the huntingtin gene. Expansion of the polyglutamine tract in the huntingtin protein results in massive cell death in the striatum of HD patients. We report that human induced pluripotent stem cells (iPSCs) derived from HD patient fibroblasts can be corrected by the replacement of the expanded CAG repeat with a normal repeat using homologous recombination, and that the correction persists in iPSC differentiation into DARPP-32-positive neurons in vitro and in vivo. Further, correction of the HD-iPSCs normalized pathogenic HD signaling pathways (cadherin, TGF-β, BDNF, and caspase activation) and reversed disease phenotypes such as susceptibility to cell death and altered mitochondrial bioenergetics in neural stem cells. The ability to make patient-specific, genetically corrected iPSCs from HD patients will provide relevant disease models in identical genetic backgrounds and is a critical step for the eventual use of these cells in cell replacement therapy.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.

Combinatorial gene perturbations provide rich information for a systematic exploration of genetic interactions. Despite successful applications to bacteria and yeast, the scalability of this approach remains a major challenge for higher organisms such as humans. Here, we report a novel experimental and computational framework to efficiently address this challenge by limiting the 'search space' for important genetic interactions. We propose to integrate rich phenotypes of multiple single gene perturbations to robustly predict functional modules, which can subsequently be subjected to further experimental investigations such as combinatorial gene silencing. We present posterior association networks (PANs) to predict functional interactions between genes estimated using a Bayesian mixture modelling approach. The major advantage of this approach over conventional hypothesis tests is that prior knowledge can be incorporated to enhance predictive power. We demonstrate in a simulation study and on biological data, that integrating complementary information greatly improves prediction accuracy. To search for significant modules, we perform hierarchical clustering with multiscale bootstrap resampling. We demonstrate the power of the proposed methodologies in applications to Ewing's sarcoma and human adult stem cells using publicly available and custom generated data, respectively. In the former application, we identify a gene module including many confirmed and highly promising therapeutic targets. Genes in the module are also significantly overrepresented in signalling pathways that are known to be critical for proliferation of Ewing's sarcoma cells. In the latter application, we predict a functional network of chromatin factors controlling epidermal stem cell fate. Further examinations using ChIP-seq, ChIP-qPCR and RT-qPCR reveal that the basis of their genetic interactions may arise from transcriptional cross regulation. A Bioconductor package implementing PAN is freely available online at http://bioconductor.org/packages/release/bioc/html/PANR.html.

Rose-comb, a classical monogenic trait of chickens, is characterized by a drastically altered comb morphology compared to the single-combed wild-type. Here we show that Rose-comb is caused by a 7.4 Mb inversion on chromosome 7 and that a second Rose-comb allele arose by unequal crossing over between a Rose-comb and wild-type chromosome. The comb phenotype is caused by the relocalization of the MNR2 homeodomain protein gene leading to transient ectopic expression of MNR2 during comb development. We also provide a molecular explanation for the first example of epistatic interaction reported by Bateson and Punnett 104 years ago, namely that walnut-comb is caused by the combined effects of the Rose-comb and Pea-comb alleles. Transient ectopic expression of MNR2 and SOX5 (causing the Pea-comb phenotype) occurs in the same population of mesenchymal cells and with at least partially overlapping expression in individual cells in the comb primordium. Rose-comb has pleiotropic effects, as homozygosity in males has been associated with poor sperm motility. We postulate that this is caused by the disruption of the CCDC108 gene located at one of the inversion breakpoints. CCDC108 is a poorly characterized protein, but it contains a MSP (major sperm protein) domain and is expressed in testis. The study illustrates several characteristic features of the genetic diversity present in domestic animals, including the evolution of alleles by two or more consecutive mutations and the fact that structural changes have contributed to fast phenotypic evolution.

Endocrine therapy resistance is a primary cause of clinical breast cancer treatment failure. The p38 mitogen activated protein kinase (MAPK) signaling pathway is known to promote ligand independent tumor growth and resistance to endocrine therapy. In this study, we investigated the therapeutic potential of the p38 inhibitor RWJ67657 in the treatment of tamoxifen resistant MDA-MB-361 cells. RWJ67657 dose-dependently decreased both basal and stimulated activation of p38 MAPK signaling in this drug resistant cell system. Decreased activation of p38 by RWJ67657 resulted in inhibition of the downstream p38 targets hsp27 and MAPKAPK. Diminished p38 signaling resulted in inhibition of p38-medated gene transcription. Furthermore, pharmacological inhibition of p38 by RWJ67657 decreased biological effects of p38, including ER-mediated gene expression and clonogenic survival in a dose-dependent manner. Animal studies revealed significantly decreased p38 signaling in vivo following exposure to RWJ67657. Treatment with the inhibitor markedly decreased phosphorylation of p38 in MDA-MB-361 tumors, leading to decreased transcription of both Fra-1 and progesterone receptor. Utilizing well-established xenograft tumor models, we demonstrated that RWJ67657 exhibits potent anti-tumor properties. Treatment with RWJ67657 markedly decreased tamoxifen resistant tumor growth, both in the presence and absence of estrogen. Taken together, our findings demonstrate the therapeutic potential of targeting the p38-MAPK signaling cascade in the treatment of endocrine resistant breast cancer.

We report a retrospective case series of four patients with genetically confirmed Huntington's disease (HD) and sporadic amyotrophic lateral sclerosis (ALS), examining the brain and spinal cord in two cases. Neuropathological assessment included a polyglutamine recruitment method to detect sites of active polyglutamine aggregation, and biochemical and immunohistochemical assessment of TDP-43 pathology. The clinical sequence of HD and ALS varied, with the onset of ALS occurring after the mid-50's in all cases. Neuropathologic features of HD and ALS coexisted in both cases examined pathologically: neuronal loss and gliosis in the neostriatum and upper and lower motor neurons, with Bunina bodies and ubiquitin-immunoreactive skein-like inclusions in remaining lower motor neurons. One case showed relatively early HD pathology while the other was advanced. Expanded polyglutamine-immunoreactive inclusions and TDP-43-immunoreactive inclusions were widespread in many regions of the CNS, including the motor cortex and spinal anterior horn. Although these two different proteinaceous inclusions coexisted in a small number of neurons, the two proteins did not co-localize within inclusions. The regional distribution of TDP-43-immunoreactive inclusions in the cerebral cortex partly overlapped with that of expanded polyglutamine-immunoreactive inclusions. In the one case examined by TDP-43 immunoblotting, similar TDP-43 isoforms were observed as in ALS. Our findings suggest the possibility that a rare subset of older HD patients is prone to develop features of ALS with an atypical TDP-43 distribution that resembles that of aggregated mutant huntingtin. Age-dependent neuronal dysfunction induced by mutant polyglutamine protein expression may contribute to later-life development of TDP-43 associated motor neuron disease in a small subset of patients with HD.

Routine integration of genotype data into drug decision making could improve patient safety, particularly if many relevant genetic variants can be assayed simultaneously before prescribing the target drug. The frequency of opportunities for pharmacogenetic prescribing and the potential adverse events (AEs) mitigated are unknown. We examined the frequency with which 56 medications with known outcomes influenced by variant alleles were prescribed in a cohort of 52,942 medical home patients at Vanderbilt University Medical Center (VUMC). Within a 5-year window, we estimated that 64.8% (95% confidence interval (CI): 64.4-65.2%) of individuals were exposed to at least one medication with an established pharmacogenetic association. Using previously published results for six medications with severe, well-characterized, genetically linked AEs, we estimated that 383 events (95% CI, 212-552) could have been prevented with an effective preemptive genotyping program. Our results suggest that multiplexed, preemptive genotyping may represent an efficient alternative approach to current single-use ("reactive") methods and may also improve safety.

Efficient genetic modification of herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) has come to rely on bacterial artificial chromosome (BAC) technology. In order to facilitate this approach, we generated a new KSHV BAC clone, called BAC16, derived from the rKSHV.219 virus, which stems from KSHV and Epstein-Barr virus-coinfected JSC1 primary effusion lymphoma (PEL) cells. Restriction enzyme and complete sequencing data demonstrate that the KSHV of JSC1 PEL cells showed a minimal level of sequence variation across the entire viral genome compared to the complete genomic sequence of other KSHV strains. BAC16 not only stably propagated in both Escherichia coli and mammalian cells without apparent genetic rearrangements, but also was capable of robustly producing infectious virions (∼5 × 10(7)/ml). We also demonstrated the utility of BAC16 by generating deletion mutants of either the K3 or K5 genes, whose products are E3 ligases of the membrane-associated RING-CH (MARCH) family. While previous studies have shown that individual expression of either K3 or K5 results in efficient downregulation of the surface expression of major histocompatibility complex class I (MHC-I) molecules, we found that K5, but not K3, was the primary factor critical for the downregulation of MHC-I surface expression during KSHV lytic reactivation or following de novo infection. The data presented here demonstrate the utility of BAC16 for the generation and characterization of KSHV knockout and mutant recombinants and further emphasize the importance of functional analysis of viral genes in the context of the KSHV genome besides the study of individual gene expression.

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.

Primary prevention guidelines recommend calculation of lifetime cardiovascular disease (CVD) predicted risk in patients who may not meet criteria for high short-term (10-year) Adult Treatment Panel III risk for coronary heart disease (CHD). Extreme obesity and bariatric surgery are more common in women who often have low short-term predicted CHD risk. The distribution and correlates of lifetime CVD predicted risk, however, have not yet been evaluated in bariatric surgical candidates. Using established 10-year (Adult Treatment Panel III) CHD and lifetime CVD risk prediction algorithms and presurgery risk factors, participants from the Longitudinal Assessment of Bariatric Surgery-2 study without prevalent CVD (n = 2,070) were stratified into 3 groups: low 10-year (<10%)/low lifetime (<39%) predicted risk, low 10-year (<10%)/high lifetime (≥39%) predicted risk, and high 10-year (≥10%) predicted risk or diagnosed diabetes. Participants were predominantly white (86%) and women (80%) with a median age of 45 years and median body mass index of 45.6 kg/m(2). High 10-year CHD predicted risk was common (36.5%) and associated with diabetes, male gender, and older age, but not with higher body mass index or high-sensitivity C-reactive protein. Most participants (76%) with low 10-year predicted risk had high lifetime CVD predicted risk, which was associated with dyslipidemia and hypertension but not with body mass index, waist circumference, high-density lipoprotein cholesterol, or high-sensitivity C-reactive protein. In conclusion, bariatric surgical candidates without diabetes or existing CVD are likely to have low short-term, but high lifetime CVD predicted risk. Current data support the need for long-term monitoring and treatment of increased CVD risk factors in bariatric surgical patients to maximize lifetime CVD risk decrease (clinical trial registration, Long-term Effects of Bariatric Surgery, indentifier NCT00465829, available at: http://www.clinicaltrials.gov/ct2/results?term=NCT00465829).

Bim is a proapoptotic BH3-only Bcl-2 family member. In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.

The hallmarks of many haematological malignancies and solid tumours are chromosomal translocations, which may lead to gene fusions. Recently, next-generation sequencing techniques at the transcriptome level (RNA-Seq) have been used to verify known and discover novel transcribed gene fusions. We present FusionFinder, a Perl-based software designed to automate the discovery of candidate gene fusion partners from single-end (SE) or paired-end (PE) RNA-Seq read data. FusionFinder was applied to data from a previously published analysis of the K562 chronic myeloid leukaemia (CML) cell line. Using FusionFinder we successfully replicated the findings of this study and detected additional previously unreported fusion genes in their dataset, which were confirmed experimentally. These included two isoforms of a fusion involving the genes BRK1 and VHL, whose co-deletion has previously been associated with the prevalence and severity of renal-cell carcinoma. FusionFinder is made freely available for non-commercial use and can be downloaded from the project website (http://bioinformatics.childhealthresearch.org.au/software/fusionfinder/).

Mitochondria are key organelles in the cell, hosting essential functions, from biosynthetic and metabolic pathways, to oxidative phosphorylation and ATP production, from calcium buffering to red-ox homeostasis and apoptotic signalling pathways. Mitochondria are also dynamic organelles, continuously fusing and dividing, and their localization, size and trafficking are finely regulated. Moreover, in recent decades, alterations in mitochondrial function and dynamics have been implicated in an increasing number of diseases. In this review, we focus on the relationship clarified hitherto between mitochondrial dynamics and cancer, neurodegenerative and neuroinflammatory diseases.

Alpha-fetoprotein (AFP), des-γ-carboxy prothrombin (DCP), and lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) have been developed with the intent to detect hepatocellular carcinoma (HCC) and for the surveillance of at-risk patients. However, at present, none of these tests can be recommended to survey cirrhotic patients at risk for HCC development because of their suboptimal ability for routine clinical practice in HCC diagnosis. Starting from these considerations, these markers have been therefore routinely and successfully used as predictors of survival and HCC recurrence in patients treated with curative intent. All these markers have been largely used as predictors in patients treated with hepatic resection or locoregional therapies, mainly in Eastern countries. In recent studies, AFP has been proposed as predictor of recurrence after liver transplantation and as selector of patients in the waiting list. Use of AFP modification during the waiting list for LT is still under investigation, potentially representing a very interesting tool for patient selection. The development of a new predictive model combining radiological and biological features based on biological markers is strongly required. New genetic markers are continuously discovered, but they are not already fully available in the clinical practice.

<h4>Background</h4>A common task in analyzing microarray data is to determine which genes are differentially expressed across two (or more) kind of tissue samples or samples submitted under experimental conditions. Several statistical methods have been proposed to accomplish this goal, generally based on measures of distance between classes. It is well known that biological samples are heterogeneous because of factors such as molecular subtypes or genetic background that are often unknown to the experimenter. For instance, in experiments which involve molecular classification of tumors it is important to identify significant subtypes of cancer. Bimodal or multimodal distributions often reflect the presence of subsamples mixtures. Consequently, there can be genes differentially expressed on sample subgroups which are missed if usual statistical approaches are used. In this paper we propose a new graphical tool which not only identifies genes with up and down regulations, but also genes with differential expression in different subclasses, that are usually missed if current statistical methods are used. This tool is based on two measures of distance between samples, namely the overlapping coefficient (OVL) between two densities and the area under the receiver operating characteristic (ROC) curve. The methodology proposed here was implemented in the open-source R software.<h4>Results</h4>This method was applied to a publicly available dataset, as well as to a simulated dataset. We compared our results with the ones obtained using some of the standard methods for detecting differentially expressed genes, namely Welch t-statistic, fold change (FC), rank products (RP), average difference (AD), weighted average difference (WAD), moderated t-statistic (modT), intensity-based moderated t-statistic (ibmT), significance analysis of microarrays (samT) and area under the ROC curve (AUC). On both datasets all differentially expressed genes with bimodal or multimodal distributions were not selected by all standard selection procedures. We also compared our results with (i) area between ROC curve and rising area (ABCR) and (ii) the test for not proper ROC curves (TNRC). We found our methodology more comprehensive, because it detects both bimodal and multimodal distributions and different variances can be considered on both samples. Another advantage of our method is that we can analyze graphically the behavior of different kinds of differentially expressed genes.<h4>Conclusion</h4>Our results indicate that the arrow plot represents a new flexible and useful tool for the analysis of gene expression profiles from microarrays.

<h4>Objectives</h4>Epigenetics can influence disease susceptibility and severity. While DNA methylation of individual genes has been explored in autoimmunity, no unbiased systematic analyses have been reported. Therefore, a genome-wide evaluation of DNA methylation loci in fibroblast-like synoviocytes (FLS) isolated from the site of disease in rheumatoid arthritis (RA) was performed.<h4>Methods</h4>Genomic DNA was isolated from six RA and five osteoarthritis (OA) FLS lines and evaluated using the Illumina HumanMethylation450 chip. Cluster analysis of data was performed and corrected using Benjamini-Hochberg adjustment for multiple comparisons. Methylation was confirmed by pyrosequencing and gene expression was determined by qPCR. Pathway analysis was performed using the Kyoto Encyclopedia of Genes and Genomes.<h4>Results</h4>RA and control FLS segregated based on DNA methylation, with 1859 differentially methylated loci. Hypomethylated loci were identified in key genes relevant to RA, such as CHI3L1, CASP1, STAT3, MAP3K5, MEFV and WISP3. Hypermethylation was also observed, including TGFBR2 and FOXO1. Hypomethylation of individual genes was associated with increased gene expression. Grouped analysis identified 207 hypermethylated or hypomethylated genes with multiple differentially methylated loci, including COL1A1, MEFV and TNF. Hypomethylation was increased in multiple pathways related to cell migration, including focal adhesion, cell adhesion, transendothelial migration and extracellular matrix interactions. Confirmatory studies with OA and normal FLS also demonstrated segregation of RA from control FLS based on methylation pattern.<h4>Conclusions</h4>Differentially methylated genes could alter FLS gene expression and contribute to the pathogenesis of RA. DNA methylation of critical genes suggests that RA FLS are imprinted and implicate epigenetic contributions to inflammatory arthritis.

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

<h4>Objectives</h4>Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages.<h4>Materials and methods</h4>SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250μM) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively.<h4>Results</h4>SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death.<h4>Conclusion</h4>SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy.

<h4>Background</h4>As dengue spreads to new geographical regions and the force of infection changes in existing endemic areas, a greater breadth of clinical presentations is being recognised. Clinical experience suggests that adults manifest a pattern of complications different from those observed in children, but few reports have described the age-related spectrum of disease in contemporaneous groups of patients recruited at the same geographical location.<h4>Methodology/principal findings</h4>Using detailed prospectively collected information from ongoing studies that encompass the full spectrum of hospitalised dengue cases admitted to a single hospital in southern Vietnam, we compared clinical and laboratory features, management, and outcome for 647 adults and 881 children with confirmed dengue. Signs of vascular leakage and shock were more frequent and more severe in children than adults, while bleeding manifestations and organ involvement were more common in adults. Additionally, adults experienced significantly more severe thrombocytopenia. Secondary infection but not serotype was independently associated with greater thrombocytopenia, although with a smaller effect than age-group. The effect of age-group on platelet count was also apparent in the values obtained several weeks after recovery, indicating that healthy adults have intrinsically lower counts compared to children.<h4>Conclusions/significance</h4>There are clear distinctions between adults and children in the pattern of complications seen in association with dengue infection, and these depend partly on intrinsic age-dependent physiological differences. Knowledge of such differences is important to inform research on disease pathogenesis, as well as to encourage development of management guidelines that are appropriate to the age-groups at risk.

The preparation of low-generation dendrimers based on a simple calix[4]arene scaffold by insertion of the iminosugar-analogue C(2)-symmetric 3,4-dihydroxypyrrolidine is described. This methodology allows a rapid incorporation of a considerable number of iminosugar-like moieties in a reduced volume and in a well-defined geometry. The inclusion of alkali-metal ions (sodium and potassium) in the polar cavity defined by the acetamide moieties at the lower rim of the calixarene was demonstrated, which allows also the rigidification of the dendrimer structure and the iminosugar presentation in the clusters. The combination of the supramolecular properties of calixarenes with the advantage of a dendrimeric presentation of repetitive units opens up the possibility of generating well-defined multivalent and multifaceted systems with more complex and/or biologically relevant iminosugars.

Obesity is a complex multifaceted disease resulting from interactions between genetics and lifestyle. The proportion of phenotypic variance ascribed to genetic variance is 0.4 to 0.7 for obesity and recent years have seen considerable success in identifying disease-susceptibility variants. Although with the advent of genome-wide association studies the list of genetic variants predisposing to obesity has significantly increased the identified variants only explain a fraction of disease heritability. Studies of gene-environment interactions can provide more insight into the biological mechanisms involved in obesity despite the challenges associated with such designs. Epigenetic changes that affect gene function without DNA sequence modifications may be a key factor explaining interindividual differences in obesity, with both genetic and environmental factors influencing the epigenome. Disentangling the relative contributions of genetic, environmental and epigenetic marks to the establishment of obesity is a major challenge given the complex interplay between these determinants.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus - another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain.

A key saggitamide intermediate corresponding to a rare sugar framework has been obtained. This approach should help to establish the overall configuration of more complex structures of the sagittamide family.

Axon injury elicits profound cellular changes, including axon regeneration. However, the full range of neuronal injury responses remains to be elucidated. Surprisingly, after axons of Drosophila dendritic arborization neurons were severed, dendrites were more resistant to injury-induced degeneration. Concomitant with stabilization, microtubule dynamics in dendrites increased. Moreover, dendrite stabilization was suppressed when microtubule dynamics was dampened, which was achieved by lowering levels of the microtubule nucleation protein γ-tubulin. Increased microtubule dynamics and global neuronal stabilization were also activated by expression of expanded polyglutamine (poly-Q) proteins SCA1, SCA3, and huntingtin. In all cases, dynamics were increased through microtubule nucleation and depended on JNK signaling, indicating that acute axon injury and long-term neuronal stress activate a common cytoskeleton-based stabilization program. Reducing levels of γ-tubulin exacerbated long-term degeneration induced by SCA3 in branched sensory neurons and in a well established Drosophila eye model of poly-Q-induced neurodegeneration. Thus, increased microtubule dynamics can delay short-term injury-induced degeneration, and, in the case of poly-Q proteins, can counteract progressive longer-term degeneration. We conclude that axon injury or stress triggers a microtubule-based neuroprotective pathway that stabilizes neurons against degeneration.

MicroRNAs (miRNAs) represent small noncoding RNAs that play a role in many diseases, including diabetes. miRNAs target genes important for pancreas development, β-cell proliferation, insulin secretion, and exocytosis. Previously, we documented that microRNA-30d (miR-30d), one of miRNAs up-regulated by glucose, induces insulin gene expression in pancreatic β-cells. Here, we found that the induction of insulin production by overexpression of miR-30d is associated with increased expression of MafA, a β-cell-specific transcription factor. Of interest, overexpression of miR-30d prevented the reduction in both MafA and insulin receptor substrate 2 (IRS2) with TNF-α exposure. Moreover, we identified that mitogen-activated protein 4 kinase 4 (MAP4K4), a TNF-α-activated kinase, is a direct target of miR-30d. Overexpression of miR-30d protected β-cells against TNF-α suppression on both insulin transcription and insulin secretion through the down-regulation of MAP4K4 by the miR-30d. A decrease of miR-30d expression was observed in the islets of diabetic db/db mice, in which MAP4K4 expression level was elevated. Our data support the notion that miR-30d plays multiple roles in activating insulin transcription and protecting β-cell functions from impaired by proinflammatory cytokines and underscore the concept that miR-30d may represent a novel pharmacological target for diabetes intervention.

The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) supports multiple basic science consortia that generate high-content datasets, reagent resources, and methodologies, in the fields of kidney, urology, hematology, digestive, and endocrine diseases, as well as metabolic diseases such as diabetes and obesity. These currently include the Beta Cell Biology Consortium, the Nuclear Receptor Signaling Atlas, the Diabetic Complications Consortium, and the Mouse Metabolic Phenotyping Centers. Recognizing the synergy that would accrue from aggregating information generated and curated by these initiatives in a contiguous informatics network, we created the NIDDK Consortium Interconnectivity Network (dkCOIN; www.dkcoin.org). The goal of this pilot project, organized by the NIDDK, was to establish a single point of access to a toolkit of interconnected resources (datasets, reagents, and protocols) generated from individual consortia that could be readily accessed by biologists of diverse backgrounds and research interests. During the pilot phase of this activity dkCOIN collected nearly 2000 consortium-curated resources, including datasets (functional genomics) and reagents (mouse strains, antibodies, and adenoviral constructs) and built nearly 3000 resource-to-resource connections, thereby demonstrating the feasibility of further extending this database in the future. Thus, dkCOIN promises to be a useful informatics solution for rapidly identifying useful resources generated by participating research consortia.

Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34⁺ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS.

Bax inhibitor-1 (BI-1) is an anti-apoptotic protein located in the endoplasmic reticulum (ER). The role of BI-1 has been studied in different physiopathological models including ischemia, diabetes, liver regeneration and cancer. However, fundamental knowledge about the effects of BI-1 deletion on the proteome is lacking. To further explore this protein, we compared the levels of different proteins in bi-1 (-/-) and bi-1 (+/+) mouse tissues by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). In several bi-1 (-/-) mice, glucose-regulated protein 75 (GRP75/mortalin/ PBP74/mthsp70), peroxiredoxin6 (Prx6) and fumarylacetoacetate hydrolase (FAH) showed a pI shift that could be attributed to post-translational modifications. Selenium-binding protein 2 (SBP2) and ferritin light chain 1 levels were significantly increased. Phosphatidylethanolamine-binding protein-1 (PEBP-1) was dramatically decreased in bi-1 (-/-) mice, which was confirmed by Western blotting. The phosphorylation of GRP75, Prx6 and FAH were compared between bi-1 (+/+) and bi-1 (-/-) mice using liver tissue lysates. Of these three proteins, only one exhibited modified phosphorylation; Tyr phosphorylation of Prx6 was increased in bi-1 (-/-) mice. Our protein profiling results provide fundamental knowledge about the physiopathological function of BI-1.

Chemical synapses are highly specialized cell-cell contacts for communication between neurons in the CNS characterized by complex and dynamic protein networks at both synaptic membranes. The cytomatrix at the active zone (CAZ) organizes the apparatus for the regulated release of transmitters from the presynapse. At the postsynaptic side, the postsynaptic density constitutes the machinery for detection, integration, and transduction of the transmitter signal. Both pre- and postsynaptic protein networks represent the molecular substrates for synaptic plasticity. Their function can be altered both by regulating their composition and by post-translational modification of their components. For a comprehensive understanding of synaptic networks the entire ensemble of synaptic proteins has to be considered. To support this, we established a comprehensive database for synaptic junction proteins (SynProt database) primarily based on proteomics data obtained from biochemical preparations of detergent-resistant synaptic junctions. The database currently contains 2,788 non-redundant entries of rat, mouse, and some human proteins, which mainly have been manually extracted from 12 proteomic studies and annotated for synaptic subcellular localization. Each dataset is completed with manually added information including protein classifiers as well as automatically retrieved and updated information from public databases (UniProt and PubMed). We intend that the database will be used to support modeling of synaptic protein networks and rational experimental design.

<h4>Background</h4>Esophageal squamous cell carcinoma (ESCC) is highly prevalent in China and other Asian countries, as a major cause of cancer-related mortality. ESCC displays complex chromosomal abnormalities, including multiple structural and numerical aberrations. Chromosomal abnormalities, such as recurrent amplifications and homozygous deletions, directly contribute to tumorigenesis through altering the expression of key oncogenes and tumor suppressor genes.<h4>Methodology/principle findings</h4>To understand the role of genetic alterations in ESCC pathogenesis and identify critical amplification/deletion targets, we performed genome-wide 1-Mb array comparative genomic hybridization (aCGH) analysis for 10 commonly used ESCC cell lines. Recurrent chromosomal gains were frequently detected on 3q26-27, 5p15-14, 8p12, 8p22-24, 11q13, 13q21-31, 18p11 and 20q11-13, with frequent losses also found on 8p23-22, 11q22, 14q32 and 18q11-23. Gain of 11q13.3-13.4 was the most frequent alteration in ESCC. Within this region, CCND1 oncogene was identified with high level of amplification and overexpression in ESCC, while FGF19 and SHANK2 was also remarkably over-expressed. Moreover, a high concordance (91.5%) of gene amplification and protein overexpression of CCND1 was observed in primary ESCC tumors. CCND1 amplification/overexpression was also significantly correlated with the lymph node metastasis of ESCC.<h4>Conclusion</h4>These findings suggest that genomic gain of 11q13 is the major mechanism contributing to the amplification. Novel oncogenes identified within the 11q13 amplicon including FGF19 and SHANK2 may play important roles in ESCC tumorigenesis.

<h4>Background</h4>We previously demonstrated that 25-hydroxyvitamin D(3), the precursor of 1α,25-dihydroxyvitamin D(3), is abundant around periodontal soft tissues. Here we investigate whether 25-hydroxyvitamin D(3) is converted to 1α,25-dihydroxyvitamin D(3) in periodontal soft tissue cells and explore the possibility of an autocrine/paracrine function of 1α,25-dihydroxyvitamin D(3) in periodontal soft tissue cells.<h4>Methodology/principal findings</h4>We established primary cultures of human gingival fibroblasts and human periodontal ligament cells from 5 individual donors. We demonstrated that 1α-hydroxylase was expressed in human gingival fibroblasts and periodontal ligament cells, as was cubilin. After incubation with the 1α-hydroxylase substrate 25-hydroxyvitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 1α,25-dihydroxyvitamin D(3) that resulted in an up-regulation of CYP24A1 and RANKL mRNA. A specific knockdown of 1α-hydroxylase in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 1α,25-dihydroxyvitamin D(3) production and mRNA expression of CYP24A1 and RANKL. The classical renal regulators of 1α-hydroxylase (parathyroid hormone, calcium and 1α,25-dihydroxyvitamin D(3)) and Porphyromonas gingivalis lipopolysaccharide did not influence 1α-hydroxylase expression significantly, however, interleukin-1β and sodium butyrate strongly induced 1α-hydroxylase expression in human gingival fibroblasts and periodontal ligament cells.<h4>Conclusions/significance</h4>In this study, the expression, activity and functionality of 1α-hydroxylase were detected in human gingival fibroblasts and periodontal ligament cells, raising the possibility that vitamin D acts in an autocrine/paracrine manner in these cells.

Cell stress proteins (CSPS) are a large and heterogeneous family of proteins, sharing two main characteristics: their levels and/or location are modified under stress and most of them can exert a chaperon function inside the cells. Nonetheless, they are also involved in the modulation of several mechanisms, both at the intracellular and the extracellular compartments. There are more than 100 proteins belonging to the CSPs family, among them the thioredoxin (TRX) system, which is the focus of the present paper. TRX system is composed of several proteins such as TRX and peroxiredoxin (PRDX), two thiol-containing enzymes that are key players in redox homeostasis due to their ability to scavenge potential harmful reactive oxygen species. In addition to their main role as antioxidants, recent data highlights their function in several processes such as cell signalling, immune inflammatory responses, or apoptosis, all of them key mechanisms involved in atherothrombosis. Moreover, since TRX and PRDX are present in the pathological vascular wall and can be secreted under prooxidative conditions to the circulation, several studies have addressed their role as diagnostic, prognostic, and therapeutic biomarkers of cardiovascular diseases (CVDs).

<h4>Background</h4>Dihydroxylation of tandemly linked aromatic carbons in a cis-configuration, catalyzed by multicomponent oxygenase systems known as Rieske nonheme iron oxygenase systems (ROs), often constitute the initial step of aerobic degradation pathways for various aromatic compounds. Because such RO reactions inherently govern whether downstream degradation processes occur, novel oxygenation mechanisms involving oxygenase components of ROs (RO-Os) is of great interest. Despite substantial progress in structural and physicochemical analyses, no consensus exists on the chemical steps in the catalytic cycles of ROs. Thus, determining whether conformational changes at the active site of RO-O occur by substrate and/or oxygen binding is important. Carbazole 1,9a-dioxygenase (CARDO), a RO member consists of catalytic terminal oxygenase (CARDO-O), ferredoxin (CARDO-F), and ferredoxin reductase. We have succeeded in determining the crystal structures of oxidized CARDO-O, oxidized CARDO-F, and both oxidized and reduced forms of the CARDO-O: CARDO-F binary complex.<h4>Results</h4>In the present study, we determined the crystal structures of the reduced carbazole (CAR)-bound, dioxygen-bound, and both CAR- and dioxygen-bound CARDO-O: CARDO-F binary complex structures at 1.95, 1.85, and 2.00 Å resolution. These structures revealed the conformational changes that occur in the catalytic cycle. Structural comparison between complex structures in each step of the catalytic mechanism provides several implications, such as the order of substrate and dioxygen bindings, the iron-dioxygen species likely being Fe(III)-(hydro)peroxo, and the creation of room for dioxygen binding and the promotion of dioxygen binding in desirable fashion by preceding substrate binding.<h4>Conclusions</h4>The RO catalytic mechanism is proposed as follows: When the Rieske cluster is reduced, substrate binding induces several conformational changes (e.g., movements of the nonheme iron and the ligand residue) that create room for oxygen binding. Dioxygen bound in a side-on fashion onto nonheme iron is activated by reduction to the peroxo state [Fe(III)-(hydro)peroxo]. This state may react directly with the bound substrate, or O-O bond cleavage may occur to generate Fe(V)-oxo-hydroxo species prior to the reaction. After producing a cis-dihydrodiol, the product is released by reducing the nonheme iron. This proposed scheme describes the catalytic cycle of ROs and provides important information for a better understanding of the mechanism.

Classical hereditary hemochromatosis involves the HFE-gene and diagnostic analysis of the DNA variants HFE p.C282Y (c.845G>A; rs1800562) and HFE p.H63D (c.187C>G; rs1799945). The affected protein alters the iron homeostasis resulting in iron overload in various tissues. The aim of this study was to validate the TaqMan-based Sample-to-SNP protocol for the analysis of the HFE-p.C282Y and p.H63D variants with regard to accuracy, usefulness and reproducibility compared to an existing SNP protocol. The Sample-to-SNP protocol uses an approach where the DNA template is made accessible from a cell lysate followed by TaqMan analysis. Besides the HFE-SNPs other eight SNPs were used as well. These SNPs were: Coagulation factor II-gene F2 c.20210G>A, Coagulation factor V-gene F5 p.R506Q (c.1517G>A; rs121917732), Mitochondria SNP: mt7028 G>A, Mitochondria SNP: mt12308 A>G, Proprotein convertase subtilisin/kexin type 9-gene PCSK9 p.R46L (c.137G>T), Plutathione S-transferase pi 1-gene GSTP1 p.I105V (c313A>G; rs1695), LXR g.-171 A>G, ZNF202 g.-118 G>T. In conclusion the Sample-to-SNP kit proved to be an accurate, reliable, robust, easy to use and rapid TaqMan-based SNP detection protocol, which could be quickly implemented in a routine diagnostic or research facility.

HG-7-85-01(22) and HG-7-86-01(26) are thiazolo[5,4-b]pyridine containing type II tyrosine kinase inhibitors with potent cellular activity against both wild-type and 'gatekeeper' mutant T315I- Bcr-Abl. Here we report on the 'hybrid design' concept and subsequent structure activity guided optimization efforts that resulted in the development of these inhibitors.

Francisella tularensis causes the zoonosis tularemia in humans and is one of the most virulent bacterial pathogens. We utilized a global proteomic approach to characterize protein changes in bronchoalveolar lavage fluid from mice exposed to one of three organisms, F. tularensis ssp. novicida, an avirulent mutant of F. tularensis ssp. novicida (F.t. novicida-ΔmglA), and Pseudomonas aeruginosa. The composition of bronchoalveolar lavage fluid (BALF) proteins was altered following infection, including proteins involved in neutrophil activation, oxidative stress, and inflammatory responses. Components of the innate immune response were induced including the acute phase response and the complement system; however, the timing of their induction varied. F. tularensis ssp. novicida infected mice do not appear to have an effective innate immune response in the first hours of infection; however, within 24 h, they show an upregulation of innate immune response proteins. This delayed response is in contrast to P. aeruginosa infected animals which show an early innate immune response. Likewise, F.t. novicida-ΔmglA infection initiates an early innate immune response; however, this response is diminished by 24 h. Finally, this study identifies several candidate biomarkers, including Chitinase 3-like-1 (CHI3L1 or YKL-40) and peroxiredoxin 1, that are associated with F. tularensis ssp. novicida but not P. aeruginosa infection.

Thiol-ene photoclick hydrogels have been used for a variety of tissue engineering and controlled release applications. In this step-growth photopolymerization scheme, four-arm poly(ethylene glycol) norbornene (PEG4NB) was cross-linked with dithiol containing cross-linkers to form chemically cross-linked hydrogels. While the mechanism of thiol-ene gelation was well described in the literature, its network ideality and degradation behaviors are not well-characterized. Here, we compared the network cross-linking of thiol-ene hydrogels to Michael-type addition hydrogels and found thiol-ene hydrogels formed with faster gel points and higher degree of cross-linking. However, thiol-ene hydrogels still contained significant network nonideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEG-norbornene macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, we found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network cross-linking. In an attempt to manipulate network cross-linking and degradation of thiol-ene hydrogels, we incorporated peptide cross-linkers with different sequences and characterized the hydrolytic degradation of these PEG-peptide hydrogels. In addition, we incorporated a chymotrypsin-sensitive peptide as part of the cross-linkers to tune the mode of gel degradation from bulk degradation to surface erosion.

Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.

Induction of autophagy is known not only to regulate cellular homeostasis but also to decrease triglyceride accumulation in hepatocytes. The aim of this study is to investigate whether DMSO (dimethyl sulfoxide) has a beneficial role in free fatty acid-induced hepatic fat accumulation. In HepG2 cells, treatment with 0.5 mM palmitate for six hours significantly increased lipid and triglyceride (TG) accumulation, assessed by Oil-red O staining and TG quantification assay. Treatment with 0.01% DMSO for 16 h statistically reduced palmitate-induced TG contents. Pretreatment of 10 mM 3-methyladenine (3MA) for 2 h restored hepatocellular lipid contents, which were attenuated by treatment with DMSO. DMSO increased LC3-II conversion and decreased SQSTM1/p62 expression in a time and dose-dependent manner. In addition, the number of autophagosomes and autolysosomes, as seen under an electron microscopy, as well as the percentage of RFP-LAMP1 colocalized with GFP-LC3 dots in cells transfected with both GFP-LC3 and RFP-LAMP1, as seen under a fluorescent microscopy, also increased in DMSO-treated HepG2 cells. DMSO also suppressed p-eIF2α/p-EIF2S1, ATF4, p-AKT1, p-MTOR and p-p70s6k/p-RPS6KB2 expression as assessed by western blotting. Knockdown of ATF4 expression using siRNA suppressed ATF4 expression and phosphorylation of AKT1, MTOR and RPS6KB2, but increased LC3-II conversion. DMSO reduced not only soluble but also insoluble mtHTT (mutant huntingtin aggregates) expressions, which were masked in the presence of autophagy inhibitor. DMSO, a kind of chemical chaperone, activated autophagy by suppressing ATF4 expression and might play a protective role in the development of fatty acid-induced hepatosteatosis.

<h4>Background</h4>The role of the study coordinator (SC) in multicenter studies of long duration has received limited attention.<h4>Purpose</h4>To describe the evolution of the SC's role during the 28-year Diabetes Control and Complications Trial (DCCT) and its follow-up study, the Epidemiology of Diabetes Interventions and Complications (EDIC) study.<h4>Methods</h4>The evolution of the SC's position from the traditional role of protocol implementation to that of research collaborator and co-investigator, based on personal experience and observation, is described in detail. Findings from a survey regarding professional demographics and job satisfaction, completed by all 28 SCs in 2010, provided additional information. We used dimensions of the SC's role specific to DCCT/EDIC to construct a classification schema of functions and responsibilities that describe the SC's role.<h4>Results</h4>Among the 28 SCs, 24 were nurses, 12 held bachelor's degrees, 11 had a master's degree, 19 were certified diabetes educators (CDEs), 12 had worked with DCCT/EDIC for more than 20 years, and 5 had been with the study since its inception (>26 years). Responses confirmed a high degree of functional consistency across sites with data acquisition, performing study procedures, recruitment and consent for additional ancillary studies, regulatory management, scheduling, clinical consultation, and ongoing contact with study participants frequently reported. Study-wide leadership activities, a category not generally included in the usual SC role, were reported by approximately 30% of the SCs. The level of professional satisfaction was high with two-thirds being very satisfied, one-third moderately to quite satisfied, and none dissatisfied.<h4>Limitations</h4>The limitations include a relatively small sample size, self-reported data, and a single long-term multicenter trial and observational follow-up study on which we based our findings and conclusions.<h4>Conclusions</h4>By optimizing their organizational and scientific contributions to the overall research endeavor, SCs in DCCT/EDIC have made major contributions to the unprecedented success of the study and report high job satisfaction. The efforts of the SCs have been integral to the remarkably high participant retention and data completion rates. The DCCT/EDIC experience may serve as a model for the role of the SC in future diabetes and other multicenter clinical trials.

In retroviral infections, a copy of the viral DNA is first synthesized from genomic RNA by reverse transcription and subsequently integrated into host chromatin. This integration step, executed by the viral enzyme integrase (IN), is one of the hallmarks of retroviral infection. Although an obligate role for IN in retroviral integration has been clearly defined by numerous biochemical analysis of its recombinant protein and genetic analysis of the viral IN gene, several host cellular proteins have also been implicated as key factors involved in the integration step during viral replication. Although studies on integration cofactors have mostly emphasized factors that aid the integration process either through direct or indirect association with IN, it has become apparent that host cells may also harbor proteins that act as inhibitors of retroviral integration. Intriguingly, some of these inhibitory proteins appear to hamper the integration process via posttranslational modifications of the components of the preintegration complex including IN. A better understanding of the molecular mechanisms leading to the inhibition of integration will provide us with clues for the development of new strategies for treating retroviral infections. In this review, we draw attention to recent insights regarding potential host cellular factors that restrict integration, and illustrate how these inhibitory effects are achieved.

<h4>Rationale</h4>Peripheral blood biomarkers are needed to identify and determine the extent of idiopathic pulmonary fibrosis (IPF). Current physiologic and radiographic prognostic indicators diagnose IPF too late in the course of disease. We hypothesize that peripheral blood biomarkers will identify disease in its early stages, and facilitate monitoring for disease progression.<h4>Methods</h4>Gene expression profiles of peripheral blood RNA from 130 IPF patients were collected on Agilent microarrays. Significance analysis of microarrays (SAM) with a false discovery rate (FDR) of 1% was utilized to identify genes that were differentially-expressed in samples categorized based on percent predicted D(L)CO and FVC.<h4>Main measurements and results</h4>At 1% FDR, 1428 genes were differentially-expressed in mild IPF (D(L)CO >65%) compared to controls and 2790 transcripts were differentially- expressed in severe IPF (D(L)CO >35%) compared to controls. When categorized by percent predicted D(L)CO, SAM demonstrated 13 differentially-expressed transcripts between mild and severe IPF (< 5% FDR). These include CAMP, CEACAM6, CTSG, DEFA3 and A4, OLFM4, HLTF, PACSIN1, GABBR1, IGHM, and 3 unknown genes. Principal component analysis (PCA) was performed to determine outliers based on severity of disease, and demonstrated 1 mild case to be clinically misclassified as a severe case of IPF. No differentially-expressed transcripts were identified between mild and severe IPF when categorized by percent predicted FVC.<h4>Conclusions</h4>These results demonstrate that the peripheral blood transcriptome has the potential to distinguish normal individuals from patients with IPF, as well as extent of disease when samples were classified by percent predicted D(L)CO, but not FVC.

The existence of multiple inherited disorders of iron metabolism suggests genetic contributions to iron deficiency. We previously performed a genome-wide association study of iron-related single nucleotide polymorphisms (SNPs) using DNA from white men aged ≥ 25 y and women ≥ 50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤ 12 µg/L (cases) and controls (SF >100 µg/L in men, SF >50 µg/L in women). We report a follow-up study of white, African-American, Hispanic, and Asian HEIRS participants, analyzed for association between SNPs and eight iron-related outcomes. Three chromosomal regions showed association across multiple populations, including SNPs in the TF and TMPRSS6 genes, and on chromosome 18q21. A novel SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p = 3.7 × 10(-6)) and replicated in African Americans (p = 0.0012).Twenty SNPs in the TF gene region were associated with total iron-binding capacity in whites (p<4.4 × 10(-5)); six SNPs replicated in other ethnicities (p<0.01). SNP rs10904850 in the CUBN gene on 10p13 was associated with serum iron in African Americans (P = 1.0 × 10(-5)). These results confirm known associations with iron measures and give unique evidence of their role in different ethnicities, suggesting origins in a common founder.

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.

<h4>Background</h4>Although blood donations may reduce body iron stores, to date, prospective data on frequent blood donation and colorectal cancer risk are limited.<h4>Methodology/principal findings</h4>We tested whether frequent blood donation is associated with a lower risk of colorectal cancer in the Health Professionals Follow-up Study. We prospectively followed 35,121 men who provide the information on lifetime number of blood donations in 1992 through 2008. Serum ferritin levels were measured in a random sample of 305 men. Cox proportional hazard regression models were used to calculate the multivariable relative risks (RRs, 95%CIs) after adjusting for age and other established colorectal cancer risk factors. We documented 684 incident colorectal cancer cases and 224 deaths from colorectal cancer. The mean serum ferritin levels varied from 178 µg/L for men who did not donate blood to 98 µg/L for men who had at least 30 donations. Age-adjusted results for both incidence and mortality were essentially the same as the multivariable-adjusted results. Comparing with non-donors, the multivariable RRs (95%CIs) for colorectal cancer incidence were 0.92 (0.77, 1.11) for 1-5 donation, 0.85 (0.64, 1.11) for 6-9 donations, 0.96 (0.73, 1.26) for 10-19 donations, 0.91 (0.63, 1.32) for 20-29 donations, and 0.97 (0.68, 1.38) for at least 30 donations (P(trend) = 0.92). The multivariable RRs for colorectal cancer mortality were 0.99 (0.72, 1.36) for 1-5 donation, 0.93 (0.57, 1.51) for 6-9 donations, 0.85 (0.50, 1.42) for 10-19 donations, and 1.14 (0.72, 1.83) for at least 20 donations (P(trend) = 0.82). The results did not vary by cancer sub-sites, intake levels of total iron, heme iron, or family history of colorectal cancer.<h4>Conclusions/significance</h4>Frequent blood donations were not associated with colorectal cancer incidence and mortality in men. Our results do not support an important role of body iron stores in colorectal carcinogenesis.

Precision in auditory brainstem connectivity underlies sound localization. Cochlear activity is transmitted to the ventral cochlear nucleus (VCN) in the mammalian brainstem via the auditory nerve. VCN globular bushy cells project to the contralateral medial nucleus of the trapezoid body (MNTB), where specialized axons terminals, the calyces of Held, encapsulate MNTB principal neurons. The VCN-MNTB pathway is an essential component of the circuitry used to compute interaural intensity differences that are used for localizing sounds. When input from one ear is removed during early postnatal development, auditory brainstem circuitry displays robust anatomical plasticity. The molecular mechanisms that control the development of auditory brainstem circuitry and the developmental plasticity of these pathways are poorly understood. In this study we examined the role of EphB signaling in the development of the VCN-MNTB projection and in the reorganization of this pathway after unilateral deafferentation. We found that EphB2 and EphB3 reverse signaling are critical for the normal development of the projection from VCN to MNTB, but that successful circuit assembly most likely relies upon the coordinated function of many EphB proteins. We have also found that ephrin-B reverse signaling repels induced projections to the ipsilateral MNTB after unilateral deafferentation, suggesting that similar mechanisms regulate these two processes.

Autophagy is a conserved and highly regulated catabolic pathway, transferring cytoplasmic components in autophagosomes to lysosomes for degradation and providing amino acids during starvation. In multicellular organisms autophagy plays an important role for tissue homeostasis, and deregulation of autophagy has been implicated in a broad range of diseases, including cancer and neurodegenerative disorders. In mammals, many aspects of autophagy still need to be fully elucidated: what is the exact hierarchy and relationship between ATG proteins and other factors that lead to the formation and expansion of phagophores? Where does the membrane source for autophagosome formation originate? Which signaling events trigger amino acid starvation-induced autophagy? How are the activities of ULK1/2 and the class III PtdIns3K regulated and linked to each other? To develop therapeutic strategies to manipulate autophagy in human disease, a comprehensive understanding of the molecular protein machinery mediating and regulating autophagy is required.

<h4>Background</h4>Huntington disease (HD) is caused by an expanded CAG repeat in the HD gene. Although the length of the CAG repeat strongly correlates with the age-at-onset (AAO), AAO in HD individuals may differ dramatically in spite of similar expanded CAG repeat lengths. Additional genetic or environmental factors are thought to influence the disease onset. Several modifier genes have been discovered so far but they do not fully explain the variability of AAO in HD. To potentially identify a novel genetic modifier, we analyzed single nucleotide polymorphisms (SNPs) in the kalirin (KALRN) gene. Kalirin is a protein crucially involved in spine plasticity and its interaction with huntingtin-associated protein-1 (HAP-1) and a potential protein dysfunction might contribute to spine pathogenesis in HD.<h4>Methods</h4>The selected SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and association of SNPs with AAO was investigated with the framework of linear models in an analysis of variance and covariance.<h4>Results</h4>Eleven SNPs in the kalirin gene were examined in an association study in European HD patients. The ten coding SNPs under investigation were monomorphic, whereas SNP rs10934657 in the promoter region showed a minor allele frequency >1%. An analysis of covariance together with the influence of the expanded HD allele was applied in 680 HD patients. SNP rs10934657 did not affect the AAO of the examined HD population.<h4>Conclusions</h4>The results did not reveal an association between the analyzed kalirin polymorphisms and the AAO in HD. However, it does not exclude other SNPs of the kalirin gene as susceptible genetic modifiers.

Ag-doped nanocrystalline hydroxyapatite nanoparticles (Ag:HAp-NPs) (Ca10-xAgx(PO4)6(OH)2, xAg = 0.05, 0.2, and 0.3) with antibacterial properties are of great interest in the development of new products. Coprecipitation method is a promising route for obtaining nanocrystalline Ag:HAp with antibacterial properties. X-ray diffraction identified HAp as an unique crystalline phase in each sample. The calculated lattice constants of a = b = 9.435 Å, c = 6.876 Å for xAg = 0.05, a = b = 9.443 Å, c = 6.875 Å for xAg = 0.2, and a = b = 9.445 Å, c = 6.877 Å for xAg = 0.3 are in good agreement with the standard of a = b = 9.418 Å, c = 6.884 Å (space group P63/m). The Fourier transform infrared and Raman spectra of the sintered HAp show the absorption bands characteristic to hydroxyapatite. The Ag:HAp nanoparticles are evaluated for their antibacterial activity against Staphylococcus aureus, Klebsiella pneumoniae, Providencia stuartii, Citrobacter freundii and Serratia marcescens. The results showed that the antibacterial activity of these materials, regardless of the sample types, was greatest against S. aureus, K. pneumoniae, P. stuartii, and C. freundii. The results of qualitative antibacterial tests revealed that the tested Ag:HAp-NPs had an important inhibitory activity on P. stuartii and C. freundii. The absorbance values measured at 490 nm of the P. stuartii and C. freundii in the presence of Ag:HAp-NPs decreased compared with those of organic solvent used (DMSO) for all the samples (xAg = 0.05, 0.2, and 0.3). Antibacterial activity increased with the increase of xAg in the samples. The Ag:HAp-NP concentration had little influence on the bacterial growth (P. stuartii).

Activation of p53 upon DNA damage induces an array of target genes, leading to cell cycle arrest and/or apoptosis. However, the mechanism by which the cell fate is controlled by p53 remains to be clarified. Previously, we showed that DEC1, a basic helix-loop-helix transcription factor and a target of p53, is capable of inducing cell cycle arrest and mediating DNA damage-induced premature senescence. Here, we found that ectopic expression of DEC1 inhibits, whereas knockdown of DEC1 enhances, DNA damage-induced cell death. Surprisingly, we showed that the anti-cell-death activity of DEC1 is p53 dependent, but DEC1 does not directly modulate p53 expression. Instead, we showed that DEC1 inhibits the ability of p53 to induce macrophage inhibitory cytokine-1 (MIC-1), but not other prosurvival/proapoptotic targets, including p21 and Puma. Importantly, we showed that upon binding to their respective response elements on the MIC-1 promoter, DEC1 and p53 physically interact on the MIC-1 promoter via the basic helix-loop-helix domain in DEC1 and the tetramerization domain in p53, which likely weakens the DNA-binding activity of p53 to the MIC-1 promoter. Finally, we found that depletion of MIC-1 abrogates the ability of DEC1 to attenuate DNA damage-induced cell death. Together, we hypothesize that DEC1 controls the response of p53-dependent cell survival vs. cell death to a stress signal through MIC-1.

Bim is a BH3-only member of the Bcl-2 family that enables the death of T-cells. Partial rescue of cytokine-deprived T-cells occurs when Bim and the receptor for the T-cell growth factor, interleukin-7, are deleted, implicating Bim as a possible target of interleukin-7-mediated signaling. Alternative splicing yields three major isoforms: BimEL, BimL and BimS. To study the effect of Bim deficiency and define the function of the major isoforms, Bim-containing and Bim-deficient T-cells, dependent on interleukin-7 for growth, were used. Loss of total Bim in interleukin-7-deprived T-cells resulted in delayed apoptosis. However, loss of Bim also impeded the later degradative phase of autophagy. p62, an autophagy-adaptor protein which is normally degraded, accumulated in Bim deficient cells. To explain this, BimL was found to support acidification of lysosomes that later may associate with autophagic vesicles. Key findings showed that inhibition of lysosomal acidification accelerated death upon interleukin-7 withdrawal only in Bim-containing T-cells. intereukin-7 dependent T-cells lacking Bim were less sensitive to inhibition of lysosomal acidification. BimL co-immunoprecipitated with dynein and Lamp1-containing vesicles, indicating BimL could be an adaptor for dynein to facilitate loading of lysosomes. In Bim deficient T-cells, lysosome-tracking probes revealed vesicles of less acidic pH. Over-expression of BimL restored acidic vesicles in Bim deficient T-cells, while other isoforms, BimEL and BimS, promoted intrinsic cell death. These results reveal a novel role for BimL in lysosomal positioning that may be required for the formation of degradative autolysosomes.

<h4>Background & aims</h4>The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP-SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes.<h4>Methods</h4>We explore protein-protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells.<h4>Results</h4>Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120-139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS.<h4>Conclusions</h4>Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV.

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.

This chapter will focus on the role of microRNAs (miRs) in regulating the actions of opioid drugs through the opioid receptors. Opioids, such as morphine, are analgesics that are used for treating many forms of acute and chronic pain. However, their chronic use is limited by undesirable effects such as opioid tolerance. The μ opioid receptor (MOR) is the primary receptor responsible for opioids' analgesia and antinociceptive tolerance. The long 3'-untranslated region (3'-UTR) of MOR mRNA is of great interest since this region may contain elements for the post-transcriptional regulation of receptor expression, such as altering the stability of mRNA, influencing translational efficiency, and controlling mRNA transport. MicroRNAs are small non-coding RNA molecules that exert their functions through base-pairing with partially complementary sequences in the 3'-UTR of target mRNAs, resulting in decreased polypeptide formation from those mRNAs. Since the discovery of the first miR, lin-4 in Caenorhabditis elegans, hundreds of miRs have been identified from humans to viruses, which have provided a crucial and pervasive layer of post-transcriptional gene regulation. The nervous system is a rich source of miR expression, with a diversity of miR functions in fundamental neurobiological processes including neuronal development, plasticity, metabolism, and apoptosis. Recently, the let-7 family of miRs is found to be a critical regulator of MOR function in opioid tolerance. Let-7 is the first identified human miR. Its family members are highly conserved across species in sequence and function. In the review, we will present a brief review of the opioid receptors, their regulation, and opioid tolerance as well as an overview of miRs and a perspective how miRs may interact with MOR and serve as a regulator of opioid tolerance.

Fragile X syndrome is caused by the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein. FMRP is associated with messenger RiboNucleoParticles (mRNPs) present in polyribosomes and its absence in neurons leads to alteration in synaptic plasticity as a result of translation regulation defects. The molecular mechanisms by which FMRP plays a role in translation regulation remain elusive. Using immunoprecipitation approaches with monoclonal Ab7G1-1 and a new generation of chicken antibodies, we identified Caprin1 as a novel FMRP-cellular partner. In vivo and in vitro evidence show that Caprin1 interacts with FMRP at the level of the translation machinery as well as in trafficking neuronal granules. As an RNA-binding protein, Caprin1 has in common with FMRP at least two RNA targets that have been identified as CaMKIIα and Map1b mRNAs. In view of the new concept that FMRP species bind to RNA regardless of known structural motifs, we propose that protein interactors might modulate FMRP functions.

Regulation of iron homeostasis and the inflammatory response are tightly linked to protect the host from infection. Here we investigate how imbalanced systemic iron homeostasis in a murine disease model of hereditary hemochromatosis (Hfe(-/-) mice) affects the inflammatory responses of the lung. We induced acute pulmonary inflammation in Hfe(-/-) and wild-type mice by intratracheal instillation of 20 µg of lipopolysaccharide (LPS) and analyzed local and systemic inflammatory responses and iron-related parameters. We show that in Hfe(-/-) mice neutrophil recruitment to the bronchoalveolar space is attenuated compared to wild-type mice although circulating neutrophil numbers in the bloodstream were elevated to similar levels in Hfe(-/-) and wild-type mice. The underlying molecular mechanisms are likely multifactorial and include elevated systemic iron levels, alveolar macrophage iron deficiency and/or hitherto unexplored functions of Hfe in resident pulmonary cell types. As a consequence, pulmonary cytokine expression is out of balance and neutrophils fail to be recruited efficiently to the bronchoalveolar compartment, a process required to protect the host from infections. In conclusion, our findings suggest a novel role for Hfe and/or imbalanced iron homeostasis in the regulation of the inflammatory response in the lung and hereditary hemochromatosis.

Bone repair or regeneration is a common and complicated clinical problem in orthopedic surgery. The importance of natural polymers, such as microcrystalline chitosan, and minerals such as HAp and β-TCP, has grown significantly over the last two decades due to their renewable and biodegradable source, increasing the knowledge and functionality of composites in technological and biomedical applications. This study compares the biodegradation process, bioactivity, structure, morphology, and mechanical properties of microcrystalline chitosan and microcrystalline chitosan/β-TCP complex; the latter according to the new method of preparation. The complex showed a homogeneous network structure with regular pores, good bioactivity, even after 60 days of conducting the hydrolytic and enzymatic degradation process, showing a bacteriostatic and bactericidal activity. The complex indicates that it could be used successfully as a base for implants and scaffolds production in orthopedic surgery.

Embelin, identified primarily from the Embelia ribes plant, has been shown to be a natural small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP). It is also a potent inhibitor of NF-κB activation, which makes it a potentially effective suppressor of tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. However, embelin itself is insoluble in water, which makes it unsuitable for in vivo applications. In this work, we developed a novel micelle system through conjugating embelin to a hydrophilic polymer, poly(ethylene glycol) 3500 (PEG(3.5K)) through an aspartic acid bridge. The PEG(3.5k)-embelin(2) (PEG(3.5k)-EB(2)) conjugate readily forms micelles in aqueous solutions with a CMC of 0.0205 mg/mL. Furthermore, PEG(3.5k)-EB(2) micelles effectively solubilize paclitaxel (PTX), a model hydrophobic drug used in this study. Both drug-free and drug-loaded micelles were small in size (20-30 nm) with low polydispersity indexes. In vitro cytotoxicity studies with several tumor cell lines showed that PEG(3.5k)-EB(2) is comparable to embelin in antitumor activity and synergizes with PTX at much lower doses. Our results suggest that PEG-derivatized embelin may represent a novel and dual-functional carrier to facilitate the in vivo applications of poorly water-soluble anticancer drugs such as PTX.

<h4>Background</h4>Bats are the natural reservoir host for a range of emerging and re-emerging viruses, including SARS-like coronaviruses, Ebola viruses, henipaviruses and Rabies viruses. However, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. Massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. Although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. In this study, we describe the immune transcriptome of the Australian flying fox, Pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity.<h4>Results</h4>Towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from P. alecto. We identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. A small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts.<h4>Conclusions</h4>This study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. In addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection.

Hepatocellular cancer (HCC) represents one of the most rapidly spreading cancers in the world. Most HCC develops in cirrhotic livers after prolonged inflammation, supporting the hypothesis that inflammation contributes to cancer development. Increasing evidence suggests that inflammatory cell recruitment and activation is an important contributor to promoting cancerous malformation in hepatocytes. Intracellular signaling pathways involved in classical inflammatory pathway activation can be altered in parenchymal cells, hepatocytes, in the liver to promote HCC development. Inflammation is triggered by pathogen-derived or endogenous danger-associated molecular patterns via pattern recognition receptors. Activation of the pattern recognition receptors triggers downstream signaling cascades to induce proinflammatory cytokine production, release of reactive oxygen species and modulate cellular responses. Many of these inflammatory mediators have adverse effects on DNA repair and induce DNA methylation, both of which are important elements in HCC development. This review summarizes the key points and discusses recent findings related to the role of inflammation in cancer and HCC development.

To study the potential role of neural cell adhesion molecule (NCAM) in the development of thalamocortical (TC) axon topography, wild type, and NCAM null mutant mice were analyzed for NCAM expression, projection, and targeting of TC afferents within the somatosensory area of the neocortex. Here we report that NCAM and its α-2,8-linked polysialic acid (PSA) are expressed in developing TC axons during projection to the neocortex. Pathfinding of TC axons in wild type and null mutant mice was mapped using anterograde DiI labeling. At embryonic day E16.5, null mutant mice displayed misguided TC axons in the dorsal telencephalon, but not in the ventral telencephalon, an intermediate target that initially sorts TC axons toward correct neocortical areas. During the early postnatal period, rostrolateral TC axons within the internal capsule along the ventral telencephalon adopted distorted trajectories in the ventral telencephalon and failed to reach the neocortex in NCAM null mutant animals. NCAM null mutants showed abnormal segregation of layer IV barrels in a restricted portion of the somatosensory cortex. As shown by Nissl and cytochrome oxidase staining, barrels of the anterolateral barrel subfield (ALBSF) and the most distal barrels of the posteromedial barrel subfield (PMBSF) did not segregate properly in null mutant mice. These results indicate a novel role for NCAM in axonal pathfinding and topographic sorting of TC axons, which may be important for the function of specific territories of sensory representation in the somatosensory cortex.

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ(-/-) (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia.

MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n = 35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1-150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.

<h4>Objective</h4>Maternal smoking during pregnancy is associated with a reduction in birth size but the mechanism by which this occurs still remains unclear. The purpose of this study was to evaluate the effect of tobacco smoking on concentrations of pregnancy-associated plasma protein A (PAPP-A), insulin-like growth factor I (IGF-I), II (IGF-II) and binding proteins BP-3 and BP-4 in pregnant women and correlations between these parameters.<h4>Methods</h4>Sixty healthy pregnant women were divided into smoking and tobacco-abstinent group according to results of serum cotinine concentration. The current smokers were defined as those who had smoked five or more cigarettes per day during pregnancy.<h4>Results</h4>The mean serum concentrations of PAPP-A, IGF-I and IGF-II were significantly lower in smoking than in non-smoking pregnant women (p < 0.01). The level of PAPP-A correlated positively with the IGF-II concentration in both studied group (non-smoking: r = 0.54; p < 0.001; smoking: r = 0.40; p < 0.05). In tobacco-abstinent group negative correlation between IGF-II and IGFBP-4 concentrations was found (r = -0.35; p < 0.05).<h4>Conclusion</h4>Tobacco smoking during pregnancy decreases the pregnancy-associated plasma protein A and insulin growth factors I and II levels. The correlation between PAPP-A and IGF-II may suggest function of this protein as a protease and regulator in the IGF system.

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at --80 °C or with 10 % DMSO and control rate freezing to --80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.

The role of the gut microbiota in the induction of metabolic diseases has now been increasingly recognized worldwide. Indeed, a specific gut microbiota has been shown to characterize lean versus obese phenotypes both in humans and mice. We have also recently demonstrated that a precise gut microbiota is associated with the host's responsiveness to a high-fat diet. Therefore, we hypothesized that insulin resistance in humans could also be linked to a specific gut microbiota. To this aim, microbial DNA and RNA were extracted from the appendix contents of insulin-resistant versus insulin-sensitive obese subjects, matched for body mass index and age, and analyzed by DNA- and RNA-DGGE. Microbial DNA analysis showed that the patients fully segregated according to their degree of insulin action. Conversely, microbial RNA investigation showed that some degree of homology still existed between insulin-sensitive and insulin-resistant patients. Quantitative trait analysis, ordinary least squares regression, principal components regression, partial least squares, canonical correlation analysis, and canonical correspondence analysis also showed a net separation of the two phenotypes analyzed. We conclude that a specific gut microbial profile is associated with insulin action in humans.

<h4>Background</h4>Gain of chromosome 20q is an important factor in the progression from colorectal adenomas to carcinomas. Genes that drive 20q gain are expected to show correlation of mRNA and protein expression levels with 20q DNA copy number status while functionally influencing cancer processes. CSE1L, DIDO1 and RBM39 are located on the 20q amplicon and affect processes such as cell viability and anchorage-independent growth in colorectal cancer. This study aimed to investigate whether CSE1L, DIDO1 and RBM39 may drive 20q amplification.<h4>Methods</h4>Protein expression levels were examined by immunohistochemical evaluation of tissue microarrays containing a series of colorectal adenoma and carcinoma samples, which were characterized by genome-wide (microarray-based) DNA and mRNA profiling.<h4>Results</h4>CSE1L, DIDO1 and RBM39 mRNA expression levels correlated with chromosome 20q DNA copy number status. CSE1L protein expression was not associated with 20q gain, although its expression was increased in carcinomas compared to adenomas. DIDO1 and RBM39 protein expression was quite strong in the majority of tumors irrespective of 20q DNA copy number status.<h4>Conclusion</h4>The lack of correlation between protein expression levels and 20q DNA copy number status implies that CSE1L, DIDO1 and RBM39 are merely passengers rather than drivers of chromosome 20q gain in colorectal adenoma-to-carcinoma progression.

<h4>Background</h4>Genetic variants are likely to contribute to a portion of prostate cancer risk. Full elucidation of the genetic etiology of prostate cancer is difficult because of incomplete penetrance and genetic and phenotypic heterogeneity. Current evidence suggests that genetic linkage to prostate cancer has been found on several chromosomes including the X; however, identification of causative genes has been elusive.<h4>Methods</h4>Parametric and non-parametric linkage analyses were performed using 26 microsatellite markers in each of 11 groups of multiple-case prostate cancer families from the International Consortium for Prostate Cancer Genetics (ICPCG). Meta-analyses of the resultant family-specific linkage statistics across the entire 1,323 families and in several predefined subsets were then performed.<h4>Results</h4>Meta-analyses of linkage statistics resulted in a maximum parametric heterogeneity lod score (HLOD) of 1.28, and an allele-sharing lod score (LOD) of 2.0 in favor of linkage to Xq27-q28 at 138 cM. In subset analyses, families with average age at onset less than 65 years exhibited a maximum HLOD of 1.8 (at 138 cM) versus a maximum regional HLOD of only 0.32 in families with average age at onset of 65 years or older. Surprisingly, the subset of families with only 2-3 affected men and some evidence of male-to-male transmission of prostate cancer gave the strongest evidence of linkage to the region (HLOD = 3.24, 134 cM). For this subset, the HLOD was slightly increased (HLOD = 3.47 at 134 cM) when families used in the original published report of linkage to Xq27-28 were excluded.<h4>Conclusions</h4>Although there was not strong support for linkage to the Xq27-28 region in the complete set of families, the subset of families with earlier age at onset exhibited more evidence of linkage than families with later onset of disease. A subset of families with 2-3 affected individuals and with some evidence of male to male disease transmission showed stronger linkage signals. Our results suggest that the genetic basis for prostate cancer in our families is much more complex than a single susceptibility locus on the X chromosome, and that future explorations of the Xq27-28 region should focus on the subset of families identified here with the strongest evidence of linkage to this region.

<h4>Background</h4>Proteins functioning in the same biological pathway tend to be transcriptionally co-regulated or form protein-protein interactions (PPI). Multiple spatially and temporally regulated events are coordinated during mitosis to achieve faithful chromosome segregation. The molecular players participating in mitosis regulation are still being unravelled experimentally or using in silico methods.<h4>Results</h4>An extensive literature review has led to a compilation of 196 human centromere/kinetochore proteins, all with experimental evidence supporting the subcellular localization. Sixty-four were designated as "core" centromere/kinetochore components based on peak expression and/or well-characterized functions during mitosis. By interrogating and integrating online resources, we have mined for genes/proteins that display transcriptional co-expression or PPI with the core centromere/kinetochore components. Top-ranked hubs in either co-expression or PPI network are not only enriched with known mitosis regulators, but also contain candidates whose mitotic functions are not yet established. Experimental validation found that KIAA1377 is a novel centrosomal protein that also associates with microtubules and midbody; while TRIP13 is a novel kinetochore protein and directly interacts with mitotic checkpoint silencing protein p31(comet).<h4>Conclusions</h4>Transcriptional co-expression and PPI network analyses with known human centromere/kinetochore proteins as a query group help identify novel potential mitosis regulators.

<h4>Background</h4>Promoter methylation of the tumor suppressor gene Cyclin A1 could be associated with Human Papillomavirus 16 (HPV16) induced Head and Neck Squamous Cell Carcinoma (HNSCC) and Cervical Carcinoma. There is disagreement about the impact of this epigenetic event on protein expression of Cyclin A1 in malignant and non-malignant tissue and there hardly exists any information about possible relationships between Cyclin A1 expression and clinicopathological characteristics in HNSCC.<h4>Methods</h4>We analyzed protein expression of Cyclin A1 in 81 HNSCC and 74 benign tonsils by immunohistochemistry and correlated it to Cyclin A1 methylation status, presence of HPV16 infection and other clinicopathological characteristics.<h4>Results</h4>Overexpression of Cyclin A1 was more present in HNSCC than in tonsils (p < 0.001). In both entities, HNSCC and benign tonsils, expression of Cyclin A1 significantly correlated with the expression of Cyclin-dependent kinase-inhibitor p16 (p = 0.000672 and 0.00495). In tonsils, expression of Cyclin A1 was inversely proportional to age (p = 0.00000396), and further correlated with expression of tumor suppressor gene p53 (p = 0.000228). In HNSCC Cyclin A1 expression was associated with the presence of HPV16 DNA (p = 0.0014) and a lower recurrence rate in univariate and multivariate analysis (p = 0.002 and 0.013). Neither in HNSCC nor in tonsils Cyclin A1 expression correlated with promoter methylation.<h4>Conclusions</h4>Cyclin A1 is an important cell cycle regulator with age-related increased expression in tonsils of children. HPV16 induces overexpression of Cyclin A1 in HNSCC despite promoter methylation. Overexpression of Cyclin A1 predicts a lower recurrence rate in HNSCC independently of HPV16.

Ultraconserved elements in the human genome likely harbor important biological functions as they are dosage sensitive and are able to direct tissue-specific expression. Because they are under purifying selection, variants in these elements may have a lower frequency in the population but a higher likelihood of association with complex traits. We tested a set of highly constrained SNPs (hcSNPs) distributed genome-wide among ultraconserved and nearly ultraconserved elements for association with seven traits related to reproductive (age at natural menopause, number of children, age at first child, and age at last child) and overall [longevity, body mass index (BMI), and height] fitness. Using up to 24,047 European-American samples from the National Heart, Lung, and Blood Institute Candidate Gene Association Resource (CARe), we observed an excess of associations with BMI and height. In an independent replication panel the most strongly associated SNPs showed an 8.4-fold enrichment of associations at the nominal level, including three variants in previously identified loci and one in a locus (DENND1A) previously shown to be associated with polycystic ovary syndrome. Finally, using 1430 family trios, we showed that the transmissions from heterozygous parents to offspring of the derived alleles of rare (frequency ≤ 0.5%) hcSNPs are not biased, particularly after adjusting for the rates of genotype missingness and error in the data. The lack of transmission bias ruled out an immediately and strongly deleterious effect due to the rare derived alleles, consistent with the observation that mice homozygous for the deletion of ultraconserved elements showed no overt phenotype. Our study also illustrated the importance of carefully modeling potential technical confounders when analyzing genotype data of rare variants.

CCG repeats are highly over-represented in exons of the human genome. Usually they are located in the 5' UTR but are also abundant in translated sequences. The CCG repeats are associated with three tri-nucleotide repeat disorders: Huntington's disease, myotonic dystrophy type 1 and chromosome X-linked mental retardation (FRAXE). In this study, we present two crystal structures containing double-stranded CCG repeats: one of an RNA in the native form, and one containing LNA nucleotides. Both duplexes form A-helices but with strands slipped in the 5' (native structure) or the 3' direction (LNA-containing structure). As a result, one of two expected C-C pairs is eliminated from the duplex. Each of the three observed C-C pairs interacts differently, forming either one weak H-bond or none. LNA nucleotides have no apparent effect on the helical parameters but the base stacking is increased compared to the native duplex and the distribution of electrostatic potential in the major groove is changed. The CCG crystal structures explain the thermodynamic fragility of CCG runs and throw light on the observation that the MBNL1 protein recognises CCG runs, as well as CUG and CAG, but not the relatively stable CGG repeats.

<h4>Objectives</h4>REGULATE trial was designed to compare the efficacy and safety of benfluorex versus pioglitazone in type 2 diabetes mellitus (DM) patients.<h4>Methods</h4>Double-blind, parallel-group, international, randomised, non-inferiority trial. More than half of the 196 participating centres were primary care centres. Patients eligible had type 2 DM uncontrolled on sulfonylurea. 846 were randomised. They received study treatment for 1 year. 423 patients were allocated to benfluorex (150 to 450 mg/day) and 423 were allocated to pioglitazone (30 to 45 mg/day). Primary efficacy criterion was HbA(1c). Safety assessment included blinded echocardiographic evaluation of cardiac and valvular status.<h4>Results</h4>At baseline, patients were 59.1 ± 10.5 years old with HbA1c 8.3 ± 0.8%, and DM duration 7.1 ± 6.0 years. During the study, mean HbA1c significantly decreased in both groups (benfluorex: from 8.30 ± 0.80 to 7.77 ± 1.31 versus pioglitazone: from 8.30 ± 0.80 to 7.45 ± 1.30%). The last HbA1c value was significantly lower with pioglitazone than with benfluorex (p<0.001) and non-inferiority of benfluorex was not confirmed (p = 0.19). Among the 615 patients with assessable paired echocardiography (310 benfluorex, 305 pioglitazone), 314 (51%) had at least one morphological valvular abnormality and 515 (84%) at least one functional valvular abnormality at baseline. Emergent morphological abnormalities occurred in 8 patients with benfluorex versus 4 with pioglitazone (OR 1.99), 95% CI (0.59 to 6.69). Emergent regurgitation (new or increased by one grade or more) occurred more frequently with benfluorex (82 patients, 27%) than with pioglitazone (33 patients, 11%) (OR 2.97), 95% CI (1.91 to 4.63) and were mainly rated grade 1; grade 2 (mild) was detected in 2 patients with benfluorex and 3 with pioglitazone. There was no moderate or severe regurgitation.<h4>Conclusion</h4>After 1 year of exposure, our results show a 2.97 fold increase in the incidence of valvular regurgitation with benfluorex and provide evidence for the valvular toxicity of this drug.

Growing evidence indicates that Rab GTPases, key regulators of intracellular transport in eukaryotic cells, play an important role in cancer. We analysed the deregulation at the transcriptional level of the genes encoding Rab proteins and Rab-interacting proteins in bladder cancer pathogenesis, distinguishing between the two main progression pathways so far identified in bladder cancer: the Ta pathway characterized by a high frequency of FGFR3 mutation and the carcinoma in situ pathway where no or infrequent FGFR3 mutations have been identified. A systematic literature search identified 61 genes encoding Rab proteins and 223 genes encoding Rab-interacting proteins. Transcriptomic data were obtained for normal urothelium samples and for two independent bladder cancer data sets corresponding to 152 and 75 tumors. Gene deregulation was analysed with the SAM (significant analysis of microarray) test or the binomial test. Overall, 30 genes were down-regulated, and 13 were up-regulated in the tumor samples. Five of these deregulated genes (LEPRE1, MICAL2, RAB23, STXBP1, SYTL1) were specifically deregulated in FGFR3-non-mutated muscle-invasive tumors. No gene encoding a Rab or Rab-interacting protein was found to be specifically deregulated in FGFR3-mutated tumors. Cluster analysis showed that the RAB27 gene cluster (comprising the genes encoding RAB27 and its interacting partners) was deregulated and that this deregulation was associated with both pathways of bladder cancer pathogenesis. Finally, we found that the expression of KIF20A and ZWINT was associated with that of proliferation markers and that the expression of MLPH, MYO5B, RAB11A, RAB11FIP1, RAB20 and SYTL2 was associated with that of urothelial cell differentiation markers. This systematic analysis of Rab and Rab effector gene deregulation in bladder cancer, taking relevant tumor subgroups into account, provides insight into the possible roles of Rab proteins and their effectors in bladder cancer pathogenesis. This approach is applicable to other group of genes and types of cancer.

<h4>Background</h4>The plasma cholesterol and triacylglycerol lowering effects of Tamarindus indica extract have been previously described. We have also shown that the methanol extract of T. indica fruit pulp altered the expression of lipid-associated genes including ABCG5 and APOAI in HepG2 cells. In the present study, effects of the same extract on the release of proteins from the cells were investigated using the proteomics approach.<h4>Methodology/principal findings</h4>When culture media of HepG2 cells grown in the absence and presence of the methanol extract of T. indica fruit pulp were subjected to 2-dimensional gel electrophoresis, the expression of seven proteins was found to be significantly different (p<0.03125). Five of the spots were subsequently identified as alpha enolase (ENO1), transthyretin (TTR), apolipoprotein A-I (ApoA-I; two isoforms), and rab GDP dissociation inhibitor beta (GDI-2). A functional network of lipid metabolism, molecular transport and small molecule biochemistry that interconnects the three latter proteins with the interactomes was identified using the Ingenuity Pathways Analysis software.<h4>Conclusion/significance</h4>The methanol extract of T. indica fruit pulp altered the release of ENO1, ApoA-I, TTR and GDI-2 from HepG2 cells. Our results provide support on the effect of T. indica extract on cellular lipid metabolism, particularly that of cholesterol.

We examined the humoral immune response to the unglycosylated central region of the respiratory syncytial virus (RSV) attachment (G) protein in mice following intranasal challenge at day 0 (primary) and day 21 (secondary) with subtype A (A2 strain) or B (B1 strain) RSV preparations. Our serological screening reagents included bacterially derived glutathione S-transferase (GST) fusion proteins, each bearing a portion of the RSV G central core (CC; residues 151-190), proximal central core (PCC; residues 151-172), and the distal central core (DCC; residues 173-190) and purified RSV G proteins from subtype A and B viruses. Convalescent sera collected on day 21 following primary RSV infection bore robust IgG response primarily against the homosubtypic RSV G DCC with relatively modest antigen affinity/avidity as demonstrated by brief incubation with 6M urea. In contrast, sera collected on day 42 following secondary homosubtypic RSV infection bore IgG titers of higher magnitudes and antigen affinity/avidity against the homosubtypic RSV G CC, PCC, and/or the DCC regions and full-length RSV G protein but not against the heterosubtypic RSV G protein or recombinant CC subdomains. In contrast, heterosubtypic secondary RSV infection elicits a broad array of IgG responses with titers of varying magnitudes to homo- and heterosubtypic RSV G CC regions as well as to purified F, Ga, and Gb proteins with the notable exception of minimal response to the RSV G DCC domain associated with the secondary RSV challenge. Our results have implications for RSV G-based serological assays as well as prophylactic immunotherapy and RSV vaccine development.

The aggregation of alpha-synuclein (α-syn) and huntingtin (htt) into fibrillar assemblies in nerve and glial cells is a molecular hallmark of Parkinson's and Huntington's diseases. Within the aggregation process, prefibrillar and fibrillar oligomeric species form. Prefibrillar assemblies rather than fibrils are nowadays considered cytotoxic. However, recent reports describing spreading of fibrillar assemblies from one cell to another, in cell cultures, animal models, and brains of grafted patients suggest a critical role for fibrillar assemblies in pathogenesis. Here we compare the cytotoxic effect of defined and comparable particle concentrations of on-assembly pathway oligomeric and fibrillar α-syn and Htt fragment corresponding to the first exon of the protein (HttEx1). We show that homogeneous populations of α-syn and HttEx1 fibrils, rather than their precursor on-assembly pathway oligomers, are highly toxic to cultured cells and induce apoptotic cell death. We document the reasons that make fibrils toxic. We show that α-syn and HttEx1 fibrils bind and permeabilize lipid vesicles. We also show that fibrils binding to the plasma membrane in cultured cells alter Ca(2+) homeostasis. Overall, our data indicate that fibrillar α-syn and HttEx1, rather than their precursor oligomers, are highly cytotoxic, the toxicity being associated to their ability to bind and permeabilize the cell membranes.

Differential gene expression is the fundamental mechanism underlying animal development and cell differentiation. However, it is a challenge to identify comprehensively and accurately the DNA sequences that are required to regulate gene expression: namely, cis-regulatory modules (CRMs). Three major features, either singly or in combination, are used to predict CRMs: clusters of transcription factor binding site motifs, non-coding DNA that is under evolutionary constraint and biochemical marks associated with CRMs, such as histone modifications and protein occupancy. The validation rates for predictions indicate that identifying diagnostic biochemical marks is the most reliable method, and understanding is enhanced by the analysis of motifs and conservation patterns within those predicted CRMs.

High-throughput biological experiments commonly result in a list of genes or proteins of interest. In order to understand the observed changes of the genes and to generate new hypotheses, one needs to understand the functions and roles of the genes and how those functions relate to the experimental conditions. Typically, statistical tests are performed in order to detect enriched Gene Ontology categories or pathways, i.e. the categories are observed in the genes of interest more often than is expected by chance. Depending on the number of genes and the complexity and quantity of functions in which they are involved, such an analysis can easily result in hundreds of enriched terms. To this end we developed DEFOG, a web-based application that facilitates the functional analysis of gene sets by hierarchically organizing the genes into functionally related modules. Our computational pipeline utilizes three powerful tools to achieve this goal: (1) GeneMANIA creates a functional consensus network of the genes of interest based on gene-list-specific data fusion of hundreds of genomic networks from publicly available sources; (2) Transitivity Clustering organizes those genes into a clear hierarchy of functionally related groups, and (3) Ontologizer performs a Gene Ontology enrichment analysis on the resulting gene clusters. DEFOG integrates this computational pipeline within an easy-to-use web interface, thus allowing for a novel visual analysis of gene sets that aids in the discovery of potentially important biological mechanisms and facilitates the creation of new hypotheses. DEFOG is available at http://www.mooneygroup.org/defog.

The health disparities literature has identified a common pattern among middle-aged African Americans that includes high rates of chronic disease along with low rates of psychiatric disorders despite exposure to high levels of cumulative socioeconomic status (SES) risk. The current study was designed to test hypotheses about the developmental precursors to this pattern. Hypotheses were tested with a representative sample of 443 African American youths living in the rural South. Cumulative SES risk and protective processes were assessed at ages 11-13 years; psychological adjustment was assessed at ages 14-18 years; genotyping at the 5-HTTLPR was conducted at age 16 years; and allostatic load (AL) was assessed at age 19 years. A latent profile analysis identified 5 profiles that evinced distinct patterns of SES risk, AL, and psychological adjustment, with 2 relatively large profiles designated as focal profiles: a physical health vulnerability profile characterized by high SES risk/high AL/low adjustment problems, and a resilient profile characterized by high SES risk/low AL/low adjustment problems. The physical health vulnerability profile mirrored the pattern found in the adult health disparities literature. Multinomial logistic regression analyses indicated that carrying an s allele at the 5-HTTLPR and receiving less peer support distinguished the physical health vulnerability profile from the resilient profile. Protective parenting and planful self-regulation distinguished both focal profiles from the other 3 profiles. The results suggest the public health importance of preventive interventions that enhance coping and reduce the effects of stress across childhood and adolescence.

<h4>Background</h4>The chemical instability of antiangiogenic fumagillin, combined with its poor retention during intravascular transit, requires an innovative solution for clinical translation. We hypothesized that an Sn-2 lipase-labile fumagillin prodrug, in combination with a contact-facilitated drug delivery mechanism, could be used to address these problems.<h4>Methods</h4>α(v)β(3)-targeted and nontargeted nanoparticles with and without fumagillin in the prodrug or native forms were evaluated in vitro and in vivo in the Matrigel™ (BD Biosciences, CA, USA) plug model of angiogenesis in mice.<h4>Results</h4>In vitro experiments demonstrated that the new fumagillin prodrug decreased viability at least as efficacious as the parent compound, on an equimolar basis. In the Matrigel mouse angiogenesis model, α(v)β(3)-fumagillin prodrug decreased angiogenesis as measured by MRI (3T), while the neovasculature was unaffected with the control nanoparticles.<h4>Conclusion</h4>The present approach resolved the previously intractable problems of drug instability and premature release in transit to target sites.

<h4>Purpose</h4>Stereotactic Ablative Body Radiotherapy (SABR) is a non-invasive therapy option for inoperable liver oligometastases. Outcome and toxicity were retrospectively evaluated in a single-institution patient cohort who had undergone ultrasound-guided breath-hold SABR.<h4>Patients and methods</h4>19 patients with liver metastases of various primary tumors consecutively treated with SABR (image-guidance with stereotactic ultrasound in combination with computer-controlled breath-hold) were analysed regarding overall-survival (OS), progression-free-survival (PFS), progression pattern, local control (LC), acute and late toxicity.<h4>Results</h4>PTV (planning target volume)-size was 108 ± 109cm3 (median 67.4 cm3). BED2 (Biologically effective dose in 2 Gy fraction) was 83.3 ± 26.2 Gy (median 78 Gy). Median follow-up and median OS were 12 months. Actuarial 2-year-OS-rate was 31%. Median PFS was 4 months, actuarial 1-year-PFS-rate was 20%. Site of first progression was predominantly distant. Regression of irradiated lesions was observed in 84% (median time to detection of regression was 2 months). Actuarial 6-month-LC-rate was 92%, 1- and 2-years-LC-rate 57%, respectively. BED2 influenced LC. When a cut-off of BED2 = 78 Gy was used, the higher BED2 values resulted in improved local control with a statistical trend to significance (p = 0.0999). Larger PTV-sizes, inversely correlated with applied dose, resulted in lower local control, also with a trend to significance (p-value = 0.08) when a volume cut-off of 67 cm3 was used.No local relapse was observed at PTV-sizes < 67 cm3 and BED2 > 78 Gy. No acute clinical toxicity > °2 was observed. Late toxicity was also ≤ °2 with the exception of one gastrointestinal bleeding-episode 1 year post-SABR. A statistically significant elevation in the acute phase was observed for alkaline-phosphatase; in the chronic phase for alkaline-phosphatase, bilirubine, cholinesterase and C-reactive protein.<h4>Conclusions</h4>A trend to statistically significant correlation of local progression was observed for BED2 and PTV-size. Dose-levels BED2 > 78 Gy cannot be reached in large lesions constituting a significant fraction of this series. Image-guided SABR (igSABR) is therefore an effective non-invasive treatment modality with low toxicity in patients with small inoperable liver metastases.

It is well known that cytokinins are a class of phytohormones that promote cell division in plant roots and shoots. However, their targets, biological functions, and implications in mammalian systems have rarely been examined. In this study, we show that one cytokinin, zeatin riboside, can prevent pheochromocytoma (PC12) cells from serum deprivation-induced apoptosis by acting on the adenosine A(2A) receptor (A(2A)-R), which was blocked by an A(2A)-R antagonist and a protein kinase A (PKA) inhibitor, demonstrating the functional ability of zeatin riboside by mediating through A(2A)-R signaling event. Since the A(2A)-R was implicated as a therapeutic target in treating Huntington's disease (HD), a cellular model of HD was applied by transfecting mutant huntingtin in PC12 cells. By using filter retardation assay and confocal microscopy we found that zeatin riboside reversed mutant huntingtin (Htt)-induced protein aggregations and proteasome deactivation through A(2A)-R signaling. PKA inhibitor blocked zeatin riboside-induced suppression of mutant Htt aggregations. In addition, PKA activated proteasome activity and reduced mutant Htt protein aggregations. However, a proteasome inhibitor blocked both zeatin riboside-and PKA activator-mediated suppression of mutant Htt aggregations, confirming mediation of the A(2A)-R/PKA/proteasome pathway. Taken together, zeatin riboside might have therapeutic potential as a novel neuroprotectant and a lead for treating neurodegenerative disorders.

Cancer of the urinary bladder is the fifth most common neoplasm in the industrialized countries. Diagnosis and surveillance are dependent on invasive evaluation with cystoscopy and to some degree cytology as an adjunct analysis. Nomuscle invasive bladder cancer is characterized by frequent recurrences after resection, and up to 30% will develop an aggressive phenotype. The journey towards a noninvasive test for diagnosing bladder cancer, in order to replace or extend time between cystoscopy, has been ongoing for more than a decade. However, only a handful of tests that aid in clinical decision making are commercially available. Recent reports of DNA methylation in urine specimens highlight a possible clinical use of this marker type, as high sensitivities and specificities have been shown. This paper will focus on the currently available markers NMP22, ImmunoCyt, and UroVysion as well as novel DNA methylation markers for diagnosis and surveillance of bladder cancer.

The humoral innate immune response consists of multiple components, including the naturally occurring antibodies (NAb), pentraxins and the complement and contact cascades. As soluble, plasma components, these innate proteins provide key elements in the prevention and control of disease. However, pathogens and cells with altered self proteins utilize multiple humoral components to evade destruction and promote pathology. Many studies have examined the relationship between humoral immunity and autoimmune disorders. This review focuses on the interactions between the humoral components and their role in promoting the pathogenesis of bacterial and viral infections and chronic diseases such as atherosclerosis and cancer. Understanding the beneficial and detrimental aspects of the individual components and the interactions between proteins which regulate the innate and adaptive response will provide therapeutic targets for subsequent studies.

Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

Ellagic acid (1) was synthesized for the first time from methyl gallate through 3-pentagalloylglucose (α-PGG), and ellagic acid peracetate (3,4,3',4'-tetra-O-acetylellagic acid, 2) was derived from 1 by acetylation. Oral administration of 2 suppressed melanoma growth significantly in C7BL/6 immunocompetent mice without having any effect on natural killer (NK) cell activity. Comparison of the immunoenhancing activities of 1 and 2 indicated that the latter compound increased white blood cell quantities in peripheral blood and immune cells enriched from the bone marrow and liver of mice. Therefore, both the antitumor efficacy and the immunity enhancement by 2 were greater than those by 1. In addition, on oral administration neither 1 nor 2 resulted in whole body, liver, or spleen weight changes of normal, tumor-free mice, indicating that these compounds are potentially non-toxic to mice. It was shown that ellagic acid peracetate (2) inhibits B16 melanoma cell growth in vitro, and induces B16 cell apoptosis, corresponding to BCL-2 down-regulation. Collectively, the present data imply that 2 can suppress tumor growth by enhancing mouse immunity and inducing tumor cell apoptosis without apparent side effects.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathology of HD; however, the precise mechanisms by which mutant huntingtin modulates levels of oxidative damage in turn resulting in mitochondrial dysfunction are not known. We hypothesize that mutant huntingtin increases oxidative mtDNA damage leading to mitochondrial dysfunction. We measured nuclear and mitochondrial DNA lesions and mitochondrial bioenergetics in the STHdhQ7 and STHdhQ111 in vitro striatal model of HD. Striatal cells expressing mutant huntingtin show higher basal levels of mitochondrial-generated ROS and mtDNA lesions and a lower spare respiratory capacity. Silencing of APE1, the major mammalian apurinic/apyrimidinic (AP) endonuclease that participates in the base excision repair (BER) pathway, caused further reductions of spare respiratory capacity in the mutant huntingtin-expressing cells. Localization experiments show that APE1 increases in the mitochondria of wild-type Q7 cells but not in the mutant huntingtin Q111 cells after treatment with hydrogen peroxide. Moreover, these results are recapitulated in human HD striata and HD skin fibroblasts that show significant mtDNA damage (increased lesion frequency and mtDNA depletion) and significant decreases in spare respiratory capacity, respectively. These data suggest that mtDNA is a major target of mutant huntingtin-associated oxidative stress and may contribute to subsequent mitochondrial dysfunction and that APE1 (and, by extension, BER) is an important target in the maintenance of mitochondrial function in HD.

Coffee beans contain numerous bioactive components that exhibit antioxidant capacity when assessed using both chemical, cell free, and biological, cell-based model systems. However, the mechanisms underlying the antioxidant effects of coffee in biological systems are not totally understood and in some cases vary considerably from results obtained with simpler in vitro chemical assays. In the present study, the physicochemical characteristics and antioxidant activity of roasted and non-roasted coffee extracts were investigated in both cell free (ORAC(FL)) and cell-based systems. A profile of antioxidant gene expression in cultured human colon adenocarcinoma Caco-2 cells treated with both roasted and non-roasted coffee extracts, respectively, was investigated using Real-Time polymerase chain reaction (PCR) array technology. Results demonstrated that the mechanisms of the antioxidant activity associated with coffee constituents assessed by the ORAC(FL) assay were different to those observed using an intracellular oxidation assay with Caco-2 cells. Moreover, roasted coffee (both light and dark roasted) extracts produced both increased- and decreased-expressions of numerous genes that are involved in the management of oxidative stress via the antioxidant defence system. The selective and specific positive induction of antioxidant response element (ARE)-dependent genes, including gastrointestinal glutathione peroxidase (GPX2), sulfiredoxin (SRXN1), thioredoxin reductase 1 (TXNRD1), peroxiredoxin 1 (PRDX1), peroxiredoxin 4 (PDRX4) and peroxiredoxin 6 (PDRX6) were identified with the activation of the endogenous antioxidant defence system in Caco-2 cells.

<h4>Background</h4>SOX genes play an important role in a number of developmental processes. Potential roles of SOXs have been demonstrated in various neoplastic tissues as tumor suppressors or promoters depending on tumor status and types. The aim of this study was to investigate the involvement of SOXs in the progression and prognosis of human prostate cancer (PCa).<h4>Methods</h4>The gene expression changes of SOXs in human PCa tissues compared with non-cancerous prostate tissues was detected using gene expression microarray, and confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) analysis and immunohositochemistry. The roles of these genes in castration resistance were investigated in LNCaP xenograft model of PCa.<h4>Results</h4>The microarray analysis identified three genes (SOX7, SOX9 and SOX10) of SOX family that were significantly dis-regulated in common among four PCa specimens. Consistent with the results of the microarray, differential mRNA and protein levels of three selected genes were found in PCa tissues by QRT-PCR analysis and immunohistochemistry. Additionally, we found that the immunohistochemical staining scores of SOX7 in PCa tissues with higher serum PSA level (P = 0.02) and metastasis (P = 0.03) were significantly lower than those with lower serum PSA level and without metastasis; the increased SOX9 protein expression was frequently found in PCa tissues with higher Gleason score (P = 0.02) and higher clinical stage (P < 0.0001); the down-regulation of SOX10 tend to be found in PCa tissues with higher serum PSA levels (P = 0.03) and advanced pathological stage (P = 0.01). Moreover, both univariate and multivariate analyses showed that the down-regulation of SOX7 and the up-regulation of SOX9 were independent predictors of shorter biochemical recurrence-free survival. Furthermore, we discovered that SOX7 was significantly down-regulated and SOX9 was significantly up-regulated during the progression to castration resistance.<h4>Conclusions</h4>Our data offer the convince evidence that the dis-regulation of SOX7, SOX9 and SOX10 may be associated with the aggressive progression of PCa. SOX7 and SOX9 may be potential markers for prognosis in PCa patients. Interestingly, the down-regulation of SOX7 and the up-regulation of SOX9 may be important mechanisms for castration-resistant progression of PCa.

Among serotonin (5-HT) receptors, the 5-HT(3) receptor is the only ligand-gated ion-channel. Little is known about the interaction between the 5-HT(3) receptor and other 5-HT receptors and influence of 5-HT(3) chronic activation on other 5-HT receptors and the expression of key genes of 5-HT system. Chronic activation of 5-HT(3) receptor with intracerebroventricularly administrated selective agonist 1-(3-chlorophenyl)biguanide hydrochloride (m-CPBG) (14 days, 40 nmol, i.c.v.) produced significant desensitization of 5-HT(3) and 5-HT(1A) receptors. The hypothermic responses produced by acute administration of selective agonist of 5-HT(3) receptor (m-CPBG, 40 nmol, i.c.v.) or selective agonist of 5-HT(1A) receptor (8-hydroxy-2-(di-n-propylamino)tetralin) (8-OH-DPAT, 1mg/kg, i.p.) was significantly lower in m-CPBG treated mice compared with the mice of control groups. Chronic m-CPBG administration failed to induce any significant change in the 5-HT(2A) receptor functional activity and in the expression of the gene encoding 5-HT(2A) receptor. Chronic activation of 5-HT(3) receptor produced no considerable effect on the expression on 5-HT(3), 5-HT(1A), and 5-HT transporter (5-HTT) and tryptophan hydroxylase-2 (TPH-2) genes - the key genes of brain 5-HT system, in the midbrain, frontal cortex and hippocampus. In conclusion, chronic activation of ionotropic 5-HT(3) receptor produced significant desensitization of 5-HT(3) and postsynaptic 5-HT(1A) receptors but caused no considerable changes in the expression of key genes of the brain 5-HT system.

<h4>Objective</h4>Thrombosis is a serious complication of systemic lupus erythematosus (SLE). We investigated whether genetic variants implicated in thrombosis pathways are associated with thrombosis among 2 ethnically diverse SLE cohorts.<h4>Methods</h4>Our discovery cohort consisted of 1698 patients with SLE enrolled in the University of California, San Francisco, Lupus Genetics Project and our replication cohort included 1361 patients with SLE enrolled in the PROFILE cohort. Patients fulfilled American College of Rheumatology SLE criteria, and data relevant to thrombosis were available. Thirty-three single nucleotide polymorphisms (SNP) previously shown to be associated with risk of deep venous thrombosis in the general population or implicated in thrombosis pathways were genotyped and tested for association with thrombosis in bivariate allelic analyses. SNP with p < 0.1 in the bivariate analyses were further tested in multivariable logistic regression models adjusted for age, sex, disease duration, antiphospholipid antibody status, smoking, nephritis, and medications.<h4>Results</h4>In the discovery cohort, 23% of patients with SLE experienced a thrombotic event. SNP in the following genes demonstrated association with thrombosis risk overall in the discovery or replication cohorts and were assessed using metaanalytic methods: factor V Leiden (FVL) rs6025 (OR 1.85, p = 0.02) and methylenetetrahydrofolate reductase (MTHFR) rs1801133 (OR 0.75, p = 0.04) in whites, and fibrinogen gamma (FGG) rs2066865 (OR 1.91, p = 0.01) in Hispanic Americans. SNP in these genes showed association with venous thrombosis risk in whites: MTHFR rs1801131 (OR 1.51, p = 0.01), MTHFR rs1801133 (OR 0.70, p = 0.04), FVL rs6025 (OR 2.69, p = 0.002), and FGG rs2066865 (OR 1.49, p = 0.02) in whites. A SNP in FGG rs2066865 (OR 2.19, p = 0.003) demonstrated association with arterial thrombosis risk in Hispanics.<h4>Conclusion</h4>Our results implicate specific genetic risk factors for thrombosis in patients with SLE and suggest that genetic risk for thrombosis differs across ethnic groups.

The present investigation was designed to evaluate the renal microvascular endothelial cell responses following exposure to preformed antibodies against human leukocyte antigens (HLA) in the recipient. We hypothesize that activation of endothelial cell genes has a pivotal role in renal allograft survival. In this study, we used cultured human umbilical cord vein endothelial cells (HUVEC), human microvascular glomerular endothelial cells (HMGEC), activated with and without IFN-γ and TNF-α, and pre-transplant blood group O patient sera containing multispecific HLA class I and class II antibodies. Molecular HLA typing revealed the HMGEC haplotype to be HLA-A*01, HLA-A*68, HLA-B*14, HLA-B*35, HLA-C*04, HLA-C*08, HLA-DRβ1*13, and HLA-DRβ1*15. Flow cytometry was used for phenotypic characterization of both inactivated and activated HUVECs and HMGECs with IFN-γ and TNF-α. HUVECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, PECAM, ICAM, MCAM, integrin beta-3, thrombomodulin, E-selectin, VCAM-1, and tissue factor, and negative for alpha smooth muscle actin and P-selectin antibodies. HMGECs were positive for HLA-ABC, HLA-DR/DQ, von Willebrand factor, endoglin, ICAM, MCAM, integrin beta-3, thrombomodulin, VCAM-1, and tissue factor; and negative for PECAM, E-selectin, P-selectin, and for blood group antigens A and B. 42 samples were analyzed by real time PCR and categorized into the following groups: the control group (HMGEC only, n=12), group 1 (HMGECs incubated with patient sera, n=15), and group 2 (HMGECs activated by TNF-α and IFN-γ and incubated with patient sera, n=15). Expression levels of the vasoconstriction genes endothelin 1 (EDN1), endothelin 2 (EDN2), and endothelin receptor type A (EDNRA) were up-regulated in both groups 1 and 2 compared to the control group. The thrombomodulin (THBD) gene was also up-regulated in both groups 1 and 2 compared to the control. Chemokine genes CCL5 and CX3CL1 were up-regulated in both groups 1 and 2 compared to the controls; whereas, CCL2 was up-regulated only in group 2. Cytokine activity genes colony stimulating factor 2 (CSF2), tumor necrosis factor (TNF), tumor necrosis factor (ligand) superfamilymember 10 (TNFSF10), interleukin 1 beta (IL1B), and interleukin 6 (IL6) were up-regulated in both groups 1 and 2 compared to the control; whereas, IL11 was up-regulated only in group 1 and IFNB1 in group 2. Adhesion molecule genes intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and integrin beta 3 (ITGB3) were up-regulated in both groups 1 and 2 compared to the control; whereas, CDH5 and COL18A1 were up-regulated only in group 2. Anti-apoptosis genes BCL2A1, CFLAR, and SPHK1 were up-regulated only in group 2 compared to controls. Apoptosis and caspase-activation genes CASP, RIPK1, and FAS were up-regulated only in group 2 compared to the control. Angiopoietin 1 (ANGPT1) and prostaglandin I2 (prostacyclin) synthase (PTGIS) were down-regulated in both groups 1 and 2 compared to the control group. Our results indicate that expression of the endothelin gene in endothelial cells may contribute to vasoconstriction of blood vessels in post-renal allograft transplantation. In addition, thrombomodulin, by reducing thrombogenic activity, and interleukin 11, through its cytoprotective effects, may have a role in transplant accommodation in the presence of pre-formed HLA antibodies. This study showed that activation of the vasoconstriction genes, thrombomodulin gene, chemokine genes, cytokine activity genes, adhesion genes, anti-apoptosis genes, and apoptosis and caspase-activation genes could have consequential effects on renal allograft survival.

Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.

<h4>Introduction</h4>Progesterone receptors (PR) are emerging as important breast cancer drivers. Phosphorylation events common to breast cancer cells impact PR transcriptional activity, in part by direct phosphorylation. PR-B but not PR-A isoforms are phosphorylated on Ser294 by mitogen activated protein kinase (MAPK) and cyclin dependent kinase 2 (CDK2). Phospho-Ser294 PRs are resistant to ligand-dependent Lys388 SUMOylation (that is, a repressive modification). Antagonism of PR small ubiquitin-like modifier (SUMO)ylation by mitogenic protein kinases suggests a mechanism for derepression (that is, transcriptional activation) of target genes. As a broad range of PR protein expression is observed clinically, a PR gene signature would provide a valuable marker of PR contribution to early breast cancer progression.<h4>Methods</h4>Global gene expression patterns were measured in T47D and MCF-7 breast cancer cells expressing either wild-type (SUMOylation-capable) or K388R (SUMOylation-deficient) PRs and subjected to pathway analysis. Gene sets were validated by RT-qPCR. Recruitment of coregulators and histone methylation levels were determined by chromatin immunoprecipitation. Changes in cell proliferation and survival were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and western blotting. Finally, human breast tumor cohort datasets were probed to identify PR-associated gene signatures; metagene analysis was employed to define survival rates in patients whose tumors express a PR gene signature.<h4>Results</h4>'SUMO-sensitive' PR target genes primarily include genes required for proliferative and pro-survival signaling. DeSUMOylated K388R receptors are preferentially recruited to enhancer regions of derepressed genes (that is, MSX2, RGS2, MAP1A, and PDK4) with the steroid receptor coactivator, CREB-(cAMP-response element-binding protein)-binding protein (CBP), and mixed lineage leukemia 2 (MLL2), a histone methyltransferase mediator of nucleosome remodeling. PR SUMOylation blocks these events, suggesting that SUMO modification of PR prevents interactions with mediators of early chromatin remodeling at 'closed' enhancer regions. SUMO-deficient (phospho-Ser294) PR gene signatures are significantly associated with human epidermal growth factor 2 (ERBB2)-positive luminal breast tumors and predictive of early metastasis and shortened survival. Treatment with antiprogestin or MEK inhibitor abrogated expression of SUMO-sensitive PR target-genes and inhibited proliferation in BT-474 (estrogen receptor (ER)+/PR+/ERBB2+) breast cancer cells.<h4>Conclusions</h4>We conclude that reversible PR SUMOylation/deSUMOylation profoundly alters target gene selection in breast cancer cells. Phosphorylation-induced PR deSUMOylation favors a permissive chromatin environment via recruitment of CBP and MLL2. Patients whose ER+/PR+ tumors are driven by hyperactive (that is, derepressed) phospho-PRs may benefit from endocrine (antiestrogen) therapies that contain an antiprogestin.

We report here that spermatozoa of mice lacking both the sperm nucleus glutathione peroxidase 4 (snGPx4) and the epididymal glutathione peroxidase 5 (GPx5) activities display sperm nucleus structural abnormalities including delayed and defective nuclear compaction, nuclear instability and DNA damage. We show that to counteract the GPx activity losses, the epididymis of the double KO animals mounted an antioxydant response resulting in a strong increase in the global H(2)O(2)-scavenger activity especially in the cauda epididymis. Quantitative RT-PCR data show that together with the up-regulation of epididymal scavengers (of the thioredoxin/peroxiredoxin system as well as glutathione-S-transferases) the epididymis of double mutant animals increased the expression of several disulfide isomerases in an attempt to recover normal disulfide-bridging activity. Despite these compensatory mechanisms cauda-stored spermatozoa of double mutant animals show high levels of DNA oxidation, increased fragmentation and greater susceptibility to nuclear decondensation. Nevertheless, the enzymatic epididymal salvage response is sufficient to maintain full fertility of double KO males whatever their age, crossed with young WT female mice.

The purpose of this study was to investigate the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in lung squamous cell carcinoma tissue and its correlation with the clinical pathology of lung squamous cell carcinoma. RKIP and E-cadherin mRNA (by RT-PCR) and protein (by western blotting) levels were monitored in carcinoma tissues and surrounding normal tissues from 86 lung squamous cell carcinoma cases, and their positive rates were calculated. The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The positive expression rates were significantly lower in those with lymph node metastasis than in those without (P < 0.05). The lower the degree of tumor differentiation, the lower the E-cadherin mRNA positive expression rate (P < 0.05). The rates of positive RKIP and E-cadherin mRNA expression were significantly lower in patients at advanced (III, IV) stages than in patients at early (I, II) stages (p < 0.05); this rate, however, was independent of gender, age, and tumor size (P > 0.05). The protein levels of RKIP and E-cadherin were significantly lower in lung squamous cell carcinoma than in the surrounding normal tissues (P < 0.05). The levels were significantly lower in patients with lymph node metastasis than in those without it (P < 0.05). The lower the degree of tumor differentiation, the lower the protein level of E-cadherin (P < 0.05). Both RKIP and E-cadherin are tumor suppressors, their low expression levels may be associated with initiation, invasion and/or metastasis, as well as with the inhibition of lung squamous cell carcinoma differentiation.

The immediate-early gene early growth response 3 (Egr3) is associated with schizophrenia and expressed at reduced levels in postmortem patients' brains. We have previously reported that Egr3-deficient (Egr3(-/-)) mice display reduced sensitivity to the sedating effects of clozapine compared with wild-type (WT) littermates, paralleling the heightened tolerance of schizophrenia patients to antipsychotic side effects. In this study, we have used a pharmacological dissection approach to identify a neurotransmitter receptor defect in Egr3(-/-) mice that may mediate their resistance to the locomotor suppressive effects of clozapine. We report that this response is specific to second-generation antipsychotic agents (SGAs), as first-generation medications suppress the locomotor activity of Egr3(-/-) and WT mice to a similar degree. Further, in contrast to the leading theory that sedation by clozapine results from anti-histaminergic effects, we show that H1 histamine receptors are not responsible for this effect in C57BL/6 mice. Instead, selective serotonin 2A receptor (5HT(2A)R) antagonists ketanserin and MDL-11939 replicate the effect of SGAs, repressing the activity in WT mice at a dosage that fails to suppress the activity of Egr3(-/-) mice. Radioligand binding revealed nearly 70% reduction in 5HT(2A)R expression in the prefrontal cortex of Egr3(-/-) mice compared with controls. Egr3(-/-) mice also exhibit a decreased head-twitch response to 5HT(2A)R agonist 1-(2,5-dimethoxy 4-iodophenyl)-2-amino propane (DOI). These findings provide a mechanism to explain the reduced sensitivity of Egr3(-/-) mice to the locomotor suppressive effects of SGAs, and suggest that 5HT(2A)Rs may also contribute to the sedating properties of these medications in humans. Moreover, as the deficit in cortical 5HT(2A)R in Egr3(-/-) mice aligns with numerous studies reporting decreased 5HT(2A)R levels in the brains of schizophrenia patients, and the gene encoding the 5HT(2A)R is itself a leading schizophrenia candidate gene, these findings suggest a potential mechanism by which putative dysfunction in EGR3 in humans may influence risk for schizophrenia.

<h4>Background</h4>Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways.<h4>Results</h4>We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2).<h4>Conclusions</h4>We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.

<h4>Background</h4>Pulmonary hypertension (PH) is a complication of chronic obstructive pulmonary disease (COPD). This study examined genetic variations in mediators of vascular remodelling and their association with PH in patients with COPD. In patients with COPD, we genotyped 7 SNPs in 6 candidate PH genes (NOS3, ACE, EDN1, PTGIS, SLC6A4, VEGFA). We tested for association with right ventricular systolic pressure (RVSP), spirometry and gas transfer, and hypoxemia.<h4>Methods</h4>In patients with COPD, we genotyped 7 SNPs in 6 candidate PH genes (NOS3, ACE, EDN1, PTGIS, SLC6A4, VEGFA). We tested for association with right ventricular systolic pressure (RVSP), spirometry and gas transfer, and hypoxemia.<h4>Results</h4>580 COPD patients were recruited, 341 patients had a transthoracic echocardiogram, with RVSP measurable in 278 patients (mean age 69  years, mean FEV1 50% predicted, mean RVSP 44  mmHg, median history of 50 pack-years). Of the 7 tested SNPs, the NOS3-VNTR polymorphism was significantly associated with RVSP in a dose-dependent fashion for the risk allele: mean RVSP for a/a and a/b genotypes were 52.0 and 46.6  mmHg respectively, compared to 43.2  mmHg for b/b genotypes (P = 0.032). No associations were found between RVSP and other polymorphisms. ACE II or ID genotypes were associated with a lower FEV1% predicted than the ACE DD genotype (P = 0.028). The NOS3-298 TT genotype was associated with lower KCO % predicted than the NOS3-298 GG or GT genotype (P = 0.031).<h4>Conclusions</h4>The NOS3-VNTR polymorphism was associated with RVSP in patients with COPD, supporting its involvement in the pathogenesis of PH in COPD. ACE and NOS3 genotypes were associated with COPD disease severity, but not with the presence of PH. Further study of these genes could lead to the development of prognostic and screening tools for PH in COPD.

<h4>Background</h4>Thalidomide is an anti-inflammatory and anti-angiogenic drug currently used for the treatment of several diseases, including erythema nodosum leprosum, which occurs in patients with lepromatous leprosy. In this research, we use DNA microarray analysis to identify the impact of thalidomide on gene expression responses in human cells after lipopolysaccharide (LPS) stimulation. We employed a two-stage framework. Initially, we identified 1584 altered genes in response to LPS. Modulation of this set of genes was then analyzed in the LPS stimulated cells treated with thalidomide.<h4>Results</h4>We identified 64 genes with altered expression induced by thalidomide using the rank product method. In addition, the lists of up-regulated and down-regulated genes were investigated by means of bioinformatics functional analysis, which allowed for the identification of biological processes affected by thalidomide. Confirmatory analysis was done in five of the identified genes using real time PCR.<h4>Conclusions</h4>The results showed some genes that can further our understanding of the biological mechanisms in the action of thalidomide. Of the five genes evaluated with real time PCR, three were down regulated and two were up regulated confirming the initial results of the microarray analysis.

Oocyte loss has a significant impact on fertility and somatic health. Yet, we know little about factors that impact this process. We sought to identify genetic variants associated with ovarian reserve (oocyte number as measured by antral follicle count, AFC). Based on recently published genome-wide scans that identified loci associated with age of menopause, we also sought to test our hypothesis that follicle number and menopausal age share underlying genetic associations. We analyzed menopause-related variants for association with follicle number in an independent population of approximately 450 reproductive-aged women of European and African ancestry; these women were assessed for AFC, anthropometric, clinical, and lifestyle factors. One SNP strongly associated with later menopausal age in Caucasian women (+1.07 ± 0.11 years) in previous work was also associated with higher follicle counts in Caucasians (+2.79 ± 1.67 follicles) in our study. This variant is within the Minichromosome Maintenance Complex Component 8 (MCM8) gene, which we found was expressed within oocytes in follicles of the human ovary. In genome-wide scans of AFC, we also identified one marginally genome-wide and several nominally significant SNPs within several other genes associated with follicle number in both ethnic groups. Further, there were overlapping variants associated with multiple ovarian reserve markers (AFC, serum hormone levels, menopausal age). This study provides the first evidence for direct genetic associations underlying both follicle number and menopause and identifies novel candidate genes. Genetic variants associated with ovarian reserve may facilitate discovery of genetic markers to predict reproductive health and lifespan in women.

Neurodegenerative diseases are a large group of disabling disorders of the nervous system, characterized by the relative selective death of neuronal subtypes. In most cases, there is overwhelming evidence of impaired mitochondrial function as a causative factor in these diseases. More recently, evidence has emerged for impaired mitochondrial dynamics (shape, size, fission-fusion, distribution, movement etc.) in neurodegenerative diseases such as Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. Here, we provide a concise overview of the major findings in recent years highlighting the importance of healthy mitochondria for a healthy neuron.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (T(FH)) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the "san" allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of T(FH) cells preceded tumor development, and clonal rearrangements in the TCR-β genes were present in most tumors. Furthermore, T(FH) cells exhibited increased clonality compared with non-T(FH) cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent T(FH) development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a T(FH)-derived origin. Roquin(san/+) mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated T(FH) cells or their consequences.

Older adults show extensive variability in cognitive performance, including episodic memory. A portion of this variability could potentially be explained by genetic factors. Recent literature shows that the neurotransmitter serotonin plays an important role in memory processes, as enhancements of brain serotonin have led to memory improvement. Here, we have begun to explore genetic contributions to the performance and underlying brain activity associated with source memory monitoring. Using a source recognition memory task during fMRI scanning, this study offers evidence that older adults who carry a short allele (S-car) of the serotonin transporter linked polymorphic region (5-HTTLPR) in the SLC6A4 gene show specific deficits in source memory monitoring relative to older adults who are homozygous for the long allele (LL). These deficits are accompanied by less neural activity in regions of prefrontal cortex that have been shown to support accurate memory monitoring. Moreover, while the older adult LL group's behavioral performance does not differ from younger adults, their brain activation reveals evidence of compensatory activation that likely supports their higher performance level. These results provide preliminary evidence that the long-allele homozygous profile is cognitively beneficial to older adults, particularly for memory functioning.

The tumour suppressor Adenomatous Polyposis Coli (APC) is required for proper mitosis; however, the exact role of APC in mitosis is not understood. Using demembranated sperm chromatin exposed to meiotic Xenopus egg extract and HeLa cells expressing fluorescently labelled histones, we established that APC contributes to chromatin compaction. Sperm chromatin in APC-depleted Xenopus egg extract frequently formed tight round or elongated structures. Such abnormally compacted chromatin predominantly formed spindles with low microtubule content. Furthermore, in mitotic HeLa cells expressing GFP- and mCherry-labelled H2B histones, depletion of APC caused a decrease in the donor fluorescence lifetime of neighbouring fluorophores, indicative of excessive chromatin compaction. Profiling the chromatin-associated proteome of sperm chromatin incubated with Xenopus egg extracts revealed temporal APC-dependent changes in the abundance of histones, closely mirrored by chromatin-associated Topoisomerase IIa, condensin I complex and Kif4. In the absence of APC these factors initially accumulated on chromatin, but then decreased faster than in controls. We also found and validated significant APC-dependent changes in chromatin modifiers Set-a and Rbbp7. Both were decreased on chromatin in APC-depleted extract; in addition, the kinetics of association of Set-a with chromatin was altered in the absence of APC.

The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (<19 years) had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.

Bone marrow stromal cell cultures contain multipotent cells that may have therapeutic utility for tissue restoration; however, the identity of the cell that maintains this function remains poorly characterized. We have utilized a unique model of murine bone marrow stroma in combination with liquid chromatography mass spectrometry to compare the nuclear, cytoplasmic and membrane associated proteomes of multipotent (MSC) (CD105+) and non-multipotent (CD105-) stromal cells. Among the 25 most reliably identified proteins, 10 were verified by both real-time PCR and Western Blot to be highly enriched, in CD105+ cells and were members of distinct biological pathways and functional networks. Five of these proteins were also identified as potentially expressed in human MSC derived from both standard and serum free human stromal cultures. The quantitative amount of each protein identified in human stromal cells was only minimally affected by media conditions but varied highly between bone marrow donors. This study provides further evidence of heterogeneity among cultured bone marrow stromal cells and identifies potential candidate proteins that may prove useful for identifying and quantifying both murine and human MSC in vitro.

<h4>Aim</h4>A growing body of evidence has shown that increased formation of oxidized molecules and reactive oxygen species within the vasculature (i.e., the extracellular space) plays a crucial role in the initiation and progression of atherosclerosis and in the formation of unstable plaques. Peroxiredoxin 4 (PRDX4) is the only known secretory member of the antioxidant PRDX family. However, the relationship between PRDX4 and susceptibility to atherosclerosis has remained unclear.<h4>Results</h4>To define the role of PRDX4 in hyperlipidemia-induced atherosclerosis, we generated hPRDX4 transgenic (Tg) and apolipoprotein E (apoE) knockout mice (hPRDX4(+/+)/apoE(-/-)). After feeding the mice a high-cholesterol diet, they showed fewer atheromatous plaques, less T-lymphocyte infiltration, lower levels of oxidative stress markers, less necrosis, a larger number of smooth muscle cells, and a larger amount of collagen, resulting in thickened fibrous cap formation and possible stable plaque phenotype as compared with apoE(-/-) mice. We also detected greater suppression of apoptosis and decreased Bax expression in hPRDX4(+/+)/apoE(-/-) mice than in apoE(-/-) mice. Bone marrow transplantation from hPRDX4(+/+) donors to apoE(-/-) mice confirmed the antiatherogenic aspects of PRDX4, revealing significantly suppressed atherosclerotic progression.<h4>Innovation</h4>In this study, we demonstrated for the first time that PRDX4 suppressed the development of atherosclerosis in apoE(-/-) mice fed a high-cholesterol diet.<h4>Conclusion</h4>These data indicate that PRDX4 is an antiatherogenic factor and, by suppressing oxidative damage and apoptosis, that it may protect against the formation of vulnerable (unstable) plaques.

A large number of individuals are at risk for deep venous thrombosis (DVT) due to alterations in multiple coagulation factors and inhibitors secondary to malignancy, drug interactions, or other general medical conditions. Traditional metrics of haemostasis such as prothrombin time, partial thromboplastin time, and bleeding time, generally estimate anticoagulation status and bleeding risk rather than thrombosis risk. The objective of this study was to correlate a novel, systems-based metric of clotting potential to risk of DVT from a database derived from the Leiden Thrombophilia Study (LETS). We utilised a computational model of blood coagulation, which addresses the interplay between biochemical factors, blood flow, and physiologic surface initiation of coagulation, to calculate an individualised, systems-based metric of clotting potential, termed the flow-simulated thrombin generation (FSTG), for 210 pre-menopausal women in LETS. Both DVT and oral contraceptive (OC) use were associated with higher values of FSTG. We demonstrated a nearly three-fold increased risk of DVT for each standard deviation increase above the mean in FSTG determined under venous flow conditions, which remained highly predictive after adjustment for age and OC status (adjusted odds ratio 2.66; 95% confidence interval 1.69-4.19; p<0.0001). In conclusion, a systems-based screening approach that integrates biochemical factors and flow haemodynamics identifies small subgroups of patients at risk of thrombosis that may benefit from oral anticoagulants.

Alu repeats within human genes may potentially alter gene expression. Here, we show that 3'-UTR-located inverted Alu repeats significantly reduce expression of an AcGFP reporter gene. Mutational analysis demonstrates that the secondary structure, but not the primary nucleotide sequence, of the inverted Alu repeats is critical for repression. The expression levels and nucleocytoplasmic distribution of reporter mRNAs with or without 3'-UTR inverted Alu repeats are similar; suggesting that reporter gene repression is not due to changes in mRNA levels or mRNA nuclear sequestration. Instead, reporter gene mRNAs harboring 3'-UTR inverted Alu repeats accumulate in cytoplasmic stress granules. These findings may suggest a novel mechanism whereby 3'-UTR-located inverted Alu repeats regulate human gene expression through sequestration of mRNAs within stress granules.

Both environmental and genetic factors impact lipid traits. Environmental modifiers of known genotype-phenotype associations may account for some of the "missing heritability" of these traits. To identify such modifiers, we genotyped 23 lipid-associated variants identified previously through genome-wide association studies (GWAS) in 2,435 non-Hispanic white, 1,407 non-Hispanic black, and 1,734 Mexican-American samples collected for the National Health and Nutrition Examination Surveys (NHANES). Along with lipid levels, NHANES collected environmental variables, including fat-soluble macronutrient serum levels of vitamin A and E levels. As part of the Population Architecture using Genomics and Epidemiology (PAGE) study, we modeled gene-environment interactions between vitamin A or vitamin E and 23 variants previously associated with high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels. We identified three SNP × vitamin A and six SNP × vitamin E interactions at a significance threshold of p < 2.2 × 10(-3). The most significant interaction was APOB rs693 × vitamin E (p = 8.9 × 10(-7)) for LDL-C levels among Mexican-Americans. The nine significant interaction models individually explained 0.35-1.61% of the variation in any one of the lipid traits. Our results suggest that vitamins A and E may modify known genotype-phenotype associations; however, these interactions account for only a fraction of the overall variability observed for HDL-C, LDL-C, and TG levels in the general population.

<h4>Background</h4>Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis.<h4>Methods</h4>Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients.<h4>Results</h4>We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian.<h4>Conclusions</h4>Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis.

<h4>Background</h4>Identification of driver mutations among numerous genomic alternations remains a critical challenge to the elucidation of the underlying mechanisms of cancer. Because driver mutations by definition are associated with a greater number of cancer phenotypes compared to other mutations, we hypothesized that driver mutations could more easily be identified once the genotype-phenotype correlations are detected across tumor samples.<h4>Results</h4>In this study, we describe a novel network analysis to identify the driver mutation through integrating both cancer genomes and transcriptomes. Our method successfully identified a significant genotype-phenotype change correlation in all six solid tumor types and revealed core modules that contain both significantly enriched somatic mutations and aberrant expression changes specific to tumor development. Moreover, we found that the majority of these core modules contained well known cancer driver mutations, and that their mutated genes tended to occur at hub genes with central regulatory roles. In these mutated genes, the majority were cancer-type specific and exhibited a closer relationship within the same cancer type rather than across cancer types. The remaining mutated genes that exist in multiple cancer types led to two cancer type clusters, one cluster consisted of three neural derived or related cancer types, and the other cluster consisted of two adenoma cancer types.<h4>Conclusions</h4>Our approach can successfully identify the candidate drivers from the core modules. Comprehensive network analysis on the core modules potentially provides critical insights into convergent cancer development in different organs.

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent '+4' cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5(+) CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+) stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5(+)stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find-and confirm by independent approaches-that the proposed quiescent/'+4' stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.

miR-125 microRNAs, such as lin-4 in Caenorhabditis elegans, were among the first microRNAs discovered, are phylogenetically conserved, and have been implicated in regulating developmental timing. Here, we showed that loss-of-function mutations in lin-4 microRNA increased axon attraction mediated by the netrin homolog UNC-6. The absence of lin-4 microRNA suppressed the axon guidance defects of anterior ventral microtubule (AVM) neurons caused by loss-of-function mutations in slt-1, which encodes a repulsive guidance cue. Selective expression of lin-4 microRNA in AVM neurons of lin-4-null animals indicated that the effect of lin-4 on AVM axon guidance was cell-autonomous. Promoter reporter analysis suggested that lin-4 was likely expressed strongly in AVM neurons during the developmental time frame that the axons are guided to their targets. In contrast, the lin-4 reporter was barely detectable in anterior lateral microtubule (ALM) neurons, axon guidance of which is insensitive to netrin. In AVM neurons, the transcription factor LIN-14, a target of lin-4 microRNA, stimulated UNC-6-mediated ventral guidance of the AVM axon. LIN-14 promoted attraction of the AVM axon through the UNC-6 receptor UNC-40 [the worm homolog of vertebrate Deleted in Colorectal Cancer (DCC)] and its cofactor MADD-2, which signals through both the UNC-34 (Ena) and the CED-10 (Rac1) downstream pathways. LIN-14 stimulated UNC-6-mediated axon attraction in part by increasing UNC-40 abundance. Our study indicated that lin-4 microRNA reduced the activity of LIN-14 to terminate UNC-6-mediated axon guidance of AVM neurons.

Huntington's disease (HD) is a devastating autosomal-dominant neurodegenerative disorder initiated by an abnormally expanded polyglutamine in the huntingtin protein. Determining the contribution of specific factors to the pathogenesis of HD should provide rational targets for therapeutic intervention. One suggested contributor is the type 2 transglutaminase (TG2), a multifunctional calcium dependent enzyme. A role for TG2 in HD has been suggested because a polypeptide-bound glutamine is a rate-limiting factor for a TG2-catalyzed reaction, and TG2 can cross-link mutant huntingtin in vitro. Further, TG2 is up regulated in brain areas affected in HD. The objective of this study was to further examine the contribution of TG2 as a potential modifier of HD pathogenesis and its validity as a therapeutic target in HD. In particular our goal was to determine whether an increase in TG2 level, as documented in human HD brains, modulates the well-characterized phenotype of the R6/2 HD mouse model. To accomplish this objective a genetic cross was performed between R6/2 mice and an established transgenic mouse line that constitutively expresses human TG2 (hTG2) under control of the prion promoter. Constitutive expression of hTG2 did not affect the onset and progression of the behavioral and neuropathological HD phenotype of R6/2 mice. We found no alterations in body weight changes, rotarod performances, grip strength, overall activity, and no significant effect on the neuropathological features of R6/2 mice. Overall the results of this study suggest that an increase in hTG2 expression does not significantly modify the pathology of HD.

α-Synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson's disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation.

<h4>Background</h4>SFTS virus (SFTSV) is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS) in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection.<h4>Methodology/principal findings</h4>We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs) recognized the nucleocapsid (N) protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs.<h4>Conclusions/significance</h4>The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.

In this work, oxidized nanocellulose (ONC) was synthesized and chemically coupled with amino acids and peptides using a two step coupling method at room temperature. First, ONC was activated by N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride, forming a stable active ester in the presence of N-hydroxysuccinimide. Second, the active ester was reacted with the amino group of the amino acid or peptide, forming an amide bond between ONC and the grafted molecule. Using this method, the intermolecular interaction of amino acids and peptides was avoided and uniform coupling of these molecules on ONC was achieved. The coupling reaction was very fast in mild conditions and without alteration of the polysaccharide. The coupling products (ONC-amino acids and ONC-peptides) were characterized by transmission electron microscopy and by the absorption, emission, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) spectroscopic techniques.

No abstract available.

<h4>Unlabelled</h4>Pegylated interferon-α (PEG-IFN-α) forms an integral part of the current treatment for hepatitis C virus (HCV) infection. PEG-IFN-α suppresses HCV production by augmenting the innate antiviral immune response. Recent studies have reported the induction of hepcidin, the iron regulatory hormone, by IFN-α in vitro. As hepcidin plays an important role in innate immunity, we hypothesized that this finding may be of clinical relevance to HCV and investigated the changes in iron homeostasis during the first 24 hours of treatment. Blood samples were obtained from HCV patients immediately prior to and 6, 12, and 24 hours following the first dose of PEG-IFN-α/ribavirin (RBV). Samples were analyzed for hepcidin, cytokine, iron levels, and HCV viral load, and hepcidin messenger RNA (mRNA) expression was quantified in peripheral blood mononuclear cells. Hepcidin induction by IFN-α was further analyzed in cell culture. In HCV patients a single dose of PEG-IFN-α/RBV resulted in a significant increase in serum hepcidin, peaking at 12 hours, coinciding with a 50% reduction in serum iron and transferrin saturation over the 24-hour period. Patients with a ≥ 2 log decline in HCV viral load over the first 24 hours had significantly lower SI and TS levels at 12 and 24 hours. Moreover, 24-hour SI levels were an independent predictor of the immediate HCV viral decline, an indicator of ultimate treatment outcome. In cell culture, a direct induction of hepcidin by IFN-α was seen, controlled by the STAT3 transcription factor.<h4>Conclusion</h4>Hepcidin induction occurs following the initiation of PEG-IFN-α treatment for HCV, and is mediated by way of STAT3 signaling. The subsequent hypoferremia was greatest in those with the most significant decline in viral load, identifying systemic iron withdrawal as a marker of immediate interferon-α efficacy in HCV patients.

<h4>Unlabelled</h4>Mutations in hemochromatosis protein (HFE) or transferrin receptor 2 (TFR2) cause hereditary hemochromatosis (HH) by impeding production of the liver iron-regulatory hormone, hepcidin (HAMP). This study examined the effects of disruption of Hfe or Tfr2, either alone or together, on liver iron loading and injury in mouse models of HH. Iron status was determined in Hfe knockout (Hfe(-/-)), Tfr2 Y245X mutant (Tfr2(mut)), and double-mutant (Hfe(-/-) ×Tfr2(mut) ) mice by measuring plasma and liver iron levels. Plasma alanine transaminase (ALT) activity, liver histology, and collagen deposition were evaluated to assess liver injury. Hepatic oxidative stress was assessed by measuring superoxide dismutase (SOD) activity and F(2)-isoprostane levels. Gene expression was measured by real-time polymerase chain reaction. Hfe(-/-) ×Tfr2(mut) mice had elevated hepatic iron with a periportal distribution and increased plasma iron, transferrin saturation, and non-transferrin-bound iron, compared with Hfe(-/-), Tfr2(mut), and wild-type (WT) mice. Hamp1 expression was reduced to 40% (Hfe(-/-) and Tfr2(mut) ) and 1% (Hfe(-/-) ×Tfr2(mut)) of WT values. Hfe(-/-) ×Tfr2(mut) mice had elevated plasma ALT activity and mild hepatic inflammation with scattered aggregates of infiltrating inflammatory cluster of differentiation 45 (CD45)-positive cells. Increased hepatic hydoxyproline levels as well as Sirius red and Masson's Trichrome staining demonstrated advanced portal collagen deposition. Hfe(-/-) and Tfr2(mut) mice had less hepatic inflammation and collagen deposition. Liver F(2) -isoprostane levels were elevated, and copper/zinc and manganese SOD activities decreased in Hfe(-/-) ×Tfr2(mut), Tfr2(mut), and Hfe(-/-) mice, compared with WT mice.<h4>Conclusion</h4>Disruption of both Hfe and Tfr2 caused more severe hepatic iron overload with more advanced lipid peroxidation, inflammation, and portal fibrosis than was observed with the disruption of either gene alone. The Hfe(-/-) ×Tfr2(mut) mouse model of iron-induced liver injury reflects the liver injury phenotype observed in human HH.

<h4>Background</h4>In ovarian cancer, cyclooxygenase-2 (COX-2) overexpression is prognostic for poor survival. We investigated the efficacy of celecoxib (C), a selective COX-2 inhibitor, added to docetaxel (Taxotere)/carboplatin (DC) in advanced ovarian cancer.<h4>Patients and methods</h4>In a phase II, randomized study, 400 mg celecoxib b.i.d. was added to first-line DC treatment (DCC). Celecoxib was to be continued after DC termination up to 3 years. Study end points were tolerability, progression-free survival (PFS) and overall survival (OS).<h4>Results</h4>151 of 196 eligible patients were diagnosed with stage IIIC/IV disease. Median follow-up for patients alive was 32.3 months. Celecoxib was used during a mean of 8.5 months. Twenty-three of 97 DCC patients stopped celecoxib prematurely, mainly due to skin reactions. Complete biochemical response was achieved in 51/78 DC patients (65%) versus 57/78 DCC patients (75%, not significant). In both study arms, median PFS was 14.3 months and median OS 34 months. COX-2 was expressed in 82% of 120 tumor samples retrospectively recovered. The PFS and OS of patients with intermediate/high COX-2 expression were similar to that in the other patients.<h4>Conclusion</h4>Celecoxib did not influence PFS and OS, but interpretation of results is hampered by premature celecoxib discontinuation.

In the present study, the application of mass spectrometry (MS) binding assays as a tool for library screening is reported. For library generation, dynamic combinatorial chemistry (DCC) was used. These libraries can be screened by means of MS binding assays when appropriate measures are taken to render the libraries pseudostatic. That way, the efficiency of MS binding assays to determine ligand binding in compound screening with the ease of library generation by DCC is combined. The feasibility of this approach is shown for γ-aminobutyric acid (GABA) transporter 1 (GAT1) as a target, representing the most important subtype of the GABA transporters. For the screening, hydrazone libraries were employed that were generated in the presence of the target by reacting various sets of aldehydes with a hydrazine derivative that is delineated from piperidine-3-carboxylic acid (nipecotic acid), a common fragment of known GAT1 inhibitors. To ensure that the library generated is pseudostatic, a large excess of the nipecotic acid derivative is employed. As the library is generated in a buffer system suitable for binding and the target is already present, the mixtures can be directly analyzed by MS binding assays-the process of library generation and screening thus becoming simple to perform. The binding affinities of the hits identified by deconvolution were confirmed in conventional competitive MS binding assays performed with single compounds obtained by separate synthesis. In this way, two nipecotic acid derivatives exhibiting a biaryl moiety, 1-{2-[2'-(1,1'-biphenyl-2-ylmethylidene)hydrazine]ethyl}piperidine-3-carboxylic acid and 1-(2-{2'-[1-(2-thiophenylphenyl)methylidene]hydrazine}ethyl)piperidine-3-carboxylic acid, were found to be potent GAT1 ligands exhibiting pK(i) values of 6.186 ± 0.028 and 6.229 ± 0.039, respectively. This method enables screening of libraries, whether generated by conventional chemistry or DCC, and is applicable to all kinds of targets including membrane-bound targets such as G protein coupled receptors (GPCRs), ion channels and transporters. As such, this strategy displays high potential in the drug discovery process.

<h4>Background</h4>Histone H3 lysine 4 (K4) methylation has been linked with transcriptional activity in mammalian cells. The WD40-repeat protein, WDR5, is an essential component of the MLL complex that induces histone H3 K4 methylation, but the role of WDR5 in human globin gene regulation has not yet been established.<h4>Design and methods</h4>To study the role of WDR5 in human globin gene regulation, we performed knockdown experiments in both K562 cells and primary human bone marrow erythroid progenitor cells (BMC). The effects of WDR5 knockdown on γ-globin gene expression were determined. Biochemical approaches were also employed to investigate WDR5 interaction molecules. Chromosomal marks in the globin locus were analyzed by ChIP.<h4>Results</h4>We found that WDR5 interacted with protein arginine methyltransferase 5 (PRMT5), a known repressor of γ-globin gene expression, and was essential for generating tri-methylated H3K4 (H3K4me3) at the γ-globin promoter in K562 cells. Enforced expression of WDR5 in K562 cells reduced γ-globin gene expression, whereas knockdown of WDR5 increased γ-globin gene expression in both K562 cells and primary human bone marrow erythroid progenitor cells. Consistent with this, both histone H3 and H4 acetylation at the γ-globin promoter were increased, while histone H4R3 and H3K9 methylation were decreased, in WDR5 knockdown cells compared to controls. We found that WDR5 interacted with HDAC1 and a PHD domaincontaining protein, ING2 (inhibitor of growth), an H3K4me3 mark reader, to enhance γ-globin gene transcriptional repression. In human BMC, levels of WDR5 were highly enriched on the γ-promoter relative to levels on other globin promoters and compared to the γ-promoter in cord blood erythroid progenitors, suggesting that WDR5 is important in the developmental globin gene expression program.<h4>Conclusions</h4>Our data are consistent with a model in which WDR5 binds the γ-globin promoter in a PRMT5-dependent manner; H3K4me3 induced at the γ-globin promoter by WDR5 may result in the recruitment of the ING2-associated HDAC1 component and consequent silencing of γ-globin gene expression.

<h4>Background</h4>Iron and its homeostasis are intimately related to inflammatory responses, but the underlying molecular mechanisms are poorly understood. We investigated the role of Steap3 in regulating iron homeostasis in macrophages, and the effects of Steap3 depletion on host inflammatory responses.<h4>Design and methods</h4>We analyzed bone marrow-derived macrophages and primary cultured hepatocytes from Steap3(-/-) mouse models to investigate the roles of Steap3 in coordinately regulating iron homeostasis and inflammatory responses. First, we examined iron distribution and iron status in cells deficient in Steap3, as well as the requirement for the Steap3 gene during inflammatory responses. Secondly, we analyzed the regulation of Steap3 expression by inflammatory stimuli and thus, the influence of these stimuli on iron distribution and homeostasis.<h4>Results</h4>We found that Steap3 mRNA was expressed at high levels in macrophages and hepatocytes. Steap3 deficiency led to impaired iron homeostasis, causing abnormal iron distribution and a decreased availability of cytosolic iron in macrophages. Among STEAP family members, Steap3 mRNA was uniquely down-regulated in macrophages stimulated by lipopolysaccharides. To determine whether Steap3 regulated iron homeostasis during inflammatory stress, we treated Steap3(-/-) mice with lipopolysaccharide, which produced greater iron accumulation in the vital tissues of these mice compared to in the tissues of wild-type controls. Furthermore, Steap3 depletion led to impaired induction of interferon-β, monocyte chemoattractant protein-5, and interferon induced protein-10 in macrophages via the TLR4-mediated signaling pathway.<h4>Conclusions</h4>Steap3 is important in regulating both iron homeostasis and TLR4-mediated inflammatory responses in macrophages. Steap3 deficiency causes abnormal iron status and homeostasis, which leads to impaired TLR4-mediated inflammatory responses in macrophages. Following inflammatory stimuli, Steap3 depletion causes dysregulated iron sequestration and distribution. Our results provide important insights into the function of Steap3 as a coordinate regulator of both iron homeostasis and innate immunity.

Using biomarkers to select the most at-risk population, to detect the disease while measurable and yet not clinically apparent has been the goal of many investigations. Recent advances in molecular strategies and analytic platforms, including genomics, epigenomics, proteomics, and metabolomics, have identified increasing numbers of potential biomarkers in the blood, urine, exhaled breath condensate, bronchial specimens, saliva, and sputum, but none have yet moved to the clinical setting. Therefore, there is a recognized gap between the promise and the product delivery in the cancer biomarker field. In this review, we define clinical contexts where risk and diagnostic biomarkers may have use in the management of lung cancer, identify the most relevant candidate biomarkers of early detection, provide their state of development, and finally discuss critical aspects of study design in molecular biomarkers for early detection of lung cancer.

Aberrant miRNA expression has been previously established in breast cancer and has clinical relevance. However, no studies so far have defined miRNAs deregulated in hereditary breast tumors. In this study we investigated the role of miRNAs in hereditary breast tumors comparing with normal breast tissue. Global miRNA expression profiling using Exiqon microarrays was performed on 22 hereditary breast tumors and 15 non-tumoral breast tissues. We identified 19 miRNAs differentially expressed, most of them down-regulated in tumors. An important proportion of deregulated miRNAs in hereditary tumors were previously identified commonly deregulated in sporadic breast tumors. Under-expression of these miRNAs was validated by qRT-PCR in additional 18 sporadic breast tumors and their normal breast tissue counterparts. Pathway enrichment analysis revealed that deregulated miRNAs collectively targeted a number of genes belonging to signaling pathways such as MAPK, ErbB, mTOR, and those regulating cell motility or adhesion. In silico prediction detected KRAS oncogene as target of several deregulated miRNAs. In particular, we experimentally validated KRAS as a miR-30c target. Luciferase assays confirmed that miR-30c binds the 3'UTR of KRAS transcripts and expression of pre-miR-30c down-regulated KRAS mRNA and protein. Furthermore, miR-30c overexpression inhibited proliferation of breast cancer cells. Our results identify miRNAs associated to hereditary breast cancer, as well as miRNAs commonly miss-expressed in hereditary and sporadic tumors, suggesting common underlying mechanisms of tumor progression. In addition, we provide evidence that KRAS is a target of miR-30c, and that this miRNA suppresses breast cancer cell growth potentially through inhibition of KRAS signaling.

Targeted delivery of contrast agents is a highly desirable strategy for enhancing diagnostic efficiency and reducing side effects and toxicity. Water-soluble and tumor-targeting superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by loading hydrophobic SPIONs into micelles assembled from an amphiphilic block copolymer poly(ethylene glycol)- poly(ε-caprolactone) (PEG-PCL) bearing folate in the distal ends of PEG chains. Compared to the water-soluble SPIONs obtained by small molecular surfactant coating, ultrasmall SPION encapsulation with PEG-PCL micelles (PEG-PCL-SPIONs) simultaneously increases transverse (r(2)) and decreases longitudinal (r(1)) magnetic resonance (MR) relaxivities of water proton in micelle solution, leading to a notably high r(2)/r(1) ratio up to 78, which makes the PEG-PCL-SPIONs a highly sensitive MR imaging (MRI) T(2) contrast agent. The mean size of folate-attached SPION micelles (Fa-PEG-PCL-SPIONs) is 44 ± 3 nm on average, ideal for in vivo MRI applications in which long circulation is greatly determined by small particle size and is highly desirable. Prussian blue staining of BEL-7402 cells over-expressing folate receptors, after incubation with micelle-containing medium, demonstrated that folate functionalization of the magnetic particles significantly enhanced their cell uptake. The potential of Fa-PEG-PCL-SPIONs as a potent MRI probe for in vivo tumor detection was assessed. At 3 hours after intravenous injection of the Fa-PEG-PCL-SPION solution into mice bearing subcutaneous xenografts of human BEL-7402 hepatoma, a 41.2% signal intensity decrease was detected in the T(2)-weighted MR images of the tumor, indicating the efficient accumulation of Fa-PEG-PCL-SPIONs in the tumor tissue.

Permanent ductal closure involves anatomic remodeling, in which transforming growth factor (TGF)-β appears to play a role. Our objective was to evaluate the relationship, if any, between blood spot TGF-β on day 3 and day 7 of life and patent ductus arteriosus (PDA) in extremely low birth weight (ELBW) infants. Prospective observational study involving ELBW infants (n = 968) in the National Institute of Child Health and Human Development Neonatal Research Network who had TGF-β measured on filter paper spot blood samples using a Luminex assay. Infants with a PDA (n = 493) were significantly more immature, had lower birth weights, and had higher rates of respiratory distress syndrome than those without PDA (n = 475). TGF-β on days 3 and 7 of life, respectively, were significantly lower among neonates with PDA (median 1,177 pg/ml [range 642-1,896]; median 1,386 pg/ml [range 868-1,913]) compared with others without PDA (median 1,334 pg/ml [range 760-2,064]; median 1,712 pg/ml [range 1,014-2,518 pg/ml]). The significant difference persisted when death or PDA was considered a composite outcome. TGF-β levels were not significantly different among subgroups of infants with PDA who were not treated (n = 51) versus those who were treated medically (n = 283) or by surgical ligation (n = 159). TGF-β was not a significant predictor of death or PDA (day 3 odds ratio [OR] 0.99, 95 % confidence interval [CI] 0.83-1.17; day 7 OR 0.88, 95 % CI 0.74-1.04) on adjusted analyses. Our results suggest that blood spot TGF-β alone is unlikely to be a reliable biomarker of a clinically significant PDA or its responsiveness to treatment.

Discovering the unintended 'off-targets' that predict adverse drug reactions is daunting by empirical methods alone. Drugs can act on several protein targets, some of which can be unrelated by conventional molecular metrics, and hundreds of proteins have been implicated in side effects. Here we use a computational strategy to predict the activity of 656 marketed drugs on 73 unintended 'side-effect' targets. Approximately half of the predictions were confirmed, either from proprietary databases unknown to the method or by new experimental assays. Affinities for these new off-targets ranged from 1 nM to 30 μM. To explore relevance, we developed an association metric to prioritize those new off-targets that explained side effects better than any known target of a given drug, creating a drug-target-adverse drug reaction network. Among these new associations was the prediction that the abdominal pain side effect of the synthetic oestrogen chlorotrianisene was mediated through its newly discovered inhibition of the enzyme cyclooxygenase-1. The clinical relevance of this inhibition was borne out in whole human blood platelet aggregation assays. This approach may have wide application to de-risking toxicological liabilities in drug discovery.

Iron homeostasis is maintained at the cellular and systemic levels to assure adequate iron supply while preventing iron overload. The identification of genes mutated in patients with iron-related disorders or animal models with imbalances of iron homeostasis gave insight into the molecular mechanisms underlying processes critical for balancing iron levels, such as iron uptake, storage, export, and monitoring of available iron. MicroRNAs control genes involved in some of these processes adding an additional level of complexity to the regulation of iron metabolism. This review summarizes recent advances how miRNAs regulate iron homeostasis.

<h4>Background</h4>Spontaneous intestinal perforation (SIP) is associated with the use of postnatal glucocorticoids and indometacin in extremely low birth weight (ELBW) infants. The authors hypothesised: 1) an association of SIP with the use of antenatal steroids (ANS) and indometacin either as prophylaxis for intraventricular hemorrhage (IVH) (P Indo) or for treatment of PDA (Indo/PDA) and 2) an increased risk of death or abnormal neurodevelopmental outcomes in infants with SIP at 18-22 months corrected age.<h4>Design/methods</h4>The authors retrospectively identified ELBW infants with SIP in the Neonatal Research Network's generic database. Unadjusted analysis identified the differences in maternal, neonatal and clinical variables between infants with and without SIP. Logistic regression analysis identified the adjusted OR for SIP with reference to ANS, P Indo and Indo/PDA. Neurodevelopmental outcomes were assessed among survivors at 18-22 months corrected age.<h4>Results</h4>Indo/PDA was associated with an increased risk of SIP (adjusted OR 1.61; 95% CI 1.25 to 2.08), while P Indo and ANS were not. SIP was independently associated with an increased risk of death or neurodevelopmental impairment (NDI) (adjusted OR 1.85; 95% CI 1.32 to 2.60) and NDI among survivors (adjusted OR 1.75, 95% CI 1.20 to 2.55).<h4>Conclusion</h4>Indometacin used for IVH prophylaxis and ANS were not associated with the occurrence of SIP in ELBW infants. Indometacin used for treatment of symptomatic PDA was however associated with an increased risk of SIP. ELBW infants with SIP have an increased risk of poor neurodevelopmental outcomes.

Here, we investigated an Immunoglobulin (Ig) superfamily protein IgSF8 which is abundantly expressed in olfactory sensory neuron (OSN) axons and their developing synapses. We demonstrate that expression of IgSF8 within synaptic neuropil is transitory, limited to the period of glomerular formation. Glomerular expression decreases after synaptic maturation and compartmental glomerular organization is achieved, although expression is maintained at high levels within the olfactory nerve layer (ONL). Immunoprecipitations indicate that IgSF8 interacts with tetraspanin CD9 in the olfactory bulb (OB). CD9 is a component of tetraspanin-enriched microdomains (TEMs), specialized microdomains of the plasma membrane known to regulate cell morphology, motility, invasion, fusion and signaling, in both the nervous and immune systems, as well as in tumors. In vitro, both IgSF8 and CD9 localize to puncta within axons and growth cones of OSNs, consistent with TEM localization. When the olfactory epithelium (OE) was lesioned, forcing OSN regeneration en masse, IgSF8 was once again able to be detected in OSN axon terminals as synapses were reestablished. Finally, we halted synaptic maturation within glomeruli by unilaterally blocking functional activity and found that IgSF8 did not undergo exclusion from this subcellular compartment and instead continued to be detected in adult glomeruli. These data support the hypothesis that IgSF8 facilitates OSN synapse formation.

The cause of multiple sclerosis (MS) is not known and the mechanism of interferon-beta, a disease-modifying treatment, is not well-understood. We studied gene expression in plasmacytoid dendritic cells (pDCs), antigen-presenting cells implicated in MS pathogenesis. PDCs were separated from healthy donors and MS patients at two time points: before and after initiation of treatment with interferon-beta. Expression of selected MS-linked and interferon-beta-regulated genes was validated with single assays. We have identified 60 genes which were abnormally expressed in MS patients and were corrected after treatment. These genes could be studied as potential MS biomarkers and possible therapeutic targets in MS.

The in vivo study of receptor binding potential in the human brain is made possible by positron emission tomography (PET) imaging. Here we review PET studies of neuroreceptor function in mood disorders - specifically, major depressive disorder (MDD) and bipolar disorder (BD). We concentrate on the most widely studied receptors of the serotonergic and dopaminergic systems. Specifically, the serotonin 1A (5-HT(1A)), serotonin 2A (5-HT(2A)), serotonin 1B (5-HT(1B)), dopamine 1 (D1), and dopamine 2/3 (D2/3) receptors. We also review PET studies of the serotonin transporter (5-HTT), the dopamine transporter (DAT), monoamine oxidase A (MAO-A), and the muscarinic 2 receptor (M2). On the basis of the PET literature as well as supporting genetic studies, postmortem data, and preclinical models of depression, and several models of how monoaminergic function is altered in mood disorders are discussed with respect to inflammation, endocrine dysfunction, depression subtypes, and altered neurocircuitry.

While surgery is the usual treatment for localized well-differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS), the therapeutic options for patients with advanced disease are limited. The classical antimitotic treatments are most often inefficient. The establishment of genetically characterized cell lines is therefore crucial for providing in vitro models for novel targeted therapies. We have used spectral karyotyping, fluorescence in situ hybridization with whole chromosome painting and locus-specific probes, and array-comparative genomic hybridization to identify the chromosomal and molecular alterations of a novel cell line established from a recurring sclerosing WDLPS. The karyotype was hypertriploid and showed multiple structural anomalies. All cells retained the presence of a giant marker chromosome that had been previously identified in the primary cell cultures. This giant chromosome contained high-level amplification of chromosomal regions 12q13-21 and lacked the alpha-satellite centromeric sequences associated with WDLPS/DDLPS. The 12q amplicon was large, containing 370 amplified genes. The DNA copy number ranged from 3 to 57. The highest levels of amplification were observed at 12q14.3 for GNS, WIF1, and HMGA2. We analyzed the mRNA expression status by real-time reverse transcription polymerase chain reaction for six genes from this amplicon: MDM2, HMGA2, CDK4, TSPAN31, WIF1, and YEATS4. mRNA overexpression was correlated with genomic amplification. A second amplicon originating from 10p11-14 was also present in the giant marker chromosome. The 10p amplicon contained 62 genes, including oncogenes such as MLLT10, previously described in chimeric fusion with MLL in leukemias, NEBL, and BMI1.

<h4>Background</h4>Liver injury due to prescription and nonprescription medications is an expanding public health concern in the United States, with drug-induced liver injury (DILI) being the single most common reason for regulatory actions instituted by the Food and Drug Administration against certain medications and supplements.<h4>Case presentation</h4>A 69-year-old Latino man was referred to Hepatology Clinic for urgent evaluation of new onset jaundice, nausea and fatigue associated with a >40-fold increase in his transaminase levels and elevated INR and alkaline phosphatase. The patient had received a new prescription for varenicline to aid with smoking cessation approximately 3 weeks prior to his evaluation in Hepatology Clinic. Within 5 days of starting the varenicline, the patient developed new onset of nausea, vomiting, malaise and deep jaundice. The varenicline was discontinued on day 5 by the patient. Hepatologic evaluation revealed no evidence of acute viral hepatitis, autoimmune, metabolic or alcohol-related liver disorders. The patient's past medical history was notable, however, for chronic hepatitis C. His liver enzymes and synthetic function completely normalized 9 weeks after discontinuation of the varenicline.<h4>Conclusion</h4>This report represents the second documented cases of drug-induced liver injury related to varenicline therapy, highlighting the need for clinician awareness regarding potential hepatotoxicity of varenicline, particularly among patients with pre-existing liver disease.

<h4>Background</h4>Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G)-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics.<h4>Methods</h4>Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA) for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman's rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts.<h4>Results</h4>Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p=0.024). EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated with ErbB1, mTOR, PTEN, and Stat5. Western blots confirmed that full-length and truncated beta-catenin expression correlated with U2AF65 expression in tumor extracts.<h4>Conclusions</h4>Increased triplex DNA-binding activity in vitro correlates with lymph node disease, metastasis, and reduced overall survival in colorectal cancer, and increased U2AF65 expression is associated with total and truncated beta-catenin expression in high-stage colorectal tumors.

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.

Tight regulation of virus-induced cytotoxic effector CD8(+) T cells is essential to prevent immunopathology. Naturally occurring effector CD8(+) T cells, with a KLRG1(hi) CD62L(lo) phenotype typical of short-lived effector CD8(+) T cells (SLECs), can be found in increased numbers in autoimmune-prone mice, most notably in mice homozygous for the san allele of Roquin. These SLEC-like cells were able to trigger autoimmune diabetes in a susceptible background. When Roquin is mutated (Roquin(san)), effector CD8(+) T cells accumulate in a cell-autonomous manner, most prominently as SLEC-like effectors. Excessive IFN-γ promotes the accumulation of SLEC-like cells, increases their T-bet expression, and enhances their granzyme B production in vivo. We show that overexpression of IFN-γ was caused by failed posttranscriptional repression of Ifng mRNA. This study identifies a novel mechanism that prevents accumulation of self-reactive cytotoxic effectors, highlighting the importance of regulating Ifng mRNA stability to maintain CD8(+) T cell homeostasis and prevent CD8-mediated autoimmunity.

APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here, we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-κB under basal but not TNFα-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate canonical NF-κB signaling. APPL1 synergized with TRAF2 to induce NF-κB activation, and both proteins were necessary for this process and function upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-κB pathway. Importantly, in the canonical pathway, APPL1 appeared to regulate the proper spatial distribution of the p65 subunit of NF-κB in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. By analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-κB target genes that encode cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-κB pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-κB activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-κB activation.

Cortical dopamine (DA) modulation of the gamma-amino butyric acid (GABA) system is closely associated with cognitive function and psychiatric disorders. We recently reported that the glycogen synthase kinase 3β (GSK-3β) pathway is required for hyperdopamine/D2 receptor-mediated inhibition of NMDA receptors in the prefrontal cortex. Here we explore whether or not GSK-3β is also involved in dopaminergic modulation of GABAA receptor-mediated inhibitory transmission. We confirmed that DA induces a dose-dependent, bidirectional regulatory effect on inhibitory postsynaptic currents (IPSCs) in prefrontal neurons. The modulatory effects of DA were differentially affected by co-application of GSK-3β inhibitors and different doses of DA. GSK-3β inhibitors completely blocked high-dose (20 μM) DA-induced depressive effects on IPSCs but exhibited limited effects on the facilitating regulation of IPSC in low-dose DA (200 nM). We also confirmed that surface expressions of GABAA receptor β2/3 subunits were significantly decreased by DA applied in cultured prefrontal neurons and in vivo administration of DA reuptake inhibitor. These effects were blocked by prior administration of GSK-3β inhibitors. We explored DA-mediated regulation of GABAA receptor trafficking and exhibited the participation of brefeldin A-inhibited GDP/GTP exchange factor 2 (BIG2) or dynamin-dependent trafficking of GABAA receptors. Together, these data suggest that DA may act through different signaling pathways to affect synaptic inhibition, depending on the concentration. The GSK-3β signaling pathway is involved in DA-induced decrease in BIG2-dependent insertion and an increase in the dynamin-dependent internalization of GABAA receptors, which results in suppression of inhibitory synaptic transmission.

<h4>Background</h4>Lung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers' lung carcinomas remain to a large extent unclear.<h4>Methods</h4>Unsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.<h4>Results</h4>Initial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.<h4>Conclusions</h4>Based on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.

<h4>Introduction</h4>Transplantation of mesenchymal stem cells (MSCs) derived from synovium is a promising therapy for cartilage regeneration. For clinical application, improvement of handling operation, enhancement of chondrogenic potential, and increase of MSCs adhesion efficiency are needed to achieve a more successful cartilage regeneration with a limited number of MSCs without scaffold. The use of aggregated MSCs may be one of the solutions. Here, we investigated the handling, properties and effectiveness of aggregated MSCs for cartilage regeneration.<h4>Methods</h4>Human and rabbit synovial MSCs were aggregated using the hanging drop technique. The gene expression changes after aggregation of synovial MSCs were analyzed by microarray and real time RT-PCR analyses. In vitro and in vivo chondrogenic potential of aggregates of synovial MSCs was examined.<h4>Results</h4>Aggregates of MSCs cultured for three days became visible, approximately 1 mm in diameter and solid and durable by manipulation; most of the cells were viable. Microarray analysis revealed up-regulation of chondrogenesis-related, anti-inflammatory and anti-apoptotic genes in aggregates of MSCs. In vitro studies showed higher amounts of cartilage matrix synthesis in pellets derived from aggregates of MSCs compared to pellets derived from MSCs cultured in a monolayer. In in vivo studies in rabbits, aggregates of MSCs could adhere promptly on the osteochondral defects by surface tension, and stay without any loss. Transplantation of aggregates of MSCs at relatively low density achieved successful cartilage regeneration. Contrary to our expectation, transplantation of aggregates of MSCs at high density failed to regenerate cartilage due to cell death and nutrient deprivation of aggregates of MSCs.<h4>Conclusions</h4>Aggregated synovial MSCs were a useful source for cartilage regeneration considering such factors as easy preparation, higher chondrogenic potential and efficient attachment.

The gene B lymphocyte kinase (BLK) is associated with rheumatoid arthritis, systemic lupus erythematosus and several other autoimmune disorders. The disease risk haplotype is known to be associated with reduced expression of BLK mRNA transcript in human B cell lines; however, little is known about cis-regulation of BLK message or protein levels in native cell types. Here, we show that in primary human B lymphocytes, cis-regulatory effects of disease-associated single nucleotide polymorphisms in BLK are restricted to naïve and transitional B cells. Cis-regulatory effects are not observed in adult B cells in later stages of differentiation. Allelic expression bias was also identified in primary human T cells from adult peripheral and umbilical cord blood (UCB), thymus and tonsil, although mRNA levels were reduced compared with B cells. Allelic regulation of Blk expression at the protein level was confirmed in UCB B cell subsets by intracellular staining and flow cytometry. Blk protein expression in CD4(+) and CD8(+) T cells was documented by western blot analysis; however, differences in protein expression levels by BLK genotype were not observed in any T cell subset. Blk allele expression differences at the protein level are thus restricted to early B cells, indicating that the involvement of Blk in the risk for autoimmune disease likely acts during the very early stages of B cell development.

Huntington's disease (HD) is characterized by a late clinical onset despite ubiquitous expression of the mutant gene at all developmental stages. How mutant huntingtin impacts on signalling pathways in the pre-symptomatic period has remained essentially unexplored in humans due to a lack of appropriate models. Using multiple human embryonic stem cell lines derived from blastocysts diagnosed as carrying the mutant huntingtin gene by pre-implantation genetic diagnosis, we explored early developmental changes in gene expression using differential transcriptomics, combined with gain and loss of function strategies. We demonstrated a down-regulation of the HTT gene itself in HD neural cells and identified three genes, the expression of which differs significantly in HD cells when compared with wild-type controls, namely CHCHD2, TRIM4 and PKIB. Similar dysregulation had been observed previously for CHCDH2 and TRIM4 in blood cells from patients. CHCHD2 is involved in mitochondrial function and PKIB in protein kinase A-dependent pathway regulation, which suggests that these functions may be precociously impacted in HD.

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = -0.16 mm per minor allele, P(meta) =2.69 × 10(-10)). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) =0.75, 95% CI: 0.68-0.84, P(meta) =4.38 × 10(-7)) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia.

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR=0.651, p=0.006 and HR=0.642, p=0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR=1.355 p=0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells.

Identifying and understanding the impact of gene regulatory variation is of considerable importance in evolutionary and medical genetics; such variants are thought to be responsible for human-specific adaptation and to have an important role in genetic disease. Regulatory variation in cis is readily detected in individuals showing uneven expression of a transcript from its two allelic copies, an observation referred to as allelic imbalance (AI). Identifying individuals exhibiting AI allows mapping of regulatory DNA regions and the potential to identify the underlying causal genetic variant(s). However, existing mapping methods require knowledge of the haplotypes, which make them sensitive to phasing errors. In this study, we introduce a genotype-based mapping test that does not require haplotype-phase inference to locate regulatory regions. The test relies on partitioning genotypes of individuals exhibiting AI and those not expressing AI in a 2×3 contingency table. The performance of this test to detect linkage disequilibrium (LD) between a potential regulatory site and a SNP located in this region was examined by analyzing the simulated and the empirical AI datasets. In simulation experiments, the genotype-based test outperforms the haplotype-based tests with the increasing distance separating the regulatory region from its regulated transcript. The genotype-based test performed equally well with the experimental AI datasets, either from genome-wide cDNA hybridization arrays or from RNA sequencing. By avoiding the need of haplotype inference, the genotype-based test will suit AI analyses in population samples of unknown haplotype structure and will additionally facilitate the identification of cis-regulatory variants that are located far away from the regulated transcript.

Food allergy (FA) is relatively a common disease in infants, but effective drug therapies are not yet available. Notably, mucosal mast cells, but not connective-tissue mast cells, play important roles in food allergic reactions via the release of inflammatory mediators. Therefore, we screened medicinal herb extracts for in vitro and in vivo antiallergic activity through inhibiting mucosal mast cell activation. As a result, both antigen-induced and calcium ionophore-induced degranulation was significantly inhibited by Zanthoxyli Fructus water extract (ZF) in mucosal-type murine bone marrow-derived mast cells (mBMMCs). ZF suppressed the antigen-induced [Ca(2+)](i) elevation and the antigen-enhanced mRNA expression of TNF-α, IL-4, and IL-13. The transcriptome and real-time PCR analyses revealed that ZF greatly decreased the antigen-enhanced expression level of sphingosine kinase 1 (Sphk1), which plays a key role in the FcεRI-mediated immune responses in mast cells. Furthermore, ZF inhibited allergic symptoms in an ovalbumin-caused murine FA model and decreased the number of infiltrating mucosal mast cells and the enhanced mRNA expression levels of IL-4 and Sphk1 in the FA mice colons. These results indicate that ZF suppresses mucosal mast cell activities mainly through Sphk1-dependent mechanism, and ZF is utilized for the development of a novel, potent anti-FA agent.

Asthma remains an important human disease that is responsible for substantial worldwide morbidity and mortality. The causes of asthma are multifactorial and include a complex mix of environmental, immunological and host genetic factors. In addition, epidemiological studies show strong associations between asthma and infection with respiratory pathogens, including common respiratory viruses such as rhinoviruses, human respiratory syncytial virus, adenoviruses, coronaviruses and influenza viruses, as well as bacteria (including atypical bacteria) and fungi. In this Review, we describe the many roles of microorganisms in the risk of developing asthma and in the pathogenesis of and protection against the disease, and we discuss the mechanisms by which infections affect the severity and prevalence of asthma.

<h4>Background</h4>Single-cell network profiling (SCNP) is a multi-parametric flow cytometry-based approach that simultaneously measures basal and modulated intracellular signaling activity in multiple cell subpopulations. Previously, SCNP analysis of a broad panel of immune signaling pathways in cell subsets within PBMCs from 60 healthy donors identified a race-associated difference in B cell anti-IgD-induced PI3K pathway activity.<h4>Methods</h4>The present study extended this analysis to a broader range of signaling pathway components downstream of the B cell receptor (BCR) in European Americans and African Americans using a subset of donors from the previously analyzed cohort of 60 healthy donors. Seven BCR signaling nodes (a node is defined as a paired modulator and intracellular readout) were measured at multiple time points by SCNP in PBMCs from 10 healthy donors [5 African Americans (36-51 yrs), 5 European Americans (36-56 yrs), all males].<h4>Results</h4>Analysis of BCR signaling activity in European American and African American PBMC samples revealed that, compared to the European American donors, B cells from African Americans had lower anti-IgD induced phosphorylation of multiple BCR pathway components, including the membrane proximal proteins Syk and SFK as well as proteins in the PI3K pathway (S6 and Akt), the MAPK pathways (Erk and p38), and the NF-κB pathway (NF-κB). In addition to differences in the magnitude of anti-IgD-induced pathway activation, racial differences in BCR signaling kinetic profiles were observed. Further, the frequency of IgD+ B cells differed by race and strongly correlated with BCR pathway activation. Thus, the race-related difference in BCR pathway activation appears to be attributable at least in part to a race-associated difference in IgD+ B cell frequencies.<h4>Conclusions</h4>SCNP analysis enabled the identification of statistically significant race-associated differences in BCR pathway activation within PBMC samples from healthy donors. Understanding race-associated contrasts in immune cell signaling responses may be one critical component for elucidation of differences in immune-mediated disease prevalence and treatment responses.

<h4>Background</h4>Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms.<h4>Results</h4>We provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing.<h4>Conclusions</h4>Our results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.

The basal ganglia-recipient thalamus receives inhibitory inputs from the pallidum and excitatory inputs from cortex, but it is unclear how these inputs interact during behavior. We recorded simultaneously from thalamic neurons and their putative synaptically connected pallidal inputs in singing zebra finches. We find, first, that each pallidal spike produces an extremely brief (∼5 ms) pulse of inhibition that completely suppresses thalamic spiking. As a result, thalamic spikes are entrained to pallidal spikes with submillisecond precision. Second, we find that the number of thalamic spikes that discharge within a single pallidal interspike interval (ISI) depends linearly on the duration of that interval but does not depend on pallidal activity prior to the interval. In a detailed biophysical model, our results were not easily explained by the postinhibitory "rebound" mechanism previously observed in anesthetized birds and in brain slices, nor could most of our data be characterized as "gating" of excitatory transmission by inhibitory pallidal input. Instead, we propose a novel "entrainment" mechanism of pallidothalamic transmission that highlights the importance of an excitatory conductance that drives spiking, interacting with brief pulses of pallidal inhibition. Building on our recent finding that cortical inputs can drive syllable-locked rate modulations in thalamic neurons during singing, we report here that excitatory inputs affect thalamic spiking in two ways: by shortening the latency of a thalamic spike after a pallidal spike and by increasing thalamic firing rates within individual pallidal ISIs. We present a unifying biophysical model that can reproduce all known modes of pallidothalamic transmission--rebound, gating, and entrainment--depending on the amount of excitation the thalamic neuron receives.

<h4>Objective</h4>To utilize high-throughput sequencing to determine the etiology of juvenile-onset neurodegeneration in a 19-year-old woman with progressive motor and cognitive decline.<h4>Methods</h4>Exome sequencing identified an initial list of 133,555 variants in the proband's family, which were filtered using segregation analysis, presence in dbSNP, and an empirically derived gene exclusion list. The filtered list comprised 52 genes: 21 homozygous variants and 31 compound heterozygous variants. These variants were subsequently scrutinized with predicted pathogenicity programs and for association with appropriate clinical syndromes.<h4>Results</h4>Exome sequencing data identified 2 GLB1 variants (c.602G>A, p.R201H; c.785G>T, p.G262V). β-Galactosidase enzyme analysis prior to our evaluation was reported as normal; however, subsequent testing was consistent with juvenile-onset GM1-gangliosidosis. Urine oligosaccharide analysis was positive for multiple oligosaccharides with terminal galactose residues.<h4>Conclusions</h4>We describe a patient with juvenile-onset neurodegeneration that had eluded diagnosis for over a decade. GM1-gangliosidosis had previously been excluded from consideration, but was subsequently identified as the correct diagnosis using exome sequencing. Exome sequencing can evaluate genes not previously associated with neurodegeneration, as well as most known neurodegeneration-associated genes. Our results demonstrate the utility of "agnostic" exome sequencing to evaluate patients with undiagnosed disorders, without prejudice from prior testing results.

In this work we expand upon a recently reported local drug delivery device, where air is used as a degradable component of our material to control drug release (J. Am. Chem. Soc. 2012, 134, 2016-2019). We consider its potential use as a drug loaded strip to provide both mechanical stability to the anastomosis, and as a means to release drug locally over prolonged periods for prevention of locoregional recurrence in colorectal cancer. Specifically, we electrospun poly(ε-caprolactone) (PCL) with the hydrophobic polymer dopant poly(glycerol monostearate-co-ε-caprolactone) (PGC-C18) and used the resultant mesh to control the release of two anticancer drugs (CPT-11 and SN-38). The increase in mesh hydrophobicity with PGC-C18 addition slows drug release both by the traditional means of drug diffusion, as well as by increasing the stability of the entrapped air layer to delay drug release. We demonstrate that superhydrophobic meshes have mechanical properties appropriate for surgical buttressing of the anastomosis, permit non-invasive assessment of mesh location and documentation of drug release via ultrasound, and release chemotherapy over a prolonged period of time (>90 days) resulting in significant tumor cytotoxicity against a human colorectal cell line (HT-29).

Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS's enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons.

4-Hydroxynonenal (4-HNE) is a reactive α,β-unsaturated aldehyde produced during oxidative stress and subsequent lipid peroxidation of polyunsaturated fatty acids. The reactivity of 4-HNE towards DNA and nucleophilic amino acids has been well established. In this report, using proteomic approaches, liver fatty acid-binding protein (L-FABP) is identified as a target for modification by 4-HNE. This lipid binding protein mediates the uptake and trafficking of hydrophobic ligands throughout cellular compartments. Ethanol caused a significant decrease in L-FABP protein (P<0.001) and mRNA (P<0.05), as well as increased poly-ubiquitinated L-FABP (P<0.001). Sites of 4-HNE adduction on mouse recombinant L-FABP were mapped using MALDI-TOF/TOF mass spectrometry on apo (Lys57 and Cys69) and holo (Lys6, Lys31, His43, Lys46, Lys57 and Cys69) L-FABP. The impact of 4-HNE adduction was found to occur in a concentration-dependent manner; affinity for the fluorescent ligand, anilinonaphthalene-8-sulfonic acid, was reduced from 0.347 µM to Kd(1) = 0.395 µM and Kd(2) = 34.20 µM. Saturation analyses revealed that capacity for ligand is reduced by approximately 50% when adducted by 4-HNE. Thermal stability curves of apo L-FABP was also found to be significantly affected by 4-HNE adduction (ΔTm = 5.44°C, P<0.01). Computational-based molecular modeling simulations of adducted protein revealed minor conformational changes in global protein structure of apo and holo L-FABP while more apparent differences were observed within the internal binding pocket, revealing reduced area and structural integrity. New solvent accessible portals on the periphery of the protein were observed following 4-HNE modification in both the apo and holo state, suggesting an adaptive response to carbonylation. The results from this study detail the dynamic process associated with L-FABP modification by 4-HNE and provide insight as to how alterations in structural integrity and ligand binding may a contributing factor in the pathogenesis of ALD.

Studies utilizing genetic and pharmacological manipulations in rodent models and neuronal cultures have revealed myriad roles of brain-derived neurotrophic factor (BDNF). Currently, this knowledge of BDNF function is being translated into improvement strategies for several debilitating neurological disorders in which BDNF abnormalities play a prominent role. Common among the BDNF-related disorders are irregular trafficking and release of mature BDNF (mBDNF) and/or its prodomain predecessor, proBDNF. Thus, investigating the conditions required for proper trafficking and release of BDNF is an essential step toward understanding and potentially improving these neurological disorders. This paper will provide examples of disorders related to BDNF release and serve as a review of the techniques being used to study the trafficking and release of BDNF.

Perinatal depression is an important public health problem affecting 10% to 20% of childbearing women. Perinatal depression is associated with significant morbidity, and has enormous consequences for the wellbeing of the mother and child. During the perinatal period, treatment of depression, which could affect the mother and child during pregnancy and lactation, poses a complex problem for both mother and clinician. Bright light therapy may be an attractive treatment for perinatal depression because it is low cost, home-based, and has a much lower side effect profile than pharmacotherapy. The antidepressant effects of bright light are well established, and there are several rationales for expecting that bright light might also be efficacious for perinatal depression. This review describes these rationales, summarizes the available evidence on the efficacy of bright light therapy for perinatal depression, and discusses future directions for investigation of bright light therapy as a treatment for perinatal depression.

Epithelial-mesenchymal transition (EMT) plays a specific role in the migration of tumor cells. Both estrogen and midkine (MK) have been thought to be important factors in promoting the progression of non-small-cell lung cancer (NSCLC) and can enhance EMT. Some evidence indicated the correlation between estradiol (E2) and MK, but the precise mechanism on their interreaction is unknown. Here, we try to clarify whether and how E2 regulates MK expression to promote EMT. We found that E2 increased MK mRNA expression in lung adenocarcinoma cells LTEP-a2 and A549 in a time-dependent manner. E2-induced MK expression was inhibited by the estrogen receptor (ER) antagonist ICI 182,780 and tamoxifen but not by phosphoinositide-3 kinase and MAPK inhibitors, suggesting a genomic mechanism of E2 on the regulation of MK transcription. Moreover, luciferase reporter and chromatin immunoprecipitation assays exhibited that E2 induced ERβ recruitment to the estrogen response element in the MK promoter. Small interfering RNA to ERα and ERβ revealed that ERβ mainly mediated E2-induced MK transcription. Interestingly, E2 enhanced MK expression in accordance with increase of EMT, whereas knockdown of MK could block EMT under E2 stimulation. Importantly, through analyzing lung adenocarcinoma tissues, there was indeed a correlation among levels of E2, MK, and EMT-related protein expression. Taken together, we reported a previously unrecognized mechanism on E2 in the regulation of MK expression and proved that MK plays a pivotal role in progression of E2-regulated EMT.

Protein misfolding and aggregation are exacerbated by aging and diseases of protein conformation including neurodegeneration, metabolic diseases, and cancer. In the cellular environment, aggregates can exist as discrete entities, or heterogeneous complexes of diverse solubility and conformational state. In this study, we have examined the in vivo dynamics of aggregation using imaging methods including fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS), to monitor the diverse biophysical states of expanded polyglutamine (polyQ) proteins expressed in Caenorhabditis elegans. We show that monomers, oligomers and aggregates co-exist at different concentrations in young and aged animals expressing different polyQ-lengths. During aging, when aggregation and toxicity are exacerbated, FCS-based burst analysis and purified single molecule FCS detected a populational shift toward an increase in the frequency of brighter and larger oligomeric species. Regardless of age or polyQ-length, oligomers were maintained in a heterogeneous distribution that spans multiple orders of magnitude in brightness. We employed genetic suppressors that prevent polyQ aggregation and observed a reduction in visible immobile species with the persistence of heterogeneous oligomers, yet our analysis did not detect the appearance of any discrete oligomeric states associated with toxicity. These studies reveal that the reversible transition from monomers to immobile aggregates is not represented by discrete oligomeric states, but rather suggests that the process of aggregation involves a more complex pattern of molecular interactions of diverse intermediate species that can appear in vivo and contribute to aggregate formation and toxicity.

We diagnosed three siblings from consanguineous east Asian parents with leukoencephalopathy with brainstem and spinal cord involvement and high lactate (LBSL) from characteristic MRI, MRS findings and a homozygous mutation in the DARS2 gene. The neurological symptoms of the three patients consisted of psychomotor developmental delay, cerebellar ataxia since infancy, spasticity in the initial phase and peripheral neuropathy in later stages. Their mental development was delayed, but did not deteriorate. MRI signal abnormalities included the same abnormalities reported previously but tended to be more extensive. Signal abnormalities in the cerebral and cerebellar white matter were homogeneous and confluent from early stages. In addition, other tract such as the central tegmental tract was involved. Furthermore, an atrophic change in the cerebral white matter was observed on follow-up in one case. Two of the patients were autopsied and neuropathological findings revealed characteristic vacuolar changes in the white matter of the cerebrum, cerebellum and the nerve tracts of the brain stem and spinal cord. The central myelin sheath showed intralamellar splitting by electron microscopy. These findings were consistent to a spongy degeneration in the diffuse white matter of the brain, or spongiform leukoencephalopathy. In addition, peripheral nerves showed both axonal degeneration and abnormal myelin structures. We discussed the relationship between deficits in mitochondrial aspartyl-tRNA synthetase activity and the neuropathology observed.

Stem cell therapy holds great promise for treating neurodegenerative disease, but major barriers to effective therapeutic strategies remain. A complete understanding of the derived phenotype is required for predicting cell response once introduced into the host tissue. We sought to identify major axonal guidance cues present in neurons derived from the transient overexpression of neurogenin-1 (Neurog1) in mouse embryonic stem cells (ESCs). Neurog1 upregulated the netrin-1 axon guidance receptors DCC (deleted in colorectal cancer) and neogenin (NEO1). Quantitative polymerase chain reaction results showed a 2-fold increase in NEO1 mRNA and a 36-fold increase in DCC mRNA in Neurog1-induced compared with control ESCs. Immunohistochemistry indicated that DCC was primarily expressed on cells positive for the neuronal marker TUJ1. DCC was preferentially localized to the cell soma and growth-cones of induced neurons. In contrast, NEO1 expression showed less specificity, labeling both TUJ1-positive and TUJ1-negative cells as well as uninduced control cells. Axonal outgrowth was directed preferentially toward aggregates of HEK293 cells secreting a recombinant active fragment of netrin-1. These data indicate that DCC and NEO1 are downstream products of Neurog1 and may guide the integration of Neurog1-induced ESCs with target cells secreting netrin-1. Differential expression profiles for netrin receptors could indicate different roles for this guidance cue on neuronal and non-neuronal cells.

An aptamer switch probe (ASP) linking chlorin e6 (Ce6), a photosensitizer molecule, to the surface of gold nanorods (AuNRs) was used to target cancer cells for photodynamic therapy (PDT) and photothermal therapy (PTT). In the presence of target cancer cells, the ASP changes conformation to drive Ce6 away from the gold surface, thereby producing singlet oxygen for PDT upon light irradiation. Since each AuNR is modified with many ASP-Ce6 molecules, the AuNR-ASP-Ce6 conjugate yields enhanced binding and therapeutic effect by the added ability to carry many photosensitizers. In addition, absorption of radiation by the gold nanorods enables further cell destruction by the photothermal effect. Consequently, this multimodal AuNR-ASP-Ce6 conjugate offers a remarkably improved and synergistic therapeutic effect compared to PTT or PDT alone, providing high specificity and therapeutic efficiency, which can be generalized to other types of cancer therapies.

Smallpox is a deadly and debilitating disease that killed hundreds of millions of people in the past century alone. The use of Vaccinia virus-based smallpox vaccines led to the eradication of smallpox. These vaccines are remarkably effective, inducing the characteristic pustule or "take" at the vaccine site in >97 % of recipients, and inducing a wide spectrum of long-lasting humoral and cellular immune responses. The mechanisms behind inter-individual vaccine-response variability are likely to involve host genetic variation, but have not been fully characterized. We report here the first smallpox vaccine response genome-wide association study of over 1,000 recent recipients of Dryvax(®). The data presented here focus on cellular immune responses as measured by both production of secreted IFNγ and quantitation of IFNγ secreting cells by ELISPOT assay. We identified multiple significant SNP associations in genes (RASA1, ADRA1D, TCF7L1, FAS) that are critical components of signaling pathways that directly control lymphocyte IFNγ production or cytotoxic T cell function. Similarly, we found many associations with SNPs located in genes integral to nerve cell function; findings that, given the complex interplay between the nervous and immune systems, deserve closer examination in follow-up studies.

Dysregulation of glycogen synthase kinase (GSK)-3β contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3β regulation on IFN-α-induced 5-HTT functions. GSK-3β short hairpin RNAs (shRNAs) or GSK-3β inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3β (Tyr216), which regulated 5-HT uptake. GSK-3β knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3β cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3β activation and GSK-3β-regulated 5-HT uptake. GSK-3β signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3β cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

<h4>Background and aim</h4>Expression profiling of genes specific to pediatric Crohn's Disease (CD) patients was performed to elucidate the molecular mechanisms underlying disease cause and pathogenesis at disease onset.<h4>Methods</h4>We used suppressive subtractive hybridization (SSH) and differential screening analysis to profile the mRNA expression patterns of children with CD and age- and sex-matched controls without inflammatory bowel disease (IBD).<h4>Results</h4>Sequence analysis of 1000 clones enriched by SSH identified 75 functionally annotated human genes, represented by 430 clones. The 75 genes have potential involvement in gene networks, such as antigen presentation, inflammation, infection mechanism, connective tissue development, cell cycle and cancer. Twenty-eight genes were previously described in association with CD, while 47 were new genes not previously reported in the context of IBD. Additionally, 29 of the 75 genes have been previously implicated in bacterial and viral infections. Quantitative real-time reverse transcription polymerase chain reaction performed on ileal-derived RNA from 13 CD and nine non-IBD patients confirmed the upregulation of extracellular matrix gene MMP2 (P = 0.001), and cell proliferation gene REG1A (P = 0.063) in our pediatric CD cohort.<h4>Conclusion</h4>The retrieval of 28 genes previously reported in association with adult CD emphasizes the importance of these genes in the pediatric setting. The observed upregulation of REG1A and MMP2, and their known impact on cell proliferation and extracellular matrix remodeling, agrees with the clinical behavior of the disease. Moreover, the expressions of bacterial- and virus-related genes in our CD-patient tissues support the concept that microbial agents are important in the etiopathogenesis of CD.

<h4>Background and aim</h4>HFE mutations, a common cause of hereditary hemochromatosis (HH), are reportedly associated with hepatic iron overload, severe liver fibrosis, and good response to interferon treatment in European patients with chronic hepatitis C (CHC). HH shows ethnicity-based differences and little is known about the effects of HH mutations on CHC in the Japanese. Thus, the aim of this study was to clarify the clinical influence of HFE mutations in Japanese CHC patients.<h4>Methods</h4>In a total of 251 patients with CHC, we analyzed the frequencies of H63D and S65C mutations in the HFE gene, and the influence of these mutations on clinical parameters and response to pegylated-interferon-alpha 2b (PEG-IFN) plus ribavirin therapy.<h4>Results</h4>Fourteen patients (5.6%) carried the H63D mutation; all were heterozygotes. No S65C mutations were found. Only hemoglobin levels in the H63D heterozygotes were higher than in wild-type patients. Eleven of 14 H63D heterozygotes achieved sustained virological response (SVR). On univariate analysis, factors associated with SVR were interleukin 28B (IL28B) polymorphism, age, hepatitis C virus (HCV) genotype, HCV viral load, white blood cell count, stage of fibrosis and H63D mutation. All patients with both TT genotype in IL28B (rs8099917) and H63D mutation in HFE (n = 10) achieved SVR.<h4>Conclusions</h4>The H63D mutation has little impact on the clinical characteristics of CHC, but is related to favorable response to PEG-IFN plus ribavirin therapy, particularly in patients with the TT allele in IL28B.

<h4>Background and purpose</h4>Ca(v) 3.2 T-type calcium channels, targeted by H(2) S, are involved in neuropathic hyperalgesia in rats and ascorbic acid inhibits Ca(v) 3.2 channels. Therefore, we evaluated the effects of intraplantar (i.pl.) administration of ascorbic acid or topical application of disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), a skin-permeable ascorbate derivative on hyperalgesia induced by NaHS, an H(2) S donor, and on neuropathic hyperalgesia.<h4>Experimental approach</h4>In rats mechanical hyperalgesia was evoked by i.pl. NaHS, and neuropathic hyperalgesia was induced by L5 spinal nerve cutting (L5SNC) or by repeated administration of paclitaxel, an anti-cancer drug. Dermal ascorbic acid levels were determined colorimetrically.<h4>Key results</h4>The NaHS-evoked Ca(v) 3.2 channel-dependent hyperalgesia was inhibited by co-administered ascorbic acid. Topical application of DI-VCP, but not ascorbic acid, prevented the NaHS-evoked hyperalgesia, and also increased dermal ascorbic acid levels. Neuropathic hyperalgesia induced by L5SNC or paclitaxel was reversed by i.pl. NNC 55-0396, a selective T-type calcium channel blocker, ascorbic acid or DI-VCP, and by topical DI-VCP, but not by topical ascorbic acid. The effects of i.pl. ascorbic acid and topical DI-VCP in the paclitaxel-treated rats were characterized by the faster onset and greater magnitude, compared with their effects in the L5SNC rats. Dermal ascorbic acid levels in the hindpaw significantly decreased after paclitaxel treatment, but not L5SNC, which was reversed by topical DI-VCP.<h4>Conclusions and implications</h4>Ascorbic acid, known to inhibit Ca(v) 3.2 channels, suppressed neuropathic hyperalgesia. DI-VCP ointment for topical application may be of benefit in the treatment of neuropathic pain.

Several lines of evidence indicate that the etiology of late-onset Alzheimer's disease (LOAD) is complex, with significant contributions from both genes and environmental factors. Recent research suggests the importance of epigenetic mechanisms in defining the relationship between environmental exposures and LOAD. In epidemiologic studies of adults, cumulative lifetime lead (Pb) exposure has been associated with accelerated declines in cognition. In addition, research in animal models suggests a causal association between Pb exposure during early life, epigenetics, and LOAD. There are multiple challenges to human epidemiologic research evaluating the relationship between epigenetics, LOAD, and Pb exposure. Epidemiologic studies are not well-suited to accommodate the long latency period between exposures during early life and onset of Alzheimer's disease. There is also a lack of validated circulating epigenetics biomarkers and retrospective biomarkers of Pb exposure. Members of our research group have shown bone Pb is an accurate measurement of historical Pb exposure in adults, offering an avenue for future epidemiologic studies. However, this would not address the risk of LOAD attributable to early-life Pb exposures. Future studies that use a cohort design to measure both Pb exposure and validated epigenetic biomarkers of LOAD will be useful to clarify this important relationship.

Variability and modification of the symptoms of Huntington’s disease (HD) are commonly observed in both patient populations and animal models of the disease. Utilizing a stable line of the R6/2 HD mouse model, the present study investigated the role of genetic background in the onset and severity of HD symptoms in a transgenic mouse. R6/2 congenic C57BL/6J and C57BL/6J×DBA/2J F1 (B6D2F1) mice were evaluated for survival and a number of behavioral phenotypes. This study reports that the presence of the DBA/2J allele results in amelioration or exacerbation of several HD-like phenotypes characteristic of the R6/2 mouse model and indicates the presence of dominant genetic modifiers of HD symptoms. This study is the first step in identifying genes that confer natural genetic variation and modify the HD symptoms. This identification may lead to novel targets for treatment and help elucidate the molecular mechanisms of HD pathogenesis.

12(S)-Hydroxyheptadeca-5Z,8E,10E-trienoic acid (12- HHT) is an enzymatic product of prostaglandin H(2) (PGH(2)) derived from cyclooxygenase (COX)-mediated arachidonic acid metabolism. Despite the high level of 12-HHT present in tissues and bodily fluids, its precise function remains largely unknown. In this study, we found that 12-HHT treatment in HaCaT cells remarkably down-regulated the ultraviolet B (UVB) irradiation-induced synthesis of interleukin-6 (IL-6), a pro-inflammatory cytokine associated with cutaneous inflammation. In an approach to identify the down-stream signaling mechanism by which 12-HHT down-regulates UVB-induced IL-6 synthesis in keratinocytes, we observed that 12-HHT inhibits the UVB-stimulated activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB). In addition, we found that 12-HHT markedly up-regulates MAPK phosphatase-1 (MKP-1), a critical negative regulator of p38 MAPK. When MKP-1 was suppressed by siRNA knock-down, the 12-HHT-mediated inhibitory effects on the UVB-stimulated activation of p38 MAPK and NF-κB, as well as the production of IL-6, were attenuated in HaCaT cells. Taken together, our results suggest that 12-HHT exerts anti-inflammatory effect via up-regulation of MKP-1, which negatively regulates p38 MAPK and NF-κB, thus attenuating IL-6 production in UVB-irradiated HaCaT cells. Considering the critical role of IL-6 in cutaneous inflammation, our findings provide the basis for the application of 12-HHT as a potential anti-inflammatory therapeutic agent in UV-induced skin diseases.

<h4>Objective</h4>The perimenopausal increase in circulating dehydroepiandrosterone sulfate (DHEAS) levels during the menopausal transition (MT) is accompanied by other adrenal steroids that have the potential to alter estrogen/androgen balance and explain the wide interwoman range of estrogen-related symptoms experienced during the MT.<h4>Methods</h4>Annual serum samples from the Study of Women's Health Across the Nation, which had previously been analyzed for immunoreactive estradiol (E2), testosterone, DHEAS, and sex hormone-binding globulin, were selected based on DHEAS concentration and analyzed for immunoreactive and bioactive estrogens and androgens, including immunoreactive androstenedione, dehydroepiandrosterone, and 5-androstene-3β,17β-diol (androstenediol [Adiol]).<h4>Results</h4>A two-fold increase in circulating androstenedione and testosterone was found to rise in parallel with the rise in circulating DHEAS, whereas dehydroepiandrosterone and Adiol concentrations rose seven- to eight-fold. Circulating Adiol, which has both androgenic and estrogenic biological activity, was significantly associated (P < 0.02) with circulating estrogen bioactivity only when E2 concentrations were low and Adiol levels were high.<h4>Conclusions</h4>The wide range of circulating levels of Adiol and its contribution to total circulating estrogenicity during the MT is consistent with the observed interwoman difference in symptoms at this time. Therefore, we conclude that Adiol contributes to circulating estrogenicity when E2 production falls at menopause and may contribute significantly to the endocrine changes experienced by midlife women.

<h4>Background</h4>Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α(2) -antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied.<h4>Objectives</h4>To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α(2) -antiplasmin.<h4>Methods</h4>Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition.<h4>Results and conclusions</h4>We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α(2) -antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.

<h4>Background</h4>Many studies have reported that drainage after thyroidectomy does not decrease the rate of local postoperative complications. We sought to review the safety of thyroidectomy combined with cervical neck dissection (CND) without drainage.<h4>Methods</h4>The medical records of consecutive patients who underwent thyroidectomy without drainage were retrospectively reviewed. Two groups were defined depending on whether CND was or was not performed. The main outcome was identification of patients with cervical bleeding, hematoma or seroma.<h4>Results</h4>We included 1127 patients (139 who had CND and 988 who did not). Of these, 207 patients (18%) had transient postoperative hypocalcemia, 9 (0.8%) had permanent postoperative hypoparathyroidism, 56 (5%) had transient postoperative hoarseness and 7 (0.6%) had permanent vocal cord paralysis. A total of 44 patients (4%) experienced postoperative hematoma and/or seroma: 8 patients (6%) who had CND and 36 (4%) who did not. There was no major bleeding in the 2 groups; all patients had minor bleeding or seroma not requiring surgical intervention. The postoperative stay in hospital for both groups was 1 day in 92% of patients. Wound infection occurred in 0.8% of all patients: 1 (0.7%) who had CND and 8 (0.8%) who did not. There was no significant difference between the groups in overall perioperative complications or in time of hospital discharge.<h4>Conclusion</h4>Thyroidectomy without drains is safe and effective, even in combination with CND.

Liver is the central organ of iron metabolism. During acute-phase-response (APR), serum iron concentration rapidly decreases. The current study aimed to compare expression and localization of iron transport protein ferroportin-1 (Fpn-1) and of other iron import proteins after experimental tissue damage induced by injecting turpentine oil in the hind limbs of rats and mice. Serum and spleen iron concentration decreased with an increase in total liver, cytoplasmic and nuclear iron concentration. In liver, mRNA amount of Fpn-1, Fpn-1a, Fpn-1b, HFE, hemojuvelin (HJV) and hephaestin (heph) genes showed a rapid decrease. Hepcidin, divalent metal transporter-1 (DMT-1), transferrin (Tf) and Tf-receptor-1 (TfR1), TfR-2 (TfR2) gene expression was increased. Western blot analysis of liver tissue lysate confirmed the changes observed at mRNA level. In spleen, a rapid decrease in gene expression of Fpn-1, Fpn-1a, Fpn-1b, DMT-1, Tf, TfR1 and TfR2, and an increase in hepcidin was observed. Immunohistochemistry of DMT-1 and TfR2 were mainly detected in the nucleus of rat liver and spleen, whereas TfR1 was clearly localized in the plasma membrane. Fpn-1 was mostly found in the nuclei of liver cells, whereas in spleen, the protein was mainly detected in the cell membrane. Western blot analysis of liver fractions confirmed immunohistochemical results. In livers of wild-type mice, gene expression of Fpn-1, Fpn-1a and Fpn-1b was downregulated, whereas hepcidin gene expression was increased. In contrast, these changes were less pronounced in IL-6ko-mice. Cytokine (IL-6, IL-1b and TNF-a) treatment of rat hepatocytes showed a downregulation of Fpn-1, Fpn-1a and Fpn-1b, and upregulation of hepcidin gene expression. Moreover, western blot analysis of cell lysate of IL-6-treated hepatocytes detected, as expected, an increase of a2-macroglobulin (positive acute-phase protein), whereas albumin (negative acute-phase protein) and Fpn-1 were downregulated. Our results demonstrate that liver behaves as a 'sponge' for iron under acute-phase conditions, and Fpn-1 behaves as a negative acute-phase protein in rat hepatocytes mainly, but not exclusively, because of the effect of IL-6. These changes could explain iron retention in the cytoplasm and in the nucleus of hepatocytes during APR.

Epigenetic alterations are a common event in lung cancer and their identification can serve to inform on the carcinogenic process and provide clinically relevant biomarkers. Using paired tumor and non-tumor lung tissues from 146 individuals from three independent populations we sought to identify common changes in DNA methylation associated with the development of non-small cell lung cancer. Pathologically normal lung tissue taken at the time of cancer resection was matched to tumorous lung tissue and together were probed for methylation using Illumina GoldenGate arrays in the discovery set (n = 47 pairs) followed by bisulfite pyrosequencing for validation sets (n = 99 pairs). For each matched pair the change in methylation at each CpG was calculated (the odds ratio), and these ratios were averaged across individuals and ranked by magnitude to identify the CpGs with the greatest change in methylation associated with tumor development. We identified the top gene-loci representing an increase in methylation (HOXA9, 10.3-fold and SOX1, 5.9-fold) and decrease in methylation (DDR1, 8.1-fold). In replication testing sets, methylation was higher in tumors for HOXA9 (p < 2.2 × 10 (-16) ) and SOX1 (p < 2.2 × 10 (-16) ) and lower for DDR1 (p < 2.2 × 10 (-16) ). The magnitude and strength of these changes were consistent across squamous cell and adenocarcinoma tumors. Our data indicate that the identified genes consistently have altered methylation in lung tumors. Our identified genes should be included in translational studies that aim to develop screening for early disease detection.

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.

Huntington's disease (HD) is caused by a CAG triplet repeat expansion in exon 1 of the Huntingtin (Htt) gene, encoding an abnormal expanded polyglutamine (polyQ) tract that confers toxicity to the mutant Htt (mHtt) protein. Recent data suggest that posttranslational modifications of mHtt modulate its cytotoxicity. To further understand the cytotoxic mechanisms of mHtt, we have generated HEK293 cell models stably expressing Strep- and FLAG-tagged Htt containing either 19Q (wild-type Htt), 55Q (mHtt), or 94Q (mHtt) repeats. Following tandem affinity purification, the tagged Htt and associated proteins were subjected to tandem mass spectrometry or 2D nano-LC tandem mass spectrometry and several novel modification sites of mHtt containing 55Q or 94Q were identified. These were phosphorylation sites located at Ser431 and Ser432, and ubiquitination site located at Lys444. The two phosphorylation sites were confirmed by Western blot analysis using phosphorylation site-specific antibodies. In addition, prevention of phosphorylation at the two serine sites altered mHtt toxicity and accumulation. These modifications of mHtt may provide novel therapeutic targets for effective treatment of the disorder.

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC(50) 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increased with treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion-associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells.

Addictions are common, chronic, and relapsing diseases that develop through a multistep process. The impact of addictions on morbidity and mortality is high worldwide. Twin studies have shown that the heritability of addictions ranges from 0.39 (hallucinogens) to 0.72 (cocaine). Twin studies indicate that genes influence each stage from initiation to addiction, although the genetic determinants may differ. Addictions are by definition the result of gene × environment interaction. These disorders, which are in part volitional, in part inborn, and in part determined by environmental experience, pose the full range of medical, genetic, policy, and moral challenges. Gene discovery is being facilitated by a variety of powerful approaches, but is in its infancy. It is not surprising that the genes discovered so far act in a variety of ways: via altered metabolism of drug (the alcohol and nicotine metabolic gene variants), via altered function of a drug receptor (the nicotinic receptor, which may alter affinity for nicotine but as discussed may also alter circuitry of reward), and via general mechanisms of addiction (genes such as monoamine oxidase A and the serotonin transporter that modulate stress response, emotion, and behavioral control). Addiction medicine today benefits from genetic studies that buttress the case for a neurobiologic origin of addictive behavior, and some general information on familially transmitted propensity that can be used to guide prevention. A few well-validated, specific predictors such as OPRM1, ADH1B, ALDH2, CHRNA5, and CYP26 have been identified and can provide some specific guidance, for example, to understand alcohol-related flushing and upper GI cancer risk (ADH1B and AKLDH2), variation in nicotine metabolism (CYP26), and, potentially, naltrexone treatment response (OPRM1). However, the genetic predictors available are few in number and account for only a small portion of the genetic variance in liability, and have not been integrated into clinical nosology or care.

Recently, autophagy has been found to be strongly activated in colon cancer cells, but few studies have addressed the normal colon mucosa. The aim of this study was to characterize autophagy in normal human intestinal cells. We used the expression of LC3-II and BECN1 as well as SQSTM1 as markers of autophagy activity. Using the normal human intestinal epithelial crypt (HIEC) cell experimental model, we found that autophagy was much more active in undifferentiated cells than in differentiated cells. In the normal adult colonic mucosa, BECN1 was found in the proliferative epithelial cells of the lower part of the gland while SQSTM1 was predominantly found in the differentiated cells of the upper part of the gland and surface epithelium. Interestingly, the weak punctate pattern of SQSTM1 expression in the lower gland colocalized with BECN1-labeled autophagosomes. The usefulness of SQSTM1 as an active autophagy marker was confirmed in colon cancer specimens at the protein and transcript levels. In conclusion, our results show that autophagy is active in the colonic gland and is associated with the intestinal proliferative/undifferentiated and progenitor cell populations.

Autophagy, a highly conserved lysosomal degradation pathway, was initially characterized as a bulk degradation system induced in response to starvation. In recent years, autophagy has emerged also as a highly selective pathway, targeting various cargoes such as aggregated proteins and damaged organelles for degradation. The key factors involved in selective autophagy are autophagy receptors and adaptor proteins, which connect the cargo to the core autophagy machinery. In this review, we discuss the current knowledge about the only mammalian adaptor protein identified thus far, autophagy-linked FYVE protein (ALFY). ALFY is a large, scaffolding, multidomain protein implicated in the selective degradation of ubiquitinated protein aggregates by autophagy. We also comment on the possible role of ALFY in the context of disease.

Major depressive disorder (MDD) is etiologically complex and has a heterogeneous presentation. This heterogeneity hinders the ability of molecular genetic research to reliably detect the small effects conferred by common genetic variation. As a result, significant research efforts have been directed at investigating more homogenous intermediate phenotypes believed to be more proximal to gene function and lie between genes and/or environmental effects and disease processes. In the current review we survey and integrate research on two promising intermediate phenotypes linked to depression: reward processing and stress sensitivity. A synthesis of this burgeoning literature indicates that a molecular genetic approach focused on intermediate phenotypes holds significant promise to fundamentally improve our understanding of the pathophysiology and etiology of depression, which will be required for improved diagnostic definitions and the development of novel and more efficacious treatment and prevention strategies. We conclude by highlighting challenges facing intermediate phenotype research and future development that will be required to propel this pivotal research into new directions.

The aim of this study was to investigate the changes induced by high tidal volume ventilation (HVTV) in pulmonary expression of micro-RNAs (miRNAs) and identify potential target genes and corresponding miRNA-gene networks. Using a real-time RT-PCR-based array in RNA samples from lungs of mice subjected to HVTV for 1 or 4 h and control mice, we identified 65 miRNAs whose expression changed more than twofold upon HVTV. An inflammatory and a TGF-β-signaling miRNA-gene network were identified by in silico pathway analysis being at highest statistical significance (P = 10(-43) and P = 10(-28), respectively). In the inflammatory network, IL-6 and SOCS-1, regulated by miRNAs let-7 and miR-155, respectively, appeared as central nodes. In TGF-β-signaling network, SMAD-4, regulated by miR-146, appeared as a central node. The contribution of miRNAs to the development of lung injury was evaluated in mice subjected to HVTV treated with a precursor or antagonist of miR-21, a miRNA highly upregulated by HVTV. Lung compliance was preserved only in mice treated with anti-miR-21 but not in mice treated with pre-miR-21 or negative-control miRNA. Both alveolar-arterial oxygen difference and protein levels in bronchoalveolar lavage were lower in mice treated with anti-miR-21 than in mice treated with pre-miR-21 or negative-control miRNA (D(A-a): 66 ± 27 vs. 131 ± 22, 144 ± 10 mmHg, respectively, P < 0.001; protein concentration: 1.1 ± 0.2 vs. 2.3 ± 1, 2.1 ± 0.4 mg/ml, respectively, P < 0.01). Our results show that HVTV induces changes in miRNA expression in mouse lungs. Modulation of miRNA expression can affect the development of HVTV-induced lung injury.

Aberrant promoter methylation of biologically relevant genes in cervical cancer and uneven CpG distribution within the human papillomavirus 16 (HPV 16) enhancer region have been reported. Cervical samples and questionnaires from 151 women screened for cervical cancer in Appalachian Ohio were analyzed. Methylation was measured by bisulfite sequencing in candidate gene sites in ESR1, DCC, p16, and LINE1 elements. Among 89 HPV 16-positive women, CpG sites in the E6 promoter and enhancer regions and the L1 region of the HPV 16 genome were measured. Methylation levels were compared by cervical cytology and HPV 16 status. HPV methylation was low regardless of cytology status, however E6 methylation was significantly higher in women with normal cytology. ESR1 and DCC methylation were significantly higher in HPV 16-positive women. Increased methylation at sites in the E6 promoter region was associated with lower odds of abnormal cytology. Increased methylation in candidate genes was associated with higher odds of abnormal cytology, particularly DCC region 2.4, DCC region 2.6, ESR1 region 3.2, and LINE1 site 1.2. HPV 16 genome CpG methylation was low except for the L1 region. In general, lower HPV 16 methylation and higher candidate gene methylation levels were associated with higher odds of abnormal cytology.

Post-transcriptional gene regulation by microRNAs (miRNAs) and RNA-binding proteins (RBPs) is central to many biological functions. Aberrant gene expression patterns underlie many metabolic diseases that represent major public health concerns and formidable therapeutic challenges. Several studies have established a number of post-transcriptional regulators implicated in metabolic diseases such as diabetes and obesity. In addition, emerging knowledge of metabolically active and insulin-sensitive organs, such as the pancreas, liver, muscle and adipose compartment, is rapidly expanding the panel of potential therapeutic targets for the treatment of metabolic diseases. Here, we review our current understanding of miRNAs and RBPs that affect glucose and lipid homeostasis, and their roles in normal physiology and metabolic disorders, especially type 2 diabetes and obesity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have come under scrutiny because of the gastrointestinal, renal, and cardiovascular toxicity associated with prolonged use of these drugs. The purpose of this study was to identify molecular targets for NSAIDs related to cellular toxicity with a view to optimize drug efficacy in the clinic. Coronary artery smooth muscle cells and endothelial cells were treated with low (clinically achievable) and high (typically used in preclinical studies) concentrations of celecoxib, NS398, and ibuprofen for 24 hours. NSAIDs-induced gene expression changes were evaluated by microarray analysis and validated by real-time reverse-transcription polymerase chain reaction and western blotting. The functional significance of differentially expressed genes was evaluated by Ingenuity Pathway Analysis. At high concentrations, NSAIDs altered the expression of genes regulating cell proliferation and cell death. NSAIDs also altered genes associated with cardiovascular functions including inflammation, thrombosis, fibrinolysis, coronary artery disease, and hypertension. The gene expression was most impacted by ibuprofen, celecoxib, and NS398, in that order. This study revealed that NSAIDs altered expression of an array of genes associated with cardiovascular events and emphasizes the potential for fingerprinting drugs in preclinical studies to assess the potential drug toxicity and to optimize the drug efficacy in clinical settings.

<h4>Objectives</h4>Equisetum arvense preparations have long been used to promote bone healing. The aim of this work was to evaluate osteogenic and antibacterial effects of E. arvense hydromethanolic extracts.<h4>Materials and methods</h4>Dried aerial components of E. arvense were extracted using a mixture of methanol:water (1:1), for 26 days, yielding three extracts that were tested (10-1000 μg/ml) in human osteoblastic cells: E1, E2 and EM (a mixture of E1 and E2, 1:1). Cell cultures, performed on cell culture plates or over hydroxyapatite (HA) substrates, were assessed for osteoblastic markers. In addition, effects of the extracts on Staphylococcus aureus were addressed.<h4>Results</h4>Solution E1 caused increased viability/proliferation and ALP activity at 50-500 μg/ml, and deleterious effects at levels ≥1000 μg/ml. E2 inhibited cell proliferation at levels ≥500 μg/ml. EM presented a profile between those observed with E1 and E2. In addition, E1, E2 and EM, 10-1000 μg/ml, inhibited expansion of S. aureus. Furthermore, E1, tested in HA substrates colonized with osteoblastic cells, causing increase in cell population growth (10-100 μg/ml). E1 also exhibited antibacterial activity against S. aureus cultured over HA.<h4>Conclusions</h4>Results showed that E. arvense extracts elicited inductive effects on human osteoblasts while inhibiting activity of S. aureus, suggesting a potentially interesting profile regarding bone regeneration strategies.

Identification of marker genes expressed in specific cell types is essential for the genetic dissection of neural circuits. Here we report a new strategy for classifying heterogeneous populations of neurons into functionally distinct types and for identifying associated marker genes. Quantitative single-cell expression profiling of genes related to neurotransmitters and ion channels enables functional classification of neurons; transcript profiles for marker gene candidates identify molecular handles for manipulating each cell type. We apply this strategy to the mouse medial vestibular nucleus (MVN), which comprises several types of neurons subserving cerebellar-dependent learning in the vestibulo-ocular reflex. Ion channel gene expression differed both qualitatively and quantitatively across cell types and could distinguish subtle differences in intrinsic electrophysiology. Single-cell transcript profiling of MVN neurons established six functionally distinct cell types and associated marker genes. This strategy is applicable throughout the nervous system and could facilitate the use of molecular genetic tools to examine the behavioral roles of distinct neuronal populations.

Hepatocyte nuclear factor 4α (HNF4α) and CCAAT/enhancer-binding protein α (C/EBPα) are important for the transcriptional control of coagulation factors. To determine in vivo the direct role of HNF4α and C/EBPα in control of genes encoding coagulation factors, a synthetic small interfering (si)RNA approach was used that enabled strong reduction of mouse hepatic HNF4α and C/EBPα under conditions that minimized target-related secondary effects. For both HNF4α and C/EBPα, intravenous injection of specific synthetic siRNAs (siHNF4α and siC/EBPα) resulted in more than 75% reduction in their liver transcript and protein levels 2 days post-injection. For siHNF4α, this coincided with marked and significantly reduced transcript levels of the coagulation genes Hrg, Proz, Serpina5, F11, F12, F13b, Serpinf2, F5, and F9 (in order of magnitude of effect) as compared to levels in control siRNA injected animals. Significant decreases in HNF4α target gene mRNA levels were also observed at 5 days post-siRNA injection, despite a limited level of HNF4α knockdown at this time point. Compared to HNF4α, C/EBPα knockdown had a modest impact on genes encoding coagulation factors. A strong reduction in C/EBPα transcript and protein levels resulted in significantly affected transcript levels of the control genes Pck1 and Fasn and a modest downregulation for coagulation genes Fba, Fbg and F5. F5 and F11 were the sole coagulation genes that were significantly affected upon prolonged (5 day) C/EBPα knockdown. We conclude that in the mouse, HNF4α has a direct and essential regulatory role for multiple hepatic coagulation genes, while a role for C/EBPα is more restricted. In addition, this study demonstrates that synthetic siRNA provides a simple and fast means for determining liver transcription factor involvement in vivo.

Antitumor cytolytic T lymphocytes (CTLs) recognize peptides derived from cellular proteins and presented on MHC class I. One category of peptides recognized by these CTLs is derived from proteins encoded by "cancer-germline" genes, which are specifically expressed in tumors, and therefore represent optimal targets for cancer immunotherapy. Here, we identify an antigenic peptide, which is derived from the MAGE-A1-encoded protein (160-169) and presented to CTLs by HLA-B*44:02. Although this peptide is encoded by MAGE-A1, processed endogenously and presented by tumor cells, the corresponding synthetic peptide is hardly able to sensitize target cells to CTL recognition when pulsed exogenously. Endogenous processing and presentation of this peptide is strictly dependent on the presence of tapasin, which is believed to help peptide loading by stabilizing a peptide-receptive form of HLA-B*44:02. Exogenous loading of the peptide can be dramatically improved by paraformaldehyde fixation of surface molecules or by peptide loading at acidic pH. Either strategy allows efficient exogenous loading of the peptide, presumably by generating or stabilizing a peptide-receptive, empty conformation of the HLA. Altogether, our results indicate a potential drawback of short peptide-based vaccination strategies and offer possible solutions regarding the use of problematic epitopes such as the one described here.

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.