Gene Literature Dashboard

Despite recent advances in the understanding of ethanol's biological action, many of the molecular targets of ethanol and mechanisms behind ethanol's effect on behavior remain poorly understood. In an effort to identify novel genes, the products of which regulate behavioral responses to ethanol, we recently identified a mutation in the dtao gene that confers resistance to the locomotor stimulating effect of ethanol in Drosophila. dtao encodes a member of the Ste20 family of serine/threonine kinases implicated in MAP kinase signaling pathways. In this study, we report that conditional ablation of the mouse dtao homolog, Taok2, constitutively and specifically in the nervous system, results in strain-specific and overlapping alterations in ethanol-dependent behaviors. These data suggest a functional conservation of dtao and Taok2 in mediating ethanol's biological action and identify Taok2 as a putative candidate gene for ethanol use disorders in humans.

During acute Lyme disease, bacteria can disseminate to the central nervous system (CNS), leading to the development of meningitis and other neurologic symptoms. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing a deep view into the proteome for patients diagnosed with early disseminated Lyme disease and CSF inflammation. Additionally, we analyzed individual patient samples and quantified differences in protein abundance employing label-free quantitative mass spectrometry-based methods. We identified 108 proteins that differ significantly in abundance in patients with acute Lyme disease from controls. Comparison between infected patients and control subjects revealed differences in proteins in the CSF associated with cell death localized to brain synapses and others that likely originate from brain parenchyma.

<h4>Aims</h4>Manganese superoxide dismutase (MnSOD), one of the most crucial antioxidant enzymes in the central nervous system, is thought to be one of the major mechanisms by which cells counteract the injuries of reactive oxygen species after cerebral ischemia. In this study, we used a novel synthesized compound (MnTm4PyP) with highly effective superoxide dismutase activity to study the therapeutic potential of MnSOD and the possible underlying mechanisms in cerebral ischemia.<h4>Methods</h4>Primary cultured cortical neurons were used to examine the protective effect of the compounds. Mice with middle cerebral artery occlusion were used as ischemic stroke animal model. Animals were pretreated with MnTm4PyP intravenously 30 min before surgery. At 24 h after surgery, neurological behavior and histological function were observed. Infarcted cortex tissues and cultured neurons were collected for investigation of the oxidative stress signaling pathways.<h4>Results</h4>In vitro studies revealed that MnSOD mimic MnTm4PyP pretreatment significantly increased viability of neurons after injury by H(2) O(2) . Intracellular superoxide radical levels were eliminated. In vivo experiments demonstrated MnTm4PyP pretreatment reduced infarct volume and improved neurological function. The MnSOD mimic alleviated oxidative stress and apoptosis.<h4>Conclusion</h4>MnSOD is an effective therapeutic target in ischemic stroke prevention because of its antioxidant effects and oxidative stress regulation.

<h4>Background</h4>The use of mother's own breast milk during initial hospitalization has a positive impact not only in reducing potential serious neonatal morbidities but also contribute to improvements in neurodevelopmental outcomes. Mothers of very preterm infants struggle to maintain a supply of breast milk during their infants' prolonged hospitalization. Galactogogues are medications that induce lactation by exerting its effects through oxytocin or prolactin enhancement. Domperidone is a potent dopamine D2 receptor antagonist which stimulates the release of prolactin. Small trials have established its ability in enhancing breast milk production. EMPOWER was designed to determine the safety and efficacy of domperidone in mothers experiencing an inadequate milk supply.<h4>Methods/design</h4>EMPOWER is a multicenter, double masked, randomized controlled phase-II trial to evaluate the safety and effectiveness of domperidone in those mothers identified as having difficulty in breast milk production. Eligible mothers will be randomized to one of two allocated groups: Group A: domperidone 10 mg orally three times daily for 28 days; and Group B: identical placebo 10 mg orally three times daily for 14 days followed by domperidone 10 mg orally three times daily for 14 days. The primary outcome will be determined at the completion of the first 2-week period; the second 2-week period will facilitate answering the secondary questions regarding timing and duration of treatment. To detect an estimated 30% change between the two groups (from 40% to 28%, corresponding to an odds ratio of 0.6), a total sample size of 488 mothers would be required at 80% power and alpha=0.05. To account for a 15% dropout, this number is increased to 560 (280 per group). The duration of the trial is expected to be 36-40 months.<h4>Discussion</h4>The use of a galactogogue often becomes the measure of choice for mothers in the presence of insufficient breast milk production, particularly when the other techniques are unsuccessful. EMPOWER is designed to provide valuable information in guiding the practices for this high-risk group of infants and mothers. The results of this trial will also inform both mothers and clinicians about the choices available to increase and maintain sufficient breast milk.<h4>Trial registration</h4>Clinical Trials.gov Identifier: NCT01512225.

<h4>Background</h4>The 70% methanol extract of Terminalia chebula Retz. fruit (TCME) was investigated for its in vitro iron chelating property and in vivo ameliorating effect on hepatic injury of iron overloaded mice.<h4>Methods</h4>The effect of fruit extract on Fe2+-ferrozine complex formation and Fe2+ mediated pUC-18 DNA breakdown was studied in order to find the in vitro iron chelating activity. Thirty-six Swiss Albino mice were divided into six groups of: blank, patient control and treated with 50, 100, 200 mg/kg b.w. of TCME and desirox (standard iron chelator drug with Deferasirox as parent compound). Evaluations were made for serum markers of hepatic damage, antioxidant enzyme, lipid per oxidation and liver fibrosis levels. The reductive release of ferritin iron by the extract was further studied.<h4>Results</h4>In vitro results showed considerable iron chelation with IC50 of 27.19 ± 2.80 μg/ml, and a significant DNA protection with [P]50 of 1.07 ± 0.03 μg/ml along with about 86% retention of supercoiled DNA. Iron-dextran injection (i.p.) caused significant increase in the levels of the serum enzymes, viz., alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP) and Bilirubin, which were subsequently lowered by oral administration of 200 mg/kg b.w. dose of the fruit extract by 81.5%, 105.88%, 188.08% and 128.31%, respectively. Similarly, treatment with the same dose of the extract was shown to alleviate the reduced levels of liver antioxidant enzyme superoxide dismutase, catalase, glutathione S-transferase and non-enzymatic reduced glutathione, by 49.8%, 53.5%, 35.4% and 11% respectively, in comparison to the iron overloaded mice. At the same time, the fruit extract effectively lowered the iron-overload induced raised levels of lipid per oxidation, protein carbonyl, hydroxyproline and liver iron by 49%, 67%, 67% and 26%, respectively, with oral treatment of 200 mg/kg b.w. dose of TCME. The fruit extract also showed potential activity for reductive release of ferritin iron.<h4>Conclusions</h4>These findings suggest that Terminalia chebula extract may contain active substances capable of lessening iron overload induced toxicity, and hence possibly be useful as iron chelating drug for iron overload diseases.

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others' supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2-8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.

<h4>Objective</h4>Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), shares clinical and immunological features with psoriasis. Genome-wide association studies have found common susceptibility genes. However, epidemiologic data evaluating the association between psoriasis, psoriatic arthritis and risk of IBD are sparse. We aimed to evaluate the association between psoriasis, psoriatic arthritis and incident CD and UC among women in the USA.<h4>Methods</h4>174 476 women were enrolled in the Nurses' Health Study (NHS) (1996-2008) and NHS II (1991-2007). Lifetime history of physician-diagnosed psoriasis and psoriatic arthritis was confirmed by supplementary questionnaires. Information on CD and UC was obtained by self-reported questionnaires and confirmed by medical record review.<h4>Results</h4>We documented 188 incident cases of CD and 240 incident cases of UC during follow-up. Psoriasis was associated with a significantly increased risk of subsequent CD with a multivariate-adjusted relative risk (RR) of 4.00 (95% CI 1.72 to 9.27) for NHS and 3.76 (1.82 to 7.74) for NHS II. By contrast, we did not observe a significant increase in risk of UC associated with psoriasis. In a pooled analysis of both cohorts, women with psoriasis experienced a significantly increased risk of CD (RR, 3.86, 95% CI 2.23 to 6.67), but not UC (RR, 1.17, 95% CI 0.41 to 3.36). The risk of CD was especially pronounced among psoriatics with concomitant psoriatic arthritis (RR, 6.43, 95% CI 2.04 to 20.32).<h4>Conclusions</h4>Psoriasis with concomitant psoriatic arthritis is associated with an increased risk of incident CD.

Netrin and semaphorin axon guidance cues have been found to function in the genesis of several mammalian organs; however, little is known about the underlying molecular mechanisms involved. A genetic approach could help to reveal the underpinnings of these mechanisms. The most anterior ray sensillum (ray 1) in the Caenorhabditis elegans male tail is frequently displaced anterior to its normal position in smp-1/semaphorin-1a and plexin-1/plx-1 mutants. Here we report that UNC-6/netrin and its UNC-40/DCC receptor signal in parallel to SMP-1/semaphorin-1a and its PLX-1/plexin-1 receptor to prevent the anterior displacement of ray 1 and that UNC-6 plus SMP-1 signaling can account entirely for this function. We also report that mab-20/semaphorin-2a mutations, which prevent the separation of neighboring rays and cause ray fusions, suppress the anterior displacements of ray 1 caused by deficiencies in SMP-1 and UNC-6 signaling and this is independent of the ray fusion phenotype, whereas overexpression of UNC-40 and PLX-1 cause ray fusions. This suggests that for ray 1 positioning, a balance is struck between a tendency of SMP-1 and UNC-6 signaling to prevent ray 1 from moving away from ray 2 and a tendency of MAB-20/semaphorin-2a signaling to separate all rays from each other. Additional evidence suggests this balance involves the relative adhesion of the ray 1 structural cell to neighboring SET and hyp 7 hypodermal cells. This finding raises the possibility that changes in ray 1 positioning depend on passive movements caused by attachment to the elongating SET cell in opposition to the morphologically more stable hyp 7 cell. Several lines of evidence indicate that SMP-1 and UNC-6 function permissively in the context of ray 1 positioning.

Epidemiological studies indicate that the exogenous sex steroid medroxyprogesterone acetate (MPA) can impair cell-mediated immunity, but mechanisms responsible for this observation are not well defined. In this study, MPA administered to mice 1 wk prior to HSV type 1 (HSV-1) infection of their corneal mucosa impaired initial expansion of viral-specific effector and memory precursor T cells and reduced the number of viral-specific memory T cells found in latently infected mice. MPA treatment also dampened expression of the costimulatory molecules CD40, CD70, and CD80 by dendritic cells (DC) in lymph nodes draining acute infection, whereas coculture of such DC with T cells from uninfected mice dramatically impaired ex vivo T cell proliferation compared with the use of DC from mice that did not receive MPA prior to HSV-1 infection. In addition, T cell expansion was comparable to that seen in untreated controls if MPA-treated mice were administered recombinant soluble CD154 (CD40L) concomitant with their mucosal infection. In contrast, the immunomodulatory effects of MPA were infection site dependent, because MPA-treated mice exhibited normal expansion of virus-specific T cells when infection was systemic rather than mucosal. Taken together, our results reveal that the administration of MPA prior to viral infection of mucosal tissue impairs DC activation, virus-specific T cell expansion, and development of virus-specific immunological memory.

Deconstructing the mechanistic basis of neurodegenerative disorders, such as Huntington's disease (HD), has been a particularly challenging undertaking, relying mostly on post-mortem tissue samples, non-neural cell lines from affected individuals, and model organisms. Two articles recently published in Cell Stem Cell report first the generation and characterization of induced pluripotent stem cell (iPSC)-derived models for HD, and second, the genetic correction of a disease-causing CAG expansion mutation in iPSCs from individuals with HD. Taken together, these two studies provide a framework for the production and validation of iPSC materials for human neurodegenerative disease research and yield crucial tools for investigating future therapies.

<h4>Background</h4>Serotonin (5-HT) is highly involved in pain regulation and serotonin-1A (5-HT1A) receptors are important in determining central 5-HT tone. Accordingly, variation in the 5-HT1A receptor gene (HTR1A) may contribute to inter-individual differences in human pain sensitivity. The minor G-allele of the HTR1A single nucleotide polymorphism (SNP) rs6295 attenuates firing of serotonergic neurons and reduces postsynaptic expression of the receptor. Experiments in rodents suggest that 5-HT1A-agonism modulates pain in opposite directions at mild compared to high noxious intensities. Based upon this and several other similar observations, we hypothesized that G-carriers would exhibit a relative hypoalgesia at mild thermal stimuli but tend towards hyperalgesia at higher noxious intensities.<h4>Methods</h4>Fourty-nine healthy individuals were selectively genotyped for rs6295. Heat- and cold-pain thresholds were assessed along with VAS-ratings of a range of suprathreshold noxious heat intensities (45°C-49°C). Nociceptive-flexion reflex (NFR) thresholds were also assessed.<h4>Results</h4>Volunteers did not deviate significantly from Hardy-Weinberg equilibrium. G-carriers were less sensitive to threshold-level thermal pain. This relative hypoalgesia was abolished at suprathreshold noxious intensities where G-carriers instead increased their ratings of heat-pain significantly more than C-homozygotes. No differences with regard to NFR-thresholds emerged.<h4>Conclusion/significance</h4>To the best of our knowledge this is the first study of human pain perception on the basis of variation in HTR1A. The results illustrate the importance of including a range of stimulus intensities in assessments of pain sensitivity. In speculation, we propose that an attenuated serotonergic tone may be related to a 'hypo- to hyperalgesic' response-pattern. The involved mechanisms could be of clinical interest as variation in pain regulation is known to influence the risk of developing pain pathologies. Further investigations are therefore warranted.

Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3' bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.

Leber congenital amaurosis (LCA) is one of the most severe forms of inherited retinal degeneration and can be caused by mutations in at least 15 different genes. To clarify the proteomic differences in LCA eyes, a cohort of retinal degeneration 12 (rd12) mice, an LCA2 model caused by a mutation in the RPE65 gene, were injected subretinally with an AAV vector (scAAV5-smCBA-hRPE65) in one eye, while the contralateral eye served as a control. Proteomics were compared between untreated rd12 and normal control retinas on P14 and P21, and among treated and untreated rd12 retinas and control retinas on P42. Gene therapy in rd12 mice restored retinal function in treated eyes, which was demonstrated by electroretinography (ERG). Proteomic analysis successfully identified 39 proteins expressed differently among the 3 groups. The expression of 3 proteins involved in regulation of apoptosis and neuroptotection (alpha A crystallin, heat shock protein 70 and peroxiredoxin 6) were investigated further. Immunofluorescence, Western blot and real-time PCR confirmed the quantitative changes in their expression. Furthermore, cell culture studies suggested that peroxiredoxin 6 could act in an antioxidant role in rd12 mice. Our findings support the feasibility of gene therapy in LCA2 patients and support a role for alpha A crystallin, heat shock protein 70 and peroxiredoxin 6 in the pathogenetic mechanisms involved in LCA2 disease process.

The inhibitory Smad7 acts as a critical suppressor of hepcidin, the major regulator of systemic iron homeostasis. In this study we define the mRNA expression of the two functionally related Smad proteins, Smad6 and Smad7, within pathways known to regulate hepcidin levels. Using mouse models for hereditary hemochromatosis (Hfe-, TfR2-, Hfe/TfR2-, Hjv- and hepcidin1-deficient mice) we show that hepcidin, Smad6 and Smad7 mRNA expression is coordinated in such a way that it correlates with the activity of the Bmp/Smad signaling pathway rather than with liver iron levels. This regulatory circuitry is disconnected by iron treatment of Hfe-/- and Hfe/TfR2 mice that significantly increases hepatic iron levels as well as hepcidin, Smad6 and Smad7 mRNA expression but fails to augment pSmad1/5/8 levels. This suggests that additional pathways contribute to the regulation of hepcidin, Smad6 and Smad7 under these conditions which do not require Hfe.

Human Campylobacter jejuni infection can result in an asymptomatic carrier state, watery or bloody diarrhea, bacteremia, meningitis, or autoimmune neurological sequelae. Infection outcomes of C57BL/6 IL-10(-/-) mice orally infected with twenty-two phylogenetically diverse C. jejuni strains were evaluated to correlate colonization and disease phenotypes with genetic composition of the strains. Variation between strains was observed in colonization, timing of development of clinical signs, and occurrence of enteric lesions. Five pathotypes of C. jejuni in C57BL/6 IL-10(-/-) mice were delineated: little or no colonization, colonization without disease, colonization with enteritis, colonization with hemorrhagic enteritis, and colonization with neurological signs with or without enteritis. Virulence gene content of ten sequenced strains was compared in silico; virulence gene content of twelve additional strains was compared using a C. jejuni pan-genome microarray. Neither total nor virulence gene content predicted pathotype; nor was pathotype correlated with multilocus sequence type. Each strain was unique with regard to absences of known virulence-related loci and/or possession of point mutations and indels, including phase variation, in virulence-related genes. An experiment in C. jejuni 11168-infected germ-free mice showed that expression levels of ninety open reading frames (ORFs) were significantly up- or down-regulated in the mouse cecum at least two-fold compared to in vitro growth. Genomic content of these ninety C. jejuni 11168 ORFs was significantly correlated with the capacity to colonize and cause enteritis in C57BL/6 IL-10(-/-) mice. Differences in gene expression levels and patterns are thus an important determinant of pathotype in C. jejuni strains in this mouse model.

Huntington's disease (HD) is a devastating neurodegenerative disorder whose main hallmark is brain atrophy. However, several peripheral organs are considerably affected and their symptoms may, in fact, manifest before those resulting from brain pathology. HD is of genetic origin and caused by a mutation in the huntingtin gene. The mutated protein has detrimental effects on cell survival, but whether the mutation leads to a gain of toxic function or a loss of function of the altered protein is still highly controversial. Most currently used in vitro models have been designed, to a large extent, to investigate the effects of the aggregation process in neuronal-like cells. However, as the pathology involves several other organs, new in vitro models are critically needed to take into account the deleterious effects of mutant huntingtin in peripheral tissues, and thus to identify new targets that could lead to more effective clinical interventions in the early course of the disease. This review aims to present current in vitro models of HD pathology and to discuss the knowledge that has been gained from these studies as well as the new in vitro tools that have been developed, which should reflect the more global view that we now have of the disease.

The formation and solution properties, including stability in mixed aqueous-Me(2)SO media, have been investigated for an [Fe(4)S(4)](2+) cluster derived from β-cyclodextrin (CD) dithiolate. Clusters of the type [Fe(4)S(4)(SAr)(4)](2-) (Ar = Ph, C(6)H(4)-3-F) are generated in Me(2)SO by redox reactions of [Fe(4)S(4)(SEt)(4)](2-) with 2 equiv of ArSSAr. An analogous reaction with the intramolecular disulfide of 6(A),6(D)-(3-NHCOC(6)H(4)-1-SH)(2)-6(A),6(D)-dideoxy-β-cyclodextrin (14), whose synthesis is described, affords a completely substituted cluster formulated as [Fe(4)S(4){β-CD-(1,3-NHCOC(6)H(4)S)(2)}(2)](2-) (15). Ligand binding is indicated by a circular dichroism spectrum and also by UV-visible and isotropically shifted (1)H NMR spectra and redox behavior convincingly similar to [Fe(4)S(4)(SPh)(4)](2-). One formulation of 15 is a single cluster to which two dithiolates are bound, each in bidentate coordination. With there being no proven precedent for this binding mode, we show that the cluster [Fe(4)S(4)(S(2)-m-xyl)(2)](2-) is a single cubane whose m-xylyldithiolate ligands are bound in a bidentate arrangement. This same structure type was proposed for a cluster formulated as [Fe(4)S(4){β-CD-(1,3-SC(6)H(4)S)(2)}(2)](2-) (16; Kuroda et al. J. Am. Chem. Soc.1988, 110, 4049-4050) and reported to be water-stable. Clusters 15 and 16 are derived from similar ligands differing only in the spacer group between the thiolate binding site and the CD platform. In our search for clusters stable in aqueous or organic-aqueous mixed solvents that are potential candidates for the reconstitution of scaffold proteins implicated in cluster biogenesis, 15 is the most stable cluster that we have thus far encountered under anaerobic conditions in the absence of added ligand.

Tissue-targeted expression is of major interest for studying the contribution of cellular subpopulations to neurodegenerative diseases. However, in vivo methods to investigate this issue are limited. Here, we report an analysis of the cell specificity of expression of fluorescent reporter genes driven by six neuronal promoters, with the ubiquitous phosphoglycerate kinase 1 (PGK) promoter used as a reference. Quantitative analysis of AcGFPnuc expression in the striatum and hippocampus of rodents showed that all lentiviral vectors (LV) exhibited a neuronal tropism; however, there was substantial diversity of transcriptional activity and cell-type specificity of expression. The promoters with the highest activity were those of the 67 kDa glutamic acid decarboxylase (GAD67), homeobox Dlx5/6, glutamate receptor 1 (GluR1), and preprotachykinin 1 (Tac1) genes. Neuron-specific enolase (NSE) and dopaminergic receptor 1 (Drd1a) promoters showed weak activity, but the integration of an amplification system into the LV overcame this limitation. In the striatum, the expression profiles of Tac1 and Drd1a were not limited to the striatonigral pathway, whereas in the hippocampus, Drd1a and Dlx5/6 showed the expected restricted pattern of expression. Regulation of the Dlx5/6 promoter was observed in a disease condition, whereas Tac1 activity was unaffected. These vectors provide safe tools that are more selective than others available, for the administration of therapeutic molecules in the central nervous system (CNS). Nevertheless, additional characterization of regulatory elements in neuronal promoters is still required.

Tumor suppressors are frequently downregulated in human cancers and understanding of the mechanisms through which tumor cells restrict the expression of tumor suppressors is important for the prognosis and intervention of diseases. The promyelocytic leukemia (PML) protein plays a critical role in multiple tumor suppressive functions, such as growth inhibition, apoptosis, replicative senescence, suppression of oncogenic transformation, and inhibition of migration and angiogenesis. These tumor suppression functions are recapitulated in several mouse models. The expression of PML protein is frequently downregulated in diverse types of human tumors and this downregulation often correlates with tumor progression. Recent evidence has emerged that PML is aberrantly degraded in various types of tumors through ubiquitination-dependent mechanisms. Here, we summarize our current understanding of the PML ubiquitination/degradation pathways in human cancers. We point out that multiple pathways lead to PML ubiquitination and degradation. Furthermore, the PML ubiquitination processes are often dependent on other types of posttranslational modifications, such as phosphorylation, prolylisomerization, and sumoylation. Such feature indicates a highly regulated nature of PML ubiquitination in different cellular conditions and cell contexts, thus providing many avenues of opportunity to intervene PML ubiquitination pathways. We discuss the potential of targeting PML ubiquitination pathways for anti-cancer therapeutic strategies.

No abstract available.

We hypothesized that absence of the 5-HT(1A) receptor would negatively affect the development of cardiorespiratory control. In conscious wild type (WT) and 5-HT(1A) receptor knockout (KO) mice, we measured resting ventilation (Ve), oxygen consumption (Vo(2)), heart rate (HR), breathing and HR variability, and the hypercapnic ventilatory response (HCVR) at postnatal day 5 (P5), day 15 (P15), and day 25 (P25). In KO mice compared with WT, we found a 17% decrease in body weight at only P5 (P < 0.01) and no effect on Vo(2). Ve was significantly (P < 0.001) lower at P5 and P25, but there was no effect on the HCVR. Breathing variability (interbreath interval), measured by standard deviation, the root mean square of the standard deviation (RMSSD), and the product of the major (L) and minor axes (T) of the Poincaré first return plot, was 57% to 187% higher only at P5 (P < 0.001). HR was 6-10% slower at P5 (P < 0.001) but 7-9% faster at P25 (P < 0.001). This correlated with changes in the spectral analysis of HR variability; the low frequency to high frequency ratio was 47% lower at P5 but 68% greater at P25. The RMSSD and (L × T) of HR variability were ~2-fold greater at P5 only (P < 0.001; P < 0.05). We conclude that 5-HT(1A) KO mice have a critical period of potential vulnerability at P5 when pups hypoventilate and have a slower respiratory frequency and HR with enhanced variability of both, suggesting abnormal maturation of cardiorespiratory control.

Proper development of the central nervous system (CNS) requires the establishment of appropriate connections between neurons. Recent work suggests that this process is controlled by a balance between synaptogenic molecules and proteins that negatively regulate synapse formation and plasticity. Surprisingly, many of these newly identified synapse-limiting molecules are classic 'immune' proteins. In particular, major histocompatibility complex class I (MHCI) molecules regulate neurite outgrowth, the establishment and function of cortical connections, activity-dependent refinement in the visual system, and long-term and homeostatic plasticity. This review summarizes our current understanding of MHCI expression and function in the CNS, as well as the potential mechanisms used by MHCI to regulate brain development and plasticity.

Thymic stromal lymphopoietin (TSLP), an interleukin 7-like cytokine, can trigger dendritic cell (DC)-mediated T-helper type 2 (Th2) inflammatory responses. Recent evidence demonstrates that cytokines TSLP and OX40 (CD134)/OX40 ligand seem to be important players in the maintenance of Th2 memory pool in the pathogenesis of asthma. Accumulating data reveal that the pathogenic T cells involved in asthma are likely to be inflammatory Th2 cells. TSLP is involved in the development of asthma through crosstalk with nuclear factor NF-ĸB. Progression of skin fibrosis in atopic dermatitis occurs via TSLP/TSLP receptor. TSLP-mediated dermal inflammation aggravates experimental allergic asthma. Also, TSLP polymorphisms are associated with susceptibility to asthma, atopic dermatitis, and eczema herpeticum. These findings suggest a master switch of TSLP in the initiation of allergic and adaptive inflammation through innate pathways at the epithelial cell-DC interface. The TSLP pathway is therefore a promising target for immunotherapy of allergic diseases.

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the "measles paradox". Here we show that MV preferentially infects CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.

Degeneration of synaptic and axonal compartments of neurons is an early event contributing to the pathogenesis of many neurodegenerative diseases, but the underlying molecular mechanisms remain unclear. Here, we demonstrate the effectiveness of a novel "top-down" approach for identifying proteins and functional pathways regulating neurodegeneration in distal compartments of neurons. A series of comparative quantitative proteomic screens on synapse-enriched fractions isolated from the mouse brain following injury identified dynamic perturbations occurring within the proteome during both initiation and onset phases of degeneration. In silico analyses highlighted significant clustering of proteins contributing to functional pathways regulating synaptic transmission and neurite development. Molecular markers of degeneration were conserved in injury and disease, with comparable responses observed in synapse-enriched fractions isolated from mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 5. An initial screen targeting thirteen degeneration-associated proteins using mutant Drosophila lines revealed six potential regulators of synaptic and axonal degeneration in vivo. Mutations in CALB2, ROCK2, DNAJC5/CSP, and HIBCH partially delayed injury-induced neurodegeneration. Conversely, mutations in DNAJC6 and ALDHA1 led to spontaneous degeneration of distal axons and synapses. A more detailed genetic analysis of DNAJC5/CSP mutants confirmed that loss of DNAJC5/CSP was neuroprotective, robustly delaying degeneration in axonal and synaptic compartments. Our study has identified conserved molecular responses occurring within synapse-enriched fractions of the mouse brain during the early stages of neurodegeneration, focused on functional networks modulating synaptic transmission and incorporating molecular chaperones, cytoskeletal modifiers, and calcium-binding proteins. We propose that the proteins and functional pathways identified in the current study represent attractive targets for developing therapeutics aimed at modulating synaptic and axonal stability and neurodegeneration in vivo.

The ubiquitously expressed phosphatidylinositol binding clathrin assembly (PICALM) protein associates with the plasma membrane, binds clathrin, and plays a role in clathrin-mediated endocytosis. Alterations of the human PICALM gene are present in aggressive hematopoietic malignancies, and genome-wide association studies have recently linked the PICALM locus to late-onset Alzheimer's disease. Inactivating and hypomorphic Picalm mutations in mice cause different degrees of severity of anemia, abnormal iron metabolism, growth retardation and shortened lifespan. To understand PICALM's function, we studied the consequences of PICALM overexpression and characterized PICALM-deficient cells derived from mutant fit1 mice. Our results identify a role for PICALM in transferrin receptor (TfR) internalization and demonstrate that the C-terminal PICALM residues are critical for its association with clathrin and for the inhibitory effect of PICALM overexpression on TfR internalization. Murine embryonic fibroblasts (MEFs) that are deficient in PICALM display several characteristics of iron deficiency (increased surface TfR expression, decreased intracellular iron levels, and reduced cellular proliferation), all of which are rescued by retroviral PICALM expression. The proliferation defect of cells that lack PICALM results, at least in part, from insufficient iron uptake, since it can be corrected by iron supplementation. Moreover, PICALM-deficient cells are particularly sensitive to iron chelation. Taken together, these data reveal that PICALM plays a critical role in iron homeostasis, and offer new perspectives into the pathogenesis of PICALM-associated diseases.

The eukaryotic nucleus is both spatially and functionally partitioned. This organization contributes to the maintenance, expression, and transmission of genetic information. Though our ability to probe the physical structure of the genome within the nucleus has improved substantially in recent years, relatively little is known about the factors that regulate its organization or the mechanisms through which specific organizational states are achieved. Here, we show that Drosophila melanogaster Condensin II induces axial compaction of interphase chromosomes, globally disrupts interchromosomal interactions, and promotes the dispersal of peri-centric heterochromatin. These Condensin II activities compartmentalize the nucleus into discrete chromosome territories and indicate commonalities in the mechanisms that regulate the spatial structure of the genome during mitosis and interphase.

Linker histones are essential components of chromatin, but the distributions and functions of many during cellular differentiation are not well understood. Here, we show that H1.5 binds to genic and intergenic regions, forming blocks of enrichment, in differentiated human cells from all three embryonic germ layers but not in embryonic stem cells. In differentiated cells, H1.5, but not H1.3, binds preferentially to genes that encode membrane and membrane-related proteins. Strikingly, 37% of H1.5 target genes belong to gene family clusters, groups of homologous genes that are located in proximity to each other on chromosomes. H1.5 binding is associated with gene repression and is required for SIRT1 binding, H3K9me2 enrichment, and chromatin compaction. Depletion of H1.5 results in loss of SIRT1 and H3K9me2, increased chromatin accessibility, deregulation of gene expression, and decreased cell growth. Our data reveal for the first time a specific and novel function for linker histone subtype H1.5 in maintenance of condensed chromatin at defined gene families in differentiated human cells.

General parameters of selection, such as the frequency and strength of positive selection in natural populations or the role of introgression, are still insufficiently understood. The house mouse (Mus musculus) is a particularly well-suited model system to approach such questions, since it has a defined history of splits into subspecies and populations and since extensive genome information is available. We have used high-density single-nucleotide polymorphism (SNP) typing arrays to assess genomic patterns of positive selection and introgression of alleles in two natural populations of each of the subspecies M. m. domesticus and M. m. musculus. Applying different statistical procedures, we find a large number of regions subject to apparent selective sweeps, indicating frequent positive selection on rare alleles or novel mutations. Genes in the regions include well-studied imprinted loci (e.g. Plagl1/Zac1), homologues of human genes involved in adaptations (e.g. alpha-amylase genes) or in genetic diseases (e.g. Huntingtin and Parkin). Haplotype matching between the two subspecies reveals a large number of haplotypes that show patterns of introgression from specific populations of the respective other subspecies, with at least 10% of the genome being affected by partial or full introgression. Using neutral simulations for comparison, we find that the size and the fraction of introgressed haplotypes are not compatible with a pure migration or incomplete lineage sorting model. Hence, it appears that introgressed haplotypes can rise in frequency due to positive selection and thus can contribute to the adaptive genomic landscape of natural populations. Our data support the notion that natural genomes are subject to complex adaptive processes, including the introgression of haplotypes from other differentiated populations or species at a larger scale than previously assumed for animals. This implies that some of the admixture found in inbred strains of mice may also have a natural origin.

<h4>Unlabelled</h4><h4>Background</h4>Methamphetamine (meth) is a stimulant used illegally around the world, including in Iran. Cardiomyopathy and cardiac failure may occur following chronic meth use and may cause the patients referred to the emergency department.<h4>Case reports</h4>A 28-year old man and two women, ages 29 and 31-year-old, with a history of meth use, were admitted to the emergency department with severe dyspnea at rest. Each had sinus tachycardia with tachypnea and an echocardiogram that showed severe systolic dysfunction consistent with heart failure. Additional evaluation in the hospital revealed cardiomyopathy with no other etiology other than the meth use.<h4>Conclusion</h4>There are several reports that show an increase in frequency of meth use, suggesting that cardiomyopathy and acute heart failure may be a new medical concern.

A previous report [Abeyweera, T. P., and Rotenberg, S. A. (2007) Biochemistry 46, 2364-2370] described the application of the traceable kinase method in identifying substrates of protein kinase Cα (PKC-α) in nontransformed human breast MCF-10A cells. Here, a nonradioactive variation of this method compared the phosphoprotein profiles of three traceable PKC isoforms (α, δ, and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high-affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N(6)-phenyl-ATP, and the resulting phosphoproteins were analyzed by Western blotting with an antibody that recognizes the phosphorylated PKC consensus site. Phosphoprotein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, -δ, and -ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (moles of phospho-CEP4 per mole of CEP4). Following knockdown with isoform-specific shRNA-encoding plasmids, the level of phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC-α, -δ, and -ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and -δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms.

<h4>Aims/hypothesis</h4>Manoeuvres aimed at increasing beta cell mass have been proposed as regenerative medicine strategies for diabetes treatment. Raf-1 kinase inhibitor protein 1 (RKIP1) is a common regulatory node of the mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways and therefore may be involved in regulation of beta cell homeostasis. The aim of this study was to investigate the involvement of RKIP1 in the control of beta cell mass and function.<h4>Methods</h4>Rkip1 (also known as Pebp1) knockout (Rkip1 (-/-)) mice were characterised in terms of pancreatic and glucose homeostasis, including morphological and functional analysis. Glucose tolerance and insulin sensitivity were examined, followed by assessment of glucose-induced insulin secretion in isolated islets and beta cell mass quantification through morphometry. Further characterisation included determination of endocrine and exocrine proliferation, apoptosis, MAPK activation and whole genome gene expression assays. Capacity to reverse a diabetic phenotype was assessed in adult Rkip1 (-/-) mice after streptozotocin treatment.<h4>Results</h4>Rkip1 (-/-) mice exhibit a moderately larger pancreas and increased beta cell mass and pancreatic insulin content, which correlate with an overall improvement in whole body glucose tolerance. This phenotype is established in young postnatal stages and involves enhanced cellular proliferation without significant alterations in cell death. Importantly, adult Rkip1 (-/-) mice exhibit rapid reversal of streptozotocin-induced diabetes compared with control mice.<h4>Conclusions/interpretation</h4>These data implicate RKIP1 in the regulation of pancreatic growth and beta cell expansion, thus revealing RKIP1 as a potential pharmacological target to promote beta cell regeneration.

Restless legs syndrome (RLS) is a common multifactorial disease. Some genetic risk factors have been identified. RLS susceptibility also has been related to iron. We therefore asked whether known iron-related genes are candidates for association with RLS and, vice versa, whether known RLS-associated loci influence iron parameters in serum. RLS/control samples (n = 954/1814 in the discovery step, 735/736 in replication 1, and 736/735 in replication 2) were tested for association with SNPs located within 4 Mb intervals surrounding each gene from a list of 111 iron-related genes using a discovery threshold of P = 5 × 10(-4). Two population cohorts (KORA F3 and F4 with together n = 3447) were tested for association of six known RLS loci with iron, ferritin, transferrin, transferrin-saturation, and soluble transferrin receptor. Results were negative. None of the candidate SNPs at the iron-related gene loci was confirmed significantly. An intronic SNP, rs2576036, of KATNAL2 at 18q21.1 was significant in the first (P = 0.00085) but not in the second replication step (joint nominal P-value = 0.044). Especially, rs1800652 (C282Y) in the HFE gene did not associate with RLS. Moreover, SNPs at the known RLS loci did not significantly affect serum iron parameters in the KORA cohorts. In conclusion, the correlation between RLS and iron parameters in serum may be weaker than assumed. Moreover, in a general power analysis, we show that genetic effects are diluted if they are transmitted via an intermediate trait to an end-phenotype. Sample size formulas are provided for small effect sizes.

<h4>Background</h4>Epidemiological studies have suggested the benefits of omega-3 polyunsaturated fatty acids (n-3 PUFAs) on cardiovascular health, but only limited data are available describing n-3 PUFA regulated pathways in humans. The aim of this study was to investigate the effects of n-3 PUFA administration on whole genome expression profiles in the blood of normo- and dyslipidemic subjects.<h4>Methods</h4>Differentially expressed genes were detected after four hours, one week and twelve weeks of supplementation with either fish oil (FO) or corn oil in normo- and dyslipidemic men using whole genome microarrays.<h4>Results</h4>Independent of the oil, a significantly higher number of genes was regulated in dyslipidemic subjects compared to normolipidemic subjects. Pathway analyses discovered metabolisms dominantly affected by FO after twelve weeks of supplementation, including the lipid metabolism, immune system and cardiovascular diseases. Several pro-inflammatory genes, in particular, were down-regulated in dyslipidemic subjects, indicating the immune-modulatory and anti-inflammatory capability of FO and its bioactive FAs, eicosapentaenoic acid and docosahexaenoic acid.<h4>Conclusions</h4>This is the first study showing significant differences in gene expression profiles between normo- and dyslipidemic men after FO supplementation. Further studies need to clarify the exact role of n-3 PUFAs in pathways and metabolisms which were identified as being regulated after FO supplementation in this study.<h4>Trial registration</h4>ClinicalTrials.gov (ID: NCT01089231).

<h4>Background</h4>Role of KRAS, BRAF and PIK3CA mutations in pathogenesis of colorectal cancer (CRC) has been recently investigated worldwide. In this population-based study, we evaluated the incidence rates and distribution of such somatic mutations in genetically isolated population from Sardinia.<h4>Methods</h4>From April 2009 to July 2011, formalin-fixed paraffin-embedded tissues (N = 478) were prospectively collected from Sardinian CRC patients at clinics across the entire island. Genomic DNA was isolated from tissue sections and screened for mutations in KRAS, BRAF, and PIK3CA genes by automated DNA sequencing.<h4>Results</h4>Overall, KRAS tumour mutation rate was 30% (145/478 positive cases). Distribution of mutation carriers was surprisingly different within the island: 87/204 (43%) in North Sardinia vs. 58/274 (21%) in Middle-South Sardinia (p<0.001). Among 384 CRC cases whose DNA was available, only one (0.3%) patient carried a mutation in BRAF gene; PIK3CA was found mutated in 67 (17%) patients. A significant inverse distribution of PIK3CA mutation rates was observed within Sardinian population: 19/183 (10%) cases from northern vs. 48/201 (24%) cases from central-southern island (p<0.001). This heterogeneity in frequencies of KRAS/PIK3CA somatic mutations is consistent with already-reported discrepancies in distribution of germline mutations for other malignancies within Sardinian population. Preliminary clinical evaluation of 118 KRAS wild-type patients undergoing anti-EGFR-based treatment indicated lack of role for PIK3CA in predicting response to therapy.<h4>Conclusions</h4>Our findings support the hypothesis that differences in patients' origins and related genetic backgrounds may contribute to even determine the incidence rate of somatic mutations in candidate cancer genes.

Silica nanoparticles have been functionalized by click chemistry and atom transfer radical polymerization (ATRP) simultaneously. First, the silanized silica nanoparticles were modified with bromine end group, and then the azide group was grafted onto the surface via covalent coupling. 3-Bromopropyl propiolate was synthesized, and then the synthesized materials were used to react with azide-modified silica nanoparticles via copper-mediated click chemistry and bromine surface-initiated ATRP. Transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis were performed to characterize the functionalized silica nanoparticles. We investigated the enrichment efficiency of bare silica and poly(ethylene glycol) methacrylate (PEGMA)-functionalized silica nanoparticles in Pb(II) aqueous solution. The results demonstrated that PEGMA-functionalized silica nanoparticles can enrich Pb(II) more quickly than pristine silica nanoparticles within 1 h.

Huntington disease (HD) results from CAG repeats that encode expanded polyglutamine tracts in the HTT/huntingtin protein. HD belongs to a large category of inherited and sporadic neurodegenerative disorders in which production of a misfolded protein initiates the pathogenic cascade. Previous studies have shown that misfolded proteins become resistant to cellular protein turnover pathways by eluding and disabling the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway. Based upon earlier work implicating impaired PPARGC1A function in HD, we derived inducible PPARGC1A mice and crossed them with HD mice. We found that PPARGC1A overexpression can rescue HD neurological phenotypes and neurodegeneration. An unexpected outcome of the rescue was the virtual elimination of huntingtin aggregates, and we found that PPARGC1A-mediated aggregate elimination required the autophagy pathway. Moreover, we observed decreased expression of transcription factor EB (TFEB), a master regulator of the autophagy-lysosome pathway, in HD cells and mice, and documented PPARGC1A co-activation of TFEB in these model systems, noting that PPARGC1A is upstream of TFEB in promoting proteostasis. These findings underscore the importance of bioenergetics and autophagy in neurodegeneration, and indicate that PPARGC1A promotes mitochondrial quality control to support high-energy production states in cells, such as neurons. As impaired energy production and altered protein-organelle quality control appear inextricably linked in disorders such as HD, Parkinson disease, and Alzheimer disease, efforts directed at enhancing PPARGC1A and TFEB action may represent viable strategies for therapy development in neurodegeneration.

<h4>Objective</h4>Rapid-onset dystonia-parkinsonism (RDP) is caused by a variety of missense mutations in the ATP1A3 gene. Psychiatric comorbidity has been reported, although systematic examination of psychiatric disease in individuals with RDP is lacking. This study examines psychiatric morbidity for 23 patients with RDP in 10 families with family member control subjects and in 3 unrelated patients with RDP, totaling 56 individuals.<h4>Methods</h4>Twenty-nine ATP1A3 mutation-positive individuals were examined; 26 exhibited motor symptoms (motor manifesting carrier [MMC]) and 3 did not (nonmotor manifesting carriers [NMC]). Twenty-seven ATP1A3 mutation-negative participants (noncarriers [NC]) were included. Rates of psychiatric illness for patients with RDP and related asymptomatic gene mutation carriers were compared with those for related nonmutation carriers. Outcome measures included the Unified Parkinson's Disease Rating Scale, Burke-Fahn-Marsden Dystonia Rating Scale, Instrumental Activities of Daily Living, Composite International Diagnostic Interview, Structured Clinical Interview for DSM-IV, Hamilton Depression Scale, Hamilton Anxiety Scale, and Yale-Brown Obsessive-Compulsive Scale.<h4>Results</h4>NMC participants did not report any history of psychiatric disorder. Findings in MMC and NC groups included anxiety (MMC 48, NC 41%), mood (MMC 50%, NC 22%), psychotic (MMC 19%, NC 0%), and substance abuse/dependence (MMC 38%, NC 27%).<h4>Conclusions</h4>ATP1A3 mutations cause a wide spectrum of motor and nonmotor features. Psychotic symptoms tended to emerge before or concurrent with motor symptom onset, suggesting that this could be another expression of the ATP1A3 gene mutation.

The development of pulmonary hypertension in COPD adversely affects survival and exercise capacity and is associated with an increased risk of severe acute exacerbations. Unfortunately not all patients with COPD who meet criteria for long term oxygen therapy benefit from it. Even in those who benefit from long term oxygen therapy, such therapy may reverse the elevated pulmonary artery pressure but cannot normalize it. Moreover, the recent discovery of the key roles of endothelial dysfunction and inflammation in the pathogenesis of PH provides the rationale for considering specific pulmonary vasodilators that also possess antiproliferative properties and statins.

The titled approaches were effected with various 2-substituted benzoylacetic acid oximes 3 (Beckmann) and 2-substituted malonamic acids 9 (Hofmann), their carboxyl groups being masked as a 2,4,10-trioxaadamantane unit (an orthoacetate). The oxime mesylates have been rearranged with basic Al(2)O(3) in refluxing CHCl(3), and the malonamic acids with phenyliodoso acetate and KOH/MeOH. Both routes are characterized by excellent overall yields. Structure confirmation of final products was conducted with X-ray diffraction in selected cases. The final N-benzoyl and N-(methoxycarbonyl) products are α-amino acids with both carboxyl and amino protection; hence, they are of great interest in peptide synthesis.

<h4>Background</h4>Atlantic cod (Gadus morhua) reared in sea-cages can experience large variations in temperature, and these have been shown to affect their immune function. We used the new 20K Atlantic cod microarray to investigate how a water temperature change which, simulates that seen in Newfoundland during the spring-summer (i.e. from 10°C to 16°C, 1°C increase every 5 days) impacted the cod spleen transcriptome response to the intraperitoneal injection of a viral mimic (polyriboinosinic polyribocytidylic acid, pIC).<h4>Results</h4>The temperature regime alone did not cause any significant increases in plasma cortisol levels and only minor changes in spleen gene transcription. However, it had a considerable impact on the fish spleen transcriptome response to pIC [290 and 339 significantly differentially expressed genes between 16°C and 10°C at 6 and 24 hours post-injection (HPI), respectively]. Seventeen microarray-identified transcripts were selected for QPCR validation based on immune-relevant functional annotations. Fifteen of these transcripts (i.e. 88%), including DHX58, STAT1, IRF7, ISG15, RSAD2 and IκBα, were shown by QPCR to be significantly induced by pIC.<h4>Conclusions</h4>The temperature increase appeared to accelerate the spleen immune transcriptome response to pIC. We found 41 and 999 genes differentially expressed between fish injected with PBS vs. pIC at 10°C and sampled at 6HPI and 24HPI, respectively. In contrast, there were 656 and 246 genes differentially expressed between fish injected with PBS vs. pIC at 16°C and sampled at 6HPI and 24HPI, respectively. Our results indicate that the modulation of mRNA expression of genes belonging to the NF-κB and type I interferon signal transduction pathways may play a role in controlling temperature-induced changes in the spleen's transcript expression response to pIC. Moreover, interferon effector genes such as ISG15 and RSAD2 were differentially expressed between fish injected with pIC at 10°C vs. 16°C at 6HPI. These results substantially increase our understanding of the genes and molecular pathways involved in the negative impacts of elevated ambient temperature on fish health, and may also be valuable to our understanding of how accelerated global climate change could impact cold-water marine finfish species.

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.

<h4>Background</h4>Huntington's Disease (HD) is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt) containing an expanded polyglutamine (polyQ) tract. Activation of the channel responsible for the inositol-induced Ca²⁺ release from ensoplasmic reticulum (ER), was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) has been shown to reduce mutant Htt aggregation.<h4>Results</h4>In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632.<h4>Conclusions</h4>Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases.

<h4>Background</h4>EBV DNA is found within the malignant cells of 10% of gastric cancers. Modern molecular technology facilitates identification of virus-related biochemical effects that could assist in early diagnosis and disease management.<h4>Methods</h4>In this study, RNA expression profiling was performed on 326 macrodissected paraffin-embedded tissues including 204 cancers and, when available, adjacent non-malignant mucosa. Nanostring nCounter probes targeted 96 RNAs (20 viral, 73 human, and 3 spiked RNAs).<h4>Results</h4>In 182 tissues with adequate housekeeper RNAs, distinct profiles were found in infected versus uninfected cancers, and in malignant versus adjacent benign mucosa. EBV-infected gastric cancers expressed nearly all of the 18 latent and lytic EBV RNAs in the test panel. Levels of EBER1 and EBER2 RNA were highest and were proportional to the quantity of EBV genomes as measured by Q-PCR. Among protein coding EBV RNAs, EBNA1 from the Q promoter and BRLF1 were highly expressed while EBNA2 levels were low positive in only 6/14 infected cancers. Concomitant upregulation of cellular factors implies that virus is not an innocent bystander but rather is linked to NFKB signaling (FCER2, TRAF1) and immune response (TNFSF9, CXCL11, IFITM1, FCRL3, MS4A1 and PLUNC), with PPARG expression implicating altered cellular metabolism. Compared to adjacent non-malignant mucosa, gastric cancers consistently expressed INHBA, SPP1, THY1, SERPINH1, CXCL1, FSCN1, PTGS2 (COX2), BBC3, ICAM1, TNFSF9, SULF1, SLC2A1, TYMS, three collagens, the cell proliferation markers MYC and PCNA, and EBV BLLF1 while they lacked CDH1 (E-cadherin), CLDN18, PTEN, SDC1 (CD138), GAST (gastrin) and its downstream effector CHGA (chromogranin). Compared to lymphoepithelioma-like carcinoma of the uterine cervix, gastric cancers expressed CLDN18, EPCAM, REG4, BBC3, OLFM4, PPARG, and CDH17 while they had diminished levels of IFITM1 and HIF1A. The druggable targets ERBB2 (Her2), MET, and the HIF pathway, as well as several other potential pharmacogenetic indicators (including EBV infection itself, as well as SPARC, TYMS, FCGR2B and REG4) were identified in some tumor specimens.<h4>Conclusion</h4>This study shows how modern molecular technology applied to archival fixed tissues yields novel insights into viral oncogenesis that could be useful in managing affected patients.

<h4>Objective</h4>Elevated serum ferritin has been known to be associated with the prevalence of metabolic syndrome (MetS). However, there was no research to examine whether serum ferritin levels have been actually associated with the prospective development of MetS. Accordingly, we carried out a prospective study to evaluate the longitudinal effects of baseline serum ferritin levels on the development of MetS.<h4>Research design and methods</h4>A MetS-free cohort of 18,022 healthy Korean men, who had participated in a medical health checkup program in 2005, was followed until 2010. MetS was defined according to the joint interim statement of the International Diabetes Federation Task Force on Epidemiology and Prevention. Cox proportional hazards models were performed.<h4>Results</h4>During 45,919.3 person-years of follow-up, 2,127 incident cases of MetS developed between 2006 and 2010. After adjusting for multiple covariates, the hazard ratios (95% CI) for incident MetS comparing the second quintile to the fifth quintile of serum ferritin levels versus the first quintile were 1.19 (0.98-1.45), 1.17 (0.96-1.43), 1.36 (1.12-1.65), and 1.66 (1.38-2.01), respectively (P for trend <0.001). These associations were apparent in the clinically relevant subgroup analyses.<h4>Conclusions</h4>Elevated serum ferritin levels were independently associated with future development of MetS during the 5-year follow-up period.

<h4>Objectives</h4>Loss of epithelial cells is one of the key factors that lead to airway fibrosis. Loss of epithelial cells may decrease the barrier to host cell infiltration into the lumen, allowing deposition of extracellular matrix, with subsequent obliteration of the airway. The objective of this study was to determine whether injection of epithelial cells/progenitor cells from the recipient into the lumen of the donor trachea could prevent bronchiolitis obliterans (BO) in a mouse heterotopic tracheal transplantation (HTT) model.<h4>Methods</h4>A major histocompatibility complex class I and class II mismatch of mouse HTT model of BO was used. Epithelial cells from recipient mice were isolated and reinjected into the lumen of the allografts on day 3 after transplantation. Rag-1 knock-out and isografts were also performed as controls. The grafts were analyzed by immunohistochemistry and densitometric analysis.<h4>Results</h4>The results demonstrated that tracheal epithelium was lost by day 3, regenerated between 3 to 7 days, and was lost again in all allografts, but not in the isografts or in Rag-1 knock-out groups by day 12. The reconstituted epithelium was donor originated on day 7 based on green fluorescent protein staining. Furthermore, with the injection of recipient cells into the tracheal lumen, loss of the epithelium was not observed and the luminal obliteration was significantly less in the allografts.<h4>Conclusions</h4>Injection of recipient epithelial cells prevents the second phase of epithelial loss and significantly decreases BO development in an HTT model. Clinically, the use of injected recipient epithelial cells could be a novel treatment for BO.

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

Human enteroviruses usually cause self-limited infections except polioviruses and enterovirus 71 (EV71), which frequently involve neurological complications. EV71 vaccines are being evaluated in humans. However, several challenges to licensure of EV71 vaccines need to be addressed. Firstly, EV71 and coxsackievirus A (CA) are frequently found to co-circulate and cause hand-foot-mouth disease (HFMD). A polyvalent vaccine that can provide protection against EV71 and prevalent CA are desirable. Secondly, infants are the target population of HFMD vaccines and it would need multi-national efficacy trials to prove clinical protection and speed up the licensure and usage of HFMD vaccines in children. An international network for enterovirus surveillance and clinical trials is urgently needed. Thirdly, EV71 is found to evolve quickly in the past 15 years. Prospective cohort studies are warranted to clarify clinical and epidemiological significances of the antigenic and genetic variations between different EV71 genogroups, which is critical for vaccine design.

In analogy with the accepted view that behaviour is shaped by gene×environment (G×E) interactions, G×E studies are exponentially increasing in the field of psychiatry. Whereas research was primarily driven by the premature view that negative environmental stimuli can trigger psychopathology in those subjects that are genetically predisposed, a closer look at the available data shows that G×E interactions are much more complex than initially thought. Here, we discuss G×E studies focussing on serotonin transporter (5-HTT, SERT, SLC6A4) gene variation in humans, monkeys, and rodents. Recent studies, across species, confirm the theorized 'for-better-and-for-worse' effect of low activity serotonin transporter genotypes. In addition, while 5-HTT×E interactions were thought to take place in early life, recent evidence illustrates that these interactions are also manifested in adulthood. Therefore, we discuss data based on 5-HTT×E interactions, and propose a model in which predictive adaptive responses (PARs), as shaped by early life 5-HTT×E interactions, shape responses to environmental challenges in later life, i.e. reflecting 5-HTT×E×E interactions.

Neurodegenerative diseases affect the lives of millions of patients and their families. Due to the complexity of these diseases and our limited understanding of their pathogenesis, the design of therapeutic agents that can effectively treat these diseases has been challenging. Huntington disease (HD) is one of several neurological disorders with few therapeutic options. HD, like numerous other neurodegenerative diseases, involves extensive neuronal cell loss. One potential strategy to combat HD and other neurodegenerative disorders is to intervene in the execution of neuronal cell death. Inhibiting neuronal cell death pathways may slow the development of neurodegeneration. However, discovering small molecule inhibitors of neuronal cell death remains a significant challenge. Here, we review candidate therapeutic targets controlling cell death mechanisms that have been the focus of research in HD, as well as an emerging strategy that has been applied to developing small molecule inhibitors-fragment-based drug discovery (FBDD). FBDD has been successfully used in both industry and academia to identify selective and potent small molecule inhibitors, with a focus on challenging proteins that are not amenable to traditional high-throughput screening approaches. FBDD has been used to generate potent leads, pre-clinical candidates, and has led to the development of an FDA approved drug. This approach can be valuable for identifying modulators of cell-death-regulating proteins; such compounds may prove to be the key to halting the progression of HD and other neurodegenerative disorders.

The cranial base exerts a supportive role for the brain and includes the occipital, sphenoid and ethmoid bones that arise from cartilaginous precursors in the early embryo. As the occipital bone and the posterior part of the sphenoid are mesoderm derivatives that arise in close proximity to the notochord and floor plate, it has been assumed that their development, like the axial skeleton, is dependent on Sonic hedgehog (Shh) and modulation of bone morphogenetic protein (Bmp) signalling. Here we examined the development of the cranial base in chick and mouse embryos to compare the molecular signals that are required for chondrogenic induction in the trunk and head. We found that Shh signalling is required but the molecular network controlling cranial base development is distinct from that in the trunk. In the absence of Shh, the presumptive cranial base did not undergo chondrogenic commitment as determined by the loss of Sox9 expression and there was a decrease in cell survival. In contrast, induction of the otic capsule occurred normally demonstrating that induction of the cranial base is uncoupled from formation of the sensory capsules. Lastly, we found that the early cranial mesoderm is refractory to Shh signalling, likely accounting for why development of the cranial base occurs after the axial skeleton. Our data reveal that cranial and axial skeletal induction is controlled by conserved, yet spatiotemporally distinct mechanisms that co-ordinate development of the cranial base with that of the cranial musculature and the pharyngeal arches.

Selective androgen receptor modulators (SARMs) are a class of drugs that control the activity of the androgen receptor (AR), which mediates the response to androgens, in a tissue-selective fashion. They are specifically designed to reduce the possible complications that result from the systemic inhibition or activation of AR in patients with diseases that involve androgen signalling. However, there are no ideal in vivo models for evaluating candidate SARMs. Therefore, we created a panel of androgen-responsive genes in clinically relevant AR expressing tissues including prostate, skin, bone, fat, muscle, brain and kidney. We used select genes from this panel to compare transcriptional changes in response to the full agonist dihydrotestosterone (DHT) and the SARM bolandiol at 16 h and 6 weeks. We identified several genes in each tissue whose expression at each of these time points correlates with the known tissue-specific effects of these compounds. For example, in the prostate we found four genes whose expression was much lower in animals treated with bolandiol compared with animals treated with DHT for 6 weeks, which correlated well with differences in prostate weight. We demonstrate that adding molecular measurements (androgen-regulated gene expression) to the traditional physiological measurements (tissue weights, etc.) makes the evaluation of potential SARMs more accurate, thorough and perhaps more rapid by allowing measurement of selectivity after only 16 h of drug treatment.

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

Aberrant mitochondrial function, morphology, and transport are main features of neurodegenerative diseases. To date, mitochondrial transport within neurons is thought to rely mainly on microtubules, whereas actin might mediate short-range movements and mitochondrial anchoring. Here, we analyzed the impact of actin on neuronal mitochondrial size and localization. F-actin enhanced mitochondrial size and mitochondrial number in neurites and growth cones. In contrast, raising G-actin resulted in mitochondrial fragmentation and decreased mitochondrial abundance. Cellular F-actin/G-actin levels also regulate serum response factor (SRF)-mediated gene regulation, suggesting a possible link between SRF and mitochondrial dynamics. Indeed, SRF-deficient neurons display neurodegenerative hallmarks of mitochondria, including disrupted morphology, fragmentation, and impaired mitochondrial motility, as well as ATP energy metabolism. Conversely, constitutively active SRF-VP16 induced formation of mitochondrial networks and rescued huntingtin (HTT)-impaired mitochondrial dynamics. Finally, SRF and actin dynamics are connected via the actin severing protein cofilin and its slingshot phosphatase to modulate neuronal mitochondrial dynamics. In summary, our data suggest that the SRF-cofilin-actin signaling axis modulates neuronal mitochondrial function.

The African black rhinoceros (Diceros bicornis) has adapted to a low iron diet during evolution and is thus prone to iron overload in captivity, which is associated with a number of serious disorders. A S88T polymorphism in the HFE gene has been suggested as a potential genetic basis of increased iron uptake in the black rhinoceros, while the Indian rhinoceros is thought to be unaffected by iron overload in captivity. In the present study, the histopathology and distribution of iron accumulations in five black rhinoceroses with iron overload syndrome were characterized and compared with three Indian rhinoceroses (Rhinoceros unicornis) and one African white rhinoceros (Ceratotherium simum). At necropsy examination, iron storage in black rhinoceroses was not associated with gross lesions. Microscopically, the most consistent and highest degree of iron load was found in the spleen, liver, small intestine and lung. There was minimal fibrosis and single cell necrosis in the liver. Endocrine organs, lymph nodes, heart and kidney were less often and less markedly affected. Unexpectedly, Indian rhinoceroses also showed iron load in the spleen and smaller amounts in organs similar to the black rhinoceros except for in the heart, while the white rhinoceros had only minor detectable iron storage in intestine, liver and lung. Sequence analysis confirmed the HFE S88T polymorphism in black but not in Indian rhinoceroses. The results indicate that Indian rhinoceroses may also be affected by iron storage in captivity, although in a milder form than the black rhinoceros, and therefore challenge the relevance of the S88T polymorphism in the HFE gene of black rhinoceroses as the underlying cause for iron overload.

Embryonic implantation to establish a pregnancy is a complex process that requires appropriate communication between the embryo and the maternal endometrium. Inadequate uterine receptivity may contribute to the majority of implantation failures. To provide a comprehensive inventory of genes and functional networks that represent the maternal input of the embryo-maternal cross-talk, a longitudinal, holistic study of the endometrial transcriptome in relation to the days of estrous and to the receptivity of the endometrium was performed in bovine. At day 3 of estrous, genes related to cell communication and mitochondrial energy metabolism were differentially expressed among high- and low-receptive endometria (HR, LR); at day 7, transcripts functioning in immune and inflammatory pathways, oxidative stress, and angiogenesis had different abundances. Additionally, temporal transcriptional changes between days 3 and 7 differed considerably among HR and LR. Further, several transcription factors were predicted as relevant for receptivity because they were either differentially expressed among HR and LR animals or are known to be associated with genes we detected to have differential expression. Finally, global DNA methylation varied according to the interaction of receptivity group and day of estrous, and a divergent trend, which correlated with abundance of DNMT1 transcript, was observed in LR and HR along the estrous cycle days. The study revealed that, even in early estrous, transcripts related to cell communication and response to exogenous stimuli, vascularization, and energy supply show divergent expression and longitudinal temporal regulation in HR and LR. Key components of these molecular pathways are known to be dependent on ovarian hormones that promote uterine receptivity.

Metabolic transformations have been reported as involved in neoplasms survival. This suggests a role of metabolic pathways as potential cancer pharmacological targets. Modulating tumor's energy production pathways may become a substantial research area for cancer treatment. The significant role of metabolic deregulation as inducing transcriptional instabilities and consequently whole-system failure, is thus of foremost importance. By using a data integration approach that combines experimental evidence for high-throughput genome wide gene expression, a non-equilibrium thermodynamics analysis, nonlinear correlation networks as well as database mining, we were able to outline the role that transcription factors MEF2C and MNDA may have as main master regulators in primary breast cancer phenomenology, as well as the possible interrelationship between malignancy and metabolic dysfunction. The present findings are supported by the analysis of 1191 whole genome gene expression experiments, as well as probabilistic inference of gene regulatory networks, and non-equilibrium thermodynamics of such data. Other evidence sources include pathway enrichment and gene set enrichment analyses, as well as motif comparison with a comprehensive gene regulatory network (of homologue genes) in Arabidopsis thaliana. Our key finding is that the non-equilibrium free energies provide a realistic description of transcription factor activation that when supplemented with gene regulatory networks made us able to find deregulated pathways. These analyses also suggest a novel potential role of transcription factor energetics at the onset of primary tumor development. Results are important in the molecular systems biology of cancer field, since deregulation and coupling mechanisms between metabolic activity and transcriptional regulation can be better understood by taking into account the way that master regulators respond to physicochemical constraints imposed by different phenotypic conditions.

Our understanding of the molecular mechanisms of many neurological disorders has been greatly enhanced by the discovery of mutations in genes linked to familial forms of these diseases. These have facilitated the generation of cell and animal models that can be used to understand the underlying molecular pathology. Recently, there has been a surge of interest in the use of patient-derived cells, due to the development of induced pluripotent stem cells and their subsequent differentiation into neurons and glia. Access to patient cell lines carrying the relevant mutations is a limiting factor for many centres wishing to pursue this research. We have therefore generated an open-access collection of fibroblast lines from patients carrying mutations linked to neurological disease. These cell lines have been deposited in the National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research and can be requested by any research group for use in in vitro disease modelling. There are currently 71 mutation-defined cell lines available for request from a wide range of neurological disorders and this collection will be continually expanded. This represents a significant resource that will advance the use of patient cells as disease models by the scientific community.

In mammals, DNA methylation is restricted to cytosines of CpG dinucleotides, which are frequently found in short genomic regions including gene promoters. Methylation within CpG-rich regions around promoters tends to repress gene expression; thus, the CpG islands of housekeeping genes are normally unmethylated. We previously described a testis-specific single-exon gene containing a CpG-rich sequence that is methylated and thus repressed in somatic cells, whereas its expression in spermatogenic cells requires that it be hypomethylated. However, the relationship among the specific expression of spermatogenic genes, their methylation dynamics, and their CpG frequencies are poorly understood. Here, we analyzed the methylation patterns of the sphort genomic region around the transcription start site in spermatogenic cell-specific single-exon genes of various CpG contents. By using UniGene and Ensembl database analyses of the mouse genome and reverse transcription-PCR, we identified 39 single-exon genes that are exclusively expressed in spermatogeniccells. Regardless of their specific expression characteristics, genes containing higher (7 to 14 CpGs in 200 bp; mean = 12) and lower (2 to 6 CpGs in 200 bp; mean = 3.1) number ofCpG were hypo- and hyper-methylated, respectively, in all cell types examined, including spermatogeniccells. We found that genes with intermediate number of CpG (2 to 11 CpGs in 200 bp; mean = 6.9) are methylated in somatic cells, but not in male germ cells. These results suggest that DNA methylation dynamics of spermatogenic cell-specific single-exon genes are associated with CpG content, and the methylation status are stably maintained throughout male germ cell development.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15), a divergent member of the TGF-β superfamily, is over-expressed by many common cancers including those of the prostate (PCa) and its expression is linked to cancer outcome. We have evaluated the effect of MIC-1/GDF15 overexpression on PCa development and spread in the TRAMP transgenic model of spontaneous prostate cancer. TRAMP mice were crossed with MIC-1/GDF15 overexpressing mice (MIC-1(fms)) to produce syngeneic TRAMP(fmsmic-1) mice. Survival rate, prostate tumor size, histopathological grades and extent of distant organ metastases were compared. Metastasis of TC1-T5, an androgen independent TRAMP cell line that lacks MIC-1/GDF15 expression, was compared by injecting intravenously into MIC-1(fms) and syngeneic C57BL/6 mice. Whilst TRAMP(fmsmic-1) survived on average 7.4 weeks longer, had significantly smaller genitourinary (GU) tumors and lower PCa histopathological grades than TRAMP mice, more of these mice developed distant organ metastases. Additionally, a higher number of TC1-T5 lung tumor colonies were observed in MIC-1(fms) mice than syngeneic WT C57BL/6 mice. Our studies strongly suggest that MIC-1/GDF15 has complex actions on tumor behavior: it limits local tumor growth but may with advancing disease, promote metastases. As MIC-1/GDF15 is induced by all cancer treatments and metastasis is the major cause of cancer treatment failure and cancer deaths, these results, if applicable to humans, may have a direct impact on patient care.

The transcription factor Foxp3 is indispensible for the differentiation and function of regulatory T cells (T(reg) cells). To gain insights into the molecular mechanisms of Foxp3-mediated gene expression, we purified Foxp3 complexes and explored their composition. Biochemical and mass-spectrometric analyses revealed that Foxp3 forms multiprotein complexes of 400-800 kDa or larger and identified 361 associated proteins, ∼30% of which were transcription related. Foxp3 directly regulated expression of a large proportion of the genes encoding its cofactors. Some transcription factor partners of Foxp3 facilitated its expression. Functional analysis of the cooperation of Foxp3 with one such partner, GATA-3, provided additional evidence for a network of transcriptional regulation afforded by Foxp3 and its associates to control distinct aspects of T(reg) cell biology.

<h4>Background</h4>To globally characterize the cancer stroma expression profile of muscle-invasive transitional cell carcinoma and to discuss the cancer biology as well as biomarker discovery from stroma. Laser capture micro dissection was used to harvest purified muscle-invasive bladder cancer stromal cells and normal urothelial stromal cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature.<h4>Results</h4>We identified 868/872 commonly expressed proteins and 978 differential proteins from 4 paired cancer and normal stromal samples using laser capture micro dissection coupled with two-dimensional liquid chromatography tandem mass spectrometry. 487/491 proteins uniquely expressed in cancer/normal stroma. Differential proteins were compared with the entire list of the international protein index (IPI), and there were 42/42 gene ontology (GO) terms exhibited as enriched and 8/5 exhibited as depleted in cellular Component, respectively. Significantly altered pathways between cancer/normal stroma mainly include metabolic pathways, ribosome, focal adhesion, etc. Finally, descriptive statistics show that the stromal proteins with extremes of PI and MW have the same probability to be a biomarker.<h4>Conclusions</h4>Based on our results, stromal cells are essential component of the cancer, biomarker discovery and network based multi target therapy should consider neoplastic cells itself and corresponding stroma as whole one.

Genome-wide association studies (GWAS) in diverse populations are needed to reveal variants that are more common and/or limited to defined populations. We conducted a GWAS of breast cancer in women of African ancestry, with genotyping of >1,000,000 SNPs in 3,153 African American cases and 2,831 controls, and replication testing of the top 66 associations in an additional 3,607 breast cancer cases and 11,330 controls of African ancestry. Two of the 66 SNPs replicated (p < 0.05) in stage 2, which reached statistical significance levels of 10(-6) and 10(-5) in the stage 1 and 2 combined analysis (rs4322600 at chromosome 14q31: OR = 1.18, p = 4.3 × 10(-6); rs10510333 at chromosome 3p26: OR = 1.15, p = 1.5 × 10(-5)). These suggestive risk loci have not been identified in previous GWAS in other populations and will need to be examined in additional samples. Identification of novel risk variants for breast cancer in women of African ancestry will demand testing of a substantially larger set of markers from stage 1 in a larger replication sample.

<h4>Background</h4>Dysfunctions of serotonergic neurotransmission are supposed to be involved in the pathogenesis of psychiatric disorders such as major depressive disorder (MDD). The concentration of serotonin (5-hydroxytryptamine, 5-HT) in the synaptic cleft is essentially regulated by the 5-HT transporter (5-HTT). A length polymorphism repeat in the 5-HTT promoter region, termed 5-HTTLPR, has been commonly investigated for an association with psychiatric disorders.<h4>Methods</h4>Genotyping of the 5-HTTLPR is time-consuming and technically challenging. Recently, a two-SNP haplotype was identified that tags the 5-HTTLPR at r(2)=0.775. This allows extraction of 5-HTTLPR genotype information from large genome-wide association study (GWAS) data sets. In the present study we performed haplotype analysis using a German GWAS case-control dataset to test for an association between MDD and the two-SNP tagging haplotype for 5-HTTLPR.<h4>Results</h4>We detected a significant association between the TA haplotype (tagging the S-allele of the 5-HTTLPR) and MDD. Our result is consistent with previous findings of an association between the 5-HTTLPR S-allele and MDD.<h4>Limitations</h4>Using the two-SNP tagging haplotype did not allow testing of the tri-allelic genotype (but only the two-allelic genotype). This and the fact that the haplotype tags the 5-HTTLPR with an imperfect linkage disequilibrium of r(2)=0.775 may lead to some loss of power.<h4>Conclusions</h4>Our results provide further support for an involvement of the 5-HTTLPR in MDD and represent the first example of demonstrating association between MDD and the S-allele of the length polymorphism repeat using common SNP information from SNP-array data.

Nanoparticle-based therapeutics are emerging technologies that have the potential to greatly impact the treatment of many human diseases. However, drug instability and premature release from the nanoparticles during circulation currently preclude clinical translation. Herein, we use a lipase-labile (Sn 2) fumagillin prodrug platform coupled with a unique lipid surface-to-surface targeted delivery mechanism, termed contact-facilitated drug delivery, to counter the premature drug release and overcome the inherent photo-instability of fumagillin, an established anti-angiogenic agent. We show that α(v)β(3)-integrin targeted fumagillin prodrug nanoparticles, administered at 0.3 mg of fumagillin prodrug/kg of body weight suppress the clinical disease indices of KRN serum-mediated arthritis in a dose-dependent manner when compared to treatment with the control nanoparticles with no drug. This study demonstrates the effectiveness of this lipase-labile prodrug nanocarrier in a relevant preclinical model that approximates human rheumatoid arthritis. The lipase-labile prodrug paradigm offers a translatable approach that is broadly applicable to many targeted nanosystems and increases the translational potential of this platform for many diseases.

<h4>Background</h4>There have been few genome-wide association studies (GWAS) of prostate cancer among diverse populations. To search for novel prostate cancer risk variants, we conducted GWAS of prostate cancer in Japanese and Latinos. In addition, we tested prostate cancer risk variants and developed genetic risk models of prostate cancer for Japanese and Latinos.<h4>Methods</h4>Our first-stage GWAS of prostate cancer included Japanese (cases/controls = 1,033/1,042) and Latino (cases/controls = 1,043/1,057) from the Multiethnic Cohort (MEC). Significant associations from stage I (P < 1.0 × 10(-4)) were examined in silico in GWAS of prostate cancer (stage II) in Japanese (cases/controls = 1,583/3,386) and Europeans (cases/controls = 1,854/1,894).<h4>Results</h4>No novel stage I single-nucleotide polymorphism (SNP) outside of known risk regions reached genome-wide significance. For Japanese, in stage I, the most notable putative novel association was seen with 10 SNPs (P ≤ 8.0 × 10(-6)) at chromosome 2q33; however, this was not replicated in stage II. For Latinos, the most significant association was observed with rs17023900 at the known 3p12 risk locus (stage I: OR = 1.45; P = 7.01 × 10(-5) and stage II: OR = 1.58; P = 3.05 × 10(-7)). The majority of the established risk variants for prostate cancer, 79% and 88%, were positively associated with prostate cancer in Japanese and Latinos (stage I), respectively. The cumulative effects of these variants significantly influence prostate cancer risk (OR per allele = 1.10; P = 2.71 × 10(-25) and OR = 1.07; P = 1.02 × 10(-16) for Japanese and Latinos, respectively).<h4>Conclusion and impact</h4>Our GWAS of prostate cancer did not identify novel genome-wide significant variants. However, our findings show that established risk variants for prostate cancer significantly contribute to risk among Japanese and Latinos.

The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.

Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods.

Recent studies suggest a variety of factors characterize substantia nigra neurons vulnerable to Parkinson's disease, including the transcription factors pituitary homeobox 3 (Pitx3) and orthodenticle homeobox 2 (Otx2) and the trophic factor receptor deleted in colorectal cancer (DCC), but there is limited information on their expression and localization in adult humans. Pitx3, Otx2, and DCC were immunohistochemically localized in the upper brainstem of adult humans and mice and protein expression assessed using relative intensity measures and online microarray data. Pitx3 was present and highly expressed in most dopamine neurons. Surprisingly, in our elderly subjects no Otx2 immunoreactivity was detected in dopamine neurons, although Otx2 gene expression was found in younger cases. Enhanced DCC gene expression occurred in the substantia nigra, and higher amounts of DCC protein characterized vulnerable ventral nigral dopamine neurons. Our data show that, at the age when Parkinson's disease typically occurs, there are no significant differences in the expression of transcription factors in brainstem dopamine neurons, but those most vulnerable to Parkinson's disease rely more on the trophic factor receptor DCC than other brainstem dopamine neurons.

Transposable elements, including short interspersed repetitive elements (SINEs), comprise nearly half the mammalian genome. Moreover, they are a major source of conserved non-coding elements (CNEs), which play important functional roles in regulating development-related genes, such as enhancing and silencing, serving for the diversification of morphological and physiological features among species. We previously reported a novel SINE family, AmnSINE1, as part of mammalian-specific CNEs. One AmnSINE1 locus, named AS071, showed an enhancer property in the developing mouse diencephalon. Indeed, AS071 appears to recapitulate the expression of diencephalic fibroblast growth factor 8 (Fgf8). Here we established three independent lines of AS071-transgenic mice and performed detailed expression profiling of AS071-enhanced lacZ in comparison with that of Fgf8 across embryonic stages. We demonstrate that AS071 is a distal enhancer that directs Fgf8 expression in the developing diencephalon. Furthermore, enhancer assays with constructs encoding partially deleted AS071 sequence revealed a unique modular organization in which AS071 contains at least three functionally distinct sub-elements that cooperatively direct the enhancer activity in three diencephalic domains, namely the dorsal midline and the lateral wall of the diencephalon, and the ventral midline of the hypothalamus. Interestingly, the AmnSINE1-derived sub-element was found to specify the enhancer activity to the ventral midline of the hypothalamus. To our knowledge, this is the first discovery of an enhancer element that could be separated into respective sub-elements that determine regional specificity and/or the core enhancing activity. These results potentiate our understanding of the evolution of retroposon-derived cis-regulatory elements as well as the basis for future studies of the molecular mechanism underlying the determination of domain-specificity of an enhancer.

Betel Quid (BQ) chewing independently contributes to oral, hepatic and esophageal carcinomas. Strong association of breast cancer risk with BQ chewing in Northeast Indian population has been reported where this habit is prodigal. We investigated genomic alterations in breast cancer patients with and without BQ chewing exposure. Twenty six BQ chewers (BQC) and 17 non BQ chewer (NBQC) breast cancer patients from Northeast India were analyzed for genomic alterations and pathway networks using SNP array and IPA. BQC tumors showed significantly (P<0.01) higher total number of alterations, as compared with NBQC tumors, 48 ± 17% versus 32 ± 25 respectively. Incidence of gain in fragile sites in BQC tumors were significantly (P<0.001) higher as compared with NBQC tumors, 34 versus 23% respectively. Two chromosomal regions (7q33 and 21q22.13) were significantly (p<0.05) associated with BQC tumors while two regions (19p13.3-19p12 and 20q11.22) were significantly associated with NBQC tumors. GO terms oxidoreductase and aldo-keto reductase activity in BQC tumors in contrast to G-protein coupled receptor protein signaling pathway and cell surface receptor linked signal transduction in NBQC tumors were enriched in DAVID. One network "Drug Metabolism, Molecular Transport, Nucleic Acid Metabolism" including genes AKR1B1, AKR1B10, ETS2 etc in BQC and two networks "Molecular Transport, Nucleic Acid Metabolism, Small Molecule Biochemistry" and "Cellular Development, Embryonic Development, Organismal Development" including genes RPN2, EMR3, VAV1, NNAT and MUC16 etc were seen in NBQC. Common alterations (>30%) were seen in 27 regions. Three networks were significant in common regions with key roles of PTK2, RPN2, EMR3, VAV1, NNAT, MUC16, MYC and YWHAZ genes. These data show that breast cancer arising by environmental carcinogens exemplifies genetic alterations differing from those observed in the non exposed ones. A number of genetic changes are shared in both tumor groups considered as crucial in breast cancer progression.

No abstract available.

Autism spectrum disorder (ASD) is a complex disorder that appears to be caused by interactions between genetic changes and environmental insults during early development. A wide range of factors have been linked to the onset of ASD, but recently both genetic associations and environmental factors point to a central role for immune-related genes and immune responses to environmental stimuli. Specifically, many of the proteins encoded by the major histocompatibility complex (MHC) play a vital role in the formation, refinement, maintenance, and plasticity of the brain. Manipulations of levels of MHC molecules have illustrated how disrupted MHC signaling can significantly alter brain connectivity and function. Thus, an emerging hypothesis in our field is that disruptions in MHC expression in the developing brain caused by mutations and/or immune dysregulation may contribute to the altered brain connectivity and function characteristic of ASD. This review provides an overview of the structure and function of the three classes of MHC molecules in the immune system, healthy brain, and their possible involvement in ASD.

Recurrent pregnancy loss (RPL) is a multifactorial condition. The effect of antithrombin (SERPINC1), protein C (PROC), thrombomodulin (THBD) and tissue factor pathway inhibitor (TFPI) single nucleotide polymorphisms (SNPs) on the risk of RPL is thus far unknown. Our objective was to determine the association of SNPs in the above mentioned genes with RPL. We included 117 non-pregnant women with three or more consecutive losses prior to 20 weeks of pregnancy without a previous history of carrying a fetus to viability, and 264 healthy fertile non-pregnant women who had at least two term deliveries and no known pregnancy losses. The PROC (rs1799809 and rs1799808), SERPINC1 (rs2227589), THBD (rs1042579) and TFPI (rs10931292, rs8176592 and rs10153820) SNPs were analysed by Real Time PCR. Genotype frequencies for PROC 2418A>G, PROC 2405C>T, THBD 1418C>T, TFPI (T-33C and TFPI C-399T) SNPs were similar in cases and controls. The carriers of SERPINC1 786A allele (GA + AA genotypes) had an increased risk for RPL (odds ratio [OR]: 1.77, 95% confidence interval [CI]: 1.05-3.00, p= 0.034) while women carrying the TFPI -287C allele (TC + CC genotypes) had a protection effect on having RPL (OR: 0.46, 95% CI: 0.26-0.83, p= 0.009). The TCC haplotype for TFPI T-33C/ TFPI T-287C/ TFPI C-399T SNPs was less frequent in cases (5.7%) than in controls (11.6%) (OR: 0.45, 95% CI: 0.23-0.90, p= 0.025). In conclusion, our data indicate that SERPINC1 786G>A variant increases the risk for RPL, while TFPI T-287C variant is protective; however, further studies are required to confirm our findings.

Both neuroprotective and neurotoxic roles have previously been described for histone deacetylase-1 (HDAC1). Here we report that HDAC1 expression is elevated in vulnerable brain regions of two mouse models of neurodegeneration, the R6/2 model of Huntington disease and the Ca(2+)/calmodulin-dependent protein kinase (CaMK)/p25 double-transgenic model of tauopathic degeneration, suggesting a role in promoting neuronal death. Indeed, elevating HDAC1 expression by ectopic expression promotes the death of otherwise healthy cerebellar granule neurons and cortical neurons in culture. The neurotoxic effect of HDAC1 requires interaction and cooperation with HDAC3, which has previously been shown to selectively induce the death of neurons. HDAC1-HDAC3 interaction is greatly elevated under conditions of neurodegeneration both in vitro and in vivo. Furthermore, the knockdown of HDAC3 suppresses HDAC1-induced neurotoxicity, and the knockdown of HDAC1 suppresses HDAC3 neurotoxicity. As described previously for HDAC3, the neurotoxic effect of HDAC1 is inhibited by treatment with IGF-1, the expression of Akt, or the inhibition of glycogen synthase kinase 3β (GSK3β). In addition to HDAC3, HDAC1 has been shown to interact with histone deacetylase-related protein (HDRP), a truncated form of HDAC9, whose expression is down-regulated during neuronal death. In contrast to HDAC3, the interaction between HDRP and HDAC1 protects neurons from death, an effect involving acquisition of the deacetylase activity of HDAC1 by HDRP. We find that elevated HDRP inhibits HDAC1-HDAC3 interaction and prevents the neurotoxic effect of either of these two proteins. Together, our results suggest that HDAC1 is a molecular switch between neuronal survival and death. Its interaction with HDRP promotes neuronal survival, whereas interaction with HDAC3 results in neuronal death.

KSHV is the etiological agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentricCastleman's disease (MCD). The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHV miRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3'UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.

Only one low-density lipoprotein cholesterol (LDL-C) genome-wide association study (GWAS) has been previously reported in -African Americans. We performed a GWAS of LDL-C in African Americans using data extracted from electronic medical records (EMR) in the eMERGE network. African Americans were genotyped on the Illumina 1M chip. All LDL-C measurements, prescriptions, and diagnoses of concomitant disease were extracted from EMR. We created two analytic datasets; one dataset having median LDL-C calculated after the exclusion of some lab values based on comorbidities and medication (n= 618) and another dataset having median LDL-C calculated without any exclusions (n= 1,249). SNP rs7412 in APOE was strongly associated with LDL-C in both datasets (p < 5 × 10(-8) ). In the dataset with exclusions, a decrease of 20.0 mg/dL per minor allele was observed. The effect size was attenuated (12.3 mg/dL) in the dataset without any lab values excluded. Although other signals in APOE have been detected in previous GWAS, this large and important SNP association has not been well detected in large GWAS because rs7412 was not included on many genotyping arrays. Use of median LDL-C extracted from EMR after exclusions for medications and comorbidities increased the percentage of trait variance explained by genetic variation.

Crystallization of struvite from artificial urine in the presence of Proteus mirabilis microorganisms depends strongly on pH value. At small value of pH, struvite yields crystals of coffin-like habit with very specific structure. The analysis using scanning electron microscopy shows that the crystals possess well-defined faces, but higher magnifications show very specific structuration as if the crystals were built from small three-dimensional subunits. The possible role of microorganisms in the formation of such a structuration is analyzed. At higher pH value, the crystals exhibit dendritic growth with main trunk and branches. Although the formation mechanism of the specific structuration as well as dendritic structures is unknown, the nature of forces for such an alignment is analyzed. The revealed porous internal structure of struvite is also analyzed. The investigations provide evidence for the importance of biological regulation in crystallization process.

<h4>Background</h4>Gankyrin was originally purified and characterized as the p28 component of the 26S proteasome, and later identified as an oncogenic protein in hepatocellular carcinomas (HCC). It has recently been found to be highly expressed in several other malignancies, and compelling evidence show gankyrin plays important roles in tumorigenesis. However, its mechanism of action remains unclear.<h4>Methods</h4>In order to further clarify the functions of gankyrin and better understand its molecular mechanisms, we generated a gankyrin null cell line, HCT116 gankyrin-/- , by targeted homologous recombination in human colon cancer cells, and then employed two-dimensional electrophoresis (2-DE) based proteomic approaches followed by MS identification to investigate alterations in the proteome due to the gankyrin knockout. Western blot and qRT-PCR assays were also used to examine the protein and mRNA levels of some identified proteins.<h4>Results</h4>Compared with wild-type control cells, gankyrin null cells were impaired in terms of their proliferation, migration and anchorage-independent growth. A total of 21 altered proteins were identified, which included 18 proteins that had not previously been reported to be related to gankyrin. Notably, eight metastasis-related proteins were identified. Western blot analyses confirmed that the changes in three examined proteins were consistent with 2-DE gel analysis.<h4>Conclusions</h4>In summary, we have generated a useful cell tool to clarify the functions of gankyrin. Our proteomic data provide novel information to better understand the roles and underlying mechanisms by which gankyrin is involved in tumorigenesis and cancer metastasis.

d-Propranolol (d-Pro: 2-8 mg·(kg body mass)(-1)·day(-1)) protected against cardiac dysfunction and oxidative stress during 3-5 weeks of iron overload (2 mg Fe-dextran·(g body mass)(-1)·week(-1)) in Sprague-Dawley rats. At 3 weeks, hearts were perfused in working mode to obtain baseline function; red blood cell glutathione, plasma 8-isoprostane, neutrophil basal superoxide production, lysosomal-derived plasma N-acetyl-β-galactosaminidase (NAGA) activity, ventricular iron content, and cardiac iron deposition were assessed. Hearts from the Fe-treated group of rats exhibited lower cardiac work (26%) and output (CO, 24%); end-diastolic pressure rose 1.8-fold. Further, glutathione levels increased 2-fold, isoprostane levels increased 2.5-fold, neutrophil superoxide increased 3-fold, NAGA increased 4-fold, ventricular Fe increased 4.9-fold; and substantial atrial and ventricular Fe-deposition occurred. d-Pro (8 mg) restored heart function to the control levels, protected against oxidative stress, and decreased cardiac Fe levels. After 5 weeks of Fe treatment, echocardiography revealed that the following were depressed: percent fractional shortening (%FS, 31% lower); left ventricular (LV) ejection fraction (LVEF, 17%), CO (25%); and aortic pressure maximum (P(max), 24%). Mitral valve E/A declined by 18%, indicating diastolic dysfunction. Cardiac CD11b+ infiltrates were elevated. Low d-Pro (2 mg) provided modest protection, whereas 4-8 mg greatly improved LVEF (54%-75%), %FS (51%-81%), CO (43%-78%), P(max) (56%-100%), and E/A >100%; 8 mg decreased cardiac inflammation. Since d-Pro is an antioxidant and reduces cardiac Fe uptake as well as inflammation, these properties may preserve cardiac function during Fe overload.

Macrophage-induced bystander effects have been implicated as an important mediator of chromosomal instability and colon cancer triggered by Enterococcus faecalis, a human intestinal commensal bacteria. There is little understanding about how inflammatory cytokines mediate bystander effects, but questions in this area are important because of the pivotal contributions made by inflammatory processes to cancer initiation and progression. Here, we report that the central proinflammatory cytokine TNF-α acts as a diffusible mediator of the bystander effects induced by macrophages, an effect caused by a proliferation of macrophages that trigger epithelial cell production of Netrin-1, a neuronal guidance molecule. TNF-α-mediated bystander assays used a murine coculture system of primary colonic epithelial cells and E. faecalis-infected macrophages (in vitro), with an interleukin 10 (IL-10)-deficient mouse model of colon cancer that involves long-term colonization with E. faecalis (in vivo). In cell cocultures, we observed increased expression of the TNF-α receptor Tnfrsf1b and Netrin-1. These effects were blocked by anti-TNF-α antibody or by pretreatment with an inhibitor of NF-κB signaling. RNAi-mediated attenuation of Tnfrsf1b decreased TNF-α-induced netrin-1 production and augmented epithelial cell apoptosis in culture. Extending these observations, colon biopsies from E. faecalis-colonized IL-10(-/-) mice exhibited crypt hyperplasia and increased staining for macrophages, TNF-α, netrin-1, NF-κB, Tnfrsf1b, and the proliferation marker proliferating cell nuclear antigen while also displaying a reduction in epithelial cell apoptosis. Together, our results define a pathway for macrophage-induced bystander effects in which TNF-α triggers TNFRSF1b receptor signaling leading to increased production of Netrin-1, crypt hyperplasia, and decreased epithelial cell apoptosis. In elucidating an important commensal-associated proinflammatory mechanism in the intestinal microenvironment, our work highlights the role of Netrin-1 and a specific TNF-α receptor as candidate targets to prevent or treat colorectal cancer.

<h4>Background</h4>Atopic dermatitis (AD) and psoriasis are common inflammatory diseases canonically described as involving distinct T(H) polarization and granulocytic infiltration. Acute AD lesions are associated with T(H)2 and eosinophilic inflammation, whereas psoriatic lesions are associated with T(H)1/T(H)17 and neutrophilic inflammation. Despite intensive investigation, these pathways remain incompletely understood in vivo in human subjects.<h4>Objective</h4>Using AD and psoriatic lesional skin as exemplar T(H)2 and T(H)1/T(H)17 diseased tissue, we sought to clarify common and unique molecular and pathophysiologic features in inflamed skin with different types of inflammatory polarization.<h4>Methods</h4>We conducted gene expression microarray analyses to identify distinct and commonly dysregulated expression in AD (based on Hanifin and Rajka criteria) and psoriatic lesions. We defined gene sets (GSs) as comprising genes encoding cytokines, chemokines, and growth factors that were uniquely or jointly dysregulated in patients with AD and those with psoriasis and calculated aggregate GS expression scores for lesional skin of patients with these dermatoses and healthy control skin.<h4>Results</h4>The atopic dermatitis gene set (AD-GS) score correlated with systemic and local measures of allergic inflammation, including serum IgE levels, blood eosinophil counts, and tissue eosinophil counts. Unexpectedly, genes encoding neutrophil chemoattractants among the common GS were highly expressed in AD lesional skin. Hematoxylin and eosin and immunohistochemical analyses showed the numbers of neutrophils in AD lesional skin were comparable with those in psoriatic lesional skin, and both were correlated with the extent of expression of neutrophil chemoattractant genes.<h4>Conclusion</h4>These data are evidence that neutrophilic inflammation is a feature of lesional AD pathology comorbid with allergic inflammation.

<h4>Background</h4>A high prevalence of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) infections have been reported among persons with severe mental illness. In October, 2009, the Cook County Department of Public Health (CCDPH) initiated an investigation following notification of a cluster of HBV infections among mentally ill residents at a long term care facility (LTCF).<h4>Methods</h4>LTCF staff were interviewed and resident medical records were reviewed. Residents were offered testing for HBV, HCV, and HIV. Serum specimens from residents diagnosed with HBV or HIV infection were sent to the Centers for Disease Control and Prevention (CDC) for analysis.<h4>Results</h4>Eleven newly diagnosed HBV infections were identified among mentally ill residents at the LTCF. Of these 11 infections, 4 serum specimens were available for complete HBV genome sequencing; all 4 genomes were found to be closely related. Four newly diagnosed HIV infections were identified within this same population. Upon molecular analysis, 2 of 4 HIV sequences from these new infections were found to be nearly identical and formed a tight phylogenetic cluster.<h4>Conclusions</h4>HBV and HIV transmission was identified among mentally ill residents of this LTCF. Continued efforts are needed to prevent bloodborne pathogen transmission among mentally ill residents in LTCFs.

Multicomponent RNA-peptide complexes are attractive from the viewpoint of artificial design of functional biomacromolecular systems. We have developed self-folding and self-assembling RNAs that serve as templates to assist chemical ligation between two reactive peptides with RNA-binding capabilities. The design principle of previous templates, however, can be applied only to limited classes of RNA-binding peptides. In this study, we employed a two-piece derivative of a group I intron RNA from the Tetrahymena large subunit ribosomal RNA (LSU rRNA) as a platform for new template RNAs. In this group I intron-based self-assembling platform, modules for the recognition of substrate peptides can be installed independently from modules holding the platform structure. The new self-assembling platform allows us to expand the repertoire of substrate peptides in template RNA design.

<h4>Introduction</h4>Transfusion in trauma is often empiric or based on traditional lab tests. Viscoelastic tests such as thromboelastography (TEG®) and rotational thromboelastometry (ROTEM®) have been proposed as superior to traditional lab tests. Due to the similarities between the two tests, general opinion seems to consider them equivalent with interchangeable interpretations. However, it is not clear whether the results can be similarly interpreted. This review evaluates the comparability between TEG and ROTEM and performs a descriptive review of the parameters utilized in each test in adult trauma patients.<h4>Methods</h4>PUBMED database was reviewed using the keywords "thromboelastography" and "compare", between 2000 and 2011. Original studies directly comparing TEG® with ROTEM® in any area were retrieved. To verify the individual test parameter used in studies involving trauma patients, we further performed a review using the keywords "thromboelastography" and "trauma" in the PUBMED database.<h4>Results</h4>Only 4 studies directly compared TEG® with ROTEM®. One in liver transplantation found that transfusion practice could differ depending on the device in use. Another in cardiac surgery concluded that all measurements are not completely interchangeable. The third article using commercially available plasma detected clinically significant differences in the results from the two devices. The fourth one was a head-to-head comparison of the technical aspects. The 24 articles reporting the use of viscoelastic tests in trauma patients, presented considerable heterogeneity.<h4>Conclusion</h4>Both tests are potentially useful as means to rapidly diagnose coagulopathy, guide transfusion and determine outcome in trauma patients. Differences in the activators utilized in each device limit the direct comparability. Standardization and robust clinical trials comparing the two technologies are needed before these tests can be widely recommended for clinical use in trauma.

Large-scale protein-protein interaction data sets have been generated for several species including yeast and human and have enabled the identification, quantification, and prediction of cellular molecular networks. Affinity purification-mass spectrometry (AP-MS) is the preeminent methodology for large-scale analysis of protein complexes, performed by immunopurifying a specific "bait" protein and its associated "prey" proteins. The analysis and interpretation of AP-MS data sets is, however, not straightforward. In addition, although yeast AP-MS data sets are relatively comprehensive, current human AP-MS data sets only sparsely cover the human interactome. Here we develop a framework for analysis of AP-MS data sets that addresses the issues of noise, missing data, and sparsity of coverage in the context of a current, real world human AP-MS data set. Our goal is to extend and increase the density of the known human interactome by integrating bait-prey and cocomplexed preys (prey-prey associations) into networks. Our framework incorporates a score for each identified protein, as well as elements of signal processing to improve the confidence of identified protein-protein interactions. We identify many protein networks enriched in known biological processes and functions. In addition, we show that integrated bait-prey and prey-prey interactions can be used to refine network topology and extend known protein networks.

A methyl-deficient diet (MD) lacking folic acid and the associated methyl donors choline and methionine, fed to the laboratory rat during the periods of oocyte and embryo development, has been shown to programme glucose metabolism in the offspring. The hepatic proteome of the male offspring of female rats fed MD diets for 3 weeks prior to mating and for the first 5 days of gestation has been examined by 2-dimensional gel electrophoresis. Three groups of differentially abundant proteins associated with energy metabolism, amino acid metabolism and antioxidant defence were identified in the soluble proteins extracted from the liver from the MD offspring at both 6 and 12 months of age. Altered mitochondrial activity in other programming models leads to a similar pattern of differential protein abundance. Two of the differentially abundant proteins were identified as GAPDH and PGK-1 by mass spectrometry. Western blotting showed that there were multiple isoforms of both proteins with similar molecular weights but different isoelectric points. The differentially abundant spots reduced in the MD offspring corresponded to minor isoforms of GAPDH and PGK-1. The levels of PPAR-alpha, SREBP and glucocorticoid receptor mRNAs associated with other models of prenatal programming were unchanged in the MD offspring. The data suggest that a diet deficient in folic acid and associated methyl donors fed during the peri-conception and early preimplantation periods of mammalian development affects mitochondrial function in the offspring and that the posttranslational modification of proteins may be important.

The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 phenotype is characterized by cerebellar ataxia, neuropathy and slow saccades. SCA2 foreshortens life span and is currently without symptomatic or disease-modifying treatments. Identifying function-specific therapeutics for SCA2 is problematic due to the limited knowledge of ATXN2 function. As SCA2 is likely caused by a gain-of-toxic or gain-of-normal function like other polyglutamine disorders, targeting ATXN2 expression may represent a valid therapeutic approach. This study characterized aspects of ATXN2 expression control using an ATXN2 promoter-luciferase (luc) reporter construct. We verified the fidelity of construct expression by generating transgenic mice expressing the reporter construct. High reporter expression was seen in the cerebellum and olfactory bulb in vivo but there was relatively low expression in other tissues, similar to the expression of endogenous ataxin-2. We verified the second of two possible start codons as the functional start codon in ATXN2. By evaluating deletions in the ATXN2 promoter, we identified an E-twenty six (ETS)-binding site required for ATXN2 expression. We verified that endogenous ETS1 interacted with the ATXN2 promoter by an electromobility supershift assay and chromatin immunoprecipitation polymerase chain reaction. ETS1 overexpression increased ATXN2-luc (ATXN2-luciferase) as well as endogenous ATXN2 expression. Deletion of the putative ETS1-binding site abrogated the effects on the expression of ATXN2-luc. A dominant negative ETS1 and an ETS1 short-hairpin RNA both reduced ATXN2-luc expression. Our study broadens the understanding on the transcriptional control of ATXN2 and reveals specific regulatory features of the ATXN2 promoter that can be exploited therapeutically.

<h4>Objectives</h4>Allogeneic blood transfusion and reoperation for postoperative bleeding after the coronary artery bypass grafting have a negative impact on the patient outcome. This study aimed at evaluating the effects of reduced doses of heparin and protamine on the patient outcome, using a heparin-coated mini-cardiopulmonary bypass (CPB) system.<h4>Methods</h4>Sixty patients undergoing elective first-time CPB were prospectively randomized either to have a reduced systemic heparinization [activated clotting time (ACT) = 250 s] or to a control group perfused with a full heparin dose (ACT = 420 s). Blood transfusions, ventilation time, early postoperative bleeding, ICU stay, reoperations for bleeding, postoperative cognitive status and the level of mobilization were registered.<h4>Results</h4>Twenty-nine patients were randomized to the control group, 27 patients to the low-dose group and 4 patients were excluded because of protocol violations. Four patients in the control group received a total of 10 units of packed red blood cells, and in the low-dose group, no transfusions were given, P = 0.046. No patient was reoperated because of bleeding. The ICU stay was significantly shorter in the low-dose group (8.4 vs 13.7 h, P = 0.020), less dependent on oxygen on the first postoperative day (78 vs 97%, P = 0.034), better mobilized (89 vs 59%, P = 0.006) and had less pain (visual analogue scale 2.0 vs 3.5, P = 0.019) compared with the control group.<h4>Conclusions</h4>The use of a mini-CPB system combined with a low dose of heparin reduced the need for blood transfusions and may facilitate the faster mobilization of the patients.

Netrin-1 is a well-characterised chemoattractant involved in neuronal guidance in the developing enteric nervous system (ENS), but it is also a regulator of tumorigenesis. Two of its well-characterised receptors, deleted in colorectal cancer (DCC) and uncoordinated-5 homolog (UNC-5H), belong to a family of dependence receptors that transmit either pro- or anti-apoptosis signals depending on the availability of ligand, in this case netrin-1. This review summarises these two effects of netrin-1 and highlights the additional research needed information about to allow better utilisation of netrin-1 as a therapeutic target for axonal regeneration in the context of colorectal cancer.

<h4>Background</h4>Dupuytren's contracture (DC) and HFE hemochromatosis occur in some of the same at-risk populations and present with similar comorbid conditions.<h4>Methods</h4>We estimated DC prevalence in two cohorts of white Alabama hemochromatosis probands (294 C282Y homozygotes, 67 C282Y/H63D compound heterozygotes) in a retrospective study. We performed logistic regressions on DC using the following independent variables: age, body mass index, heavy ethanol consumption, serum ferritin, elevated serum AST/ALT, non-alcoholic fatty liver disease, viral hepatitis, cirrhosis, and diabetes.<h4>Results</h4>One man and two women with C282Y homozygosity had DC (prevalence 1.02%; 95% CI 0.35%-2.96%). A man with C282Y/H63D had DC (prevalence 1.49%; 95% CI 0.26%-7.98%). DC occurred as an autosomal dominant trait in his kinship. In regression analyses, no single variable predicted DC. We observed no new DC cases after the diagnosis of hemochromatosis (mean follow-up 12.9 ± 7.5 years (1 SD), and 9.0 ± 5.1 years, respectively).<h4>Conclusions</h4>Our prevalence estimates of DC in white Alabama hemochromatosis probands are similar to those found in the white US population cohorts. DC risk was unrelated to the variables we studied.

The cyclic peptide core of the antifungal and antibiotic cyclic depsipeptide LI-F04a was synthesised by using a modified Yamaguchi macrolactonization approach. Alternative methods of macrolactonization (e.g., Corey-Nicolaou) resulted in significant epimerization of the C-terminal amino acid during the cyclization reaction. The D-stereochemistry of the alanine residue in the naturally occurring cyclic peptide may be required for the antifungal activity of this natural product.

It is not known how 5-HTTLPR genotype x childhood adversity (CA) interactions that are associated with an increased risk for affective disorders in population studies operate at the neural systems level. We hypothesized that healthy adolescents at increased genetic and environmental risk for developing mood disorders (depression and anxiety) would demonstrate increased amygdala reactivity to emotional stimuli compared to those with only one such risk factor or those with none. Participants (n=67) were classified into one of 4 groups dependent on being homozygous for the long or short alleles within the serotonin-transporter-linked polymorphic region (5-HTTLPR) of the SLC6A4 gene and exposure to CA in the first 11 years of life (present or absent). A functional magnetic resonance imaging investigation was undertaken which involved viewing emotionally-salient face stimuli. In addition, we assessed the role of other variables hypothesized to influence amygdala reactivity, namely recent negative life-events (RNLE) assessed at ages 14 and 17, current anxiety symptoms and psychiatric history. We replicated prior findings demonstrating moderation by gene variants in 5-HTTLPR, but found no support for an effect of CA on amygdala reactivity. We also found a significant effect of RNLE aged 17 with amygdala reactivity demonstrating additive, but not interactive effects with 5-HTTLPR. A whole-brain analysis found a 5-HTTLPR×CA interaction in the lingual gyrus whereby CA appears to differentially modify neural reactivity depending on genotype. These results demonstrate that two different forms of environmental adversities interplay with 5-HTTLPR and thereby differentially impact amygdala and cortical reactivity.

Cellular apoptosis susceptibility (chromosome segregation 1-like, CSE1L) gene maps to chromosomal region 20q13.13, a region frequently amplified in solid tumours. In this study, we investigated the roles played by CSE1L in colorectal cancer by examining CSE1L expression and clinico-pathological parameters in colorectal cancer and investigating the effect of CSE1L on the viability, adhesion and migration of colorectal cancer cells. RT-PCR showed that CSE1L mRNA was over-expressed in colorectal cancer. CSE1L depletion by knock-down with CSE1L-specific siRNA significantly reduced viability in HCT116 cells (p = 0.004) and SW480 cells (p = 0.003) whilst significantly increasing the proportion of apoptotic HCT116 cells (p < 0.001) and SW480 cells (p < 0.001). Furthermore, CSE1L depletion significantly reduced the adhesive capacity of HCT116 (p = 0.003) and SW480 cells (p = 0.004). Analysis by qRT-PCR following CSE1L siRNA treatment of HCT116 and SW480 cells showed significant modulation of key apoptotic (p53, p73 and BAK) and adhesive (E-cadherin, Ep-CAM and ICAM-1) molecules. Immunohistochemistry of a colorectal cancer tissue microarray showed that CSE1L had a significantly increased level in colorectal cancer compared to normal colorectal epithelium (p < 0.001). There were significant decreases in both nuclear (p = 0.006) and cytoplasmic (p = 0.003) staining of CSE1L in tumours with lymph node metastasis (stage 3 tumours) compared with lymph node-negative tumours (stage 1 and 2 tumours). In lymph node-negative patients, poor survival was associated with increased CSE1L cytoplasmic expression (p = 0.042). These results indicate that CSE1L is associated with viability and apoptosis, cellular adhesion and invasion, thus implicating CSE1L in the progression of colorectal cancer.

<h4>Background</h4>Genetic selection of Atlantic salmon families better adapted to alternative feed formulations containing high levels of vegetable ingredients has been suggested to ensure sustainable growth of aquaculture. The present study aimed to identify molecular pathways that could underlie phenotypic differences in flesh n-3 long-chain polyunsaturated fatty acid (LC-PUFA) levels when fish are fed vegetable oil diets. Liver transcriptome was analyzed and compared in four families presenting higher or lower n-3 LC-PUFA contents at two contrasting flesh total lipid levels.<h4>Results</h4>The main effect of n-3 LC-PUFA contents was in the expression of immune response genes (38% of all significantly affected genes), broadly implicated in the modulation of inflammatory processes and innate immune response. Although genetic evaluations of traits used in the breeding program revealed that the chosen families were not balanced for viral disease resistance, this did not fully explain the preponderance of immune response genes in the transcriptomic analysis. Employing stringent statistical analysis no lipid metabolism genes were detected as being significantly altered in liver when comparing families with high and low n-3 LC-PUFA flesh contents. However, relaxing the statistical analysis enabled identification of potentially relevant effects, further studied by RT-qPCR, in cholesterol biosynthesis, lipoprotein metabolism and lipid transport, as well as eicosanoid metabolism particularly affecting the lipoxygenase pathway. Total lipid level in flesh also showed an important effect on immune response and 8% of significantly affected genes related to lipid metabolism, including a fatty acyl elongase (elovl2), an acyl carrier protein and stearoyl-CoA desaturase.<h4>Conclusions</h4>Inter-family differences in n-3 LC-PUFA content could not be related to effects on lipid metabolism, including transcriptional modulation of the LC-PUFA biosynthesis pathway. An association was found between flesh adiposity and n-3 LC-PUFA in regulation of cholesterol biosynthesis, which was most likely explained by variation in tissue n-3 LC-PUFA levels regulating transcription of cholesterol metabolism genes through srebp2. A preponderance of immune response genes significantly affected by n-3 LC-PUFA contents could be potentially associated with disease resistance, possibly involving anti-inflammatory actions of tissue n-3 LC-PUFA through eicosanoid metabolism. This association may have been fortuitous, but it is important to clarify if this trait is included in future salmon breeding programmes.

<h4>Background</h4>The outcome of severely injured or ill patients can be time dependent. Short activation and approach times for emergency medical service (EMS) units are widely recognized to be important quality indicators. The use of a helicopter emergency medical service (HEMS) can significantly shorten rescue missions especially in mountainous areas. We aimed to analyze the HEMS characteristics that influence the activation and approach times.<h4>Methods</h4>In a multi-centre retrospective study, we analyzed 6121 rescue missions from nine HEMS bases situated in mountainous regions of four European countries.<h4>Results</h4>We found large differences in mean activation and approach times among HEMS bases. The shortest mean activation time was 2.9 minutes; the longest 17.0 minutes. The shortest mean approach time was 10.4 minutes; the longest 45.0 minutes. Short times are linked (p < 0.001) to the following conditions: helicopter operator is not state owned; HEMS is integrated in EMS; all crew members are at the same location; doctors come from state or private health institutions; organization performing HEMS is privately owned; helicopters are only for HEMS; operation area is around 10.000 km2; HEMS activation is by a dispatching centre of regional government who is in charge of making decisions; there is only one intermediator in the emergency call; helicopter is equipped with hoist or fixed line; HEMS has more than one base with helicopters, and one team per base; closest neighboring base is 90 km away; HEMS is about 20 years old and has more than 650 missions per year; and modern helicopters are used.<h4>Conclusions</h4>An improvement in HEMS activation and approach times is possible. We found 17 factors associated with shorter times.

<h4>Unlabelled</h4><h4>Background</h4>A giant congenital melanocytic nevus (GCMN) is a malformation of the pigment cells. It is a distress to the patients for two reasons: one is disfigurement, and the other is the possibility of malignant changes. However, the underlying mechanisms of the development of GCMN and melanotumorigenesis in GCMN are unknown. Hence, the aim of this study was to identify the proteomic alterations and associated functional pathways in GCMN.<h4>Results</h4>Proteomic differences between GCMN (n = 3) and normal skin samples (n = 3) were analyzed by one-dimensional-liquid chromatography-tandem mass spectrometry Relative levels of the selected proteins were validated using western blot analysis. The biological processes associated with the abundance modified proteins were analyzed using bioinformatic tools. Among the 46 abundance modified proteins, expression of 4 proteins was significantly downregulated and expression of 42 proteins was significantly upregulated in GCMN compared to normal skin samples (p < 0.05). More importantly, 31% of the upregulated proteins were implicated in various cancers, with five proteins being specifically related with melanoma. The abundance modified proteins in GCMN were involved in the biological processes of neurotrophin signaling, melanosome, and downregulated of MTA-3 in ER-negative breast tumors. In particular, an increase in the expression of the 14-3-3 protein family members appeared to be associated with key cellular biological functions in GCMN. Western blot analysis confirmed the upregulation of 14-3-3epsilon, 14-3-3 tau, and prohibitin in GCMN.<h4>Conclusion</h4>These findings suggest that GCMN exhibits potential proteomic alterations, which may play a role in melanotumorigenesis, and the significant alteration of 14-3-3 family proteins could be a key regulator of the biological pathway remodeling in GCMN.

We examined the relationship between a functional polymorphism of the serotonin transporter gene (5-HTTLPR) and individual differences in emotional reactivity in two laboratory studies. In Study 1, empathic responding and physiological reactivity to viewing films of others in distress were assessed in healthy adults in three age groups. In Study 2, emotional responding to watching oneself in an embarrassing situation was assessed in healthy adults and in patients with neurodegenerative diseases. In Study 1, participants with two short alleles of 5-HTTLPR reported more personal distress and showed higher levels of physiological responses in response to the films than participants with long alleles. In Study 2, participants with two short alleles reported more anger and amusement and displayed more emotional expressive behaviors in response to the embarrassing situation than participants with long alleles. These two findings from diverse samples of participants converge to indicate that individuals who are homozygous for the short allele variant of 5-HTTLPR have greater levels of emotional reactivity in two quite different socially embedded contexts.

As the understanding of the genetic basis of the inherited thrombophilias has increased over recent years, their routine diagnostic genetic analysis has also matured. This review considers methods used to test for the factor V (F5) Leiden mutation and prothrombin 20210A (F2 c.*97G>A) allele, and analysis of the SERPINC1, PROC, and PROS1 genes in cases of antithrombin, protein C (PC), and protein S (PS) deficiency, respectively. Issues relating to quality are explored, highlighting where analytical and sample handling errors may occur. Detection of the factor V Leiden mutation and the prothrombin c.*97G>A allele are best performed using real-time polymerase chain reaction analysis as this relatively simple technique allows their discrimination from rare variants of neighboring nucleotides; not possible using the more time-consuming restriction digestion assays. With the advent of low-cost and high-throughput sequence analysis, direct sequencing has become the first-line method to provide a definitive diagnosis of inherited, rather than acquired, deficiencies. Large cohort studies have shown that antithrombin and PC mutations are identified in between 61 and 87% of patients, whereas the detection rate in PS deficiency is substantially lower in around 40% of patients. Large gene deletions make up between 7 and 10% of PS and antithrombin mutations and only 1% of PC mutations, but it is suggested that dosage analysis techniques such as multiplex ligation-dependent probe amplification should be used for all three genes as part of routine analysis to ensure mutations are not missed. Best practice guidelines are available from EuroGentest covering a wide variety of the issues raised in this review and all laboratories should participate in appropriate external quality assurance schemes to ensure they continue to offer high quality service.

Regulation of transposable elements (TEs) is critical to the integrity of the host genome. The fission yeast Schizosaccharomyces pombe homologs of mammalian CENP-B perform a host genome surveillance role by controlling Tf2 long terminal repeat (LTR) retrotransposons. However, the mechanisms by which CENP-Bs effect their functions are ill defined. Here, we show that the multifaceted roles of Abp1, the prominent member of fission yeast CENP-Bs, are mediated in part via recognition of a 10-bp AT-rich motif present in most LTRs and require the DNA-binding, transposase, and dimerization domains of Abp1 to maintain transcriptional repression and genome organization. Expression profiling analyses indicated that Abp1 recruits class I/II histone deacetylases (HDACs) to repress Tf2 retrotransposons and genes activated in response to stresses. We demonstrate that class I/II HDACs and sirtuins mediate the clustering of dispersed Tf2 retrotransposons into Tf bodies. Intriguingly, we uncovered an unexpected cooperation between Abp1 and the histone H3K4 methyltransferase Set1 in regulating sense and antisense transcriptional silencing of Tf2 retrotransposons and Tf body integrity. Moreover, Set1-mediated regulation of Tf2 expression and nuclear organization appears to be largely independent of H3K4 methylation. Our study illuminates a molecular pathway involving a transposase-containing transcription factor that cooperates with chromatin modifiers to regulate TE activities.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin β1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin β1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin β1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin β1 action and actin functions.

Viral double-stranded RNA (dsRNA) activates protein kinase R (PKR), which phosphorylates eIF2α and inhibits translation. In response, viruses have evolved various strategies to evade the antiviral impact of PKR. We investigated whether guinea pig cytomegalovirus (GPCMV), a useful model of congenital CMV infection, encodes a gene that interferes with the PKR pathway. Using a proteomic screen, we identified several GPCMV dsRNA-binding proteins, among which only gp145 rescued replication of a vaccinia virus mutant that lacks E3L. gp145 also reversed the inhibitory effects of PKR on expression of a cotransfected reporter gene. Mapping studies demonstrated that the gp145 dsRNA-binding domain has homology to the PKR antagonists of other CMVs. However, dsRNA-binding by gp145 is not sufficient for it to block PKR. gp145 differs from the PKR antagonists of murine CMV in that it functions alone and from those encoded by human CMV in functioning in cells from both primates and rodents.

The ventrolateral dermomyotome gives rise to all muscles of the limbs through the delamination and migration of cells into the limb buds. These cells proliferate and form myoblasts, withdraw from the cell cycle and become terminally differentiated. The myogenic lineage colonizes pre-patterned regions to form muscle anlagen as muscle fibers are assembled. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Gene expression arrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors, labeled by Lbx1(EGFP), which resulted from the loss of Pitx2. Target genes of Pitx2 were clustered using the "David Bioinformatics Functional Annotation Tool" to bin genes according to enrichment of gene ontology keywords. This provided a way to both narrow the target genes and identify potential gene families regulated by Pitx2. Representative target genes in the most enriched bins were analyzed for the presence and evolutionary conservation of Pitx2 consensus binding sequence, TAATCY, on the -20kb, intronic, and coding regions of the genes. Fifteen Pitx2 target genes were selected based on the above analysis and were identified as having functions involving cytoskeleton organization, tissue specification, and transcription factors. Data from these studies suggest that Pitx2 acts to regulate cell motility and expression of muscle specific genes in the muscle precursors during forelimb muscle development. This work provides a framework to develop the gene network leading to skeletal muscle development, growth and regeneration.

The hemochromatosis gene (HFE) has been involved in the etiology of type 2 diabetes mellitus and investigated in numerous epidemiologic studies. The current meta-analysis was conducted to evaluate the gene-disease association in relevant studies. Electronic literature search was performed on June 18, 2011, from databases of PubMed/MEDLINE, EMBASE, and HuGE Navigator. Articles were inspected by 2 authors independently, and data were extracted by identical extraction form. A total of 5,528 type 2 diabetes cases and 6,920 controls in relation to HFE polymorphisms (a cysteine to tyrosine substitution at amino acid position 282 (C282Y) and a histidine to aspartate substitution at amino acid position 63 (H63D)) were included in the meta-analysis (1997-2011). A fixed- or random-effect model was used to calculate the pooled odds ratios based on the results from the heterogeneity tests. An increased odds ratio for type 2 diabetes mellitus was observed in persons carrying a D allele at the H63D polymorphism compared with those with an H allele (odds ratio (OR) = 1.21, 95% confidence interval (CI): 1.03, 1.41; P = 0.02). Moreover, carriers of a D allele had a modestly increased risk compared with persons with the wild genotype (OR = 1.12, 95% CI: 1.00, 1.25; P = 0.04). The C282Y variant was not significantly associated with diabetes risk. In summary, persons with a D allele may have a moderately increased risk of type 2 diabetes mellitus.

RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain.

<h4>Background</h4>Raf-1 kinase inhibitor protein (RKIP) inhibits Raf (a key element in the ERK/MAPK pathway) and is regarded as anti-tumoral. In contrast, 14-3-3 is considered protumoral. However, the pathogenetic role of RKIP and 14-3-3ε in gastric cancer is unclear.<h4>Aim</h4>The purpose of this study was to examine the influence of 14-3-3ε and RKIP on SGC7901, the regulation of the ERK/MAKP pathway by both, and the interaction between the two proteins.<h4>Methods</h4>RKIP and 14-3-3ε genes were introduced into SGC7901 cells using gene cloning technique, then, the bioactivities including the proliferation, migration and invasion of the cells were assessed by MTT and migration assays. ERK/MAKP pathway's activity was examined using real-time quantitative RT-PCR, western blot, immunoprecipitation and 3D-immunolocalization techniques.<h4>Results</h4>Our results showed that RKIP inhibited SGC7901 cells' bioactivities whereas 14-3-3ε upregulated them through the involvement of the ERK/MAPK pathway. RKIP inactivated this pathway, but 14-3-3ε activated it. RKIP and 14-3-3ε were co-localized in the cells and interacted with each other; this attributed to their opposite influence on the ERK/MAPK pathway and the cells bioactivities.<h4>Conclusions</h4>The ERK/MAPK pathway is involved in the pathogenesis of gastric cancer; RKIP and 14-3-3ε exert an opposite effect on this pathway and the cells possibly via both direct and indirect reactions with the elements in this pathway. The interaction between RKIP and 14-3-3ε may also contribute to their pathogenetic roles in gastric cancer.

Neonatal hemochromatosis is a rare disease that causes fetal loss and neonatal death in the 1st weeks of life and is one of the most common causes of liver failure in the neonate. The diagnosis is mostly made retrospectively, based on histopathologic features of severe liver fibrosis associated with hepatic and extrahepatic siderosis. Several etiologies may underlie this phenotype, including a recently hypothesized gestational alloimmune disease. Fifty-one cases of liver failure with intrahepatic siderosis in fetuses and neonates were analyzed retrospectively. Maternal and infant data were collected from hospitalization and autopsy reports. All available slides were reviewed independently by 3 pathologists. Immunologic studies were performed on maternal sera collected immediately after delivery. The diagnosis of neonatal haemochromatosis was retained in 33 cases, including 1 case with Down syndrome and 1 case with myofibromas. Liver siderosis was inversely proportional to fibrosis progression. In fetuses, iron storage was more frequent in the thyroid than in the pancreas. Perls staining in labial salivary glands was positive in 1 of 5 cases. Abnormal low signal intensity by magnetic resonance imaging was detected in the pancreas in 2 of 7 cases. Renal tubular dysgenesis was observed in 7 of 23 autopsy cases. Chronic villitis was seen in 7 of 15 placentas. Half of the mothers presented with an autoimmune background and/or autoantibodies in their sera. Our work highlights the importance of autopsy in cases of neonatal hemochromatosis and marshals additional data in support of the hypothesis that neonatal hemochromatosis could reflect maternal immune system dysregulation.

<h4>Context</h4>Polycystic ovary syndrome (PCOS) is a heterogeneous, genetically complex, endocrine disorder of uncertain etiology in women.<h4>Objective</h4>Our aim was to compare the gene expression profiles in stimulated granulosa cells of PCOS women with and without insulin resistance vs. matched controls.<h4>Research design and methods</h4>This study included 12 normal ovulatory women (controls), 12 women with PCOS without evidence for insulin resistance (PCOS non-IR), and 16 women with insulin resistance (PCOS-IR) undergoing in vitro fertilization. Granulosa cell gene expression profiling was accomplished using Affymetrix Human Genome-U133 arrays. Differentially expressed genes were classified according to gene ontology using ingenuity pathway analysis tools. Microarray results for selected genes were confirmed by real-time quantitative PCR.<h4>Results</h4>A total of 211 genes were differentially expressed in PCOS non-IR and PCOS-IR granulosa cells (fold change≥1.5; P≤0.001) vs. matched controls. Diabetes mellitus and inflammation genes were significantly increased in PCOS-IR patients. Real-time quantitative PCR confirmed higher expression of NCF2 (2.13-fold), TCF7L2 (1.92-fold), and SERPINA1 (5.35-fold). Increased expression of inflammation genes ITGAX (3.68-fold) and TAB2 (1.86-fold) was confirmed in PCOS non-IR. Different cardiometabolic disease genes were differentially expressed in the two groups. Decreased expression of CAV1 (-3.58-fold) in PCOS non-IR and SPARC (-1.88-fold) in PCOS-IR was confirmed. Differential expression of genes involved in TGF-β signaling (IGF2R, increased; and HAS2, decreased), and oxidative stress (TXNIP, increased) was confirmed in both groups.<h4>Conclusions</h4>Microarray analysis demonstrated differential expression of genes linked to diabetes mellitus, inflammation, cardiovascular diseases, and infertility in the granulosa cells of PCOS women with and without insulin resistance. Because these dysregulated genes are also involved in oxidative stress, lipid metabolism, and insulin signaling, we hypothesize that these genes may be involved in follicular growth arrest and metabolic disorders associated with the different phenotypes of PCOS.

The level of fetal hemoglobin (HbF) modifies the severity of the common β-globin disorders. Knowledge of the normal mechanisms that repress HbF in the adult stage has remained limited until recently despite nearly 3 decades of molecular investigation, in part because of imperfect model systems. Recent studies have provided new insights into the developmental regulation of globin genes and identified specific transcription factors and epigenetic regulators responsible for physiologic silencing of HbF. Most prominent among these regulators is BCL11A, a transcriptional repressor that inhibits adult-stage HbF expression. KLF1 and c-Myb are additional critical HbF-regulating erythroid transcription factors more broadly involved in erythroid gene expression programs. Chromatin modifiers, including histone deacetylases and DNA methyltransferases, also play key roles in orchestrating appropriate globin gene expression. Taken together, these discoveries present novel therapeutic targets for further consideration. Although substantial hurdles remain, opportunities are now rich for the rational design of HbF inducers.

Many of the models of neurodevelopmental processes such as cell migration, axon outgrowth, and dendrite arborization involve cell adhesion and chemoattraction as critical physical or mechanical aspects of the mechanism. However, the prevention of adhesion or attraction is under-appreciated as a necessary, active process that balances these forces, insuring that the correct cells are present and adhering in the correct place at the correct time. The phenomenon of not adhering is often viewed as the passive alternative to adhesion, and in some cases this may be true. However, it is becoming increasingly clear that active signaling pathways are involved in preventing adhesion. These provide a balancing force during development that prevents overly exuberant adhesion, which would otherwise disrupt normal cellular and tissue morphogenesis. The strength of chemoattractive signals may be similarly modulated. Recent studies, described here, suggest that Down Syndrome Cell Adhesion Molecule (DSCAM), and closely related proteins such as DSCAML1, may play an important developmental role as such balancers in multiple systems.

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.

Brain serotonin (5-HT) is implicated in a wide range of functions from basic physiological mechanisms to complex behaviors, including neuropsychiatric conditions, as well as in developmental processes. Increasing evidence links 5-HT signaling alterations during development to emotional dysregulation and psychopathology in adult age. To further analyze the importance of brain 5-HT in somatic and brain development and function, and more specifically differentiation and specification of the serotonergic system itself, we generated a mouse model with brain-specific 5-HT deficiency resulting from a genetically driven constitutive inactivation of neuronal tryptophan hydroxylase-2 (Tph2). Tph2 inactivation (Tph2-/-) resulted in brain 5-HT deficiency leading to growth retardation and persistent leanness, whereas a sex- and age-dependent increase in body weight was observed in Tph2+/- mice. The conserved expression pattern of the 5-HT neuron-specific markers (except Tph2 and 5-HT) demonstrates that brain 5-HT synthesis is not a prerequisite for the proliferation, differentiation and survival of raphe neurons subjected to the developmental program of serotonergic specification. Furthermore, although these neurons are unable to synthesize 5-HT from the precursor tryptophan, they still display electrophysiological properties characteristic of 5-HT neurons. Moreover, 5-HT deficiency induces an up-regulation of 5-HT(1A) and 5-HT(1B) receptors across brain regions as well as a reduction of norepinephrine concentrations accompanied by a reduced number of noradrenergic neurons. Together, our results characterize developmental, neurochemical, neurobiological and electrophysiological consequences of brain-specific 5-HT deficiency, reveal a dual dose-dependent role of 5-HT in body weight regulation and show that differentiation of serotonergic neuron phenotype is independent from endogenous 5-HT synthesis.

Polyglutamine (PolyQ) diseases have common features that include progressive selective neurodegeneration and the formation of protein aggregates. There is growing evidence to suggest that critical nuclear events lead to transcriptional alterations in PolyQ diseases such as spinocerebellar ataxia type 7 (SCA7) and Huntington's disease (HD), conditions which share a cerebellar degenerative phenotype. Taking advantage of laser capture microdissection technique, we compared the Purkinje cell (PC) gene expression profiles of two transgenic polyQ mouse models (HD: R6/2; SCA7: P7E) by microarray analysis that was validated by real time quantitative PCR. A large number of transcriptional alterations were detected in the R6/2 transgenic model of HD. Similar decreases in the same mRNAs, such as phospholipase C, β 3, purkinje cell protein 2 (Pcp2) and aldolase C, were found in both models. A decrease in aldolase C and phospholipase C, β 3, may lead to an increase in the vulnerability of PCs to excitotoxic events. Furthermore, downregulation of mRNAs mediated by the Pcp2-promoter is common in both models. Thus, our data reveal shared molecular abnormalities in different polyQ disorders.

Oxidative stress has been implicated in the etiology of atherosclerosis and even held responsible for plaque calcification. Transition metals such as iron aggravate oxidative stress. To understand the relation between calcium and iron in atherosclerotic lesions, a sensitive technique is required that is quantitatively accurate and avoids isolation of plaques or staining/fixing tissue, because these processes introduce contaminants and redistribute elements within the tissue. In this study, the three ion-beam techniques of scanning transmission ion microscopy, Rutherford backscattering spectrometry, and particle-induced X-ray emission have been combined in conjunction with a high-energy (MeV) proton microprobe to map the spatial distribution of the elements and quantify them simultaneously in atherosclerotic rabbit arteries. The results show that iron and calcium within the atherosclerotic lesions exhibit a highly significant spatial inverse correlation. It may be that iron accelerates the progression of atherosclerotic lesion development, but suppresses calcification. Alternatively, calcification could be a defense mechanism against atherosclerotic progression by excluding iron.

Silica nanoparticles are being investigated for a number of medical applications; however, their use in vivo has been questioned because of the potential for bioaccumulation. To obviate this problem, silica nanoshells were tested for enhanced biodegradability by doping iron(III) into the nanoshells. Exposure of the doped silica to small molecule chelators and mammalian serum was explored to test whether the removal of iron(III) from the silica nanoshell structure would facilitate its degradation. Iron chelators, such as EDTA, desferrioxamine, and deferiprone, were found to cause the nanoshells to degrade on the removal of iron(III) within several days at 80 °C. When the iron(III)-doped, silica nanoshells were submerged in fetal bovine and human serums at physiological temperature, they also degrade via removal of the iron by serum proteins, such as transferrin, over a period of several weeks.

The BTB-ZF (broad-complex, tramtrack and bric-à-brac--zinc finger) proteins are encoded by at least 49 genes in mouse and man and commonly serve as sequence-specific silencers of gene expression. This review will focus on the known physiological functions of mammalian BTB-ZF proteins, which include essential roles in the development of the immune system. We discuss their function in terminally differentiated lymphocytes and the progenitors that give rise to them, their action in hematopoietic malignancy and roles beyond the immune system.

<h4>Background</h4>The pathogenesis of natural scrapie and other prion diseases is still poorly understood. Determining the variations in the transcriptome in the early phases of the disease might clarify some of the molecular mechanisms of the prion-induced pathology and allow for the development of new biomarkers for diagnosis and therapy. This study is the first to focus on the identification of genes regulated during the preclinical phases of natural scrapie in the ovine medulla oblongata (MO) and the association of these genes with prion deposition, astrocytosis and spongiosis.<h4>Results</h4>A custom microarray platform revealed that 86 significant probes had expression changes greater than 2-fold. From these probes, we identified 32 genes with known function; the highest number of regulated genes was included in the phosphoprotein-encoding group. Genes encoding extracellular marker proteins and those involved in the immune response and apoptosis were also differentially expressed. In addition, we investigated the relationship between the gene expression profiles and the appearance of the main scrapie-associated brain lesions. Quantitative Real-time PCR was used to validate the expression of some of the regulated genes, thus showing the reliability of the microarray hybridization technology.<h4>Conclusions</h4>Genes involved in protein and metal binding and oxidoreductase activity were associated with prion deposition. The expression of glial fibrillary acidic protein (GFAP) was associated with changes in the expression of genes encoding proteins with oxidoreductase and phosphatase activity, and the expression of spongiosis was related to genes encoding extracellular matrix components or transmembrane transporters. This is the first genome-wide expression study performed in naturally infected sheep with preclinical scrapie. As in previous studies, our findings confirm the close relationship between scrapie and other neurodegenerative diseases.

For cytosine (C) demethylation of vertebrate DNA, it is known that the TET proteins could convert 5-methyl C (5-mC) to 5-hydroxymethyl C (5-hmC). However, DNA dehydroxymethylase(s), or enzymes able to directly convert 5-hmC to C, have been elusive. We present in vitro evidence that the mammalian de novo DNA methyltransferases DNMT3A and DNMT3B, but not the maintenance enzyme DNMT1, are also redox-dependent DNA dehydroxymethylases. Significantly, intactness of the C methylation catalytic sites of these de novo enzymes is also required for their 5-hmC dehydroxymethylation activity. That DNMT3A and DNMT3B function bidirectionally both as DNA methyltransferases and as dehydroxymethylases raises intriguing and new questions regarding the structural and functional aspects of these enzymes and their regulatory roles in the dynamic modifications of the vertebrate genomes during development, carcinogenesis, and gene regulation.

Genetic studies of complex diseases often collect multiple phenotypes relevant to the disorders. As these phenotypes can be correlated and share common genetic mechanisms, jointly analyzing these traits may bring more power to detect genes influencing individual or multiple phenotypes. Given the advancement brought by the multivariate phenotype approaches and the multimarker kernel machine regression, we construct a multivariate regression based on kernel machine to facilitate the joint evaluation of multimarker effects on multiple phenotypes. The kernel machine serves as a powerful dimension-reduction tool to capture complex effects among markers. The multivariate framework incorporates the potentially correlated multidimensional phenotypic information and accommodates common or different environmental covariates for each trait. We derive the multivariate kernel machine test based on a score-like statistic, and conduct simulations to evaluate the validity and efficacy of the method. We also study the performance of the commonly adapted strategies for kernel machine analysis on multiple phenotypes, including the multiple univariate kernel machine tests with original phenotypes or with their principal components. Our results suggest that none of these approaches has the uniformly best power, and the optimal test depends on the magnitude of the phenotype correlation and the effect patterns. However, the multivariate test retains to be a reasonable approach when the multiple phenotypes have none or mild correlations, and gives the best power once the correlation becomes stronger or when there exist genes that affect more than one phenotype. We illustrate the utility of the multivariate kernel machine method through the Clinical Antipsychotic Trails of Intervention Effectiveness antibody study.

B chromosomes are centric chromosomal fragments present in thousands of eukaryotic genomes. Because most B chromosomes are non-essential, they can be lost without consequence. In order to persist, however, some B chromosomes can impose strong forms of intra-genomic conflict. An extreme case is the paternal sex ratio (PSR) B chromosome in the jewel wasp Nasonia vitripennis. Transmitted solely via the sperm, PSR 'imprints' the paternal chromatin so that it is destroyed during the first mitosis of the embryo. Owing to the haplo-diploid reproduction of N. vitripennis, PSR-induced loss of the paternal chromatin converts embryos that should become females into PSR-transmitting males. This conversion is key to the persistence of PSR, although the underlying mechanisms are largely unexplored. We assessed how PSR affects the paternal chromatin and then investigated how PSR is transmitted efficiently at the cellular level. We found that PSR does not affect progression of the paternal chromatin through the cell cycle but, instead, alters its normal Histone H3 phosphorylation and loading of the Condensin complex. PSR localizes to the outer periphery of the paternal nucleus, a position that we propose is crucial for it to escape from the defective paternal set. In sperm, PSR consistently localizes to the extreme anterior tip of the elongated nucleus, while the normal wasp chromosomes localize broadly across the nucleus. Thus, PSR may alter or bypass normal nuclear organizational processes to achieve its position. These findings provide new insights into how selfish genetic elements can impact chromatin-based processes for their survival.

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.

Asymmetric cell divisions are a fundamental feature of neural development, and misregulation can lead to brain abnormalities or tumor formation. During an asymmetric cell division, molecular determinants are segregated preferentially into one daughter cell to specify its fate. An important goal is to identify the asymmetric determinants in neural progenitor cells, which could be tumor suppressors or inducers of specific neural fates. Here, we show that the double-stranded RNA-binding protein Stau2 is distributed asymmetrically during progenitor divisions in the developing mouse cortex, preferentially segregating into the Tbr2(+) neuroblast daughter, taking with it a subset of RNAs. Knockdown of Stau2 stimulates differentiation and overexpression produces periventricular neuronal masses, demonstrating its functional importance for normal cortical development. We immunoprecipitated Stau2 to examine its cargo mRNAs, and found enrichment for known asymmetric and basal cell determinants, such as Trim32, and identified candidates, including a subset involved in primary cilium function.

Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the "knocksideways" system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins.

<h4>Background & aims</h4>We aimed to identify risk factors for hepatocellular carcinoma (HCC) in patients with cirrhosis in the United States. We performed a prospective study to identify associations between etiologies of cirrhosis and ethnicity with HCC incidence.<h4>Methods</h4>We used convenience sampling to select a cohort of 379 patients with cirrhosis who visited the liver clinic at the Stanford University Medical Center from 2001 to 2009 (65% male, 75% white or Hispanic, and 20% Asian). Study end points were HCC diagnosis by histology or noninvasive criteria, liver transplantation, or last screening without HCC. Patients were followed up, with ultrasound or computed tomographic imaging analyses and measurements of serum levels of α-fetoprotein, approximately every 6 months, for a median time of 34 months (range, 6-99 mo).<h4>Results</h4>The etiologies of cirrhosis in the cohort were 68% hepatitis C, 7% hepatitis B, and 25% nonviral. Forty-four patients (12%) were diagnosed with HCC during the follow-up period. Patients with cirrhosis related to viral hepatitis had a statistically significantly higher incidence of HCC than those with nonviral diseases in Kaplan-Meier analysis (P = .04). There was no statistically significant difference in HCC incidence between Asian and non-Asian patients. In a multivariate Cox proportional hazards model that included age, sex, ethnicity, etiology, and Child-Pugh-Turcotte score, viral cirrhosis was associated significantly with HCC, compared with nonviral cirrhosis (hazard ratio, 3.6; 95% confidence interval, 1.3-10.1; P = .02) but Asian ethnicity was not.<h4>Conclusions</h4>In a diverse cohort of patients in the United States with cirrhosis, a viral etiology of cirrhosis was associated with increased incidence of HCC, but Asian ethnicity was not. These findings indicate the importance of cirrhosis etiology in determining risk for HCC.

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.

Progestins induce lipid accumulation in progesterone receptor (PR)-positive breast cancer cells. We speculated that progestin-induced alterations in lipid biology confer resistance to chemotherapy. To examine the biology of lipid loaded breast cancer cells, we used a model of progestin-induced lipid synthesis. T47D (PR-positive) and MDA-MB-231 (PR-negative) cell lines were used to study progestin response. Oil red O staining of T47D cells treated with progestin showed lipid droplet formation was PR dependent, glucose dependent and reduced sensitivity to docetaxel. This protection was not observed in PR-negative MDA-MB-231 cells. Progestin treatment induced stearoyl CoA desaturase-1 (SCD-1) enzyme expression and chemical inhibition of SCD-1 diminished lipid droplets and cell viability, suggesting the importance of lipid stores in cancer cell survival. Gas chromatography/mass spectroscopy analysis of phospholipids from progestin-treated T47D cells revealed an increase in unsaturated fatty acids, with oleic acid as most abundant. Cells surviving docetaxel treatment also contained more oleic acid in phospholipids, suggesting altered membrane fluidity as a potential mechanism of chemoresistance mediated in part by SCD-1. Lastly, intact docetaxel molecules were present within progestin induced lipid droplets, suggesting a protective quenching effect of intracellular lipid droplets. Our studies suggest the metabolic adaptations produced by progestin provide novel metabolic targets for future combinatorial therapies for progestin-responsive breast cancers.

<h4>Background</h4>The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology.<h4>Scope of review</h4>In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases.<h4>Major conclusions</h4>We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis.<h4>General significance</h4>Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Polyglutamine (PolyQ)-related diseases are dominant late-onset genetic disorders that are manifested by progressive neurodegeneration, leading to behavioral and physical impairments. An increased body of evidence suggests that chromatin structure and epigenetic regulation are involved in disease pathology. PolyQ diseases often display an aberrant transcriptional regulation due to the disrupted function of histone-modifying complexes and altered interactions of the polyQ-extended proteins with chromatin-related factors. In this review we describe recent findings relating to the role of chromatin in polyQ diseases. We discuss the involvement of epigenetic-related factors and chromatin structure in genomic instability of CAG repeats; we describe changes in the expression and regulation of chromatin-related enzymes and in the levels and patterns of histone modifications in disease state; we illustrate the potential beneficial effects of different histone deacetylase (HDAC) inhibitors for the treatment of polyQ diseases, and we end by describing the potential use of human pluripotent stem cells and their differentiated derivatives for modeling polyQ diseases in vitro. Taken together, these accumulating studies strongly suggest that disrupted chromatin regulation may be directly involved with the pathophysiology of polyQ-related diseases.

<h4>Background</h4>The consequences of influenza in children and adults are mainly absenteeism from school and work. However, the risk of complications is greatest in children and people over 65 years of age.<h4>Objectives</h4>To appraise all comparative studies evaluating the effects of influenza vaccines in healthy children, assess vaccine efficacy (prevention of confirmed influenza) and effectiveness (prevention of influenza-like illness (ILI)) and document adverse events associated with influenza vaccines.<h4>Search methods</h4>We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2011, Issue 3) which includes the Acute Respiratory Infections Group's Specialised Register, OLD MEDLINE (1950 to 1965), MEDLINE (1966 to November 2011), EMBASE (1974 to November 2011), Biological Abstracts (1969 to September 2007), and Science Citation Index (1974 to September 2007).<h4>Selection criteria</h4>Randomised controlled trials (RCTs), cohort and case-control studies of any influenza vaccine in healthy children under 16 years of age.<h4>Data collection and analysis</h4>Four review authors independently assessed trial quality and extracted data.<h4>Main results</h4>We included 75 studies with about 300,000 observations. We included 17 RCTs, 19 cohort studies and 11 case-control studies in the analysis of vaccine efficacy and effectiveness. Evidence from RCTs shows that six children under the age of six need to be vaccinated with live attenuated vaccine to prevent one case of influenza (infection and symptoms). We could find no usable data for those aged two years or younger.Inactivated vaccines in children aged two years or younger are not significantly more efficacious than placebo. Twenty-eight children over the age of six need to be vaccinated to prevent one case of influenza (infection and symptoms). Eight need to be vaccinated to prevent one case of influenza-like-illness (ILI). We could find no evidence of effect on secondary cases, lower respiratory tract disease, drug prescriptions, otitis media and its consequences and socioeconomic impact. We found weak single-study evidence of effect on school absenteeism by children and caring parents from work. Variability in study design and presentation of data was such that a meta-analysis of safety outcome data was not feasible. Extensive evidence of reporting bias of safety outcomes from trials of live attenuated influenza vaccines (LAIVs) impeded meaningful analysis. One specific brand of monovalent pandemic vaccine is associated with cataplexy and narcolepsy in children and there is sparse evidence of serious harms (such as febrile convulsions) in specific situations.<h4>Authors' conclusions</h4>Influenza vaccines are efficacious in preventing cases of influenza in children older than two years of age, but little evidence is available for children younger than two years of age. There was a difference between vaccine efficacy and effectiveness, partly due to differing datasets, settings and viral circulation patterns. No safety comparisons could be carried out, emphasising the need for standardisation of methods and presentation of vaccine safety data in future studies. In specific cases, influenza vaccines were associated with serious harms such as narcolepsy and febrile convulsions. It was surprising to find only one study of inactivated vaccine in children under two years, given current recommendations to vaccinate healthy children from six months of age in the USA, Canada, parts of Europe and Australia. If immunisation in children is to be recommended as a public health policy, large-scale studies assessing important outcomes, and directly comparing vaccine types are urgently required. The degree of scrutiny needed to identify all global cases of potential harms is beyond the resources of this review. This review includes trials funded by industry. An earlier systematic review of 274 influenza vaccine studies published up to 2007 found industry-funded studies were published in more prestigious journals and cited more than other studies independently from methodological quality and size. Studies funded from public sources were significantly less likely to report conclusions favourable to the vaccines. The review showed that reliable evidence on influenza vaccines is thin but there is evidence of widespread manipulation of conclusions and spurious notoriety of the studies. The content and conclusions of this review should be interpreted in the light of this finding.

Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. Here, we review the functions of alternative splicing events that have been experimentally determined. The overall function of alternative splicing is to increase the diversity of mRNAs expressed from the genome. Alternative splicing changes proteins encoded by mRNAs, which has profound functional effects. Experimental analysis of these protein isoforms showed that alternative splicing regulates binding between proteins, between proteins and nucleic acids as well as between proteins and membranes. Alternative splicing regulates the localization of proteins, their enzymatic properties and their interaction with ligands. In most cases, changes caused by individual splicing isoforms are small. However, cells typically coordinate numerous changes in 'splicing programs', which can have strong effects on cell proliferation, cell survival and properties of the nervous system. Due to its widespread usage and molecular versatility, alternative splicing emerges as a central element in gene regulation that interferes with almost every biological function analyzed.

Hodgkin's lymphoma represents one of the most frequent lymphoproliferative syndromes, especially in young population. Although HL is considered one of the most curable tumors, a sizeable fraction of patients recur after successful upfront treatment or, less commonly, are primarily resistant. This work tries to summarize the data on clinical, histological, pathological, and biological factors in HL, with special emphasis on the improvement of prognosis and their impact on therapeutical strategies. The recent advances in our understanding of HL biology and immunology show that infiltrated immune cells and cytokines in the tumoral microenvironment may play different functions that seem tightly related with clinical outcomes. Strategies aimed at interfering with the crosstalk between tumoral Reed-Sternberg cells and their cellular partners have been taken into account in the development of new immunotherapies that target different cell components of HL microenvironment. This new knowledge will probably translate into a change in the antineoplastic treatments in HL in the next future and hopefully will increase the curability rates of this disease.

Telomere length in Drosophila is maintained by targeted transposition of three non-LTR retrotransposons: HeT-A, TART and TAHRE (HTT), but understanding the regulation of this process is hindered by our poor knowledge of HTT associated proteins. We have identified new protein components of the HTT array: Chromator (Chro), the TRF2/DREF complex and the sumoylation machinery. Chro was localized on telomeric HTT arrays by immunostaining, where it may interact with Prod directly, as indicated by yeast two-hybrid interaction, co-IP, and colocalization on polytene chromosomes. The TRF2/DREF complex may promote the open structure of HTT chromatin. The protein interactions controlling HTT chromatin structure and telomere length may be modulated by sumoylation.

Accumulation of aberrant protein aggregates, such as amyloid beta peptide (Aβ), due to decreased proteasome activities might contribute to the neurodegeneration in Alzheimer's disease. In this study, lithocholic acid derivatives 3α-O-pimeloyl-lithocholic acid methyl ester (2) and its isosteric isomer (6) were found to activate the chymotrypsin-like activity of the proteasome at an EC(50) of 7.8 and 4.3 μM, respectively. Replacing the C24 methyl ester in 2 with methylamide resulted in a complete devoid of proteasome activating activity. Epimerizing the C3 substituent from an alpha to beta orientation transformed the activator into a proteasome inhibitor. Unlike the cellular proteasome activator PA28, proteasome activated by 2 was not inhibited by Aβ. Furthermore, 2 potently antagonized the inhibitory effect of Aβ on the proteasome. In summary, compound 2 represents a novel class of small molecules that not only activates the proteasome but also antagonizes the inhibitory effect of Aβ on the proteasome.

Neuroglobin is a hypoxia-inducible O(2)-binding protein with neuroprotective effects in cell and animal models of stroke and Alzheimer's disease. The mechanism underlying neuroglobin's cytoprotective action is unknown, although several possibilities have been proposed, including anti-oxidative and anti-apoptotic effects. We used affinity purification-mass spectrometry methods to identify neuroglobin-interacting proteins in normoxic and hypoxic murine neuronal (HN33) cell lysates, and to compare these interactions with those of a structurally and functionally related protein, myoglobin. We report that the protein interactomes of neuroglobin and myoglobin overlap substantially and are modified by hypoxia. In addition, neuroglobin-interacting proteins include partners consistent with both anti-oxidative and anti-apoptotic functions, as well as with a relationship to several neurodegenerative diseases.

<h4>Background</h4>Huntington's disease (HD) is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ) stretch within Huntingtin (htt), the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR) in modulating HD mouse model pathogenesis is currently unknown.<h4>Results</h4>We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh) encoding N-terminal hemaglutinin (HA) or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt) and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt). Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q) htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt's polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis.<h4>Conclusion</h4>In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Most pathway and gene-set enrichment methods prioritize genes by their main effect and do not account for variation due to interactions in the pathway. A portion of the presumed missing heritability in genome-wide association studies (GWAS) may be accounted for through gene-gene interactions and additive genetic variability. In this study, we prioritize genes for pathway enrichment in GWAS of bipolar disorder (BD) by aggregating gene-gene interaction information with main effect associations through a machine learning (evaporative cooling) feature selection and epistasis network centrality analysis. We validate this approach in a two-stage (discovery/replication) pathway analysis of GWAS of BD. The discovery cohort comes from the Wellcome Trust Case Control Consortium (WTCCC) GWAS of BD, and the replication cohort comes from the National Institute of Mental Health (NIMH) GWAS of BD in European Ancestry individuals. Epistasis network centrality yields replicated enrichment of Cadherin signaling pathway, whose genes have been hypothesized to have an important role in BD pathophysiology but have not demonstrated enrichment in previous analysis. Other enriched pathways include Wnt signaling, circadian rhythm pathway, axon guidance and neuroactive ligand-receptor interaction. In addition to pathway enrichment, the collective network approach elevates the importance of ANK3, DGKH and ODZ4 for BD susceptibility in the WTCCC GWAS, despite their weak single-locus effect in the data. These results provide evidence that numerous small interactions among common alleles may contribute to the diathesis for BD and demonstrate the importance of including information from the network of gene-gene interactions as well as main effects when prioritizing genes for pathway analysis.

The role of NF-κB activation in tumor initiation has not been thoroughly investigated. We generated Ikkβ(EE)(IEC) transgenic mice expressing constitutively active IκB kinase β (IKKβ) in intestinal epithelial cells (IECs). Despite absence of destructive colonic inflammation, Ikkβ(EE)(IEC) mice developed intestinal tumors after a long latency. However, when crossed to mice with IEC-specific allelic deletion of the adenomatous polyposis coli (Apc) tumor suppressor locus, Ikkβ(EE)(IEC) mice exhibited more β-catenin(+) early lesions and visible small intestinal and colonic tumors relative to Apc(+/ΔIEC) mice, and their survival was severely compromised. IEC of Ikkβ(EE)(IEC) mice expressed high amounts of inducible nitric oxide synthase (iNOS) and elevated DNA damage markers and contained more oxidative DNA lesions. Treatment of Ikkβ(EE)(IEC)/Apc(+/ΔIEC) mice with an iNOS inhibitor decreased DNA damage markers and reduced early β-catenin(+) lesions and tumor load. The results suggest that persistent NF-κB activation in IEC may accelerate loss of heterozygocity by enhancing nitrosative DNA damage.

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.

Dynamic clamp is a powerful approach for electrophysiological investigations allowing researchers to introduce artificial electrical components into target neurons to simulate ionic conductances, chemical or electrotonic inputs or connections to other cells. Due to the rapidly changing and potentially large current injections during dynamic clamp, problematic voltage artifacts appear on the electrode used to inject dynamic clamp currents into a target neuron. Dynamic clamp experiments, therefore, typically use two separate electrodes in the same cell, one for recording membrane potential and one for injecting currents. The requirement for two independent electrodes has been a limiting factor for the use of dynamic clamp in applications where dual recordings of this kind are difficult or impossible to achieve. The recent development of an active electrode compensation (AEC) method has overcome some of these prior limitations, permitting artifact-free dynamic clamp experimentation with a single electrode. Here we describe an AEC method for the free dynamic clamp software StdpC. The AEC component of StdpC is the first such system implemented for the use of non-expert users and comes with a set of semi-automated configuration and calibration procedures that facilitate its use. We briefly introduce the AEC method and its implementation in StdpC and then validate it with an electronic model cell and in two different biological preparations.

<h4>Background</h4>Two methoxyl poly(ethylene glycol)-poly(L-histidine)-poly(L-lactide) (mPEG-PH-PLLA) triblock copolymers with different poly(L-histidine) chain lengths were synthesized. The morphology and biocompatibility of these self-assembled nanoparticles was investigated.<h4>Methods</h4>Doxorubicin, an antitumor drug, was trapped in the nanoparticles to explore their drug-release behavior. The drug-loaded nanoparticles were incubated with HepG2 cells to evaluate their antitumor efficacy in vitro. The effects of poly(L-histidine) chain length on the properties, drug-release behavior, and antitumor efficiency of the nanoparticles were investigated.<h4>Results</h4>The nanoparticles were pH-sensitive. The mean diameters of the two types of mPEG-PH- PLLA nanoparticle were less than 200 nm when the pH values were 5.0 and 7.4. The nanoparticles were nontoxic to NIH 3T3 fibroblasts and HepG2 cells. The release of doxorubicin at pH 5.0 was much faster than that at pH 7.4. The release rate of mPEG(45)-PH(15)-PLLA(82) nanoparticles was much faster than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles at pH 5.0.<h4>Conclusion</h4>The inhibition effect of mPEG(45)-PH(15)-PLLA(82) nanoparticles on the growth of HepG2 cells was greater than that of mPEG(45)-PH(30)-PLLA(82) nanoparticles when the concentration of encapsulated doxorubicin was less than 15 μg/mL.

<h4>Background</h4>Cancers arise through an evolutionary process in which cell populations are subjected to selection; however, to date, the process of bladder cancer, which is one of the most common cancers in the world, remains unknown at a single-cell level.<h4>Results</h4>We carried out single-cell exome sequencing of 66 individual tumor cells from a muscle-invasive bladder transitional cell carcinoma (TCC). Analyses of the somatic mutant allele frequency spectrum and clonal structure revealed that the tumor cells were derived from a single ancestral cell, but that subsequent evolution occurred, leading to two distinct tumor cell subpopulations. By analyzing recurrently mutant genes in an additional cohort of 99 TCC tumors, we identified genes that might play roles in the maintenance of the ancestral clone and in the muscle-invasive capability of subclones of this bladder cancer, respectively.<h4>Conclusions</h4>This work provides a new approach of investigating the genetic details of bladder tumoral changes at the single-cell level and a new method for assessing bladder cancer evolution at a cell-population level.

<h4>Background</h4>Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized.<h4>Results</h4>We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of incorporation of some RNA binding (RHA and HELIC2) and DNA binding proteins (MCM5 and Ku80) in the viral cores from T cells was higher than in the cores from both mMΦ and mMN and did not correlate with the abundance of these proteins in virus producing cells.<h4>Conclusions</h4>Profiles of host proteins packaged in the cores of HIV-1 virions depend on the type of virus producing cell. The pool of proteins present in the cores of all virions is likely to contain factors important for viral functions. Incorporation ratio of certain RNA- and DNA-binding proteins suggests their more efficient, non-random packaging into virions in T cells than in mMΦ and mMN.

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

Aggregation of protein molecules is a pathological hallmark of many neurodegenerative diseases. Abnormal modifications have often been observed in the aggregated proteins, supporting the aggregation mechanism regulated by post-translational modifications on proteins. Modifications are in general assumed to occur in soluble proteins before aggregation, but actually it remains quite obscure when proteins are modified in the course of the aggregation. Here we focus upon aggregation of huntingtin (HTT), which causes a neurodegenerative disorder, Huntington disease, and we show that oxidation of a methionine residue in HTT occurs in vitro and also in vivo. Copper ions as well as added hydrogen peroxide are found to oxidize the methionine residue, but notably, this oxidative modification occurs only in the aggregated HTT but not in the soluble state. Furthermore, the methionine oxidation creates additional interactions among HTT aggregates and alters overall morphologies of the aggregates. We thus reveal that protein aggregates can be a target of oxidative modifications and propose that such a "post-aggregation" modification is a relevant factor to regulate properties of protein aggregates.

<h4>Background</h4>The sigma-2 receptor is an attractive target for tumor imaging and targeted therapy because it is overexpressed in multiple types of solid tumors, including prostate cancer, breast cancer, and lung cancer. SV119 is a synthetic small molecule that binds to sigma-2 receptors with high affinity and specificity. This study investigates the utility of SV119 in mediating the selective targeting of liposomal vectors in various types of cancer cells.<h4>Methods</h4>SV119 was covalently linked with polyethylene glycol-dioleyl amido aspartic acid conjugate (PEG-DOA) to generate a novel functional lipid, SV119-PEG-DOA. This lipid was utilized for the preparation of targeted liposomes to enhance their uptake by cancer cells. Liposomes with various SV119 densities (0, 1, 3, and 5 mole%) were prepared and their cellular uptake was investigated in several tumor cell lines. In addition, doxorubicin (DOX) was loaded into the targeted and unmodified liposomes, and the cytotoxic effect on the DU-145 cells was evaluated by MTT assay.<h4>Results</h4>Liposomes with or without SV119-PEG-DOA both have a mean diameter of approximately 90 nm and a neutral charge. The incorporation of SV119-PEG-DOA significantly increased the cellular uptake of liposomes by the DU-145, PC-3, A549, 201T, and MCF-7 tumor cells, which was shown by fluorescence microscopy and the quantitative measurement of fluorescence intensity. In contrast, the incorporation of SV119 did not increase the uptake of liposomes by the normal BEAS-2B cells. In a time course study, the uptake of SV119 liposomes by DU-145 cells was also significantly higher at each time point compared to the unmodified liposomes. Furthermore, the DOX-loaded SV119 liposomes showed significantly higher cytotoxicity to DU-145 cells compared to the DOX-loaded unmodified liposomes.<h4>Conclusion</h4>SV119 liposomes were developed for targeted drug delivery to cancer cells. The targeting efficiency and specificity of SV119 liposomes to cancer cells was demonstrated in vitro. The results of this study suggest that SV119-modified liposomes might be a promising drug carrier for tumor-targeted delivery.

BACKGROUND. Liraglutide leading to improve not only glycaemic control but also liver inflammation in non-alcoholic fatty liver disease (NAFLD) patients. AIMS. The aim of this study is to elucidate the effectiveness of liraglutide in NAFLD patients with type 2 diabetes mellitus (T2DM) compared to sitagliptin and pioglitazone. METHODS. We retrospectively enrolled 82 Japanese NAFLD patients with T2DM and divided into three groups (liraglutide: N = 26, sitagliptin; N = 36, pioglitazone; N = 20). We compared the baseline characteristics, changes of laboratory data and body weight. RESULTS. At the end of follow-up, ALT, fast blood glucose, and HbA1c level significantly improved among the three groups. AST to platelet ratio significantly decreased in liraglutide group and pioglitazone group. The body weight significantly decreased in liraglutide group (81.8 kg to 78.0 kg, P < 0.01). On the other hands, the body weight significantly increased in pioglitazone group and did not change in sitagliptin group. Multivariate regression analysis indicated that administration of liraglutide as an independent factor of body weight reduction for more than 5% (OR 9.04; 95% CI 1.12-73.1, P = 0.04). CONCLUSIONS. Administration of liraglutide improved T2DM but also improvement of liver inflammation, alteration of liver fibrosis, and reduction of body weight.

Histone gene transcription is actively downregulated after completion of DNA synthesis to avoid overproduction. However, the precise mechanistic details of the cessation of histone mRNA synthesis are not clear. We found that histone H2B phosphorylation at Tyr37 occurs upstream of histone cluster 1, Hist1, during the late S phase. We identified WEE1 as the kinase that phosphorylates H2B at Tyr37. Loss of expression or inhibition of WEE1 kinase abrogated H2B Tyr37 phosphorylation with a concomitant increase in histone transcription in yeast and mammalian cells. H2B Tyr37 phosphorylation excluded binding of the transcriptional coactivator NPAT and RNA polymerase II and recruited the histone chaperone HIRA upstream of the Hist1 cluster. Taken together, our data show a previously unknown and evolutionarily conserved function for WEE1 kinase as an epigenetic modulator that marks chromatin with H2B Tyr37 phosphorylation, thereby inhibiting the transcription of multiple histone genes to lower the burden on the histone mRNA turnover machinery.

<h4>Rationale</h4>Serotonin transporter (5-HTT) gene polymorphisms are linked with antidepressant response to selective serotonin reuptake inhibitor drugs (SSRIs), though the favorable allelic variant differs by ethnic group (Caucasian versus Korean or Japanese). In Caucasian patients, response also is linked to measures of platelet 5-HTT function.<h4>Objectives</h4>Here, we study both 5-HTT gene polymorphisms and 5-HTT function as determinants of antidepressant response to SSRIs in Korean patients.<h4>Methods</h4>We enrolled 99 patients with major depression and 48 control subjects. For statistical power, both samples were enriched with the l/l 5-HTTLPR polymorphism, which is uncommon in Koreans. Patients were treated with fluoxetine or sertraline. Response was assessed at 6 weeks. Subjects were genotyped for s/l polymorphism in the 5-HTT promoter region (5-HTTLPR). Platelet 5-HTT activity was determined as maximal uptake rate (Vmax) and affinity constant (Km).<h4>Results</h4>Response was differentially associated with the s allele of 5-HTTLPR, which also was significantly associated with Vmax. These associations are opposite to those reported in Caucasian populations. Responders had significantly higher Vmax and Km than nonresponders. In Koreans as well as Caucasians, high Vmax is related to antidepressant response to SSRIs, though the 5-HTTLPR polymorphism associations with both response and function differ by ethnicity.<h4>Conclusions</h4>Both ethnicity and function must be considered in evaluating candidate gene biomarkers of response to SSRIs in depression.

Many pathological states involve dysregulation of mitochondrial fusion, fission, or transport. These dynamic events are usually studied in cells lines because of the challenges in tracking mitochondria in tissues. To investigate mitochondrial dynamics in tissues and disease models, we generated two mouse lines with photo-activatable mitochondria (PhAM). In the PhAM(floxed) line, a mitochondrially localized version of the photo-convertible fluorescent protein Dendra2 (mito-Dendra2) is targeted to the ubiquitously expressed Rosa26 locus, along with an upstream loxP-flanked termination signal. Expression of Cre in PhAM( floxed) cells results in bright mito-Dendra2 fluorescence without adverse effects on mitochondrial morphology. When crossed with Cre drivers, the PhAM(floxed) line expresses mito-Dendra2 in specific cell types, allowing mitochondria to be tracked even in tissues that have high cell density. In a second line (PhAM(excised) ), the expression of mito-Dendra2 is ubiquitous, allowing mitochondria to be analyzed in a wide range of live and fixed tissues. By using photo-conversion techniques, we directly measured mitochondrial fusion events in cultured cells as well as tissues such as skeletal muscle. These mouse lines facilitate analysis of mitochondrial dynamics in a wide spectrum of primary cells and tissues, and can be used to examine mitochondria in developmental transitions and disease states.

<h4>Background</h4>Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain.<h4>Results</h4>The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR.<h4>Conclusion</h4>RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method.

High titer autoantibodies, which are often associated with specific clinical phenotypes, are useful diagnostically and prognostically in systemic autoimmune diseases. In several autoimmune rheumatic diseases (e.g. myositis and Sjogren's syndrome), 20-40% of patients are autoantibody negative as assessed by conventional assays. The recent discovery of new specificities (e.g., anti-MDA5) in a subset of these autoantibody-negative subjects demonstrates that additional specificities await identification. In this manuscript, we describe a rapid multidimensional method to identify new autoantigens. A central foundation of this rapid approach is the use of an antigen source in which a pathogenic pathway active in the disease is recapitulated. Additionally, the method involves a modified serological proteome analysis strategy which allows confirmation that the correct gel plug has been removed prior to sending for sequencing. Lastly, the approach uses multiple sources of information to enable rapid triangulation and identification of protein candidates. Possible permutations and underlying principles of this triangulation strategy are elaborated to demonstrate the broad utility of this approach for antigen discovery.

<h4>Background</h4>Iron involvement/imbalance is strongly suspected in multiple sclerosis (MS) etiopathogenesis, but its role is quite debated. Iron deposits encircle the veins in brain MS lesions, increasing local metal concentrations in brain parenchyma as documented by magnetic resonance imaging and histochemical studies. Conversely, systemic iron overload is not always observed. We explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes in MS patients.<h4>Methods</h4>By the pyrosequencing technique, we investigated 414 MS cases [Relapsing-remitting (RR), n=273; Progressive, n=141, of which: Secondary (SP), n=103 and Primary (PP), n=38], and 414 matched healthy controls. Five SNPs in 4 genes were assessed: hemochromatosis (HFE: C282Y, H63D), ferroportin (FPN1: -8CG), hepcidin (HEPC: -582AG), and transferrin (TF: P570S).<h4>Results</h4>The FPN1-8GG genotype was overrepresented in the whole MS population (OR=4.38; 95%CI, 1.89-10.1; P<0.0001) and a similar risk was found among patients with progressive forms. Conversely, the HEPC -582GG genotype was overrepresented only in progressive forms (OR=2.53; 95%CI, 1.34-4.78; P=0.006) so that SP and PP versus RR yielded significant outputs (P=0.009). For almost all SNPs, MS disability score (EDSS), severity score (MSSS), as well as progression index (PI) showed a significant increase when comparing homozygotes versus individuals carrying other genotypes: HEPC -582GG (EDSS, 4.24±2.87 vs 2.78±2.1; P=0.003; MSSS, 5.6±3.06 vs 3.79±2.6; P=0.001); FPN1-8GG (PI, 1.11±2.01 vs 0.6±1.31; P=0.01; MSSS, 5.08±2.98 vs 3.85±2.8; P=0.01); HFE 63DD (PI, 1.63±2.6 vs 0.6±0.86; P=0.009). Finally, HEPC -582G-carriers had a significantly higher chance to switch into the progressive form (HR=3.55; 1.83-6.84; log-rank P=0.00006).<h4>Conclusions</h4>Polymorphisms in the genes coding for iron binding and transporting proteins, in the presence of local iron overload, might be responsible for suboptimal iron handling. This might account for the significant variability peculiar to MS phenotypes, particularly affecting MS risk and progression paving the way for personalized pharmacogenetic applications in the clinical practice.

Sox2 (sex-determining region Y-Box) is one of the master transcriptional factors that are important in maintaining the pluripotency of embryonic stem cells (ESCs). In line with this function, Sox2 expression is largely restricted to ESCs and somatic stem cells. We report that Sox2 is expressed in cell lines and tumor samples derived from ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), for which the normal cellular counterpart is believed to be mature T-cells. The expression of Sox2 in ALK(+)ALCL can be attributed to nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), the oncogenic fusion protein carrying a central pathogenetic role in these tumors. By confocal microscopy, Sox2 protein was detectable in virtually all cells in ALK(+)ALCL cell lines. However, the transcriptional activity of Sox2, as assessed using a Sox2-responsive reporter construct, was detectable only in a small proportion of cells. Importantly, downregulation of Sox2 using short interfering RNA in isolated Sox2(active) cells, but not Sox2(inactive) cells, resulted in a significant decrease in cell growth, invasiveness and tumorigenicity. To conclude, ALK(+)ALCL represents the first example of a hematologic malignancy that aberrantly expresses Sox2, which represents a novel mechanism by which NPM-ALK mediates tumorigenesis. We also found that the transcriptional activity and oncogenic effects of Sox2 can be heterogeneous in cancer cells.

Genome-wide gene expression profile analyses using a cDNA microarray containing 27,648 genes or expressed sequence tags identified MMS22L (methyl methanesulfonate-sensitivity protein 22-like) to be overexpressed in the majority of clinical lung and esophageal cancers, but not expressed in normal organs except testis. Transfection of siRNAs against MMS22L into cancer cells suppressed its expression and inhibited cell growth, while exogenous expression of MMS22L enhanced the growth of mammalian cells. MMS22L protein was translocated to the nucleus and stabilized by binding to C-terminal portion of NFKBIL2 [nuclear factor of kappa (NFKB) light polypeptide gene enhancer in B-cells inhibitor-like 2]. Expression of a C-terminal portion of NFKBIL2 protein including the MMS22L-interacting site in cancer cells could reduce the levels of MMS22L in nucleus and suppressed cancer cell growth. Interestingly, reduction of MMS22L by siRNAs in cancer cells inhibited the TNF-α-dependent activation of RelA/p65 in the NFKB pathway and expression of its downstream anti-apoptotic molecules such as Bcl-XL and TRAF1. In addition, knockdown of MMS22L expression also enhanced the apoptosis of cancer cells that were exposed to DNA-damaging agents including 5-FU and CDDP. Our data strongly suggest that targeting MMS22L as well as its interaction with NFKBIL2 could be a promising strategy for novel cancer treatments, and also improve the efficacy of DNA damaging anticancer drugs.

The cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (<i>CDKAL1</i>) gene encodes methylthiotransferase, and the gene contains risk variants for type 2 diabetes in humans. In this study, we performed complementary DNA cloning for <i>Cdkal1</i> in the cat and dog and characterized the tissue expression profiles of its messenger RNA. Cat and dog <i>Cdkal1</i> complementary DNA encoded 576 and 578 amino acids, showing very high sequence homology to mammalian <i>CDKAL1</i> (>88.4%). Real-time polymerase chain reaction analyses revealed that <i>Cdkal1</i> messenger RNA is highly expressed in smooth muscle and that tissue distribution of <i>Cdkal1</i> is similar in cats and dogs. Genotyping analysis of single-nucleotide polymorphism for cat <i>Cdkal1</i> revealed that obese cats had different tendencies from normal cats. These findings suggest that the cat and dog <i>Cdkal1</i> gene is highly conserved among mammals and that cat <i>Cdkal1</i> may be a candidate marker for genetic diagnosis of obesity.

Although elevated expression of CAV1/caveolin 1 is associated with the malignant progression of various human cancers, the molecular mechanism underlying its oncogenic functions is largely unknown. We found that CAV1 is frequently overexpressed in advanced colorectal tumors due to aberrant promoter CpG site hypomethylation, and its elevation is implicated in enhanced aerobic glycolysis of tumor cells. Depletion of elevated CAV1 downregulates glucose uptake, intracellular ATP level and lactate accumulation, and triggers autophagy through activation of AMPK-TP53/p53 signaling. CAV1 elevation increases glucose uptake and ATP production by stimulating transcription of the glucose transporter SLC2A3/GLUT3 via an HMGA1-binding site within the promoter. Collectively, our study suggests that elevated CAV1 expression may contribute to colorectal tumor progression by providing tumor cells growth and survival advantages under nutritional stress conditions.

Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrP(Sc) in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrP(Sc) in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrP(Sc) in a prion-infected cell line after treatment with bafilomycin A(1). These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.

<h4>Background</h4>Cell therapy is a potential therapeutic approach for several neurodegenetative disease, including Huntington Disease (HD). To evaluate the putative efficacy of cell therapy in HD, most studies have used excitotoxic animal models with only a few studies having been conducted in genetic animal models. Genetically modified animals should provide a more accurate representation of human HD, as they emulate the genetic basis of its etiology.<h4>Results</h4>In this study, we aimed to assess the therapeutic potential of a human striatal neural stem cell line (STROC05) implanted in the R6/2 transgenic mouse model of HD. As DARPP-32 GABAergic output neurons are predominately lost in HD, STROC05 cells were also pre-differentiated using purmorphamine, a hedgehog agonist, to yield a greater number of DARPP-32 cells. A bilateral injection of 4.5x105 cells of either undifferentiated or pre-differentiated DARPP-32 cells, however, did not affect outcome compared to a vehicle control injection. Both survival and neuronal differentiation remained poor with a mean of only 161 and 81 cells surviving in the undifferentiated and differentiated conditions respectively. Only a few cells expressed the neuronal marker Fox3.<h4>Conclusions</h4>Although the rapid brain atrophy and short life-span of the R6/2 model constitute adverse conditions to detect potentially delayed treatment effects, significant technical hurdles, such as poor cell survival and differentiation, were also sub-optimal. Further consideration of these aspects is therefore needed in more enduring transgenic HD models to provide a definite assessment of this cell line's therapeutic relevance. However, a combination of treatments is likely needed to affect outcome in transgenic models of HD.

So far, the annotation of translation initiation sites (TISs) has been based mostly upon bioinformatics rather than experimental evidence. We adapted ribosomal footprinting to puromycin-treated cells to generate a transcriptome-wide map of TISs in a human monocytic cell line. A neural network was trained on the ribosomal footprints observed at previously annotated AUG translation initiation codons (TICs), and used for the ab initio prediction of TISs in 5062 transcripts with sufficient sequence coverage. Functional interpretation suggested 2994 novel upstream open reading frames (uORFs) in the 5' UTR, 1406 uORFs overlapping with the coding sequence, and 546 N-terminal protein extensions. The TIS detection method was validated on the basis of previously published alternative TISs and uORFs. Among primates, TICs in newly annotated TISs were significantly more conserved than control codons, both for AUGs and near-cognate codons. The transcriptome-wide map of novel candidate TISs derived as part of the study will shed further light on the way in which human proteome diversity is influenced by alternative translation initiation and regulation.

<h4>Background</h4>We performed a genome-wide association study (GWAS) to identify common risk variants for schizophrenia.<h4>Methods</h4>The discovery scan included 1606 patients and 1794 controls from Ireland, using 6,212,339 directly genotyped or imputed single nucleotide polymorphisms (SNPs). A subset of this sample (270 cases and 860 controls) was subsequently included in the Psychiatric GWAS Consortium-schizophrenia GWAS meta-analysis.<h4>Results</h4>One hundred eight SNPs were taken forward for replication in an independent sample of 13,195 cases and 31,021 control subjects. The most significant associations in discovery, corrected for genomic inflation, were (rs204999, p combined = 1.34 × 10(-9) and in combined samples (rs2523722 p combined = 2.88 × 10(-16)) mapped to the major histocompatibility complex (MHC) region. We imputed classical human leukocyte antigen (HLA) alleles at the locus; the most significant finding was with HLA-C*01:02. This association was distinct from the top SNP signal. The HLA alleles DRB1*03:01 and B*08:01 were protective, replicating a previous study.<h4>Conclusions</h4>This study provides further support for involvement of MHC class I molecules in schizophrenia. We found evidence of association with previously reported risk alleles at the TCF4, VRK2, and ZNF804A loci.

<h4>Background</h4>Loss of parent during childhood or loss of partner has been associated with adulthood depression. The serotonin transporter polymorphism (5-HTTLPR) has been reported to moderate stress sensitivity reflected for example in the relationship between childhood maltreatment and depression. Therefore, the effect of 5-HTT promoter variation on the relationship between the loss of parent or partner and depression was examined.<h4>Method</h4>411 depressive cases and 1347 control subjects from a large well-characterized longitudinal population-based sample of adult Swedes with data on life history and life situation, including psychiatric diagnostic instruments, were studied. Their DNA was genotyped for the mini-haplotype 5-HTTLPR-rs25531.<h4>Results</h4>Individuals with low 5-HTT activity variants had an increased risk of depression given loss of partner last year compared to those with high activity variants. Conversely, 5-HTT activity variation appeared not to strongly influence the risk of depression given loss of parent during childhood.<h4>Limitation</h4>Small sample size for those with losses of both parent and partner. Limited power to detect small interaction effects.<h4>Conclusion</h4>The increased risk of depression given last year loss of partner appeared to be influenced by genetic variation regulating 5-HTT activity. This adds to previous findings of 5-HTT x stressful life events interactions on depression and is in agreement with stronger GxE effects when using objective environmental measures.

The "Hippo" signaling pathway has emerged as a major regulator of cell proliferation and survival in metazoans. The pathway, as delineated by genetic and biochemical studies in Drosophila, consists of a kinase cascade regulated by cell-cell contact and cell polarity that inhibits the transcriptional coactivator Yorkie and its proliferative, anti-differentiation, antiapoptotic transcriptional program. The core pathway components are the GC kinase Hippo, which phosphorylates the noncatalytic polypeptide Mats/Mob1 and, with the assistance of the scaffold protein Salvador, phosphorylates the ndr-family kinase Lats. In turn phospho-Lats, after binding to phospho-Mats, autoactivates and phosphorylates Yorkie, resulting in its nuclear exit. Hippo also uses the scaffold protein Furry and a different Mob protein to control another ndr-like kinase, the morphogenetic regulator Tricornered. Architecturally homologous kinase cascades consisting of a GC kinase, a Mob protein, a scaffolding polypeptide and an ndr-like kinase are well described in yeast; in Saccharomyces cerevisiae, e.g., the MEN pathway promotes mitotic exit whereas the RAM network, using a different GC kinase, Mob protein, scaffold and ndr-like kinase, regulates cell polarity and morphogenesis. In mammals, the Hippo orthologs Mst1 and Mst2 utilize the Salvador ortholog WW45/Sav1 and other scaffolds to regulate the kinases Lats1/Lats2 and ndr1/ndr2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in the epithelial cells of the liver and gut; loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by reduction or elimination of YAP. Despite this conservation, considerable diversification in pathway composition and regulation is already evident; in skin, e.g., YAP phosphorylation is independent of Mst1Mst2 and Lats1Lats2. Moreover, in lymphoid cells, Mst1/Mst2, under the control of the Rap1 GTPase and independent of YAP, promotes integrin clustering, actin remodeling and motility while restraining the proliferation of naïve T cells. This review will summarize current knowledge of the structure and regulation of the kinases Hippo/Mst1&2, their noncatalytic binding partners, Salvador and the Rassf polypeptides, and their major substrates Warts/Lats1&2, Trc/ndr1&2, Mats/Mob1 and FOXO.

Much emphasis has been placed on the identification, functional characterization, and therapeutic potential of somatic variants in tumor genomes. However, the majority of somatic variants lie outside coding regions and their role in cancer progression remains to be determined. In order to establish a system to test the functional importance of non-coding somatic variants in cancer, we created a low-passage cell culture of a metastatic melanoma tumor sample. As a foundation for interpreting functional assays, we performed whole-genome sequencing and analysis of this cell culture, the metastatic tumor from which it was derived, and the patient-matched normal genomes. When comparing somatic mutations identified in the cell culture and tissue genomes, we observe concordance at the majority of single nucleotide variants, whereas copy number changes are more variable. To understand the functional impact of non-coding somatic variation, we leveraged functional data generated by the ENCODE Project Consortium. We analyzed regulatory regions derived from multiple different cell types and found that melanocyte-specific regions are among the most depleted for somatic mutation accumulation. Significant depletion in other cell types suggests the metastatic melanoma cells de-differentiated to a more basal regulatory state. Experimental identification of genome-wide regulatory sites in two different melanoma samples supports this observation. Together, these results show that mutation accumulation in metastatic melanoma is nonrandom across the genome and that a de-differentiated regulatory architecture is common among different samples. Our findings enable identification of the underlying genetic components of melanoma and define the differences between a tissue-derived tumor sample and the cell culture created from it. Such information helps establish a broader mechanistic understanding of the linkage between non-coding genomic variations and the cellular evolution of cancer.

dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.

Throughout life, bone tissue undergoes a continuous process of resorption and formation. Melatonin, with its antioxidant properties and its ability to detoxify free radicals, as suggested by Conconi et al. (2000) may interfere in the osteoclast function and thereby inhibit bone resorption, as suggested by Schroeder et al. (1981). Inhibition of bone resorption may be enhanced by a reaction of indoleamine in osteoclastogenesis. That it has been observed melatonin, at pharmacological doses, decrease bone mass resorption by suppressing through down regulation of the RANK-L, as suggested by Penarrocha Diago et al. (2005) and Steflik et al. (1994). These data point an osteogenic effect towards that may be of melatonin of clinical importance, as it could be used as a therapeutic agent in situations in which would be advantageous bone formation, such as in the treatment of fractures or osteoporosis or their use as, a bioactive surface on implant as suggested by Lissoni et al. (1991).

Preoperative examination for abdominal aortic aneurysms (AAAs) occasionally reveals an abnormal decrease in coagulation factors and thrombocytopenia, fulfilling the criteria for disseminated intravascular coagulation (DIC). Treatment of the underlying disorder is indispensable for alleviating DIC. We report an uncommon case of a patient with AAA and DIC who showed prolonged thrombocytopenia despite successful treatment of AAA and temporary recovery of coagulation factors. A 70-year-old man presented with AAA and shaggy aorta accompanied by DIC and underwent aneurysmectomy. Combined preoperative use of nafamostat mesilate and recombinant human soluble thrombomodulin was effective in controlling DIC. Although recovery of coagulation factors was observed after surgery, the thrombocytopenia continued throughout the postoperative course and was refractory to platelet transfusion. Because HPA antibody and PA-IgG were present, a trial administration of γ-globulin was performed; this resulted in rapid improvement of thrombocytopenia. Although DIC recurred again 2 weeks thereafter, coagulation factors subsequently recovered without any medication.

<h4>Background</h4>Iron deficiency is the most common micronutrient deficiency worldwide. Experimental animal studies suggest that mothers deficient in iron during pregnancy are more likely to have offspring who become obese with high blood pressure. C282Y mutation carriers are more likely to have higher iron stores.<h4>Methods</h4>We undertook an instrumental variable (IV) analysis, using maternal C282Y as an indicator for the mother's iron status, to examine its association with offspring blood pressure (BP), waist circumference (WC), and body mass index (BMI), and compared the results to that of ordinary least squares (OLS) regression. Offspring of a sub-cohort of mothers from the UK Women's Cohort Study (UKWCS) were recruited in 2009-2010 (n = 348, mean age = 41 years). Their blood pressure, height, and weight were measured at their local general medical practice, and they were asked to self-measure their waist circumference. About half were offspring of C282Y carriers. Maternal ferritin was used as a biomarker of maternal iron status.<h4>Results</h4>Maternal C282Y was strongly associated with maternal ferritin (mean difference per allele = 84 g/L, 95% confidence interval: 31-137, P = 0.002). Using IV analyses, maternal ferritin was not linked to offspring's BP, BMI, or WC. The first stage F-statistic for the strength of the instrument was 10 (Kleibergen-Paap rk LM P = 0.009). Maternal ferritin was linked to offspring diastolic BP, WC, and BMI in univariable, but not in multivariable OLS analysis. There was no difference between the OLS and the IV models coefficients for any of the outcomes considered.<h4>Conclusion</h4>We found no association between maternal iron status and adult offspring's BP and adiposity using both multivariable OLS and IV modeling. To our knowledge, this is the first study examining this relationship. Further exploration in larger studies that have genetic variation assessed in both mother and offspring should be considered.

The meningococcal antigen typing system (MATS) sandwich enzyme-linked immunosorbent assay (ELISA) was designed to measure the immunologic cross-reactivity and quantity of antigens in target strains of a pathogen. It was first used to measure the factor H-binding protein (fHbp), neisserial adhesin A (NadA), and neisserial heparin-binding antigen (NHBA) content of serogroup B meningococcal (MenB) isolates relative to a reference strain, or "relative potency" (RP). With the PorA genotype, the RPs were then used to assess strain coverage by 4CMenB, a multicomponent MenB vaccine. In preliminary studies, MATS accurately predicted killing in the serum bactericidal assay using human complement, an accepted correlate of protection for meningococcal vaccines. A study across seven laboratories assessed the reproducibility of RPs for fHbp, NadA, and NHBA and established qualification parameters for new laboratories. RPs were determined in replicate for 17 MenB reference strains at laboratories A to G. The reproducibility of RPs among laboratories and against consensus values across laboratories was evaluated using a mixed-model analysis of variance. Interlaboratory agreement was very good; the Pearson correlation coefficients, coefficients of accuracy, and concordance correlation coefficients exceeded 99%. The summary measures of reproducibility, expressed as between-laboratory coefficients of variation, were 7.85% (fHbp), 16.51% (NadA), and 12.60% (NHBA). The overall within-laboratory measures of variation adjusted for strain and laboratory were 19.8% (fHbp), 28.8% (NHBA), and 38.3% (NadA). The MATS ELISA was successfully transferred to six laboratories, and a further laboratory was successfully qualified.

No abstract available.

Hemojuvelin is a critical regulator of hepcidin expression and can be cleaved by proteases to form soluble hemojuvelin. Soluble hemojuvelin has been recently identified in human serum but the presence and quantity of soluble hemojuvelin in mouse serum is unknown. We developed a two-site enzyme-linked immunosorbent assay using a monoclonal anti-hemojuvelin as the capture antibody and a biotinylated polyclonal anti-hemojuvelin as the detection antibody to quantify the levels of soluble hemojuvelin in mouse serum. We validated this assay using cell-conditioned media and serum from Hemojuvelin-null and Bone morphogenetic protein 6-null mice. We also used this validated assay to measure serum soluble hemojuvelin concentrations in mice receiving an acute low iron or high iron treatment. This two-site enzyme-linked immunosorbent assay was highly specific for mouse hemojuvelin, with a lower limit of detection at 13.2-26.8 ng/mL of soluble hemojuvelin in mouse serum. The median serum soluble hemojuvelin concentration in wild-type C57BL/6J mice was 57.9 ± 22 ng/mL, which is 4- to 20-fold less than that reported in healthy human volunteers. After acute low iron diet treatment in these mice, serum soluble hemojuvelin levels were increased and correlated with lowered serum iron levels and decreased hepatic hepcidin expression. An acute high iron diet in wild-type mice or chronically iron-overloaded Bone morphogenetic protein 6-null mice did not significantly lower serum soluble hemojuvelin concentrations. Here we report reliable quantitation of mouse serum soluble hemojuvelin using a novel and validated enzyme-linked immunosorbent assay. This assay may provide a useful tool to elucidate the source and physiological role of serum soluble hemojuvelin in hepcidin regulation and iron metabolism using well-established mouse models of iron-related disorders.

The application of functional genomic analysis of breast cancer metastasis has led to the identification of a growing number of organ-specific metastasis genes, which often function in concert to facilitate different steps of the metastatic cascade. However, the gene regulatory network that controls the expression of these metastasis genes remains largely unknown. Here, we demonstrate a computational approach for the deconvolution of transcriptional networks to discover master regulators of breast cancer bone metastasis. Several known regulators of breast cancer bone metastasis such as Smad4 and HIF1 were identified in our analysis. Experimental validation of the networks revealed BACH1, a basic leucine zipper transcription factor, as the common regulator of several functional metastasis genes, including MMP1 and CXCR4. Ectopic expression of BACH1 enhanced the malignance of breast cancer cells, and conversely, BACH1 knockdown significantly reduced bone metastasis. The expression of BACH1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. This study established BACH1 as the master regulator of breast cancer bone metastasis and provided a paradigm to identify molecular determinants in complex pathological processes.

Stool samples from two healthy infant siblings collected at about weekly intervals during their first year of life were analyzed by PCR for 15 different enteric viral genera. Adenovirus, Aichi virus, Anellovirus, Astrovirus, Bocavirus, Enterovirus, Parechovirus, Picobirnavirus, and Rotavirus were detected. Not detected were Coronavirus, Cardiovirus, Cosavirus, Salivirus, Sapovirus, and Norovirus. Long-term virus shedding, lasting from one to 12 months, was observed for adenoviruses, anelloviruses, bocaviruses, enteroviruses, parechoviruses, and picobirnaviruses. Repeated administration of oral poliovirus vaccine resulted in progressively shorter periods of poliovirus detection. Four nonpolio enterovirus genotypes were also detected. An average of 1.8 distinct human viruses were found per time point. Ninety-two percent (66/72) of the fecal samples tested contained one to five different human viruses. Two British siblings in the mid-1980s showed nearly constant fecal viral shedding. Our results demonstrate that frequent enteric infections with diverse viruses occur during early childhood in the absence of severe clinical symptoms.

NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both preleukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 of 27 (30%) acquired an Flt3 mutation. Two CA10 AMLs acquired an Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation.

<h4>Background & aims</h4>Hereditary hemochromatosis (HH) is most frequently related to homozygosity for the p.C282Y HFE mutation (C282Y(+/+)), hampering hepcidin induction in response to iron. The rs855791 polymorphism, encoding for the p.A736V variant of TMPRSS6 regulating hepcidin, influences iron status in the population. The aim of this study was to assess the influence of rs855791 on the penetrance and clinical expression of HH.<h4>Methods</h4>We retrospectively considered 315 HH patients (163 C282Y(+/+), and 152 with other HFE genotypes) evaluated at the time of diagnosis, and 271 healthy controls with normal iron parameters, residents of Northern Italy; TMPRSS6 genotype was assessed by allele specific polymerase chain reaction.<h4>Results</h4>The p.736V variant determining higher hepcidin release was under-represented in the patients (p=0.0023), independently of the presence of the C282Y(+/+) genotype, and the p.736V/V genotype protected from HH independently of age and sex (OR of HH for p.736A/A: 2.57, 1.3-4.1 and for p.736A/V: 1.84, 1.1-3.2). In the 96 C282Y(+/+) male patients without chronic viral hepatitis and alcohol abuse, the "high hepcidin" p.736V allele was negatively associated with cirrhosis independently of age, ferritin, ALT levels, and alcohol intake (OR 3.93, 95% C.I. 1.17-14.61 for the p.736A variant), and with the cumulative incidence of hepatocellular carcinoma (17% p.736A/A, 4% p.736A/V, 0 p.736V/V, p=0.05).<h4>Conclusions</h4>The p.A736V TMPRSS6 polymorphism is likely a modifier of HH expression. Additional studies are warranted to validate these findings in other cohorts and test their potential relevance for the clinical management of HH patients.

Following our earlier approach to the synthesis of dihydronepetalactones, all eight stereoisomers of trans-fused iridomyrmecins were synthesized starting from the enantiomers of limonene. Combined gas chromatography and mass spectrometry including enantioselective gas chromatography revealed that volatiles released by the endohyperparasitoid wasp Alloxysta victrix contain (4S,4aR,7S,7aR)-iridomyrmecin of 95-97% ee and stereochemically pure (4S,4aS,7R,7aS)-iridomyrmecin as a minor component.

Homologous chromosomes are usually separated at the entrance of meiosis; how they become paired is one of the outstanding mysteries of the meiotic process. Reduction of spacing between homologues makes possible the occurrence of chromosomal interactions leading to homology detection and the formation of bivalents. In many organisms, telomere-led chromosome movements are generated that bring homologues together. Additional movements produced by chromatin conformational changes at early meiosis may also facilitate homologous contacts. Organisms used in the study of meiosis show a surprising variety of strategies for homology detection. In dipterans, homologous chromosomes remain paired throughout most of development. Pairing seems to arise as a balance between promoter and suppressor pairing genes. Some fungi, plants and animals, use mechanisms based on recombinational interactions. Other mechanisms leading to homology search are recombination-independent and require specialized pairing sites. In the worm Caenorhabditis elegans, each chromosome carries a pairing center consisting of a chromosome-specific DNA-protein complex, and in the fission yeast Schizosaccharomyces pombe, the sme2 locus encodes a meiosis-specific non-coding RNA that mediates on homologous recognition. In addition, mismatch correction plays a relevant role, especially in polyploids, which evolved genetic systems that suppress pairing between non-homologous related (homoeologus) chromosomes.

<h4>Purpose</h4>The purpose of this study was to investigate light damage-induced transcript changes within neurosensory retina (NSR) and isolated retinal pigment epithelium (RPE). Similar studies have been conducted previously, but were usually limited to the NSR and only a portion of the transcriptome. Herein most of the transcriptome, not just in the NSR but also in isolated RPE, was queried.<h4>Methods</h4>Mice were exposed to 10,000 lux cool white fluorescent light for 18 hours and euthanized 4 hours after photic injury. NSR and isolated RPE were collected, and RNA was isolated. DNA microarray hybridization was conducted as described in the Affymetrix GeneChip Expression Analysis Technical Manual. Microarray analysis was performed using probe intensity data derived from the Mouse Gene 1.0 ST Array. For the genes of interest, confirmation of gene expression was done using quantitative real-time PCR. Immunofluorescence assessed protein levels and localization.<h4>Results</h4>Numerous iron regulatory genes were significantly changed in the light-exposed NSR and RPE. Several of these gene expression changes favored an iron-overloaded state. For example, the transferrin receptor was upregulated in both light-exposed NSR and RPE. Consistent with this, there was stronger transferrin receptor immunoreactivity in the light-exposed retinas. Significant changes in gene expression following light damage were also observed in oxidative stress and complement system genes.<h4>Conclusions</h4>The concept of a photooxidative stress-induced vicious cycle of increased iron uptake leading to further oxidative stress was introduced.

Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

<h4>Objective</h4>As the proportion of children living low malaria transmission areas in sub-Saharan Africa increases, approaches for identifying non-malarial severe illness need to be evaluated to improve child outcomes.<h4>Design</h4>As a prospective cohort study, we identified febrile paediatric inpatients, recorded data using Integrated Management of Childhood Illness (IMCI) criteria, and collected diagnostic specimens.<h4>Setting</h4>Tertiary referral centre, northern Tanzania.<h4>Results</h4>Of 466 participants with known outcome, median age was 1.4 years (range 2 months-13.0 years), 200 (42.9%) were female, 11 (2.4%) had malaria and 34 (7.3%) died. Inpatient death was associated with: Capillary refill >3 s (OR 9.0, 95% CI 3.0 to 26.7), inability to breastfeed or drink (OR 8.9, 95% CI 4.0 to 19.6), stiff neck (OR 7.0, 95% CI 2.8 to 17.6), lethargy (OR 5.2, 95% CI 2.5 to 10.6), skin pinch >2 s (OR 4.8, 95% CI 1.9 to 12.3), respiratory difficulty (OR 4.0, 95% CI 1.9 to 8.2), generalised lymphadenopathy (OR 3.6, 95% CI 1.6 to 8.3) and oral candidiasis (OR 3.4, 95% CI 1.4 to 8.3). BCS <5 (OR 27.2, p<0.001) and severe wasting (OR 6.9, p<0.001) were independently associated with inpatient death.<h4>Conclusions</h4>In a low malaria transmission setting, IMCI criteria performed well for predicting inpatient death from non-malarial illness. Laboratory results were not as useful in predicting death, underscoring the importance of clinical examination in assessing prognosis. Healthcare workers should consider local malaria epidemiology as malaria over-diagnosis in children may delay potentially life-saving interventions in areas where malaria is uncommon.

Cytosolic aggregation of the nuclear RNA-binding protein (RBP) TDP-43 (43 kDa TAR DNA-binding domain protein) is a suspected direct or indirect cause of motor neuron deterioration in amyotrophic lateral sclerosis (ALS). In this study, we implemented a high-content, genome-wide RNAi screen to identify pathways controlling TDP-43 nucleocytoplasmic shuttling. We identified ∼60 genes whose silencing increased the cytosolic localization of TDP-43, including nuclear pore complex components and regulators of G2/M cell cycle transition. In addition, we identified the type 1 inositol-1,4,5-trisphosphate (IP3) receptor (ITPR1), an IP3-gated, endoplasmic reticulum (ER)-resident Ca(2+) channel, as a strong modulator of TDP-43 nucleocytoplasmic shuttling. Knockdown or chemical inhibition of ITPR1 induced TDP-43 nuclear export in immortalized cells and primary neurons and strongly potentiated the recruitment of TDP-43 to Ubiquilin-positive autophagosomes, suggesting that diminished ITPR1 function leads to autophagosomal clearance of TDP-43. The functional significance of the TDP-43-ITPR1 genetic interaction was tested in Drosophila, where mutant alleles of ITPR1 were found to significantly extended lifespan and mobility of flies expressing TDP-43 under a motor neuron driver. These combined findings implicate IP3-gated Ca(2+) as a key regulator of TDP-43 nucleoplasmic shuttling and proteostasis and suggest pharmacologic inhibition of ITPR1 as a strategy to combat TDP-43-induced neurodegeneration in vivo.

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the PERIOD (PER) and TIMELESS (TIM) proteins accumulate during the night, inhibit the activity of the CLOCK (CLK)/CYCLE (CYC) transcriptional complex, and are degraded in the early morning. The control of PER and TIM oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. SLMB, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated PER. We show here that CULLIN-3 (CUL-3) is required for the circadian control of PER and TIM oscillations. Expression of either Cul-3 RNAi or dominant negative forms of CUL-3 in the clock neurons alters locomotor behavior and dampens PER and TIM oscillations in light-dark cycles. In constant conditions, CUL-3 deregulation induces behavioral arrhythmicity and rapidly abolishes TIM cycling, with slower effects on PER. CUL-3 affects TIM accumulation more strongly in the absence of PER and forms protein complexes with hypo-phosphorylated TIM. In contrast, SLMB affects TIM more strongly in the presence of PER and preferentially associates with phosphorylated TIM. CUL-3 and SLMB show additive effects on TIM and PER, suggesting different roles for the two ubiquitination complexes on PER and TIM cycling. This work thus shows that CUL-3 is a new component of the Drosophila clock, which plays an important role in the control of TIM oscillations.

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT(1A)) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT(1A) receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT(1A) receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT(1A) receptors. In addition, [(35)S]guanosine 5'-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT(1A) receptors. This finding has lent support to reports that diverse partial agonists for 5-HT(1A) receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.

Small molecules with antioxidative properties have been implicated in amyloid disorders. Curcumin is the active ingredient present in turmeric and known for several biological and medicinal effects. Adequate evidence substantiates the importance of curcumin in Alzheimer's disease and recent evidence suggests its role in Prion and Parkinson's disease. However, contradictory effects have been suggested for Huntington's disease. This difference provided a compelling reason to investigate the effect of curcumin on glutamine-rich (Q-rich) and non-glutamine-rich (non Q-rich) amyloid aggregates in the well established yeast model system. Curcumin significantly inhibited the formation of htt72Q-GFP (a Q-rich) and Het-s-GFP (a non Q-rich) aggregates in yeast. We show that curcumin prevents htt72Q-GFP aggregation by down regulating Vps36, a component of the ESCRT-II (Endosomal sorting complex required for transport). Moreover, curcumin disrupted the htt72Q-GFP aggregates that were pre-formed in yeast and cured the yeast prion, [PSI(+)].

Autism is a severe neurodevelopmental disorder, diagnosed on the basis of core behavioral symptoms. Although the mechanistic basis for the disorder is not yet known, genetic analyses have suggested a role for abnormal excitatory/inhibitory signaling systems in brain, including dysregulation of glutamatergic neurotransmission. In mice, the constitutive knockdown of NMDA receptors leads to social deficits, repetitive behavior, and self-injurious responses that reflect aspects of the autism clinical profile. However, social phenotypes differ with age: mice with reduced NMDA-receptor function exhibit hypersociability in adolescence, but markedly deficient sociability in adulthood. The present studies determined whether acute disruption of NMDA neurotransmission leads to exaggerated social approach, similar to that observed with constitutive disruption, in adolescent C57BL/6J mice. The effects of MK-801, an NMDA receptor antagonist, were compared with amphetamine, a dopamine agonist, and fluoxetine, a selective serotonin reuptake inhibitor, on performance in a three-chamber choice task. Results showed that acute treatment with MK-801 led to social approach deficits at doses without effects on entry numbers. Amphetamine also decreased social preference, but increased number of entries at every dose. Fluoxetine (10 mg/kg) had selective effects on social novelty preference. Withdrawal from a chronic ethanol regimen decreased activity, but did not attenuate sociability. Low doses of MK-801 and amphetamine were also evaluated in a marble-burying assay for repetitive behavior. MK-801, at a dose that did not disrupt sociability or alter entries, led to a profound reduction in marble-burying. Overall, these findings demonstrate that moderate alteration of NMDA, dopamine, or serotonin function can attenuate social preference in wild type mice.

Reversible histone methylation has emerged in the last few years as an important mechanism of epigenetic regulation. Histone methyltransferases and demethylases have been identified as contributing factors in the development of several diseases, especially cancer. Therefore, they have been postulated to be new drug targets with high therapeutic potential. Here, we review histone demethylases with a special focus on their potential role in oncology drug discovery. We present an overview over the different classes of enzymes, their biochemistry, selected data on their role in physiology and already available inhibitors.

Synthetic hydroxyapatite (HA) has been widely used and developed as the material for bone substitute in medical applications. The addition of zirconia is needed to improve the strength of hydroxyapatite as the bone substitute. One of the drawbacks in the use of biomedical materials is the occurrence of biomaterial-centred infections. The recent method of limiting the presence of microorganism on biomaterials is by providing biomaterial-bound metal-containing compositions. In this case, S. epidermidis is the most common infectious organism in biomedical-centred infection. Objective. This study was designed to evaluate the effect of zirconia concentrations in hydroxyapatite on the growth of S. epidermidis. Methods and Materials. The subjects of this study were twenty hydroxyapatite discs, divided into four groups in which one was the control and the other three were the treatment groups. Zirconia powder with the concentrations of 20%, 30%, and 40% was added into the three different treatment groups. Scanning electron microscope analysis was performed according to the hydroxyapatite and hydroxyapatite-zirconia specimens. All discs were immersed into S. epidermidis culture for 24 hours and later on they were soaked into a medium of PBS. The cultured medium was spread on mannitol salt agar. After incubation for 24 hours at 37°C , the number of colonies was measured with colony counter. Data obtained were analyzed using the ANOVA followed by the pairwise comparison. Result. The statistical analysis showed that different concentrations of zirconia powder significantly influenced the number of S. epidermidis colony (P < 0.05) . Conclusion. The addition of zirconia into hydroxyapatite affected the growth of S. epidermidis. Hydroxyapatite with 20% zirconia proved to be an effective concentration to inhibit the growth of S. epidermidis colony.

<h4>Background</h4>Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown.<h4>Objective</h4>To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC) or caffeic acid (CA) using whole-genome microarrays.<h4>Results</h4>ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells.<h4>Conclusions</h4>Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

Starting from the enantiomers of limonene, all eight stereoisomers of trans-fused dihydronepetalactones were synthesized. Key compounds were pure stereoisomers of 1-acetoxymethyl-2-methyl-5-(2-hydroxy-1-methylethyl)-1-cyclopentene. The stereogenic center of limonene was retained at position 4a of the target compounds and used to stereoselectively control the introduction of the other chiral centers during the synthesis. Basically, this approach could also be used for the synthesis of enantiomerically pure trans-fused iridomyrmecins. Using synthetic reference samples, the combination of enantioselective gas chromatography and mass spectrometry revealed that volatiles released by the endohyperparasitoid wasp Alloxysta victrix contain the enantiomerically pure trans-fused (4R,4aR,7R,7aS)-dihydronepetalactone as a minor component, showing an unusual (R)-configured stereogenic center at position 7.

Neuropeptide and neurotrophin receptors are increasingly important molecular targets in cancer. Scientific findings indicate that compounds blocking gastrin-releasing peptide receptors (GRPR) or tropomyosin receptor kinase (Trk) receptors are likely to have antiproliferative activities against cancer cells. The present study aimed to demonstrate that, in contrast to previous findings, GRPR activation reduces, whereas its blockade increases the viability of breast, ovarian and cervical cancer cell lines. However, consistent with previous studies, Trk inhibition was demonstrated to reduce the viability of these cells. MCF-7 (breast), OVCAR-3 (ovarian) and HeLa (cervical) human cancer cell lines were treated with GRP, the GRPR antagonists RC-3095 and RC-3940-II, brain-derived neurotrophic factor (BDNF) and the Trk antagonist K252α. Cell viability was measured by the MTT assay. Expression of GRPR and BDNF was confirmed with reverse transcription-polymerase chain reaction (RT-PCR). GRP reduced, whereas RC-3940-II enhanced the viability of the three cell lines. Treatment with K252α inhibited the viability of the cell lines, while BDNF increased the viability of OVCAR-3 cells. The results supported the hypothesis that GRPR and BDNF/TrkB signaling regulates cancer cell viability. Most importantly, these findings are the first to demonstrate that GRPR blockade can stimulate, rather than inhibits the viability of breast and gynecologic cancer cell lines.

<h4>Background</h4>Although a number of intestinal inflammatory conditions pertain to the ileum, whole-genome gene expression analyses in animal models of ileal inflammation are lacking to date. Therefore, we aimed to identify and characterize alterations in gene expression in the acutely inflamed ileum of two murine models of intestinal inflammation, namely intestinal schistosomiasis and TNBS-induced ileitis, compared to healthy controls. To this end, we used whole-genome microarrays, followed by bioinformatics analyses to detect over-represented Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology categories.<h4>Results</h4>Following screening of almost all known mouse genes and transcripts represented on the array, intestinal schistosomiasis and TNBS-induced ileitis yielded 207 and 1417 differentially expressed genes, respectively, with only 30 overlapping concordantly changed genes. Functional category groups consisting of complement and coagulation cascades, extracellular matrix (ECM)-receptor interaction, Fc epsilon receptor I signaling pathways and protein activation cascade, cell adhesion categories were over-represented in the differential gene list of intestinal schistosomiasis. Antigen processing and presentation, cell adhesion molecules, ABC transporters, Toll-like receptor signaling pathways and response to chemical stimulus categories were over-represented in the differential gene list of TNBS-induced ileitis. Although cytokine-cytokine receptor interaction, intestinal immune network for IgA production, focal adhesion pathways and immune, inflammatory and defense response categories were over-represented in the differential gene lists of both inflammation models, the vast majority of the associated genes and changes were unique to each model.<h4>Conclusions</h4>This study characterized two models of ileal inflammation at a whole-genome level and outlined distinct gene expression profiles and patterns in the two models. The results indicate that intestinal schistosomiasis involves Th2 responses, complement activation, protein activation and enhanced ECM turnover, while TNBS-induced ileitis involves Th17 responses, defective antigen processing and presentation and altered Toll-like receptor-mediated responses. Signs of an impaired epithelial barrier are apparent in both inflammation models. Furthermore, the comprehensive differential gene list and functional groups provided by this study constitute an interesting starting point to explore new targets and extended functional networks dealing with small bowel inflammation.

<h4>Objective</h4>DNA aberrations that cause colorectal cancer (CRC) occur in multiple steps that involve microsatellite instability (MSI) and chromosomal instability (CIN). Herein, we studied CRCs from AA patients for their CIN and MSI status.<h4>Experimental design</h4>Array CGH was performed on 30 AA colon tumors. The MSI status was established. The CGH data from AA were compared to published lists of 41 TSG and oncogenes in Caucasians and 68 cancer genes, proposed via systematic sequencing for somatic mutations in colon and breast tumors. The patient-by-patient CGH profiles were organized into a maximum parsimony cladogram to give insights into the tumors' aberrations lineage.<h4>Results</h4>The CGH analysis revealed that CIN was independent of age, gender, stage or location. However, both the number and nature of aberrations seem to depend on the MSI status. MSI-H tumors clustered together in the cladogram. The chromosomes with the highest rates of CGH aberrations were 3, 5, 7, 8, 20 and X. Chromosome X was primarily amplified in male patients. A comparison with Caucasians revealed an overall similar aberration profile with few exceptions for the following genes; THRB, RAF1, LPL, DCC, XIST, PCNT, STS and genes on the 20q12-q13 cytoband. Among the 68 CAN genes, all showed some level of alteration in our cohort.<h4>Conclusion</h4>Chromosome X amplification in male patients with CRC merits follow-up. The observed CIN may play a distinctive role in CRC in AAs. The clustering of MSI-H tumors in global CGH data analysis suggests that chromosomal aberrations are not random.

It is apparent that non-coding transcripts are a common feature of higher organisms and encode uncharacterized layers of genetic regulation and information. We used public bovine EST data from many developmental stages and tissues, and developed a pipeline for the genome wide identification and annotation of non-coding RNAs (ncRNAs). We have predicted 23,060 bovine ncRNAs, 99% of which are un-annotated, based on known ncRNA databases. Intergenic transcripts accounted for the majority (57%) of the predicted ncRNAs and the occurrence of ncRNAs and genes were only moderately correlated (r = 0.55, p-value<2.2e-16). Many of these intergenic non-coding RNAs mapped close to the 3' or 5' end of thousands of genes and many of these were transcribed from the opposite strand with respect to the closest gene, particularly regulatory-related genes. Conservation analyses showed that these ncRNAs were evolutionarily conserved, and many intergenic ncRNAs proximate to genes contained sequence-specific motifs. Correlation analysis of expression between these intergenic ncRNAs and protein-coding genes using RNA-seq data from a variety of tissues showed significant correlations with many transcripts. These results support the hypothesis that ncRNAs are common, transcribed in a regulated fashion and have regulatory functions.

We implemented least absolute shrinkage and selection operator (LASSO) regression to evaluate gene effects in genome-wide association studies (GWAS) of brain images, using an MRI-derived temporal lobe volume measure from 729 subjects scanned as part of the Alzheimer's Disease Neuroimaging Initiative (ADNI). Sparse groups of SNPs in individual genes were selected by LASSO, which identifies efficient sets of variants influencing the data. These SNPs were considered jointly when assessing their association with neuroimaging measures. We discovered 22 genes that passed genome-wide significance for influencing temporal lobe volume. This was a substantially greater number of significant genes compared to those found with standard, univariate GWAS. These top genes are all expressed in the brain and include genes previously related to brain function or neuropsychiatric disorders such as MACROD2, SORCS2, GRIN2B, MAGI2, NPAS3, CLSTN2, GABRG3, NRXN3, PRKAG2, GAS7, RBFOX1, ADARB2, CHD4, and CDH13. The top genes we identified with this method also displayed significant and widespread post hoc effects on voxelwise, tensor-based morphometry (TBM) maps of the temporal lobes. The most significantly associated gene was an autism susceptibility gene known as MACROD2. We were able to successfully replicate the effect of the MACROD2 gene in an independent cohort of 564 young, Australian healthy adult twins and siblings scanned with MRI (mean age: 23.8 ± 2.2 SD years). Our approach powerfully complements univariate techniques in detecting influences of genes on the living brain.

A multifunctional gold nanorod (GNR)-based nanoplatform for targeted anticancer drug delivery and positron emission tomography (PET) imaging of tumors was developed and characterized. An anti-cancer drug (i.e., doxorubicin (DOX)) was covalently conjugated onto PEGylated (PEG: polyethylene glycol) GNR nanocarriers via a hydrazone bond to achieve pH-sensitive controlled drug release. Tumor-targeting ligands (i.e., the cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides, cRGD) and (64)Cu-chelators (i.e., 1,4,7-triazacyclononane-N, N', N''-triacetic acid (NOTA)) were conjugated onto the distal ends of the PEG arms to achieve active tumor-targeting and PET imaging, respectively. Based on flow cytometry analysis, cRGD-conjugated nanocarriers (i.e., GNR-DOX-cRGD) exhibited a higher cellular uptake and cytotoxicity than non-targeted ones (i.e., GNR-DOX) in vitro. However, GNR-DOX-cRGD and GNR-DOX nanocarriers had similar in vivo biodistribution according to in vivo PET imaging and biodistribution studies. Due to the unique optical properties of GNRs, this multifunctional GNR-based nanoplatform can potentially be optimized for combined cancer therapies (chemotherapy and photothermal therapy) and multimodality imaging (PET, optical, X-ray computed tomography (CT), etc.).

A review of in vitro bioaccessibility and bioavailability methods for polyphenols and selected nutrients is presented. The review focuses on in vitro solubility, dialyzability, the dynamic gastrointestinal model (TIM)™, and Caco-2 cell models, the latter primarily for uptake and transport, and a discussion of how these methods have been applied to generate data for a range of nutrients, carotenoids, and polyphenols. Recommendations are given regarding which methods are most justified for answering bioaccessibility or bioavailability related questions for specific nutrients. The need for more validation studies in which in vivo results are compared to in vitro results is also discussed.

The PICALM-MLLT10 fusion gene, generated by the t(10;11)(p12-13;q14-21) translocation, is a rare but recurrent event in acute leukemias. In this study, we assessed the characteristics and outcome of 18 PICALM-MLLT10 AML patients. As compared with non PICALM-MLLT10 patients (n=72), PICALM-MLLT10 AML were characterized by more frequent extramedullary diseases, CD7 expression and higher platelet counts. Three out of four therapy-related PICALM-MLLT10 AMLs had been previously treated for diffuse large B-cell lymphoma. The complete response rate was 71% after intensive chemotherapy. PICALM-MLLT10 patients had a shorter median overall survival than patients with favorable cytogenetics (12 months vs. not reached, p=0.07) but not significantly different from those of intermediate (26 months, p=0.32) or unfavorable cytogenetic groups (8 months, p=0.13). Long term responses were achieved in a subset of patients after allogeneic stem-cell transplantation but also after high-dose cytarabine.

Disruption of cellular cholesterol balance results in pathologic processes including atherosclerosis, metabolic syndrome, type II diabetes and Alzheimer's disease. Maintenance of cholesterol homeostasis requires constant metabolic adjustment, achieved partly through the fine regulation of the classical transcription factors (e.g., by SREBP and LXR), but also through members of a class of noncoding RNAs termed miRNAs. Some miRNAs have now been identified to be potent post-transcriptional regulators of lipid metabolism genes, including miR-122, miR-33, miR-758, and miR-106b. Different strategies have been developed to modulate miRNA effects for therapeutic purposes. The promise demonstrated by the use of anti-miRs in human preclinical studies, in the case of miR-122, raises the possibility that miR-33, miR-758, and miR-106b may become viable therapeutic targets in future. This review summarizes the evidence for a critical role of some miRNAs in regulating cholesterol metabolism and suggests novel ways to manage dyslipidemias and cardiovascular diseases.

We developed a paclitaxel-conjugated polymeric micelle, ABP-PEG3.5k-Paclitaxel (APP) consisting of poly (ethylene glycol) (PEG) and arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP) for the co-delivery of gene and drug. The APP polymer self-assembled into cationic polymeric micelles with a critical micelle concentration (CMC) value of approximately 0.062 mg/mL, which was determined from measurements of the UV absorption of pyrene. The micelles have an average size of about 3 nm and a zeta potential of about +14 mV. Due to the positive surface charge, APP micelles formed polyplexes with plasmid DNA approximately 200 nm in diameter. The luciferase gene and mouse interleukin-12 (IL-12) gene was used to monitor gene delivery potency. APP polyplexes showed increased gene delivery efficiency and cellular uptake with higher anticancer potency than paclitaxel alone. These results demonstrate that an APP micelle-based delivery system is well suitable for the co-delivery of gene and drug.

Psychologists, quality of life and well-being researchers have grown increasingly interested in understanding the factors that are associated with human happiness. Although twin studies estimate that genetic factors account for 35-50% of the variance in human happiness, knowledge of specific genes is limited. However, recent advances in molecular genetics can now provide a window into neurobiological markers of human happiness. This investigation examines association between happiness and monoamine oxidase A (MAOA) genotype. Data were drawn from a longitudinal study of a population-based cohort, followed for three decades. In women, low expression of MAOA (MAOA-L) was related significantly to greater happiness (0.261 SD increase with one L-allele, 0.522 SD with two L-alleles, P=0.002) after adjusting for the potential effects of age, education, household income, marital status, employment status, mental disorder, physical health, relationship quality, religiosity, abuse history, recent negative life events and self-esteem use in linear regression models. In contrast, no such association was found in men. This new finding may help explain the gender difference on happiness and provide a link between MAOA and human happiness.

Hereditary hemochromatosis causes iron overload and is associated with a variety of genetic and phenotypic conditions. Early diagnosis is important so that effective treatment can be administered and the risk of tissue damage avoided. Most patients are homozygous for the c.845G>A (p.C282Y) mutation in the HFE gene; however, rare forms of genetic iron overload must be diagnosed using a specific genetic analysis. We studied the genotype of 5 patients who had hyperferritinemia and an iron overload phenotype, but not classic mutations in the HFE gene. Two patients were undergoing phlebotomy and had no iron overload, 1 with metabolic syndrome and no phlebotomy had mild iron overload, and 2 patients had severe iron overload despite phlebotomy. The patients' first-degree relatives also underwent the analysis. We found 5 not previously published mutations: c.-408_-406delCAA in HFE, c.1118G>A (p.G373D), c.1473G>A (p.E491E) and c.2085G>C (p.S695S) in TFR2; and c.-428_-427GG>TT in SLC40A1. Moreover, we found 3 previously published mutations: c.221C>T (p.R71X) in HFE; c.1127C>A (p.A376D) in TFR2; and c.539T>C (p.I180T) in SLC40A1. Four patients were double heterozygous or compound heterozygous for the mutations mentioned above, and the patient with metabolic syndrome was heterozygous for a mutation in the TFR2 gene. Our findings show that hereditary hemochromatosis is clinically and genetically heterogeneous and that acquired factors may modify or determine the phenotype.

The antiporter system x(c)(-) imports the amino acid cystine, the oxidized form of cysteine, into cells with a 1:1 counter-transport of glutamate. It is composed of a light chain, xCT, and a heavy chain, 4F2 heavy chain (4F2hc), and, thus, belongs to the family of heterodimeric amino acid transporters. Cysteine is the rate-limiting substrate for the important antioxidant glutathione (GSH) and, along with cystine, it also forms a key redox couple on its own. Glutamate is a major neurotransmitter in the central nervous system (CNS). By phylogenetic analysis, we show that system x(c)(-) is a rather evolutionarily new amino acid transport system. In addition, we summarize the current knowledge regarding the molecular mechanisms that regulate system x(c)(-), including the transcriptional regulation of the xCT light chain, posttranscriptional mechanisms, and pharmacological inhibitors of system x(c)(-). Moreover, the roles of system x(c)(-) in regulating GSH levels, the redox state of the extracellular cystine/cysteine redox couple, and extracellular glutamate levels are discussed. In vitro, glutamate-mediated system x(c)(-) inhibition leads to neuronal cell death, a paradigm called oxidative glutamate toxicity, which has successfully been used to identify neuroprotective compounds. In vivo, xCT has a rather restricted expression pattern with the highest levels in the CNS and parts of the immune system. System x(c)(-) is also present in the eye. Moreover, an elevated expression of xCT has been reported in cancer. We highlight the diverse roles of system x(c)(-) in the regulation of the immune response, in various aspects of cancer and in the eye and the CNS.

Reverse transcription (RT)-PCR is now the standard method for typing group A rotaviruses (RVA) to monitor the circulating genotypes in a population. Selection of primers that can accurately type the circulating genotypes is crucial in the context of vaccine introduction and correctly interpreting the impact of vaccination on strain distribution. To our knowledge this study is the first report from Asia of misidentification of genotype G9 as G3 due to a primer-template mismatch. We tested two published G-genotype specific primers sets, designed by Gouvea and colleagues (Set A) and Iturriza-Gomara and colleagues (Set B) on RVA from Hong Kong and Sri Lanka. Among 52 rotaviruses typed as G3 by set A primers, 36 (69.2%) were identified as G9 by nucleotide sequencing and set B primers. Moreover, of 300 rotaviruses tested, 28.3% were untypable by set A primers whereas only 12.3% were untypable by set B primers. Our findings reinforce the need to periodically monitor the primers used for RVA genotyping.

Poly(2-oxazoline)s (POx) are currently discussed as an upcoming polymer platform for biomaterials design and especially for polymer therapeutics. POx meet specific requirements needed for the development of next-generation polymer therapeutics such as biocompatibility, high modulation of solubility, variation of size, architecture as well as chemical functionality. Although in the early 1990s first and promising POx-based systems were presented, the field lay dormant for almost two decades. Only very recently, POx-based polymer therapeutics came back into the focus of very intensive research. In this review, we give an overview on the chemistry and physicochemical properties of POx and summarize the research of POx-protein conjugates, POx-drug conjugates, POx-based polyplexes and POx micelles for drug delivery.

To ascertain whether reactive oxygen species (ROS) contribute to training-induced adaptation of skeletal muscle, we administered ROS-scavenging antioxidants (AOX; 140 mg/l of ascorbic acid, 12 mg/l of coenzyme Q10 and 1% N-acetyl-cysteine) via drinking water to 16 C57BL/6 mice. Sixteen other mice received unadulterated tap water (CON). One cohort of both groups (CON(EXE) and AOX(EXE) ) was subjected to treadmill exercise for 4 weeks (16-26 m/min, incline of 5°-10°). The other two cohorts (CON(SED) and AOX(SED) ) remained sedentary. In skeletal muscles of the AOX(EXE) mice, GSSG and the expression levels of SOD-1 and PRDX-6 were significantly lower than those in the CON(EXE) mice after training, suggesting disturbance of ROS levels. The peak power related to the body weight and citrate synthase activity was not significantly influenced in mice receiving AOX. Supplementation with AOX significantly altered the mRNA levels of the exercise-sensitive genes HK-II, GLUT-4 and SREBF-1c and the regulator gene PGC-1alpha but not G6PDH, glycogenin, FABP-3, MCAD and CD36 in skeletal muscle. Although the administration of AOX during endurance exercise alters the expression of particular genes of the ROS metabolism, it does not influence peak power or generally shift the metabolism, but it modulates the expression of specific genes of the carbohydrate and lipid metabolism and PGC-1alpha within murine skeletal muscle.

MLL-rearranged leukemias exemplify malignancies with perturbations of the epigenetic landscape. Specific chromatin modifications that aid in the perpetuation of MLL fusion gene driven oncogenic programs are being defined, presenting novel avenues for therapeutic intervention. Proof-of-concept studies have recently been reported, using small-molecule inhibitors targeting the histone methyltransferase disruptor of telomeric silencing 1-like (DOT1L), or the acetyl-histone binding protein bromodomain containing protein 4 (BRD4) showing potent activity against MLL-rearranged leukemias in preclinical models. It is apparent that intensive efforts will be made toward the further development of small-molecule inhibitors targeting these, and other chromatin-associated protein targets. These studies may lead to the advent of a new generation of much-needed therapeutic modalities in leukemia and other cancers.

OX40L is an important costimulatory molecule that plays a crucial role in the regulation of T-cell-mediated immunity. The interaction of OX40-OX40L is involved in the pathogenesis of multiple autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), carotid artery disease and cancer. The genetic variants of OX40L can increase the risk of SLE, atherosclerosis, systemic sclerosis and show gender-specific effects in some studies. Accordingly, we performed a case-control study including 557 breast cancer patients and 580 age- and sex-matched healthy controls to investigate whether single nucleotide polymorphisms (SNPs) in the OX40L gene are associated with sporadic breast cancer susceptibility and progression in Chinese Han women. Seven SNPs of OX40L (rs6661173, rs1234313, rs3850641, rs1234315, rs12039904, rs844648 and rs10912580) were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results indicated that rs3850641G allele could increase the susceptibility to breast cancer (P = 0.009662), even in the validation study (P = 0.0001515). A significant association between rs3850641 and breast cancer risk was observed under the additive model and dominant model (P = 0.01042 and 0.01942, respectively). The haplotype analysis showed that haplotype A(rs844648)A(rs10912580) was significantly associated with breast cancer, even after 10,000 permutations for haplotypes in block only (P = 0.0003). In clinicopathologic features analysis, the association between rs1234315 and C-erbB2 status was significant (P = 0.02541). Our data primarily indicates that rs3850641 of OX40L gene contributes to sporadic breast carcinogenesis in a northeast Chinese Han population.

While neurodegenerative diseases are characterized by steady degeneration over relatively long timelines, it is widely believed that the early stages are the most promising for therapeutic intervention, before irreversible neuronal loss occurs. Developing a therapeutic response requires a precise measure of disease progression. However, since the early stages are for the most part asymptomatic, obtaining accurate measures of disease progression is difficult. Longitudinal databases of hundreds of subjects observed during several years with tens of validated biomarkers are becoming available, allowing the use of computational methods. We propose a widely applicable statistical methodology for creating a disease progression score (DPS), using multiple biomarkers, for subjects with a neurodegenerative disease. The proposed methodology was evaluated for Alzheimer's disease (AD) using the publicly available AD Neuroimaging Initiative (ADNI) database, yielding an Alzheimer's DPS or ADPS score for each subject and each time-point in the database. In addition, a common description of biomarker changes was produced allowing for an ordering of the biomarkers. The Rey Auditory Verbal Learning Test delayed recall was found to be the earliest biomarker to become abnormal. The group of biomarkers comprising the volume of the hippocampus and the protein concentration amyloid beta and Tau were next in the timeline, and these were followed by three cognitive biomarkers. The proposed methodology thus has potential to stage individuals according to their state of disease progression relative to a population and to deduce common behaviors of biomarkers in the disease itself.