Gene Literature Dashboard

We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and FcγR1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-α, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.

<h4>Background</h4>Human Rotavirus is a significant cause of severe gastroenteritis in infants and young children worldwide. In recent years, Rotavirus genotyping by RT-PCR has provided valuable information about the diversity of Rotaviruses circulating worldwide. The purpose of the present study is to monitor the prevalence of the different G types of Rotaviruses circulating in Shiraz, Southern Iran and detect any uncommon or novel types.<h4>Methods</h4>During the period from December 2007 to November 2008, a total of 138 stool samples were collected from children less than 5 years old who were hospitalized for acute gastroenteritis. Rotavirus-associated diarrhea was investigated in fecal specimens with enzyme immunoassays (EIA). Rotavirus-positive specimens were typed by the Nested RT-PCR and by using different types of specific primers.<h4>Results</h4>Out of the 138 collected samples, 34.78% (48 cases) tested positive for Rotavirus. The frequency of G1, G2 and G4 types was 6.25%, 2.08% and 27.08%, respectively. Mixed and non-typeable infections were detected in 33.34% and 31.25% of hospitalized children with acute diarrhea, respectively. This is the first time mixed Rotavirus infections with G1/G3 have been reported in Iran.<h4>Conclusion</h4>The high frequency of Rotavirus detection indicates the severity and the burden of Rotavirus disease may be able to reduce through the implementation of an effective vaccine and continual surveillance for the detection of Rotavirus genotypes circulating in other regions of Iran. Regarding to the noticeable frequency of non-typeable and mixed infections, it is suggested to use the other specific primers and further studies to detection of other novel and unusual types.

Phase II metabolic enzymes are a battery of critical proteins that detoxify xenobiotics by increasing their hydrophilicity and enhancing their disposal. These enzymes have long been studied for their preventative and protective effects against mutagens and carcinogens and for their regulation via the Keap1 (Kelch-like ECH associated protein 1)/Nrf2 (Nuclear factor erythroid 2 related factor 2)/ARE (antioxidant response elements) pathway. Recently, a series of studies have reported the altered expression of phase II genes in postmortem tissue of patients with various neurological diseases. These observations hint at a role for phase II enzymes in the evolution of such conditions. Furthermore, promising findings reveal that overexpression of phase II genes, either by genetic or chemical approaches, confers neuroprotection in vitro and in vivo. Therefore, there is a need to summarize the current literature on phase II genes in the central nervous system (CNS). This should help guide future studies on phase II genes as therapeutic targets in neurological diseases. In this review, we first briefly introduce the concept of phase I, II and III enzymes, with a special focus on phase II enzymes. We then discuss their expression regulation, their inducers and executors. Following this background, we expand our discussion to the neuroprotective effects of phase II enzymes and the potential application of Nrf2 inducers to the treatment of neurological diseases.

Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization.

Novel benzofuran-2-carboxamide ligands, which are selective for sigma receptors, have been synthesized via a microwave-assisted Perkin rearrangement reaction and a modified Finkelstein halogen-exchange used to facilitate N-alkylation. The ligands synthesized are the 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides (KSCM-1, KSCM-5 and KSCM-11). The benzofuran-2-carboxamide structure was N-arylated and N-alkylated to include both N-phenyl and N-(3-(piperidin-1-yl)propyl substituents, respectively. These new carboxamides exhibit high affinity at the sigma-1 receptor with K(i) values ranging from 7.8 to 34nM. Ligand KSCM-1 with two methoxy substituents at C-5 and C-6 of the benzofuran ring, and K(i)=27.5nM at sigma-1 was found to be more selective for sigma-1 over sigma-2.

<h4>Introduction</h4>Tetrabenazine (TBZ) reduces chorea related to Huntington disease (HD); however, it is uncertain whether this effect improves functionally relevant motor skills such as hand coordination and balance. The objective of this study was to provide pilot data regarding three motor function tests, which might be useful in monitoring symptom progression and therapeutic response, pending formal validation.<h4>Methods</h4>The authors assessed 11 ambulatory patients with HD-related chorea on two occasions: (1) while off TBZ (either prior to starting therapy or following a >24 h washout) and (2) when on a stable dose of TBZ, titrated to optimal effect. Study evaluations included the Jebsen-Taylor Hand Function Test (JTHFT) and Berg Balance Scale, a timed 25-foot walk, the Montreal Cognitive Assessment (MoCA) and the complete United Huntington Disease Rating Scale (UHDRS).<h4>Results</h4>Maximal chorea scores (UHDRS item 12) improved from 11.1 ± 2.9 to 8.5 ± 3.9 while on TBZ (P = 0.03), but we could not detect an improvement in functional measures while on TBZ in this small cohort. Scores of the JTHFT were globally slower than published normative data and correlated with MoCA summary scores, but not UHDRS chorea scores.<h4>Conclusions</h4>This pilot study did not detect significant functional gains with chorea suppression. The fact that performance on tests of hand function correlates with MoCA but not UHDRS chorea scores highlights the need for additional treatments targeted toward the cognitive aspects of HD.

Emerging evidence indicates that microRNAs (miRNAs) may play an important role in the pathogenesis of Huntington's disease (HD). To identify the individual miRNAs that are altered in HD and may therefore regulate a gene network underlying mutant huntingtin-induced neuronal dysfunction in HD, we performed miRNA array analysis combined with mRNA profiling in the cerebral cortex from N171-82Q HD mice. Expression profiles of miRNAs as well as mRNAs in HD mouse cerebral cortex were analyzed and confirmed at different stages of disease progression; the most significant changes of miRNAs in the cerebral cortex were also detected in the striatum of HD mice. Our results revealed a significant alteration of miR-200 family members, miR-200a, and miR-200c in the cerebral cortex and the striatum, at the early stage of disease progression in N171-82Q HD mice. We used a coordinated approach to integrate miRNA and mRNA profiling, and applied bioinformatics to predict a target gene network potentially regulated by these significantly altered miRNAs that might be involved in HD disease progression. Interestingly, miR-200a and miR-200c are predicted to target genes regulating synaptic function, neurodevelopment, and neuronal survival. Our results suggest that altered expression of miR-200a and miR-200c may interrupt the production of proteins involved in neuronal plasticity and survival, and further investigation of the involvement of perturbed miRNA expression in HD pathogenesis is warranted, and may lead to reveal novel approaches for HD therapy.

Pre-natal alcohol exposure causes fetal alcohol spectrum disorders (FASD), the most common, preventable cause of developmental disability. The developing cerebellum is particularly vulnerable to the effects of ethanol. We reported that ethanol inhibits the stimulation of axon outgrowth in cerebellar granule neurons (CGN) by NAP, an active motif of activity-dependent neuroprotective protein (ADNP), by blocking NAP activation of Fyn kinase and its downstream signaling molecule, the scaffolding protein Cas. Here, we asked whether ethanol inhibits the stimulation of axon outgrowth by diverse axon guidance molecules through a common action on the Src family kinases (SFK). We first demonstrated that netrin-1, glial cell line-derived neurotrophic factor (GDNF), and neural cell adhesion molecule L1 stimulate axon outgrowth in CGNs by activating SFK, Cas, and extracellular signal-regulated kinase 1 and 2 (ERK1/2). The specific SFK inhibitor, PP2, blocked the stimulation of axon outgrowth and the activation of the SFK-Cas-ERK1/2 signaling pathway by each of these axon-guidance molecules. In contrast, brain-derived neurotrophic factor (BDNF) stimulated axon outgrowth and activated ERK1/2 without first activating SFK or Cas. Clinically relevant concentrations of ethanol inhibited axon outgrowth and the activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, and L1, but did not disrupt BDNF-induced axon outgrowth or ERK1/2 activation. These results indicate that SFK, but not ERK1/2, is a primary target for ethanol inhibition of axon outgrowth. The ability of ethanol to block the convergent activation of the SFK-Cas-ERK1/2 pathway by netrin-1, GDNF, L1, and ADNP could contribute significantly to the pathogenesis of FASD.

Many anticancer drugs have been established clinically, but their efficacy can be compromised by nonspecific toxicity and an inability to reach the desired cancerous intracellular spaces. In order to address these issues, researchers have explored the use of folic acid as a targeted moiety to increase specificity of chemotherapeutic drugs. To expand upon such research, we have conjugated folic acid to functionalized poly(ethylene glycol) and subsequently decorated the surface of l-tyrosine polyphosphate (LTP) nanoparticles. These nanoparticles possess the appropriate size (100-500 nm) for internalization as shown by scanning electron microscopy and dynamic light scattering. Under simulated physiological flow, LTP nanoparticles decorated with folic acid (targeted nanoparticles) show a 10-fold greater attachment to HeLa, a cervical cancer cell line, compared to control nanoparticles and to human dermal fibroblasts. The attachment of these targeted nanoparticles progresses at a linear rate, and the strength of this nanoparticle attachment is shown to withstand shear stresses of 3.0 dyn/cm(2). These interactions of the targeted nanoparticles to HeLa are likely a result of a receptor-ligand binding, as a competition study with free folic acid inhibits the nanoparticle attachment. Finally, the targeted nanoparticles encapsulated with a silver based drug show increased efficacy in comparison to nondecorated (plain) nanoparticles and drug alone against HeLa cells. Thus, targeted nanoparticles are a promising delivery platform for developing anticancer therapies that overexpress the folate receptors (FRs).

Several neurodegenerative diseases are associated with the polyglutamine (polyQ) repeat disorder in which a segment of consecutive glutamines in the native protein is produced with too many glutamines. Huntington's disease, for example, is related to the misfolding of the Huntingtin protein which occurs when the polyQ segment has more than approximately 36 glutamines. Experimentally, it is known that the polyQ segment alone aggregates into β-rich conformations such as amyloid fibrils. Its aggregation is modulated by the number of glutamine residues as well as by the surrounding amino acid sequences such as the 17-amino-acid N-terminal fragment of Huntingtin which increases the aggregation rate. Little structural information is available, however, regarding the first steps of aggregation and the atomistic mechanisms of oligomerization are yet to be described. Following previous coarse-grained replica-exchange molecular dynamics simulations that show the spontaneous formation of a nanotube consisting of two intertwined antiparallel strands (Laghaei, R.; Mousseau, N. J. Chem. Phys. 2010, 132, 165102), we study this configuration and some extensions of it using all-atom explicit solvent MD simulations. We compare two different lengths for the polyQ segment, 40 and 30 glutamines, and we investigate the impact of the Huntingtin N-terminal residues (htt(NT)). Our results show that the dimeric nanotubes can provide a building block for the formation of longer nanotubes (hexamers and octamers). These longer nanotubes are characterized by large β-sheet propensities and a small solvent exposure of the main-chain atoms. Moreover, the oligomerization between two nanotubes occurs through the formation of protein/protein H-bonds and can result in an elongation of the water-filled core. Our results also show that the htt(NT) enhances the structural stability of the β-rich seeds, suggesting a new mechanism by which it can increase the aggregation rate of the amyloidogenic polyQ sequence.

<h4>Background</h4>Parkinson's disease (PD) is a multifactorial disease where environmental factors act on genetically predisposed individuals. Although only 5% of PD manifestations are associated with specific mutations, majority of PD cases are of idiopathic origin, where environment plays a prominent role. Concurrent exposure to Paraquat (PQ) and Maneb (MB) in rural workers increases the risk for PD and exposure of adult mice to MB/PQ results in dopamine fiber loss and decreased locomotor activity. While PD is characterized by neuronal loss in the substantia nigra, we previously showed that accumulation of α-synuclein in the limbic system contributes to neurodegeneration by interfering with adult neurogenesis.<h4>Results</h4>We investigated the effect of pesticides on adult hippocampal neurogenesis in two transgenic models: Line 61, expressing the human wild type SNCA gene and Line LRRK2(G2019S), expressing the human LRRK2 gene with the mutation G2019S. Combined exposure to MB/PQ resulted in significant reduction of neuronal precursors and proliferating cells in non-transgenic animals, and this effect was increased in transgenic mice, in particular for Line 61, suggesting that α-synuclein accumulation and environmental toxins have a synergistic effect. We further investigated the transcription of 84 genes with direct function on neurogenesis. Overexpresion of α-synuclein resulted in the downregulation of 12% of target genes, most of which were functionally related to cell differentiation, while LRRK2 mutation had a minor impact on gene expression. MB/PQ also affected transcription in non-transgenic backgrounds, but when transgenic mice were exposed to the pesticides, profound alterations in gene expression affecting 27% of the studied targets were observed in both transgenic lines. Gene enrichment analysis showed that 1:3 of those genes were under the regulation of FoxF2 and FoxO3A, suggesting a primary role of these proteins in the response to genetic and environmental cues.<h4>Conclusions</h4>We report that adult neurogenesis is highly susceptible to multiple "risk factors" for PD, including α-synuclein accumulation, LRRK2 G2019 mutation and exposure to environmental toxins. We identified specific groups of genes that are responsive to each stressor, while uncovering a novel function for Fox transcription factors in PD.

Mucoepidermoid carcinoma is the most common salivary gland malignancy, and includes a spectrum of lesions ranging from non-aggressive low-grade tumors to aggressive high-grade tumors. To further characterize this heterogeneous group of tumors we have performed a comprehensive analysis of copy number alterations and CRTC1-MAML2 fusion status in a series of 28 mucoepidermoid carcinomas. The CRTC1-MAML2 fusion was detected by RT-PCR or fluorescence in situ hybridization in 18 of 28 mucoepidermoid carcinomas (64%). All 15 low-grade tumors were fusion-positive whereas only 3 of 13 high-grade tumors were fusion-positive. High-resolution array-based comparative genomic hybridization revealed that fusion-positive tumors had significantly fewer copy number alterations/tumor compared with fusion-negative tumors (1.5 vs 9.5; P=0.002). Twelve of 18 fusion-positive tumors had normal genomic profiles whereas only 1 out of 10 fusion-negative tumors lacked copy number alterations. The profiles of fusion-positive and fusion-negative tumors were very similar to those of low- and high-grade tumors. Thus, low-grade mucoepidermoid carcinomas had significantly fewer copy number alterations/tumor compared with high-grade mucoepidermoid carcinomas (0.7 vs 8.6; P<0.0001). The most frequent copy number alterations detected were losses of 18q12.2-qter (including the tumor suppressor genes DCC, SMAD4, and GALR1), 9p21.3 (including the tumor suppressor genes CDKN2A/B), 6q22.1-q23.1, and 8pter-p12.1, and gains of 8q24.3 (including the oncogene MAFA), 11q12.3-q13.2, 3q26.1-q28, 19p13.2-p13.11, and 8q11.1-q12.2 (including the oncogenes LYN, MOS, and PLAG1). On the basis of these results we propose that mucoepidermoid carcinoma may be subdivided in (i) low-grade, fusion-positive mucoepidermoid carcinomas with no or few genomic imbalances and favorable prognosis, (ii) high-grade, fusion-positive mucoepidermoid carcinomas with multiple genomic imbalances and unfavorable prognosis, and (iii) a heterogeneous group of high-grade, fusion-negative adenocarcinomas with multiple genomic imbalances and unfavorable outcome. Taken together, our studies indicate that molecular genetic analysis can be a useful adjunct to histologic scoring of mucoepidermoid carcinoma and may lead to development of new clinical guidelines for management of these patients.

G protein-coupled receptors (GPCRs) are major regulators of intercellular interactions. They initiate these actions by being activated by a wide variety of natural ligands. Historically, ligands were discovered first, but the advent of molecular biology reversed this trend. Most GPCRs are identified on the basis of their DNA sequences and thus are initially unmatched to known natural ligands. They are termed orphan GPCRs. Discovering their ligands-i.e., "deorphanizing" the GPCRs-gave birth to the field of reverse pharmacology. This review discusses the present status of GPCR deorphanization, presents a few examples of successes and surprises, and highlights difficulties encountered in these efforts.

<h4>Background</h4>MEDLINE(®)/PubMed(®) currently indexes over 18 million biomedical articles, providing unprecedented opportunities and challenges for text analysis. Using Medical Subject Heading Over-representation Profiles (MeSHOPs), an entity of interest can be robustly summarized, quantitatively identifying associated biomedical terms and predicting novel indirect associations.<h4>Methods</h4>A procedure is introduced for quantitative comparison of MeSHOPs derived from a group of MEDLINE(®) articles for a biomedical topic (for example, articles for a specific gene or disease). Similarity scores are computed to compare MeSHOPs of genes and diseases.<h4>Results</h4>Similarity scores successfully infer novel associations between diseases and genes. The number of papers addressing a gene or disease has a strong influence on predicted associations, revealing an important bias for gene-disease relationship prediction. Predictions derived from comparisons of MeSHOPs achieves a mean 8% AUC improvement in the identification of gene-disease relationships compared to gene-independent baseline properties.<h4>Conclusions</h4>MeSHOP comparisons are demonstrated to provide predictive capacity for novel relationships between genes and human diseases. We demonstrate the impact of literature bias on the performance of gene-disease prediction methods. MeSHOPs provide a rich source of annotation to facilitate relationship discovery in biomedical informatics.

Neurodegenerative diseases are typically late-onset, progressive disorders that affect neural function and integrity. Although most attention has been focused on the genetic underpinnings of familial disease, mechanisms are likely to be shared with more predominant sporadic forms, which can be influenced by age, environment, and genetic inputs. Previous work has largely addressed the roles of select protein-coding genes; however, disease pathogenesis is complicated and can be modulated through not just protein-coding genes, but also regulatory mechanisms mediated by the exploding world of small non-coding RNAs. Here, we focus on emerging roles of miRNAs in age-associated events impacting long-term brain integrity and neurodegenerative disease.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in both men and women in Canada and the United States and has the most dismal survival rates among any solid malignancy. Most patients are diagnosed with pancreatic cancer once the disease has progressed into an advanced or metastatic stage, making the only curative approach of resection surgery impossible. The persistent delayed or missed diagnosis of pancreatic cancer can be attributed to the absence of early symptoms and the lack of efficient non-invasive screening or diagnostic tests in clinical practice. Given that earlier diagnosis is critical for ameliorating patients' survival rates, there is an urgent need for biomarkers with enough sensitivity and specificity to help diagnose pancreatic cancer early. Serological biomarkers provide a minimally invasive and efficient way of detecting pancreatic cancer, however, there is currently no marker with sufficient diagnostic sensitivity and specificity to identify early cancer patients. This review focuses on the classical tumor markers for PDAC as well as emerging markers. In addition, we will discuss an integrative proteomic approach used in our lab to identify a panel of biomarkers that have the potential to allow the early detection of PDAC.This article is part of a Special Issue entitled: From protein structures to clinical applications.

Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human β -casein fragment (54-59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of M. bovis BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the M. bovis BCG mediated apoptosis resulting in enhanced clearance of M. bovis BCG from THP-1 cell line.

Mutations in the hyperparathyroidism type 2 (HRPT2/CDC73) gene and alterations in the parafibromin protein have been established in the majority of parathyroid carcinomas and in subsets of parathyroid adenomas. While it is known that CDC73-mutated parathyroid tumors display specific gene expression changes compared to CDC73 wild-type cases, the molecular cytogenetic profile in CDC73-mutated cases compared to unselected adenomas (with an expected very low frequency of CDC73 mutations) remains unknown. For this purpose, nine parathyroid tumors with established CDC73 gene inactivating mutations (three carcinomas, one atypical adenoma and five adenomas) were analyzed for copy number alterations and loss of heterozygosity using array-comparative genomic hybridization (a-CGH) and single nucleotide polymorphism (SNP) microarrays, respectively. Furthermore, CDC73 gene promoter methylation levels were assessed using bisulfite Pyrosequencing. The panel included seven tumors with single mutation and three with double mutations of the CDC73 gene. The carcinomas displayed copy number alterations in agreement with previous studies, whereas the CDC73-mutated adenomas did not display the same pattern of alterations at loci frequently deleted in unselected parathyroid tumors. Furthermore, gross losses of chromosomal material at 1p and 13 were significantly (p = 0.012) associated with parathyroid carcinomas as opposed to adenomas. Quantitative PCR-based copy number loss regarding CDC73 was observed in three adenomas, while all the carcinomas were diploid or showed copy number gain for CDC73 gene. Hypermethylation of the CDC73 gene promoter was not observed. Our data could suggest that CDC73-mutated parathyroid adenomas exhibit a partly unique cytogenetic profile in addition to that of carcinomas and unselected adenomas. Furthermore, CDC73-mutated carcinomas displayed losses at 1p and 13 which are not seen in CDC73-mutated adenomas, making these regions of interest for further studies regarding malignant properties in tumors from CDC73-mutated cases. However, due to the small sample size, validation of the results in a larger cohort is warranted.

Periodontitis is a chronic inflammatory disease affecting the soft tissue and bone that surrounds the teeth. Despite extensive research, distinctive genes responsible for the disease have not been identified. The objective of this study was to elucidate transcriptome changes in periodontitis, by investigating gene expression profiles in gingival tissue obtained from periodontitis-affected and healthy gingiva from the same patient, using RNA-sequencing. Gingival biopsies were obtained from a disease-affected and a healthy site from each of 10 individuals diagnosed with periodontitis. Enrichment analysis performed among uniquely expressed genes for the periodontitis-affected and healthy tissues revealed several regulated pathways indicative of inflammation for the periodontitis-affected condition. Hierarchical clustering of the sequenced biopsies demonstrated clustering according to the degree of inflammation, as observed histologically in the biopsies, rather than clustering at the individual level. Among the top 50 upregulated genes in periodontitis-affected tissues, we investigated two genes which have not previously been demonstrated to be involved in periodontitis. These included interferon regulatory factor 4 and chemokine (C-C motif) ligand 18, which were also expressed at the protein level in gingival biopsies from patients with periodontitis. In conclusion, this study provides a first step towards a quantitative comprehensive insight into the transcriptome changes in periodontitis. We demonstrate for the first time site-specific local variation in gene expression profiles of periodontitis-affected and healthy tissues obtained from patients with periodontitis, using RNA-seq. Further, we have identified novel genes expressed in periodontitis tissues, which may constitute potential therapeutic targets for future treatment strategies of periodontitis.

<h4>Purpose</h4>Since our newly synthesized potent 5-indolyl derivative, (2-(1 H-Indol-5-yl) thiazol-4-yl) 3, 4, 5-trimethoxyphenyl methanone (LY293), to treat resistant melanoma was hydrophobic, our objective was to synthesize a biodegradable copolymer for formulating this drug into nanoparticles and to determine its anticancer activity and mechanism of action.<h4>Methods</h4>Methoxy poly (ethylene glycol)-b-poly (carbonate-co-lactide) [mPEG-b-P (CB-co-LA)] was synthesized for formulating LY293 into nanoparticles by o/w emulsification and stabilization by solvent evaporation. Particle size, drug release profile, in vitro efficacy in multiple melanoma cells, and mechanism of action of drug-loaded nanoparticles were determined.<h4>Results</h4>LY293-loaded nanoparticles with 170 nm mean size and 2.2 and 4.16% drug loading efficiently inhibited proliferation of A375 and B16F10 cells with IC(50) of 12.5 nM and 25 nM, respectively. LY293 circumvented multidrug resistance and inhibited proliferation of Pgp overexpressing MDA-MB435/LCC6 MDR1 melanoma cells. Upon treatment with LY293-loaded nanoparticles, A375 cells underwent cell cycle arrest in G2/M phase and apoptotic cell death. Immunofluorescence images showed inhibition of tubulin polymerization after treatment with LY293.<h4>Conclusion</h4>LY293-loaded mPEG-b-P (CB-co-LA) nanoparticles showed excellent efficacy and induced apoptosis in melanoma cells. These polyester/polycarbonate-based nanoparticles provided an excellent platform to deliver different poorly soluble drugs to melanoma.

<h4>Introduction</h4>Pancreatogenic diabetes after pancreatectomy is of growing importance due to the increasing life expectancy of pancreatectomized patients. Although reduction of pancreatic volume is thought to affect glucose metabolism, a consistent relationship has yet to be determined. This study aimed to investigate functional consequences of distal pancreatectomy (DP) in preoperatively non-diabetic patients.<h4>Methods</h4>This study included 61 non-diabetic patients who underwent DP. Clinical data were obtained, and the percent resected volume (PRV) of each pancreas was determined via multi-detector row computed tomography volumetry.<h4>Results</h4>During the follow-up period (median 26 months), 22 patients (36 %) developed new-onset diabetes within a median onset time of 8 months (range 0.5-42 months) postoperatively. The remaining 39 patients also showed impaired glucose metabolism. Multivariate analysis identified preoperative hemoglobin A1c ≥ 5.7 % (odds ratio 15.6, p = 0.001) and PRV > 44 % (odds ratio 11.3, p = 0.004) as independent risk factors for new-onset diabetes.<h4>Conclusions</h4>Key determinants of postoperative glycemic control include preoperative functional reserve of the endocrine pancreas and the volume reduction of pancreatic parenchyma. Our findings enable reliable preoperative evaluation of the risk of postoperative diabetes and appropriate postoperative surveillance, which is helpful for early intervention in high risk patients.

No abstract available.

Down regulation of serotonin transporter (5-HTT) expression has been associated with brain function and major depression. The aim of this study was to explore the allelic variation (short and long) of the 5-HTTLPR polymorphism in attention bias associated with top-down processing of emotion. One hundred sixty-two healthy participants underwent genotyping (5-HTTLPR), background interviews, psychological screening, and a computerised test session (The Emo 1-back task). Carriers of the short 5-HTTLPR alleles in the serotonin transporter gene (SLC6A4) demonstrated less accuracy in The Emo 1-back task when presented with successive images of sad or fearful faces, but not for happy or neutral emotional faces. The study suggests an association between 5-HTTLPR variation in the serotonin transporter gene and altered emotion processing.

<h4>Background</h4>MEDLINE®/PubMed® indexes over 20 million biomedical articles, providing curated annotation of its contents using a controlled vocabulary known as Medical Subject Headings (MeSH). The MeSH vocabulary, developed over 50+ years, provides a broad coverage of topics across biomedical research. Distilling the essential biomedical themes for a topic of interest from the relevant literature is important to both understand the importance of related concepts and discover new relationships.<h4>Results</h4>We introduce a novel method for determining enriched curator-assigned MeSH annotations in a set of papers associated to a topic, such as a gene, an author or a disease. We generate MeSH Over-representation Profiles (MeSHOPs) to quantitatively summarize the annotations in a form convenient for further computational analysis and visualization. Based on a hypergeometric distribution of assigned terms, MeSHOPs statistically account for the prevalence of the associated biomedical annotation while highlighting unusually prevalent terms based on a specified background. MeSHOPs can be visualized using word clouds, providing a succinct quantitative graphical representation of the relative importance of terms. Using the publication dates of articles, MeSHOPs track changing patterns of annotation over time. Since MeSHOPs are quantitative vectors, MeSHOPs can be compared using standard techniques such as hierarchical clustering. The reliability of MeSHOP annotations is assessed based on the capacity to re-derive the subset of the Gene Ontology annotations with equivalent MeSH terms.<h4>Conclusions</h4>MeSHOPs allows quantitative measurement of the degree of association between any entity and the annotated medical concepts, based directly on relevant primary literature. Comparison of MeSHOPs allows entities to be related based on shared medical themes in their literature. A web interface is provided for generating and visualizing MeSHOPs.

<h4>Objective</h4>To investigate genetic etiologies of preterm birth (PTB) in Argentina through evaluation of single-nucleotide polymorphisms (SNPs) in candidate genes and population genetic admixture.<h4>Study design</h4>Genotyping was performed in 389 families. Maternal, paternal and fetal effects were studied separately. Mitochondrial DNA (mtDNA) was sequenced in 50 males and 50 females. Y-chromosome anthropological markers were evaluated in 50 males.<h4>Result</h4>Fetal association with PTB was found in the progesterone receptor (PGR, rs1942836; P=0.004). Maternal association with PTB was found in small conductance calcium activated potassium channel isoform 3 (KCNN3, rs883319; P=0.01). Gestational age associated with PTB in PGR rs1942836 at 32-36 weeks (P=0.0004). MtDNA sequencing determined 88 individuals had Amerindian consistent haplogroups. Two individuals had Amerindian Y-chromosome consistent haplotypes.<h4>Conclusion</h4>This study replicates single locus fetal associations with PTB in PGR, maternal association in KCNN3, and demonstrates possible effects for divergent racial admixture on PTB.

Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes. In particular, iron chelators can serve as potential pharmacological tools to alleviate diseases of iron overload. This review focuses on the pharmacology of iron transport, introducing iron transport membrane proteins and known inhibitors.

Genome cyclization through conserved RNA sequences located in the 5' and 3' terminal regions of flavivirus genomic RNA is essential for virus replication. Although the role of various cis-acting RNA elements in panhandle formation is well characterized, almost nothing is known about the potential contribution of protein cofactors to viral RNA cyclization. Proteins with nucleic acid chaperone activities are encoded by many viruses (e.g., retroviruses, coronaviruses) to facilitate RNA structural rearrangements and RNA-RNA interactions during the viral replicative cycle. Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5'-3' genomic RNA annealing in vitro. Core protein binding resulted in a dramatic, dose-dependent increase in 5'-3' complex formation. Mutations introduced in either the UAR (upstream AUG region) or CS (conserved sequence) elements of the viral RNA diminished core protein-dependent annealing, while compensatory mutations restored the 5'-3' RNA interaction. The activity responsible for stimulating RNA annealing was mapped to the C-terminal RNA-binding region of WNV core protein. These results indicate that core protein - besides its function in viral particle formation - might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication.

Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two-hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1(RNAi) enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, our results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth.

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as "functionality" and "functional relationships" are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30-75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily that exert their effects via type I and type II serine threonine kinase receptors and the SMAD intracellular signaling pathway to regulate diverse biologic processes. Recently, we discovered that the repulsive guidance molecule (RGM) family, including RGMA, RGMB, and RGMC/hemojuvelin (HJV), function as co-receptors that enhance cellular responses to BMP ligands. Here, we use surface plasmon resonance to quantitate the binding kinetics of RGM proteins for BMP ligands. We show that among the RGMs, HJV exhibits the highest affinity for BMP6, BMP5, and BMP7 with K(D) 8.1, 17, and 20 nM respectively, versus 28, 33, and 166 nM for RGMB, and 55, 83, and 63 nM for RGMA. Conversely, RGMB exhibits preferential binding to BMP4 and BMP2 with K(D) 2.6 and 5.5 nM respectively, versus 4.5 and 9.4 nM for HJV, and 14 and 22 nM for RGMA, while RGMA exhibits the lowest binding affinity for most BMPs tested. Among the BMP ligands, RGMs exhibit the highest relative affinity for BMP4 and the lowest relative affinity for BMP7, while none of the RGMs bind to BMP9. Thus, RGMs exhibit preferential binding for distinct subsets of BMP ligands. The preferential binding of HJV for BMP6 is consistent with the functional role of HJV and BMP6 in regulating systemic iron homeostasis. Our data may help explain the mechanism by which BMPs exert cell-context specific effects via a limited number of type I and type II receptors.

<h4>Background</h4>Suicide is one of the top ten leading causes of death in North America and represents a major public health burden, particularly for people with Major Depressive disorder (MD). Many studies have suggested that suicidal behavior runs in families, however, identification of genomic loci that drive this efffect remain to be identified.<h4>Methodology/principal findings</h4>Using subjects collected as part of STAR D, we genotyped 189 subjects with MD with history of a suicide attempt and 1073 subjects with Major Depressive disorder that had never attempted suicide. Copy Number Variants (CNVs) were called in Birdsuite and analyzed in PLINK. We found a set of CNVs present in the suicide attempter group that were not present in in the non-attempter group including in SNTG2 and MACROD2 - two brain expressed genes previously linked to psychopathology; however, these results failed to reach genome-wide signifigance.<h4>Conclusions</h4>These data suggest potential CNVs to be investigated further in relation to suicide attempts in MD using large sample sizes.

<h4>Background</h4>Huntington's disease (HD) is caused by expanded CAG repeats encoding a polyglutamine tract in the huntingtin (HTT) protein. A number of differentially-expressed protein molecules have been identified in striatum of HD animal models. Here we examined if the expression changes could be visualized in the peripheral leukocytes of HD patients and pre-symptomatic HD (PreHD) carriers.<h4>Methods and findings</h4>The expression levels of 17 candidate genes that differentially expressed in striatum between transgenic HD and wild-type mice in literature were measured in the peripheral leukocytes of 4 PreHD carriers, 16 HD patients and 20 healthy controls. Four genes majorly involved in metabolism and oxidative stress response, including AHCY1, ACO2, OXCT1 and CAP1, demonstrated consistent downregulation in peripheral leukocytes of both PreHD carriers and HD patients, while UCP2 was only down-regulated in HD patients.<h4>Conclusion</h4>These results provide potential peripheral biomarkers to indicate disease onset in preclinical stage, and to monitor the efficacy of early treatment. Further studies of a large series of preHD carriers and symptomatic HD patients will be warranted to verify the findings and examine if these markers correlate with clinical features.

Dental materials have to meet high standards regarding mechanical strength and handling properties. There is however only a limited amount of research that has been devoted to natural formation of hydroxyapatite (HA) in contact with the materials. The objective of the current investigation was to study the surface reactions occurring in human salvia on a novel dental cement. Ceramir Crown & Bridge, a bioceramic luting agent intended for permanent cementation of conventional oral prosthetics, was evaluated by immersing discs made from the cement in human saliva and phosphate buffered saline (PBS) for seven days, after which they were dried and analyzed. The analytical methods used in order to verify HA formation on the surface were grazing incidence X-ray diffraction (GI-XRD), scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). All results showed that HA was formed on the surfaces of samples stored in saliva as well as on samples stored in PBS. The possibility of a dental luting cement to promote natural formation of HA at the tooth interface increases the stability and durability of the system and could help prevent secondary caries.

Diabetes mellitus is characterized by chronic hyperglycemia caused by a deficiency in insulin action, insulin secretion or both. Type 1 diabetes is classified as the destruction of beta cells leading to a deficiency in insulin production. Type1 diabetes accounts for 5-10% of patients with diabetes and most commonly is caused by the autoimmune destruction of the beta cells in the pancreas. The adaptive immune system is composed of antigen specific T and B lymphocytes which play a central role in protecting the human body from infectious pathogens but occasionally autoreactive T and B cells can escape immune tolerance, become activated and induce autoimmune diseases. Naïve T cells require two distinct signals one delivered via the antigen receptor and the second through the costimulatory receptor CD28 that leads to the induction of IL-2 gene transcription. IL-2 is an important T cell growth factor that can influence both immunity and tolerance. Given its pivotal role it is not surprising that the immune system places strict regulation over Il2 gene transcription that is controlled by a number of E3 ubiquitin ligases that modulate TCR and CD28 signaling. This review will examine how different E3 ligases function to control T effector cell differentiation and how studies in gene knockout animal models has been crucial in understanding how these proteins function in vivo to regulate immune tolerance in the peripheral circulation.

Chordoma is a rare malignant tumour of bone, the molecular marker of which is the expression of the transcription factor, brachyury. Having recently demonstrated that silencing brachyury induces growth arrest in a chordoma cell line, we now seek to identify its downstream target genes. Here we use an integrated functional genomics approach involving shRNA-mediated brachyury knockdown, gene expression microarray, ChIP-seq experiments, and bioinformatics analysis to achieve this goal. We confirm that the T-box binding motif of human brachyury is identical to that found in mouse, Xenopus, and zebrafish development, and that brachyury acts primarily as an activator of transcription. Using human chordoma samples for validation purposes, we show that brachyury binds 99 direct targets and indirectly influences the expression of 64 other genes, thereby acting as a master regulator of an elaborate oncogenic transcriptional network encompassing diverse signalling pathways including components of the cell cycle, and extracellular matrix components. Given the wide repertoire of its active binding and the relative specific localization of brachyury to the tumour cells, we propose that an RNA interference-based gene therapy approach is a plausible therapeutic avenue worthy of investigation.

Leukotrienes (LTs) are lipid-signaling molecules derived from arachidonic acid (AA) that initiate and amplify inflammation. To initiate LT formation, the 5-lipoxygenase (5-LO) enzyme translocates to nuclear membranes, where it associates with its scaffold protein, 5-lipoxygenase-activating protein (FLAP), to form the core of the multiprotein LT synthetic complex. FLAP is considered to function by binding free AA and facilitating its use as a substrate by 5-LO to form the initial LT, LTA(4). We used a combination of fluorescence lifetime imaging microscopy, cell biology, and biochemistry to identify discrete AA-dependent and AA-independent steps that occur on nuclear membranes to control the assembly of the LT synthetic complex in polymorphonuclear leukocytes. The association of AA with FLAP changes the configuration of the scaffold protein, enhances recruitment of membrane-associated 5-LO to form complexes with FLAP, and controls the closeness of this association. Granulocyte monocyte colony-stimulating factor provides a second AA-independent signal that controls the closeness of 5-LO and FLAP within complexes but not the number of complexes that are assembled. Our results demonstrate that the LT synthetic complex is a signal integrator that transduces extracellular signals to modulate the interaction of 5-LO and FLAP.

Iron deficiency affects nearly 2 billion people worldwide, with pregnant women and young children being most severely impacted. Sustained anemia during the first year of life can cause cognitive, attention and motor deficits, which may persist despite iron supplementation. We conducted iTRAQ analyses on cerebrospinal fluid (CSF) from infant monkeys (Macaca mulatta) to identify differential protein expression associated with early iron deficiency. CSF was collected from 5 iron-sufficient and 8 iron-deficient anemic monkeys at weaning age (6-7 months) and again at 12-14 months. Despite consumption of iron-fortified food after weaning, which restored hematological indices into the normal range, expression of 5 proteins in the CSF remained altered. Most of the proteins identified are involved in neurite outgrowth, migration or synapse formation. The results reveal novel ways in which iron deficiency undermines brain growth and results in aberrant neuronal migration and connections. Taken together with gene expression data from rodent models of iron deficiency, we conclude that significant alterations in neuroconnectivity occur in the iron-deficient brain, which may persist even after resolution of the hematological anemia. The compromised brain infrastructure could account for observations of behavioral deficits in children during and after the period of anemia.

Neuronal voltage-gated calcium channels generate rapid, transient intracellular calcium signals in response to membrane depolarization. Neuronal Ca(V) channels regulate a range of cellular functions and are implicated in a variety of neurological and psychiatric diseases including epilepsy, Parkinson's disease, chronic pain, schizophrenia, and bipolar disorder. Each mammalian Cacna1 gene has the potential to generate tens to thousands of Ca(V) channels by alternative pre-mRNA splicing, a process that adds fine granulation to the pool of Ca(V) channel structures and functions. The precise composition of Ca(V) channel splice isoform mRNAs expressed in each cell are controlled by cell-specific splicing factors. The activity of splicing factors are in turn regulated by molecules that encode various cellular features, including cell-type, activity, metabolic states, developmental state, and other factors. The cellular and behavioral consequences of individual sites of Ca(V) splice isoforms are being elucidated, as are the cell-specific splicing factors that control splice isoform selection. Altered patterns of alternative splicing of Ca(V) pre-mRNAs can alter behavior in subtle but measurable ways, with the potential to influence drug efficacy and disease severity. This article is part of a Special Issue entitled: Calcium channels.

<h4>Introduction</h4>Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question.<h4>Methods</h4>European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication.<h4>Results</h4>THERE WERE THREE NEW ASSOCIATIONS: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR=0.76 and 1.30, P(corr) =0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci.<h4>Conclusion</h4>Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.

<h4>Background</h4>The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders.<h4>Methods</h4>Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.<h4>Results</h4>With HIVE the HIV-1 RNA load in brain tissue was three log(10) units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.<h4>Interpretation</h4>Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).

MicroRNAs (miRNAs) are small non-coding RNAs which function as endogenous silencers of various target genes. miRNAs are expressed in a tissue-specific manner and playing important roles in cell proliferation, apoptosis, and differentiation during mammalian development. Links between miRNAs and the initiation and progression of human diseases including cancer are becoming increasingly apparent. Recent studies have revealed that some miRNAs such as miR-9, miR-29 family, and miR-34 family are differentially expressed in Alzheimer's disease, Parkinson's disease, and Huntington's disease. These miRNAs are also reported to act as tumor suppressors during human carcinogenesis. In this review, we discuss about miRNAs which are important in the molecular pathogenesis of both cancer and neurodegeneration. Cancer and neurodegenerative disorder may be influenced by common miRNA pathways that regulate differentiation, proliferation, and death of cells.

Much of the excitement generated by induced pluripotent stem cell technology is concerned with the possibility of disease modeling as well as the potential for personalized cell therapy. However, to pursue this it is important to understand the 'normal' pluripotent state including its inherent variability. We have performed various molecular profiling assays for 21 hESC lines and 8 hiPSC lines to generate a comprehensive snapshot of the undifferentiated state of pluripotent stem cells. Analysis of the gene expression data revealed no iPSC-specific gene expression pattern in accordance with previous reports. We further compared cells, differentiated as embryoid bodies in 2 media proposed to initiate differentiation towards separate cell fates, as well as 20 adult tissues. From this analysis we have generated a gene list which defines pluripotency and establishes a baseline for the pluripotent state. Finally, we provide lists of genes enriched under both differentiation conditions which show the proposed bias toward independent cell fates.

In surgical disciplines, where bones have to be repaired, augmented or improved, bone substitutes are essential. Therefore, an interest has dramatically increased in application of synthetic bone grafts. As various interactions among cells, surrounding tissues and implanted biomaterials always occur at the interfaces, the surface properties of the implants are of the paramount importance in determining both the biological response to implants and the material response to the physiological conditions. Hence, a surface engineering is aimed to modify both the biomaterials, themselves, and biological responses through introducing desirable changes to the surface properties of the implants but still maintaining their bulk mechanical properties. To fulfill these requirements, a special class of artificial bone grafts has been introduced in 1976. It is composed of various mechanically stable (therefore, suitable for load bearing applications) biomaterials and/or bio-devices with calcium orthophosphate coatings, films and layers on their surfaces to both improve interactions with the surrounding tissues and provide an adequate bonding to bones. Many production techniques of calcium orthophosphate coatings, films and layers have been already invented and new promising techniques are continuously investigated. These specialized coatings, films and layers used to improve the surface properties of various types of artificial implants are the topic of this review.

Driven by the increasing economic burden associated with bone injury and disease, biomaterial development for bone repair represents the most active research area in the field of tissue engineering. This article provides an update on recent advances in the development of bioactive biomaterials for bone regeneration. Special attention is paid to the recent developments of sintered Na-containing bioactive glasses, borate-based bioactive glasses, those doped with trace elements (such as Cu, Zn, and Sr), and novel elastomeric composites. Although bioactive glasses are not new to bone tissue engineering, their tunable mechanical properties, biodegradation rates, and ability to support bone and vascular tissue regeneration, as well as osteoblast differentiation from stem and progenitor cells, are superior to other bioceramics. Recent progresses on the development of borate bioactive glasses and trace element-doped bioactive glasses expand the repertoire of bioactive glasses. Although boride and other trace elements have beneficial effects on bone remodeling and/or associated angiogenesis, the risk of toxicity at high levels must be highly regarded in the design of new composition of bioactive biomaterials so that the release of these elements must be satisfactorily lower than their biologically safe levels. Elastomeric composites are superior to the more commonly used thermoplastic-matrix composites, owing to the well-defined elastic properties of elastomers which are ideal for the replacement of collagen, a key elastic protein within the bone tissue. Artificial bone matrix made from elastomeric composites can, therefore, offer both sound mechanical integrity and flexibility in the dynamic environment of injured bone.

We characterized the α-to-β transition in α-helical coiled-coil connectors of the human fibrin(ogen) molecule using biomolecular simulations of their forced elongation and theoretical modeling. The force (F)-extension (X) profiles show three distinct regimes: (1) the elastic regime, in which the coiled coils act as entropic springs (F < 100-125 pN; X < 7-8 nm); (2) the constant-force plastic regime, characterized by a force-plateau (F ≈ 150 pN; X ≈ 10-35 nm); and (3) the nonlinear regime (F > 175-200 pN; X > 40-50 nm). In the plastic regime, the three-stranded α-helices undergo a noncooperative phase transition to form parallel three-stranded β-sheets. The critical extension of the α-helices is 0.25 nm, and the energy difference between the α-helices and β-sheets is 4.9 kcal/mol per helical pitch. The soft α-to-β phase transition in coiled coils might be a universal mechanism underlying mechanical properties of filamentous α-helical proteins.

We report on the synthesis and characterization of a PEGylated IR786 GUMBOS (Group of Uniform Materials Based on Organic Salts). The synthesis of this material was accomplished using a three step protocol: (1) substitution of chloride on the cyclohexenyl ring in the heptamethine chain of IR786 by 6-aminohexanoic acid, (2) grafting of methoxy polyethylene glycol (MeOPEG) onto the 6-aminohexanoic acid via an esterification reaction, and (3) anion exchange between [PEG786][I] and lithium bis(trifluoromethylsulfonyl)imide (LiNTf(2)) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in order to obtain PEG786 GUMBOS. Examination of spectroscopic data for this PEG786 GUMBOS indicates a large stokes shift (122 nm). It was observed that this PEG786 GUMBOS associates in aqueous solution to form nano- and mesoscale self-assemblies with sizes ranging from 100 to 220 nm. These nano- and mesoscale GUMBOS are also able to resist nonspecific binding to proteins. PEGylation of the original IR786 leads to reduced cytotoxicity. In addition, it was noted that anions, such as NTf(2) and AOT, play a significant role in improving the photostability of PEG786 GUMBOS. Irradiation-induced J-aggregation in [PEG786][NTf(2)] and to some extent in [PEG786][AOT] produced enhanced photostability. This observation was supported by use of both steady state and time-resolved fluorescence measurements.

The yeast SUMO (small ubiquitin-like modifier) orthologue SMT3 was initially discovered in a genetic suppressors screen for the centromeric protein Mif2 (Meluh and Koshland in Mol Bio Cell 6:793-807, 1). Later, it turned out that the homologous mammalian proteins SUMO1 to SUMO4 are reversible protein modifiers that can form isopeptide bonds with lysine residues of respective target proteins (Mahajan et al. in Cell 88:97-107, 2). This was the discovery of a post-translational modification called sumoylation, which enzymatically resembles ubiquitination. However, very soon it became clear that SUMO attachments served a far more diverse role than ubiquitination. Meanwhile, numerous cellular processes are known to be subject to the impact of SUMO modification, including transcription, protein targeting, protein solubility, apoptosis or activity of various enzymes. In many instances, SUMO proteins create new protein interaction surfaces or block existing interaction domains (Geiss-Friedlander and Melchior in Nat Rev in Mol Cell Biol 8:947-956, 3). For the past few years, sumoylation attracted increasing attention as a versatile regulator of toxic protein properties in neurodegenerative diseases. In this review, we summarize the growing knowledge about the involvement of sumoylation in neurodegeneration, and discuss the underlying molecular principles affected by this multifaceted and intriguing post-translational modification.

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of "stemness". These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.

Histone-modifying enzymes play a pivotal role in gene expression and repression. In human, DOT1L (Dot1-like) is the only known histone H3 lysine 79 methyltransferase. hDOT1L is associated with transcriptional activation, but the general mechanism connecting hDOT1L to active transcription remains largely unknown. Here, we report that hDOT1L interacts with the phosphorylated C-terminal domain of actively transcribing RNA polymerase II (RNAPII) through a region conserved uniquely in multicellular DOT1 proteins. Genome-wide profiling analyses indicate that the occupancy of hDOT1L largely overlaps with that of RNAPII at actively transcribed genes, especially surrounding transcriptional start sites, in embryonic carcinoma NCCIT cells. We also find that C-terminal domain binding or H3K79 methylations by hDOT1L is important for the expression of target genes such as NANOG and OCT4 and a marker for pluripotency in NCCIT cells. Our results indicate that a functional interaction between hDOT1L and RNAPII targets hDOT1L and subsequent H3K79 methylations to actively transcribed genes.

Among the known pathways of protein nuclear import, the karyopherin β2/transportin pathway is only the second to have a defined nuclear localization signal (NLS) consensus. Huntingtin, a 350-kDa protein, has defined roles in the nucleus, as well as a CRM1/exportin-dependent nuclear export signal; however, the NLS and exact pathway of import have remained elusive. Here, using a live cell assay and affinity chromatography, we show that huntingtin has a karyopherin β2-dependent proline-tyrosine (PY)-NLS in the amino terminus of the protein. This NLS comprises three consensus components: a basic charged sequence, a downstream conserved arginine, and a PY sequence. Unlike the classic PY-NLS, which has an unstructured intervening sequence between the consensus components, we show that a β sheet structured region separating the consensus elements is critical for huntingtin NLS function. The huntingtin PY-NLS is also capable of import through the importin/karyopherin β1 pathway but was not functional in all cell types tested. We propose that this huntingtin PY-NLS may comprise a new class of multiple import factor-dependent NLSs with an internal structural component that may regulate NLS activity.

We report the design and implementation of the first phase 3 trial of CoenzymeQ₁₀ (CoQ₁₀) in children with genetic mitochondrial diseases. A novel, rigorous set of eligibility criteria was established. The trial, which remains open to recruitment, continues to address multiple challenges to the recruitment of patients, including widely condoned empiric use of CoQ₁₀ by individuals with proven or suspected mitochondrial disease and skepticism among professional and lay mitochondrial disease communities about participating in placebo-controlled trials. These attitudes represent significant barriers to the ethical and scientific evaluation--and ultimate approval--of nutritional and pharmacological therapies for patients with life-threatening inborn errors of energy metabolism.

Elucidating the genetic mechanisms that underlie complex adaptive phenotypes is a central problem in evolutionary biology. For behavioral biologists, the ability to link variation in gene expression to the occurrence of specific behavioral traits has long been a largely unobtainable goal. Social interactions with conspecifics represent a fundamental component of the behavior of most animal species. Although several studies of mammals have attempted to uncover the genetic bases for social relationships using a candidate gene approach, none have attempted more comprehensive, transcriptome-based analyses using high throughout sequencing. As a first step toward improved understanding of the genetic underpinnings of mammalian sociality, we generated a reference transcriptome for the colonial tuco-tuco (Ctenomys sociabilis), a social species of subterranean rodent that is endemic to southwestern Argentina. Specifically, we analyzed over 500 million Illumina sequencing reads derived from the hippocampi of 10 colonial tuco-tucos housed in captivity under a variety of social conditions. The resulting reference transcriptome provides a critical tool for future studies aimed at exploring relationships between social environment and gene expression in this non-model species of social mammal.

<h4>Background/aims</h4>The aim of this study was to describe the types and causes of liver disease in patients from a single community hospital in Korea between April 2005 and May 2010.<h4>Methods</h4>A cohort of patients who visited the liver clinic of the hospital during the aforementioned time period were consecutively enrolled (n=6,307). Consistent diagnostic criteria for each liver disease were set by a single, experienced hepatologist, and the diagnosis of all of the enrolled patients was confirmed by retrospective review of their medical records.<h4>Results</h4>Among the 6,307 patients, 528 (8.4%) were classified as acute hepatitis, 3,957 (62.7%) as chronic hepatitis, 767 (12.2%) as liver cirrhosis, 509 (8.1%) as primary liver cancer, and 546 (8.7%) as a benign liver mass or other diseases. The etiologies in the acute hepatitis group in decreasing order of prevalence were hepatitis A (44.3%), toxic hepatitis (32.4%), other hepatitis viruses (13.8%), and cryptogenic hepatitis (9.1%). In the chronic hepatitis group, 51.2% of cases were attributed to viral hepatitis, 33.3% to nonalcoholic fatty liver disease, and 13.0% to alcoholic liver disease (ALD). Of the cirrhoses, 73.4% were attributable to viral causes and 18.1% to alcohol. Of the hepatocellular carcinoma cases, 86.6% were attributed to viral hepatitis and 11.6% to ALD. Among the benign tumors, hemangioma comprised 52.2% and cystic liver disease comprised 33.7%.<h4>Conclusions</h4>Knowledge of the current status of the type and cause of liver disease in Korea may be valuable as a basis for evaluating changing trends in liver disease in that country.

The autoimmune disorder primary Sjogren's syndrome (SS) is associated with xerostomia and xerophthalmia. SS pathogenesis involves both genetic/epigenetic and environmental factors. A major potential contributor is oxidative stress associated with damage to cellular components, including DNA. We reported previously that the green tea polyphenol epigallocatechin-3-gallate (EGCG) normalizes the elevated levels of proliferating cell nuclear antigen (PCNA), a key component of DNA repair, in the NOD mouse model for SS and type 1 diabetes. The current study examined levels of the antioxidant enzymes peroxiredoxin 6 (PRDX6), catalase and superoxide dismutase (SOD), as well as PCNA, in NOD.B10.Sn-H2 mice, a model for primary SS, and determined the effect of EGCG on their expression. PCNA elevation was detected in the submandibular gland and pancreas by 8 weeks of age in water-fed mice, and increased through 14 weeks of age, prior to overt onset of symptoms. This early PCNA elevation was followed by a decline of peroxiredoxin 6 protein. In contrast, EGCG-fed mice exhibited normal levels of PCNA and peroxiredoxin 6, comparable to healthy untreated BALB/c mice. Similar patterns were observed in the pancreas, even though these mice do not develop diabetes. Thus, elevated PCNA is an early biomarker for exocrine glandular dysfunction associated with SS-like autoimmune disease, accompanied subsequently by decreased PRDX6 antioxidant enzyme levels that could further contribute to oxidative stress, and these changes precede inflammatory cell infiltration. Importantly, EGCG consumption normalizes the expression of these biomarkers in this model. These observations could lead to early diagnosis and intervention of autoimmune disorders.

<h4>Aim</h4>Genetic factors are important for outcome after traumatic brain injury (TBI), although exact knowledge of relevant genes/pathways is still lacking. We here used an unbiased approach to define differentially activated pathways between the inbred DA and PVG rat strains. The results prompted us to study further if a naturally occurring genetic variation in glutathione-S-transferase alpha 4 (Gsta4) affects the outcome after TBI.<h4>Results</h4>Survival of neurons after experimental TBI is increased in PVG compared to the DA strain. Global expression profiling analysis shows the glutathione metabolism pathway to be the most regulated between the strains, with increased Gsta4 in PVG among top regulated transcripts. A congenic strain (R5) with a PVG genomic insert containing the Gsta4 gene on DA background displays a reversal of the strain pattern for Gsta4 expression and increased survival of neurons compared to DA. Gsta4 is known to effectively reduce 4-hydroxynonenal (4-HNE), a noxious by-product of lipid peroxidation. Immunostaining of 4-HNE was evident in both rat and human TBI. Intracerebral injection of 4-HNE resulted in neurodegeneration with increased levels of a marker for nerve injury in cerebrospinal fluid of DA compared to R5.<h4>Innovation</h4>These findings provide strong support for the notion that the inherent capability of coping with increased 4-HNE after TBI affects outcome in terms of nerve cell loss.<h4>Conclusion</h4>A naturally occurring variation in Gsta4 expression in rats affects neurodegeneration after TBI. Further studies are needed to explore if genetic variability in Gsta4 can be associated to outcome also in human TBI.

The delta-protocadherin (δ-Pcdh) family of transmembrane proteins belongs to the cadherin superfamily, which is involved in embryogenesis mediated by a homophilic binding during the embryonic development. In the present study, expression patterns of eight members of the δ-Pcdh family were investigated in the developing chicken cochlea by in situ hybridization. Our results provide a dynamical profile to show that the δ-Pcdhs are expressed spatially and temporally in the developing chicken cochleae. The earliest onset of the δ-Pcdh expression begins in the otic vesicle from embryonic incubation day (E) 3. From E11 onwards, the individual δ-Pcdh is expressed in different cell types of the cochlea. Protocadherin-1 (Pcdh1) is mainly expressed by spindle-shaped cells and acoustic ganglion cells; Pcdh7 and Pcdh17 are strongly expressed by supporting cells, cuboidal cells, hyaline cells and acoustic ganglion cells, and Pcdh9 is prominently expressed by homogene cells and acoustic ganglion cells; Pcdh8 was found to be transcribed in hair cells, spindle-shaped cells and acoustic ganglion cells; Pcdh10 mRNA is restricted to spindle-shaped cells and acoustic ganglion cells at later stages. mRNAs of Pcdh1, Pcdh18 and Pcdh19 are also expressed in blood vessels of the cochlea. The expression of the different δ-Pcdhs suggests a functional role for them during cochlear development.

<h4>Background</h4>In order to obtain a lead of the pathophysiology of endometriosis, genome-wide expressional analyses of eutopic and ectopic endometrium have earlier been reported, however, the effects of stages of severity and phases of menstrual cycle on expressional profiles have not been examined. The effect of genetic heterogeneity and fertility history on transcriptional activity was also not considered. In the present study, a genome-wide expression analysis of autologous, paired eutopic and ectopic endometrial samples obtained from fertile women (n=18) suffering from moderate (stage 3; n=8) or severe (stage 4; n=10) ovarian endometriosis during proliferative (n=13) and secretory (n=5) phases of menstrual cycle was performed.<h4>Methods</h4>Individual pure RNA samples were subjected to Agilent's Whole Human Genome 44K microarray experiments. Microarray data were validated (P<0.01) by estimating transcript copy numbers by performing real time RT-PCR of seven (7) arbitrarily selected genes in all samples. The data obtained were subjected to differential expression (DE) and differential co-expression (DC) analyses followed by networks and enrichment analysis, and gene set enrichment analysis (GSEA). The reproducibility of prediction based on GSEA implementation of DC results was assessed by examining the relative expressions of twenty eight (28) selected genes in RNA samples obtained from fresh pool of eutopic and ectopic samples from confirmed ovarian endometriosis patients with stages 3 and 4 (n=4/each) during proliferative and secretory (n=4/each) phases.<h4>Results</h4>Higher clustering effect of pairing (cluster distance, cd=0.1) in samples from same individuals on expressional arrays among eutopic and ectopic samples was observed as compared to that of clinical stages of severity (cd=0.5) and phases of menstrual cycle (cd=0.6). Post hoc analysis revealed anomaly in the expressional profiles of several genes associated with immunological, neuracrine and endocrine functions and gynecological cancers however with no overt oncogenic potential in endometriotic tissue. Dys-regulation of three (CLOCK, ESR1, and MYC) major transcription factors appeared to be significant causative factors in the pathogenesis of ovarian endometriosis. A novel cohort of twenty-eight (28) genes representing potential marker for ovarian endometriosis in fertile women was discovered.<h4>Conclusions</h4>Dysfunctional expression of immuno-neuro-endocrine behaviour in endometrium appeared critical to endometriosis. Although no overt oncogenic potential was evident, several genes associated with gynecological cancers were observed to be high in the expressional profiles in endometriotic tissue.

<h4>Background</h4>Sporadic Amyotrophic Lateral Sclerosis (sALS) is a devastating, complex disease of unknown etiology. We studied this disease with microarray technology to capture as much biological complexity as possible. The Affymetrix-focused BaFL pipeline takes into account problems with probes that arise from physical and biological properties, so we adapted it to handle the long-oligonucleotide probes on our arrays (hence LO-BaFL). The revised method was tested against a validated array experiment and then used in a meta-analysis of peripheral white blood cells from healthy control samples in two experiments. We predicted differentially expressed (DE) genes in our sALS data, combining the results obtained using the TM4 suite of tools with those from the LO-BaFL method. Those predictions were tested using qRT-PCR assays.<h4>Results</h4>LO-BaFL filtering and DE testing accurately predicted previously validated DE genes in a published experiment on coronary artery disease (CAD). Filtering healthy control data from the sALS and CAD studies with LO-BaFL resulted in highly correlated expression levels across many genes. After bioinformatics analysis, twelve genes from the sALS DE gene list were selected for independent testing using qRT-PCR assays. High-quality RNA from six healthy Control and six sALS samples yielded the predicted differential expression for 7 genes: TARDBP, SKIV2L2, C12orf35, DYNLT1, ACTG1, B2M, and ILKAP. Four of the seven have been previously described in sALS studies, while ACTG1, B2M and ILKAP appear in the context of this disease for the first time. Supplementary material can be accessed at: http://webpages.uncc.edu/~cbaciu/LO-BaFL/supplementary_data.html.<h4>Conclusion</h4>LO-BaFL predicts DE results that are broadly similar to those of other methods. The small healthy control cohort in the sALS study is a reasonable foundation for predicting DE genes. Modifying the BaFL pipeline allowed us to remove noise and systematic errors, improving the power of this study, which had a small sample size. Each bioinformatics approach revealed DE genes not predicted by the other; subsequent PCR assays confirmed seven of twelve candidates, a relatively high success rate.

<h4>Background</h4>Huntington's disease (HD) is a devastating heredoneurodegenerative disorder associated with a wide variety of neurological and psychiatric symptoms caused by an expanded CAG repeat in the HTT gene. The expansion mutation in HTT is dominantly transmitted and codes for a protein named huntingtin (htt).<h4>Hypothesis</h4>One hypothesis, according to a multistep mechanism, is that the intergenerational transmission of the normal repeat size causes small, progressive CAG stretch elongations in the general population from one generation to another, until a critical pathological CAG repeat threshold is reached. Mutations may originate in the offspring from paternally transmitted CAG repeats, falling within an intermediate alleles (IA) range of 27 to 35 in repeat length.<h4>Conclusions</h4>There has been emerging evidence that some individuals with IAs might develop an HD phenotype. This presents a challenge for genetic counseling, because these individuals are often reassured that they are "disease free." However, there are many unanswered questions related to the role of IAs in the development of the HD phenotype and in the pathogenesis of HD.

Uterine decidualization, a crucial process for implantation, is a tightly regulated process encompassing proliferation, differentiation, and polyploidization of uterine stromal cells. Hoxa (Homeobox A)-10, a homeobox transcription factor, is highly expressed in decidualizing stromal cells. Targeted gene deletion experiments have demonstrated marked infertility resulting from severely compromised decidualization in Hoxa-10(-/-) mice. However, the underlying mechanism by which Hoxa-10 regulates stromal cell differentiation remains poorly understood. Cyclin D3, a G(1) phase cell-cycle regulatory protein involved in stromal cell proliferation and decidualization, is significantly reduced in Hoxa-10(-/-) mice. The expression of cyclin D3 in the pregnant mouse uterus parallels stromal cell decidualization. Here, we show that adenovirus-driven cyclin D3 replacement in Hoxa-10(-/-) mice improves stromal cell decidualization. To address our question of whether cyclin D3 replacement in Hoxa-10(-/-) mice can improve decidualization, both in vitro and in vivo studies were completed after the addition of cyclin D3 or empty (control) viral vectors. Immunostaining demonstrated increased proliferation and decidualization in both in vitro and in vivo studies, and in situ hybridization confirmed increased expression of decidualization markers in vivo. Placentation was demonstrated as well in vivo in the cyclin D3-replaced animals. However, fertility was not restored in Hoxa-10(-/-) mice after d 10 of pregnancy. Finally, we identified several downstream targets of cyclin D3 during decidualization in vitro via proteomics experiments, and these were confirmed using in situ hybridization in vivo. Collectively, these results demonstrate that cyclin D3 expression influences a host of genes involved in decidualization and can improve decidualization in Hoxa-10(-/-) mice.

Nucleosomes often undergo extensive rearrangement when genes are activated for transcription. We have shown previously, using paired-end sequencing of yeast nucleosomes, that major changes in chromatin structure occur when genes are activated by 3-aminotriazole (3AT), an inducer of the transcriptional activator Gcn4. Here, we provide a global analysis of these data. At the genomic level, nucleosomes are regularly phased relative to the transcription start site. However, for a subset of 234 strongly induced genes, this phasing is much more irregular after induction, consistent with the loss of some nucleosomes and the re-positioning of the remaining nucleosomes. To address the nature of this rearrangement, we developed the inter-nucleosome distance auto-correlation (DAC) function. At long range, DAC analysis indicates that nucleosomes have an average spacing of 162 bp, consistent with the reported repeat length. At short range, DAC reveals a 10.25-bp periodicity, implying that nucleosomes in overlapping positions are rotationally related. DAC analysis of the 3AT-induced genes suggests that transcription activation coincides with rearrangement of nucleosomes into irregular arrays with longer spacing. Sequence analysis of the +1 nucleosomes belonging to the 45 most strongly activated genes reveals a distinctive periodic oscillation in the A/T-dinucleotide occurrence that is present throughout the nucleosome and extends into the linker. This unusual pattern suggests that the +1 nucleosomes might be prone to sliding, thereby facilitating transcription.

<h4>Purpose</h4>Prostate specific antigen and digital rectal examination have low specificity for detecting prostate cancer and they poorly predict the presence of aggressive disease. Urine is readily available and noninvasive, and it represents a promising source of biomarkers for the early detection and prediction of prostate cancer prognosis. We identified promising biomarkers for urine based prostate cancer, examined trends and outlined potential pitfalls.<h4>Materials and methods</h4>We performed PubMed® and Web of Science® database searches of the peer reviewed literature on urine based testing for prostate cancer. Original studies of this subject as well as a small number of reviews were analyzed, including the strengths and weaknesses. We provide a comprehensive review of urine based testing for prostate cancer that covers the technical aspects, including the methodology of urine collection, as well as recent developments in biomarkers spanning the fields of genomics, epigenetics, transcriptomics, proteomics and metabolomics.<h4>Results</h4>The process of urine collection is subject to variability, which may result in conflicting clinical results. Detecting prostate cancer in urine is technically feasible, as demonstrated by numerous proof of principle studies, but few markers have been validated in multiple large sample sets. Biomarker development using urine has been accelerating in recent years with numerous studies identifying DNA, RNA, protein and metabolite based biomarkers in urine. Advanced clinical studies have identified PCA3 and TMPRSS2:ERG fusion transcripts as promising RNA markers for cancer detection and possibly prognosis. DNA methylation analysis of multiple genes improves specificity and represents a promising platform for developing clinical grade assays.<h4>Conclusions</h4>Urine based testing is noninvasive and represents a rich source of novel biomarkers for prostate cancer. Although urine shows promise for detecting cancer, the ability to identify aggressive subsets of prostate cancer needs further development.

Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of ∼30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP.Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L2, a novel repressor of BMP-induced transcription. Such transcriptional changes may reflect epigenetic changes, which are part of a specific osteoporosis-associated aging process.

<h4>Background</h4>MicroRNA (miRNA) are negative regulators of gene expression, capable of exerting pronounced influences upon the translation and stability of mRNA. They are potential regulators of normal mammary gland development and of the maintenance of mammary epithelial progenitor cells. This study was undertaken to determine the role of miR-30b on the establishment of a functional mouse mammary gland. miR-30b is a member of the miR-30 family, composed of 6 miRNA that are highly conserved in vertebrates. It has been suggested to play a role in the differentiation of several cell types.<h4>Methodology/principal findings</h4>The expression of miR-30b was found to be regulated during mammary gland development. Transgenic mice overexpressing miR-30b in mammary epithelial cells were used to investigate its role. During lactation, mammary histological analysis of the transgenic mice showed a reduction in the size of alveolar lumen, a defect of the lipid droplets and a growth defect of pups fed by transgenic females. Moreover some mammary epithelial differentiated structures persisted during involution, suggesting a delay in the process. The genes whose expression was affected by the overexpression of miR-30b were characterized by microarray analysis.<h4>Conclusion/significance</h4>Our data suggests that miR-30b is important for the biology of the mammary gland and demonstrates that the deregulation of only one miRNA could affect lactation and involution.

Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.

Huntington's disease is a progressive neurodegenerative disease, caused by a polyglutamine expansion in the huntingtin protein. A prominent hallmark of the disease is the presence of intracellular aggregates initiated by N-terminal huntingtin fragments containing the polyglutamine repeat, which recruit components of the ubiquitin-proteasome system. While it is commonly thought that proteasomes are irreversibly sequestered into these aggregates leading to impairment of the ubiquitin-proteasome system, the data on proteasomal impairment in Huntington's disease is contradictory. In addition, it has been suggested that proteasomes are unable to actually cleave polyglutamine sequences in vitro, thereby releasing aggregation-prone polyglutamine peptides in cells. Here, we discuss how the proteasome is involved in the various stages of polyglutamine aggregation in Huntington's disease, and how alterations in activity may improve clearance of mutant huntingtin fragments.

<h4>Objective</h4>The contribution of genetic variants to body mass index (BMI) during adolescence across multiethnic samples is largely unknown. We selected genetic loci associated with BMI or obesity in European-descent samples and examined them in a multiethnic adolescent sample.<h4>Design and sample</h4>In 5103 European American (EA), 1748 African American (AfA), 1304 Hispanic American (HA) and 439 Asian American (AsA) participants of the National Longitudinal Study of Adolescent Health (Add Health; ages 12-21 years, 47.5% male), we assessed the association between 41 established obesity-related single-nucleotide polymorphisms (SNPs) with BMI using additive genetic models, stratified by race/ethnicity, and in a pooled meta-analysis sample. We also compared the magnitude of effect for BMI-SNP associations in EA and AfA adolescents to comparable effect estimates from 11 861 EA and AfA adults in the Atherosclerosis Risk in Communities study (ages 45-64 years, 43.2% male).<h4>Results</h4>Thirty-five of 41 BMI-SNP associations were directionally consistent with published studies in European populations, 18 achieved nominal significance (P<0.05; effect sizes from 0.19 to 0.71 kg m(-2) increase in BMI per effect allele), while 4 (FTO, TMEM18, TFAP2B, MC4R) remained significant after Bonferroni correction (P<0.0015). Of 41 BMI-SNP associations in AfA, HA and AsA adolescents, nine, three and five, respectively, were directionally consistent and nominally significant. In the pooled meta-analysis, 36 of 41 effect estimates were directionally consistent and 21 of 36 were nominally significant. In EA adolescents, BMI effect estimates were larger (P<0.05) for variants near TMEM18, PTER and MC4R and smaller for variants near MTIF3 and NRXN3 compared with EA adults.<h4>Conclusion</h4>Our findings suggest that obesity susceptibility loci may have a comparatively stronger role during adolescence than during adulthood, with variation across race/ethnic subpopulation.

We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.

Sigma (σ) receptors are unique non-opioid binding sites that are associated with a broad range of disease states. Sigma-2 receptors provide a promising target for diagnostic imaging and pharmacological interventions to curb tumor progression. Most recently, the progesterone receptor (PGRMC1, 25 kDa) has been shown to have σ2 receptor-like binding properties, thus highlighting the need to understand the biological function of an 18 kDa protein that exhibits σ2-like photoaffinity labeling (denoted here as σ2-18k) but the amino acid sequence of which is not known. In order to provide new tools for the study of the σ2-18k protein, we have developed bifunctional σ receptor ligands each bearing a benzophenone photo-crosslinking moiety and an alkyne group to which an azide-containing biotin affinity tag can be covalently attached through click chemistry after photo-crosslinking. Although several compounds showed favorable σ2 binding properties, the highest affinity (2 nM) and the greatest potency in blocking photolabeling of σ2-18k by a radioactive photoaffinity ligand was shown by compound 22. These benzophenone-alkyne σ receptor ligands might therefore be amenable for studying the σ2-18k protein through chemical biology approaches. To the best of our knowledge, these compounds represent the first reported benzophenone-containing clickable σ receptor ligands, which might potentially have broad applications based on the "plugging in" of various tags.

ADAMTS-like proteins are related to ADAMTS metalloproteases by their similarity to ADAMTS ancillary domains. Here, we have characterized ADAMTSL5, a novel member of the superfamily with a unique modular organization that includes a single C-terminal netrin-like (NTR) module. Alternative splicing of ADAMTSL5 at its 5' end generates two transcripts that encode different signal peptides, but the same mature protein. These transcripts differ in their translational efficiency. Recombinant ADAMTSL5 is a secreted, N-glycosylated 60kDa glycoprotein located in the subcellular matrix, on the cell-surface, and in the medium of transfected cells. RT-PCR and western blot analysis of adult mouse tissues showed broad expression. Western blot analysis suggested proteolytic release of the NTR module in transfected cells as well as in some mouse tissues. Immunostaining during mouse organogenesis identified ADAMTSL5 in musculoskeletal tissues such as skeletal muscle, cartilage and bone, as well as in many epithelia. Affinity-chromatography demonstrated heparin-binding of ADAMTSL5 through its NTR-module. Recombinant ADAMTSL5 bound to both fibrillin-1 and fibrillin-2, and co-localized with fibrillin microfibrils in the extracellular matrix of cultured fibroblasts, but without discernible effect on microfibril assembly. ADAMTSL5 is the first family member shown to bind both fibrillin-1 and fibrillin-2. Like other ADAMTS proteins implicated in microfibril biology through identification of human and animal mutations, ADAMTSL5 could have a role in modulating microfibril functions.

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets.

Gliomas are the most common tumor in the central nervous system. High-grade glioblastomas are characterized by their high invasiveness and resistance to radiotherapy, leading to high recurrence rate and short median survival despite radical surgical resection. Characterizations of gliomas at molecular level have revealed aberrations of various growth factor receptors, receptor tyrosine kinases, and tumor suppressor genes that lead to deregulation of multiple signaling pathways, thereby contributing to abnormal proliferation, invasion, and resistance to apoptosis in cancer cells. Recently, accumulating evidence points to the emerging role of axon guidance molecules in glioma progression. Notably, many signaling events harnessed by guidance molecules to regulate cell migration and axon navigation during development are also found to be involved in the modulation of deregulated pathways in gliomas. This paper focused on the signalings triggered by the guidance molecule semaphorins and their receptors plexins and neuropilins, and how their crosstalk with oncogenic pathways in gliomas might modulate cancer progression. The emerging role of semaphorins and plexins as tumor suppressors or oncogenes is also discussed.

The Hedgehog signaling pathway plays an essential role in embryo development and adult tissue homeostasis, in regulating stem cells and is abnormally activated in many cancers. Given the importance of this signaling pathway, we developed a novel and versatile high-throughput, cell-based screening platform using confocal imaging, based on the role of β-arrestin in Hedgehog signal transduction, that can identify agonists or antagonist of the pathway by a simple change to the screening protocol. Here we report the use of this assay in the antagonist mode to identify novel antagonists of Smoothened, including a compound (A8) with low nanomolar activity against wild-type Smo also capable of binding the Smo point mutant D473H associated with clinical resistance in medulloblastoma. Our data validate this novel screening approach in the further development of A8 and related congeners to treat Hedgehog related diseases, including the treatment of basal cell carcinoma and medulloblastoma.

<h4>Background</h4>Short bowel syndrome (SBS) may induce a plethora of clinical symptoms ranging from underweight to nutrient-, vitamin- and electrolyte deficiencies. The objective of this case report is to illustrate how demanding the management of a 60 year old patient with SBS and recurrent joint attacks was for different medical disciplines.<h4>Case presentation</h4>The patient with SBS presented with a body mass index of 16.5 kg/m2 after partial jejunoileal resection of the small intestine with a six year long history of recurrent pain attacks in multiple peripheral joints, chronic diarrhoea and food intolerances. Pain attacks occurred 4-5 times a week with a median consumption of 15 mg prednisone per day. The interdisciplinary workup after several gastroenterologic, rheumatologic, radiologic, psychiatric and orthopedic consultations is shown including successful treatment steps.Clinical diagnosis revealed no systemic inflammatory disease, but confirmed extreme hypomagnesemia (0.2 mmol/l) after reproducible pathological magnesium resorption tests as causative for chronic calcium pyrophosphate crystal inflammatory arthritis (pseudogout, chondrocalcinosis).Multidisciplinary treatment included application of colchicines, parenteral nutrition and magnesium substitution, antiperistaltic agents and avoidance of intolerant foods. Normalization of magnesium levels and a marked remission of joint attacks were achieved after six months with significant reduction of prednisone to 1.5 mg/day.<h4>Conclusion</h4>Despite the rarity of this condition, it is important to know that hypomagnesaemia may be associated with calcium pyrophosphate crystal inflammatory arthritis (chondrocalcinosis) and that SBS patients may be prone to develop extreme hypomagnesaemia causing recurrent joint attacks without systemic inflammation.

<h4>Background</h4>Environment, health behavior, and genetic background are important in the development of obesity. Adolescents spend substantial part of daily leisure time on cultural and social activities, but knowledge about the effects of participation in such activities on weight is limited.<h4>Methods</h4>A number of 1450 adolescents from the Norwegian HUNT study (1995-97) were followed-up in 2006-08 as young adults. Phenotypic data on lifestyle and anthropometric measures were assessed using questionnaires and standardized clinical examinations. Genotypic information on 12 established obesity-susceptibility loci were available for analyses. Generalized estimating equations were used to examine the associations between cultural and social activities in adolescence and adiposity measures in young adulthood. In addition, interaction effects of a genetic predisposition score by leisure time activities were tested.<h4>Results</h4>In girls, participation in cultural activities was negatively associated with waist circumference (WC) (B = -0.04, 95%CI: -0.08 to -0.00) and with waist-hip ratio (WHR) (B = -0.058, 95%CI: -0.11 to -0.01). However, participation in social activities was positively associated with WC (B = 0.040, CI: 0.00 to 0.08) in girls and with BMI (B = 0.027, CI: 0.00 to 0.05) in boys. The effect of the obesity-susceptibility genetic variants on anthropometric measures was lower in adolescents with high participation in cultural activities compared to adolescents with low participation.<h4>Conclusion</h4>This study suggests that the effects of cultural activities on body fat are different from the effects of participation in social activities. The protective influence of cultural activities in female adolescents against overweight in adulthood and their moderating effect on obesity-susceptibility genes suggest that even cultural activities may be useful in public health strategies against obesity.

<h4>Background</h4>Frequent blood transfusion in patients with beta thalassemia major can lead to iron overload especially in liver. Chronic iron overload could cause cirrhosis of the liver. Transfusion- transmitted hepatitis B and C also could develop cirrhosis in individuals.<h4>Materials and methods</h4>The present cross- sectional descriptive study is to assess hepatomegaly and liver enzymes in 100 patients with beta thalassemia major, ages between 2-18 years old. The study was carried out retrospectively. One hundred medical records have chosen from 400 samples of thalassemia major patients, who are under a regular care of the department of sarvar clinic.<h4>Results</h4>Out of these patients, 55% were male and 45% female. The mean age of thalassemia patients was 10.8± 4.4 years. The mean and S. D of hemoglobin, ferritin, deferoxamine dosage was 8.5 ± 1.5g/dl , 2183 ± 1528 ng , 30 ± 11.16 mg/kg, respectively. Forty six percent of them had hepatomegaly. The mean and S. D of AST and ALT were 95± 70 IU/L and 70 ±35U/L respectively. Splenectomy was performed on 44% of patient.<h4>Conclusion</h4>Hepatomegaly is one of the most common findings in the thalassemic patient that induced with hemosiderosis and hepatitis.

<h4>Background</h4>Iron homeostasis is chiefly regulated by hepcidin whose expression is tightly controlled by inflammation, iron stores, and hypoxia. Hemojuvelin (HJV) is a bone morphogenetic protein co-receptor that has been identified as a main upstream regulator of hepcidin expression; HJV mutations are associated with a severe form of iron overload (Juvenile haemochromatosis). Currently however, there is no information on how HJV is regulated by inflammation.<h4>Methods</h4>To study the regulation of Hjv expression by inflammation and whether Hfe has a role in that regulation, control and LPS-injected wild type and Hfe KO mice were used. Moreover, human hepatoma cells (HuH7) were used to study the effect of IL-6 and TNF-α on HJV mRNA expression.<h4>Results</h4>Here we show that LPS repressed hepatic Hjv and BMPs, while it induced hepcidin 1 expression in wild-type and Hfe KO mice with no effect on hepatic pSMAD 1, 5, 8 protein levels. In addition, exogenous TNF-α (20 ng/mL) decreased HJV mRNA and protein expression to 40% of control with no effect on hepcidin mRNA expression in 24 hours. On the other hand, IL-6 induced hepcidin mRNA and protein expression with no effect on HJV mRNA expression levels. Moreover, using the HJV promoter-luciferase reporter fusion construct (HJVP1.2-luc), we showed that the basal luciferase activity of HJVP1.2-luc was inhibited by 33% following TNF-α treatment of HuH7 transfected cells suggesting that the TNF-α down-regulation is exerted at the transcriptional level. Additionally, mutation of a canonical TNF- alpha responsive element (TNFRE) within HJVP1.2-luc abolished TNF-α response suggesting that this TNFRE is functional.<h4>Conclusions</h4>From these results, we conclude that TNF-α suppresses HJV transcription possibly via a novel TNFRE within the HJV promoter. In addition, the results suggest that the proposed link between inflammation and BMP-SMAD signalling is independent of HJV and BMP ligands.

Cardiomyopathy is the leading cause of death in Friedreich's ataxia. This autosomal recessive disease is caused by a homozygous guanine-adenine-adenine trinucleotide repeat expansion in the frataxin gene (chromosome 9q21). One untoward effect of frataxin deficiency is the lack of iron (Fe)-sulfur clusters. Progressive remodeling of the heart in FA, however, may be more specifically related to sarcoplasmic Fe overload. The Fe-containing inclusions in a small percentage of cardiomyocytes may not represent purely mitochondrial accumulation of the metal. The objective of the present study was to re-examine the contribution of Fe to cardiomyocyte hypertrophy, fiber necrosis, and myocardial scarring, using a combination of X-ray fluorescence, slide histochemistry of Fe, and immunohistochemistry of 2 Fe-related proteins. Polyethylene glycol-embedded human cardiac tissues from the left and right ventricular walls, ventricular septum, right atrium, and atrial septum were studied using qualitative and quantitative X-ray fluorescence. Tissues were recovered from the polyethylene glycol matrix, re-embedded in paraffin, and sectioned for visualization of Fe, ferritin, and ferroportin. X-ray fluorescence showed quantifiable levels of Fe and zinc. Regions of significantly increased Fe (1 to 4 mm(2)) were irregularly distributed throughout the working myocardium. Fe granules were sparse in conductive tissue. Zinc signals remained unchanged. Robust cytosolic ferritin reaction product occurred in many fibers of the affected regions. Ferroportin displayed no response except in fibers with advanced Fe overload. These observations are at variance with the concept of selective Fe overload only in cardiac mitochondria. In conclusion, Fe-mediated damage to cardiomyocytes and myocardial scarring are more likely due to cytosolic Fe excess.

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh(-/-) background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT.

Gene set analysis using biological pathways has become a widely used statistical approach for gene expression analysis. A biological pathway can be represented through a graph where genes and their interactions are, respectively, nodes and edges of the graph. From a biological point of view only some portions of a pathway are expected to be altered; however, few methods using pathway topology have been proposed and none of them tries to identify the signal paths, within a pathway, mostly involved in the biological problem. Here, we present a novel algorithm for pathway analysis clipper, that tries to fill in this gap. clipper implements a two-step empirical approach based on the exploitation of graph decomposition into a junction tree to reconstruct the most relevant signal path. In the first step clipper selects significant pathways according to statistical tests on the means and the concentration matrices of the graphs derived from pathway topologies. Then, it identifies within these pathways the signal paths having the greatest association with a specific phenotype. We test our approach on simulated and two real expression datasets. Our results demonstrate the efficacy of clipper in the identification of signal transduction paths totally coherent with the biological problem.

The BCR-ABL fusion kinase is the driving mutation of chronic myelogenous leukemias and is also expressed in a subset of acute lymphoblastic leukemias. Recent advances in elucidating the structure, regulation, and signaling of BCR-ABL have led to the identification of allosteric sites that are distant from the ATP-binding pocket and are critical for BCR-ABL-dependent oncogenic transformation. Here, we review the available data regarding the molecular mechanism of action and the specificity of ATP-competitive tyrosine kinase inhibitors targeting BCR-ABL. In addition, we discuss how targeting of allosteric sites could provide new opportunities to inhibit resistant BCR-ABL mutants, either alone or in combination with conventional ATP-competitive inhibitors.

Systemic lupus erythematosus (SLE) is a generalized autoimmune disease characterized by abnormal B cell activation and the occurrence of increased frequencies of circulating plasma cells (PC). The molecular characteristics and nature of circulating PC and B cells in SLE have not been completely characterized. Microarray analysis of gene expression was used to characterize circulating PC in subjects with active SLE. Flow cytometry was used to sort PC and comparator B cell populations from active SLE blood, normal blood and normal tonsil. The gene expression profiles of the sorted B cell populations were then compared. SLE PC exhibited a similar gene expression signature as tonsil PC. The differences in gene expression between SLE PC and normal tonsil PC and tonsil plasmablasts (PB) suggest a mature Ig secreting cell phenotype in the former population. Despite this, SLE PC differed in expression of about half the genes from previously published gene expression profiles of normal bone marrow PC, indicating that these cells had not achieved a fully mature status. Abnormal expression of several genes, including CXCR4 and S1P(1), suggests a mechanism for the persistence of SLE PC in the circulation. All SLE B cell populations revealed an interferon (IFN) gene signature previously only reported in unseparated SLE peripheral blood mononuclear cells. These data indicate that SLE PC are a unique population of Ig secreting cells with a gene expression profile indicative of a mature, but not fully differentiated phenotype.

<h4>Background</h4>One primary goal of transcriptomic studies is identifying gene expression patterns correlating with disease progression. This is usually achieved by considering transcripts that independently pass an arbitrary threshold (e.g. p<0.05). In diseases involving severe perturbations of multiple molecular systems, such as Alzheimer's disease (AD), this univariate approach often results in a large list of seemingly unrelated transcripts. We utilised a powerful multivariate clustering approach to identify clusters of RNA biomarkers strongly associated with markers of AD progression. We discuss the value of considering pairs of transcripts which, in contrast to individual transcripts, helps avoid natural human transcriptome variation that can overshadow disease-related changes.<h4>Methodology/principal findings</h4>We re-analysed a dataset of hippocampal transcript levels in nine controls and 22 patients with varying degrees of AD. A large-scale clustering approach determined groups of transcript probe sets that correlate strongly with measures of AD progression, including both clinical and neuropathological measures and quantifiers of the characteristic transcriptome shift from control to severe AD. This enabled identification of restricted groups of highly correlated probe sets from an initial list of 1,372 previously published by our group. We repeated this analysis on an expanded dataset that included all pair-wise combinations of the 1,372 probe sets. As clustering of this massive dataset is unfeasible using standard computational tools, we adapted and re-implemented a clustering algorithm that uses external memory algorithmic approach. This identified various pairs that strongly correlated with markers of AD progression and highlighted important biological pathways potentially involved in AD pathogenesis.<h4>Conclusions/significance</h4>Our analyses demonstrate that, although there exists a relatively large molecular signature of AD progression, only a small number of transcripts recurrently cluster with different markers of AD progression. Furthermore, considering the relationship between two transcripts can highlight important biological relationships that are missed when considering either transcript in isolation.

EBV and KSHV are both gamma-herpesviruses which express multiple viral microRNAs. Various methods have been used to investigate the functions of these microRNAs, largely through identification of microRNA target genes. Surprisingly, these related viruses do not share significant sequence homology in their microRNAs. A number of reports have described functions of EBV and KSHV microRNA targets, however only three experimentally validated target genes have been shown to be targeted by microRNAs from both viruses. More sensitive methods to identify microRNA targets have predicted approximately 60% of host targets could be shared by EBV and KSHV microRNAs, but by targeting different sequences in the host targets. In this review, we explore the similarities of microRNA functions and targets of these related viruses.

<h4>Background/aims</h4>Considerable evidence suggests that iron could be a comorbid factor for liver injury in chronic hepatitis C (CHC). Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy. The aim of this study was to evaluate the frequency of hemochromatosis gene mutations in Sardinian CHC patients, the association with iron overload and the impact on response to therapy.<h4>Methods</h4>Sixty-nine CHC patients were enrolled. Iron indices, hepatic and viral parameters were detected. C282Y, H63D and S65C mutations were identified through a PCR. Liver biopsy was performed for hepatic fibrosis evaluation. All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon 180 mcg once weekly and ribavirin 1000-1200 mg/daily. Sustained virological response (SVR) was defined as undetectable HCV RNA 24 weeks after the end of treatment.<h4>Results</h4>HFE gene mutation was detected in 29 patients (42%). The presence of HFE mutations was significantly associated with elevated transferrin saturation (P < 0.01). Hepatic fibrosis was more advanced in HFE mutation carriers (χ (2), P = 0.04). Among mutation carriers 27.5% achieved responses at the end of treatment compared with 60% of non-carriers (P = 0.005). Patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P = 0.03).<h4>Conclusions</h4>The literature shows discordant results about the prevalence, hepatic distribution and possible therapeutic implications of iron overload in chronic hepatitis C. Our findings shows that HFE gene mutations could favor, synergically with CHC and other genetic or acquired factors, the development of liver damage and could influence the outcome of interferon treatment with higher rate of non-response.

<h4>Background and aims</h4>Spontaneous bacterial peritonitis (SBP) requires rapid diagnosis and the initiation of antibiotics. Diagnosis of SBP is usually based on cytobacteriological examination of ascitic fluid. These tests require good laboratory facilities and reporting time of few hours to 1-2 day. However, the 24 h laboratory facilities not widely available in country like India. We evaluated the diagnostic utility of reagent strip (Multistix 10 SG(®)) for rapid diagnosis of SBP.<h4>Material and methods</h4>The study was prospectively carried out on patients of cirrhosis with ascites. Bedside leukocyte esterase reagent strip testing was performed on ascitic fluid. Cell count as determined by colorimetric scale of reagent strip was compared with counting chamber method. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated.<h4>Result</h4>Out of 100 patients with cirrhotic ascites, [72 males: 28 female; mean age 44.34 (SD 13.03) years] 18 patients were diagnosed to have SBP by counting chamber method as compared to 14 patients detected to have SBP by reagent strip test ≥++ positive. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of reagent strip ≥++ positive were 77.77%, 95.12%, 77.77%, 95.12% and 92% respectively compared to counting chamber method.<h4>Conclusion</h4>Reagent strip to diagnose SBP is very specific but less sensitive as compared to counting chamber method. This can be performed rapidly, easily and efficiently even in remote area of developing countries. This bedside test could be a useful tool for the diagnosis of SBP in country like India.

<h4>Unlabelled</h4>Hepcidin regulation is linked to both iron and inflammatory signals and may influence iron loading in nonalcoholic steatohepatitis (NASH). The aim of this study was to examine the relationships among HFE genotype, serum hepcidin level, hepatic iron deposition, and histology in nonalcoholic fatty liver disease (NAFLD). Single-nucleotide polymorphism genotyping for C282Y (rs1800562) and H63D (rs1799945) HFE mutations was performed in 786 adult subjects in the NASH Clinical Research Network (CRN). Clinical, histologic, and laboratory data were compared using nonparametric statistics and multivariate logistic regression. NAFLD patients with C282Y, but not H63D mutations, had lower median serum hepcidin levels (57 versus 65 ng/mL; P = 0.01) and higher mean hepatocellular (HC) iron grades (0.59 versus 0.28; P < 0.001), compared to wild-type (WT) subjects. Subjects with hepatic iron deposition had higher serum hepcidin levels than subjects without iron for all HFE genotypes (P < 0.0001). Hepcidin levels were highest among patients with mixed HC/reticuloendothelial system cell (RES) iron deposition. H63D mutations were associated with higher steatosis grades and NAFLD activity scores (odds ratio [OR], ≥1.4; 95% confidence interval [CI]: >1.0, ≤2.5; P ≤ 0.041), compared to WT, but not with either HC or RES iron. NAFLD patients with C282Y mutations had less ballooning or NASH (OR, ≤0.62; 95% CI: >0.39, <0.94; P ≤ 0.024), compared to WT subjects.<h4>Conclusions</h4>The presence of C282Y mutations in patients with NAFLD is associated with greater HC iron deposition and decreased serum hepcidin levels, and there is a positive relationship between hepatic iron stores and serum hepcidin level across all HFE genotypes. These data suggest that body iron stores are the major determinant of hepcidin regulation in NAFLD, regardless of HFE genotype. A potential role for H63D mutations in NAFLD pathogenesis is possible through iron-independent mechanisms.

Traumatic brain injury (TBI) is a complex and common problem resulting in the loss of cognitive function. In order to build a comprehensive knowledge base of the proteins that underlie these cognitive deficits, we employed unbiased quantitative mass spectrometry, proteomics, and bioinformatics to identify and quantify dysregulated proteins in the CA3 subregion of the hippocampus in the fluid percussion model of TBI in rats. Using stable isotope 18O-water differential labeling and multidimensional tandem liquid chromatography (LC)-MS/MS with high stringency statistical analyses and filtering, we identified and quantified 1002 common proteins, with 124 increased and 76 decreased. The ingenuity pathway analysis (IPA) bioinformatics tool identified that TBI had profound effects on downregulating global energy metabolism, including glycolysis, the Krebs cycle, and oxidative phosphorylation, as well as cellular structure and function. Widespread upregulation of actin-related cytoskeletal dynamics was also found. IPA indicated a common integrative signaling node, calcineurin B1 (CANB1, CaNBα, or PPP3R1), which was downregulated by TBI. Western blotting confirmed that the calcineurin regulatory subunit, CANB1, and its catalytic binding partner PP2BA, were decreased without changes in other calcineurin subunits. CANB1 plays a critical role in downregulated networks of calcium signaling and homeostasis through calmodulin and calmodulin-dependent kinase II to highly interconnected structural networks dominated by tubulins. This large-scale knowledge base lays the foundation for the identification of novel therapeutic targets for cognitive rescue in TBI.

<h4>Purpose</h4>Next-generation sequencing has transformed genetic research and is poised to revolutionize clinical diagnosis. However, the vast amount of data and inevitable discovery of incidental findings require novel analytic approaches. We therefore implemented for the first time a strategy that utilizes an a priori structured framework and a conservative threshold for selecting clinically relevant incidental findings.<h4>Methods</h4>We categorized 2,016 genes linked with Mendelian diseases into "bins" based on clinical utility and validity, and used a computational algorithm to analyze 80 whole-genome sequences in order to explore the use of such an approach in a simulated real-world setting.<h4>Results</h4>The algorithm effectively reduced the number of variants requiring human review and identified incidental variants with likely clinical relevance. Incorporation of the Human Gene Mutation Database improved the yield for missense mutations but also revealed that a substantial proportion of purported disease-causing mutations were misleading.<h4>Conclusion</h4>This approach is adaptable to any clinically relevant bin structure, scalable to the demands of a clinical laboratory workflow, and flexible with respect to advances in genomics. We anticipate that application of this strategy will facilitate pretest informed consent, laboratory analysis, and posttest return of results in a clinical context.

Members of the highly conserved Arf family of small GTPases serve as master regulators of vesicular transport. In yeast, Arf1 acts at the Golgi and trans-Golgi network (TGN) to recruit vesicular coat proteins and other effectors for both anterograde and retrograde transport. Arf1 is activated at the TGN by Sec7, the founding member of the Sec7 family of guanine nucleotide exchange factors (GEFs) and a close homolog of the human ARFGEF2 implicated in congenital defects in cerebral cortex development. Through the use of purified Sec7 in biochemical assays, we recently discovered that autoinhibition of Sec7 is relieved by stable recruitment to lipid membranes by activated Arf1. This interaction is mediated by a conserved domain proximal to, but not including, the GEF domain, creating a positive feedback loop in the activation of Arf1 at the TGN. We further demonstrated that this stable interaction with Arf1 plays a role in localizing Sec7 to the TGN. We elaborate here on the implications of these results to small GTPase-mediated cellular processes and coincidence detection models of GEF localization.

The composition of a dynamic mixture of similar 2,2'-bipyridine complexes of iron(II) bearing either an amide (5-benzylamido-2,2'-bipyridine and 5-(2-methoxyethane)amido-2,2'-bipyridine) or an ester (2,2'-bipyridine-5-carboxylic acid benzylester and 2,2'-bipyridine-5-carboxylic acid 2-methoxyethane ester) side chain have been evaluated by electrospray mass spectroscopy in acetonitrile. The time taken for the complexes to come to equilibrium appears to be dependent on the counteranion, with chloride causing a rapid redistribution of two preformed heteroleptic complexes (of the order of 1 hour), whereas the time it takes in the presence of tetrafluoroborate salts is in excess of 24 h. Similarly the final distribution of products is dependent on the anion present, with the presence of chloride, and to a lesser extent bromide, preferring three amide-functionalized ligands, and a slight preference for an appended benzyl over a methoxyethyl group. Furthermore, for the first time, this study shows that the distribution of a dynamic library of metal complexes monitored by ESI-MS can adapt following the introduction of a different anion, in this case tetrabutylammonium chloride to give the most favoured heteroleptic complex despite the increasing ionic strength of the solution.

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10(4) molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion.

The dosage compensation complex (DCC) binds to single X chromosomes in Drosophila males and increases the transcription level of X-linked genes by approximately twofold. Male-specific lethal 2 (MSL2) together with MSL1 mediates the initial recruitment of the DCC to high-affinity sites in the X chromosome. MSL2 contains a DNA-binding cysteine-rich CXC domain that is important for X targeting. In this study, we determined the solution structure of MSL2 CXC domain by NMR spectroscopy. We identified three zinc ions in the CXC domain and determined the metal-to-cysteine connectivities from (1)H-(113)Cd correlation experiments. The structure reveals an unusual zinc-cysteine cluster composed of three zinc ions coordinated by six terminal and three bridging cysteines. The CXC domain exhibits unexpected structural homology to pre-SET motifs of histone lysine methyltransferases, expanding the distribution and structural diversity of the CXC domain superfamily. Our findings provide novel structural insight into the evolution and function of CXC domains.

A positive inotropic responsiveness to serotonin, mediated by 5-HT(4) and 5-HT(2A) receptors, appears in the ventricle of rats with post-infarction congestive heart failure (HF) and pressure overload-induced hypertrophy. A hallmark of HF is a transition towards a foetal genotype which correlates with loss of cardiac functions. Thus, we wanted to investigate whether the foetal and neonatal cardiac ventricle displays serotonin responsiveness. Wistar rat hearts were collected day 3 and 1 before expected birth (days -3 and -1), as well as day 1, 3, 5 and 113 (age matched with Sham and HF) after birth. Hearts from post-infarction HF and sham-operated animals (Sham) were also collected. Heart tissue was examined for mRNA expression of 5-HT(4), 5-HT(2A) and 5-HT(2B) serotonin receptors, 5-HT transporter, atrial natriuretic peptide (ANP) and myosin heavy chain (MHC)-α and MHC-β (real-time quantitative RT-PCR) as well as 5-HT-receptor-mediated increase in contractile function exvivo (electrical field stimulation of ventricular strips from foetal and neonatal rats and left ventricular papillary muscle from adult rats in organ bath). Both 5-HT(4) mRNA expression and functional responses were highest at day -3 and decreased gradually to day 5, with a further decrease to adult levels. In HF, receptor mRNA levels and functional responses reappeared, but to lower levels than in the foetal ventricle. The 5-HT(2A) and 5-HT(2B) receptor mRNA levels increased to a maximum immediately after birth, but of these, only the 5-HT(2A) receptor mediated a positive inotropic response. We suggest that the 5-HT(4) receptor is a representative of a foetal cardiac gene program, functional in late foetal development and reactivated in heart failure.

<h4>Objective</h4>It is widely recognized that the diagnosis of parathyroid carcinoma (PC) is often difficult because of the overlap of characteristics between malignant and benign parathyroid tumors, especially at an early stage. Based on the identification of tumor suppressor gene HRPT2/CDC73 and its association with hereditary and sporadic PC, screening of gene mutations and detection of parafibromin immunoreactivity have been suggested as diagnostic instruments of PC in Whites. There is little information about HRPT2/CDC73 mutations and its corresponding protein expression in patients with sporadic PC in Chinese population, and the long-term follow-up data is scarce.<h4>Methods</h4>Paraffin-embedded tissues were obtained from 13 patients with PC, 13 patients with parathyroid adenoma (PA) and 7 patients with parathyroid hyperplasia(PH), and 6 normal parathyroid (NP) tissues as controls. Peripheral blood from 11 patients with PC was collected. PCR products using Genomic DNA extracted from tumor tissues or blood as template was sequenced for HRPT2/CDC73 gene. Expression of parafibromin in tumor tissues was evaluated by immunohistochemical analysis.<h4>Results</h4>Six mutations in 6 of 13 patients with PC were identified, with three being novel. Four of them were germ-line mutations. Patients with mutations were susceptible to recurrence of the PC. Complete (8/13, 61.5%) or partial (5/13, 38.5%) loss of parafibromin expression was observed in PC tissues. All of tissue samples from normal parathyroid or benign parathyroid tumors displayed positive immunostaining of parafibromin except one adenoma.<h4>Conclusions</h4>The present study supplies information on the mutations and protein expression of HRPT2/CDC73 gene and phenotypes of parathyroid carcinoma in Chinese population. And the expanded mutation database of this gene may benefit patients in the diagnosis and treatment of this disease.

Huntington disease (HD) is caused by the expansion of an unstable polymorphic trinucleotide (CAG)n repeat in exon 1 of the HTT gene, which translates into an extended polyglutamine tract in the protein. Laboratory diagnosis of HD involves estimation of the number of CAG repeats. Molecular genetic testing for HD is offered in a wide range of laboratories both within and outside the European community. In order to measure the quality and raise the standard of molecular genetic testing in these laboratories, the European Molecular Genetics Quality Network has organized a yearly external quality assessment (EQA) scheme for molecular genetic testing of HD for over 10 years. EQA compares a laboratory's output with a fixed standard both for genotyping and reporting of the results to the referring physicians. In general, the standard of genotyping is very high but the clarity of interpretation and reporting of the test result varies more widely. This emphasizes the need for best practice guidelines for this disorder. We have therefore developed these best practice guidelines for genetic testing for HD to assist in testing and reporting of results. The analytical methods and the potential pitfalls of molecular genetic testing are highlighted and the implications of the different test outcomes for the consultand and his or her family members are discussed.

The International Mouse Phenotyping Consortium (IMPC) (http://www.mousephenotype.org) will reveal the pleiotropic functions of every gene in the mouse genome and uncover the wider role of genetic loci within diverse biological systems. Comprehensive informatics solutions are vital to ensuring that this vast array of data is captured in a standardised manner and made accessible to the scientific community for interrogation and analysis. Here we review the existing EuroPhenome and WTSI phenotype informatics systems and the IKMC portal, and present plans for extending these systems and lessons learned to the development of a robust IMPC informatics infrastructure.

We report on a female patient affected by galactosemia in whom the diagnosis was obscured by the concomitant presence of manifestations suggesting a cow's milk intolerance. This case exemplifies the problems in reaching a correct diagnosis in patients with metabolic diseases.

Early life exposure to antidepressants frequently occurs when pregnant mothers take the medication during late pregnancy. Previous studies in animal models have shown that early exposure to certain antidepressants can alter some behaviors in adulthood. We examined whether the administration of clomipramine, a serotonin reuptake inhibitor, to neonatal mice could result in depression-related behavioral alterations in adult mice. In addition, in an attempt to uncover the mechanism underlying these behavioral changes, we examined the expression of candidate genes in different areas of the brain. Here we show that mice chronically injected with clomipramine specifically during early postnatal development demonstrated depression-like behavior as well as altered stress responses in adulthood. An analysis of the expression of serotonergic genes after exposure to social defeat stress revealed small but significant changes in the expression of 5-HT1A receptor gene (Htr1a) and 5-HTT gene (Slc6a4) in the mice treated with clomipramine compared with the mice injected with saline. We concluded that antidepressant exposure in early days of life could alter stress-related behavior in adulthood and that the behavioral alterations are accompanied by altered serotonergic gene expressions.

Gastric cancer (GC) is a lethal malignancy and the second most common cause of cancer-related deaths. Although treatment options such as chemotherapy, radiotherapy, and surgery have led to a decline in the mortality rate due to GC, chemoresistance remains as one of the major causes for poor prognosis and high recurrence rate. In this study, we investigated the potential effects of isorhamnetin (IH), a 3'-O-methylated metabolite of quercetin on the peroxisome proliferator-activated receptor γ (PPAR-γ) signaling cascade using proteomics technology platform, GC cell lines, and xenograft mice model. We observed that IH exerted a strong antiproliferative effect and increased cytotoxicity in combination with chemotherapeutic drugs. IH also inhibited the migratory/invasive properties of GC cells, which could be reversed in the presence of PPAR-γ inhibitor. We found that IH increased PPAR-γ activity and modulated the expression of PPAR-γ regulated genes in GC cells. Also, the increase in PPAR-γ activity was reversed in the presence of PPAR-γ-specific inhibitor and a mutated PPAR-γ dominant negative plasmid, supporting our hypothesis that IH can act as a ligand of PPAR-γ. Using molecular docking analysis, we demonstrate that IH formed interactions with seven polar residues and six nonpolar residues within the ligand-binding pocket of PPAR-γ that are reported to be critical for its activity and could competitively bind to PPAR-γ. IH significantly increased the expression of PPAR-γ in tumor tissues obtained from xenograft model of GC. Overall, our findings clearly indicate that antitumor effects of IH may be mediated through modulation of the PPAR-γ activation pathway in GC.

Antithrombin (AT) deficiency has been associated with an increased risk of pediatric cerebral sinus venous thrombosis (CSVT); but few cases of neonatal CSVT have been reported. We describe two half-siblings who presented with seizures in the first week of life and were found to have extensive CSVT with associated parenchymal and intraventricular hemorrhage. Both infants were found to have type 1 AT deficiency. Sequencing of the SerpinC1 gene revealed a novel heterozygous mutation on exon 5 (c.1009C > T p.Q337X). Both infants were treated with anticoagulation and had recanalization of the dural sinuses on follow up imaging.

A crucial question that must be addressed in the drug development process is whether the proposed therapeutic target will yield the desired effect in the clinical population. Pharmaceutical and biotechnology companies place a large investment on research and development, long before confirmatory data are available from human trials. Basic science has greatly expanded the computable knowledge of disease processes, both through the generation of large omics data sets and a compendium of studies assessing cellular and systemic responses to physiologic and pathophysiologic stimuli. Given inherent uncertainties in drug development, mechanistic systems models can better inform target selection and the decision process for advancing compounds through preclinical and clinical research.

Increased information on the encoded mammalian genome is expected to facilitate an integrated understanding of complex anatomical structure and function based on the knowledge of gene products. Determination of gene expression-anatomy associations is crucial for this understanding. To elicit the association in the three-dimensional (3D) space, we introduce a novel technique for comprehensive mapping of endogenous gene expression into a web-accessible standard space: Transcriptome Tomography. The technique is based on conjugation of sequential tissue-block sectioning, all fractions of which are used for molecular measurements of gene expression densities, and the block- face imaging, which are used for 3D reconstruction of the fractions. To generate a 3D map, tissues are serially sectioned in each of three orthogonal planes and the expression density data are mapped using a tomographic technique. This rapid and unbiased mapping technique using a relatively small number of original data points allows researchers to create their own expression maps in the broad anatomical context of the space. In the first instance we generated a dataset of 36,000 maps, reconstructed from data of 61 fractions measured with microarray, covering the whole mouse brain (ViBrism: http://vibrism.riken.jp/3dviewer/ex/index.html) in one month. After computational estimation of the mapping accuracy we validated the dataset against existing data with respect to the expression location and density. To demonstrate the relevance of the framework, we showed disease related expression of Huntington's disease gene and Bdnf. Our tomographic approach is applicable to analysis of any biological molecules derived from frozen tissues, organs and whole embryos, and the maps are spatially isotropic and well suited to the analysis in the standard space (e.g. Waxholm Space for brain-atlas databases). This will facilitate research creating and using open-standards for a molecular-based understanding of complex structures; and will contribute to new insights into a broad range of biological and medical questions.

Buyang Huanwu decoction (BYHWD) is a well-known and canonical Chinese medicine formula from "Correction on Errors in Medical Classics" in Qing dynasty. Here, we show that BYHWD could alleviate the ventricular remodeling induced by left anterior descending (LAD) artery ligation in rats. BYHWD treatment (18 g/kg/day) decreased heart weight/body weight (HW/BW), left ventricle (LV) dimension at end diastole (LVDd) and increased LV ejection fraction (LVEF) and LV fractional shortening (LVFS) significantly compared to model group at the end of 12 weeks. The collagen volume of BYHWD group was more significantly decreased than that of model group. Proteomic analysis showed that atrial natriuretic factor (ANF) was downregulated; heat shock protein beta-6 (HSPB6) and peroxiredoxin-6 (PRDX6) were upregulated in BYHWD-treated group among successfully identified proteins. The apoptotic index (AI) was reduced by BYHWD accompanied by decreased expression of Bax and caspase 3 activity, increased Bcl-2/Bax ratio, and phosphorylation of HSPB6 compared to that of model group. Taken together, these results suggest that BYHWD can alleviate ventricular remodeling induced by LAD artery ligation. The antiremodeling effects of BYHWD are conferred by decreasing AI through affecting multiple targets including increased Bcl-2/Bax ratio and decreased caspase 3 activity that might be via upregulated PRDX6, phosphorylation of HSPB6 and subsequently reduction of ANF.

<h4>Objective</h4>To determine if molecular and immunohistochemical (IHC) features of the HRPT2/CDC73 gene and its product, parafibromin, predict the natural history of parathyroid malignancy, particularly atypical adenoma, as seen in a single-centre patient cohort.<h4>Methods</h4>Matched tumor and non-tumor tissues were obtained from 46 patients with parathyroid carcinoma (CA) (n = 15), atypical adenoma (AA) (n = 14) and typical adenoma (TA) (n = 17), as defined by standardized histopathological criteria. Exons and exon-intron boundaries of the CDC73 gene were sequenced to identify germline or somatic mutations. IHC staining for parafibromin was performed and scored as positive if nuclear staining was at least partially IHC-positive.<h4>Results</h4>Mutations of CDC73 were observed in 9/15 (60 %) CA, 2/14 (14 %) AA, and 1/17 (6 %) TA tumors. A recurrent two basepair mutation in exon 7 -- c.679_680delAG -- accounted for half of all identified mutations. Absence of parafibromin nuclear staining was noted in 8/12 (67 %) CA, 2/13 (15 %) AA, and 3/17 (18 %) TA tumors. Median follow up times were 88 months for CA, 76 months for AA, and 104 months for TA patients. One patient, a member of a previously reported multiplex family with a germline CDC73 mutation was found to have a second adenoma after removal of an atypical adenoma.<h4>Conclusions</h4>Molecular screening and IHC are both useful tools in the differential diagnosis of parathyroid tumors, but both have limited sensitivity and specificity. CDC73 mutations and negative immunostaining were common in atypical adenomas, but no local recurrence was observed in any case with successful surgical removal after follow-up periods of 27 to 210 months.

There is considerable evidence that inhaled toxicants such as cigarette smoke can cause both irreversible changes to the genetic material (DNA mutations) and putatively reversible changes to the epigenetic landscape (changes in the DNA methylation and chromatin modification state). The diseases that are believed to involve genetic and epigenetic perturbations include lung cancer, chronic obstructive pulmonary disease (COPD), and cardiovascular disease (CVD), all of which are strongly linked epidemiologically to cigarette smoking. In this review, we highlight the significance of genomics and epigenomics in these major smoking-related diseases. We also summarize the in vitro and in vivo findings on the specific perturbations that smoke and its constituent compounds can inflict upon the genome, particularly on the pulmonary system. Finally, we review state-of-the-art genomics and new techniques such as high-throughput sequencing and genome-wide chromatin assays, rapidly evolving techniques which have allowed epigenetic changes to be characterized at the genome level. These techniques have the potential to significantly improve our understanding of the specific mechanisms by which exposure to environmental chemicals causes disease. Such mechanistic knowledge provides a variety of opportunities for enhanced product safety assessment and the discovery of novel therapeutic interventions.

The deficiency of hepcidin, the hormone that controls iron absorption and its tissue distribution, is the cause of iron overload in nearly all forms of hereditary hemochromatosis and in untransfused iron-loading anemias. In a recent study, we reported the development of minihepcidins, small drug-like hepcidin agonists. Here we explore the feasibility of using minihepcidins for the prevention and treatment of iron overload in hepcidin-deficient mice. An optimized minihepcidin (PR65) was developed that had superior potency and duration of action compared with natural hepcidin or other minihepcidins, and favorable cost of synthesis. PR65 was administered by subcutaneous injection daily for 2 weeks to iron-depleted or iron-loaded hepcidin knockout mice. PR65 administration to iron-depleted mice prevented liver iron loading, decreased heart iron levels, and caused the expected iron retention in the spleen and duodenum. At high doses, PR65 treatment also caused anemia because of profound iron restriction. PR65 administration to hepcidin knockout mice with pre-existing iron overload had a more moderate effect and caused partial redistribution of iron from the liver to the spleen. Our study demonstrates that minihepcidins could be beneficial in iron overload disorders either used alone for prevention or possibly as adjunctive therapy with phlebotomy or chelation.

Activation of Toll-like receptor (TLR) signaling and related pathways by microbial products drives inflammatory responses, host-defense pathways and adaptive immunity. The cost of excessive inflammation is cell and tissue damage, an underlying cause of many acute and chronic diseases. Coincident with activation of TLR signaling, a plethora of anti-inflammatory pathways and mechanisms begin to modulate inflammation until tissue repair is complete. Whereas most studies have focused on the signaling components immediately downstream of the TLRs, this Review summarizes the different levels of anti-inflammatory pathways that have evolved to abate TLR signaling and how they are integrated to prevent cell and tissue destruction.

Mitochondrial dysfunctions cause numerous human disorders. A platform technology based on biodegradable polymers for carrying bioactive molecules to the mitochondrial matrix could be of enormous potential benefit in treating mitochondrial diseases. Here we report a rationally designed mitochondria-targeted polymeric nanoparticle (NP) system and its optimization for efficient delivery of various mitochondria-acting therapeutics by blending a targeted poly(d,l-lactic-co-glycolic acid)-block (PLGA-b)-poly(ethylene glycol) (PEG)-triphenylphosphonium (TPP) polymer (PLGA-b-PEG-TPP) with either nontargeted PLGA-b-PEG-OH or PLGA-COOH. An optimized formulation was identified through in vitro screening of a library of charge- and size-varied NPs, and mitochondrial uptake was studied by qualitative and quantitative investigations of cytosolic and mitochondrial fractions of cells treated with blended NPs composed of PLGA-b-PEG-TPP and a triblock copolymer containing a fluorescent quantum dot, PLGA-b-PEG-QD. The versatility of this platform was demonstrated by studying various mitochondria-acting therapeutics for different applications, including the mitochondria-targeting chemotherapeutics lonidamine and α-tocopheryl succinate for cancer, the mitochondrial antioxidant curcumin for Alzheimer's disease, and the mitochondrial uncoupler 2,4-dinitrophenol for obesity. These biomolecules were loaded into blended NPs with high loading efficiencies. Considering efficacy, the targeted PLGA-b-PEG-TPP NP provides a remarkable improvement in the drug therapeutic index for cancer, Alzheimer's disease, and obesity compared with the nontargeted construct or the therapeutics in their free form. This work represents the potential of a single, programmable NP platform for the diagnosis and targeted delivery of therapeutics for mitochondrial dysfunction-related diseases.

Aggregation of expanded polyglutamine repeat-containing fragments of the huntingtin (htt) protein may play a key role in Huntington's disease. Consistent with this hypothesis, two Ser-to-Asp mutations in the 17-amino-acid N-terminal htt(NT) segment abrogate both visible brain aggregates and disease symptoms in a full-length Q(97) htt mouse model while compromising aggregation kinetics and aggregate morphology in an htt fragment in vitro [Gu et al. (2009). Serines 13 and 16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron64, 828-840]. The htt(NT) segment has been shown to play a critical role in facilitating nucleation of amyloid formation in htt N-terminal exon1 fragments. We show here how these Ser-to-Asp mutations dramatically affect aggregation kinetics and aggregate structural integrity. First, these negatively charged Ser replacements impair the assembly of the α-helical oligomers that play a critical role in htt amyloid nucleation, thus providing an explanation for reduced amyloid formation rates. Second, these sequence modifications alter aggregate morphology, decrease aggregate stability, and enhance the steric accessibility of the htt(NT) segment within the aggregates. Together, these changes make the sequence-modified peptides kinetically and thermodynamically less likely to aggregate and more susceptible, if they do, to posttranslational modifications and degradation. These effects also show how phosphorylation of a protein might achieve cellular effects via direct impacts on the protein's aggregation properties. In fact, preliminary studies on exon1-like molecules containing phosphoryl-Ser residues at positions 13 and 16 show that they reduce aggregation rates and generate atypical aggregate morphologies similar to the effects of the Ser-to-Asp mutants.

The sodium channel blocker mexiletine is considered the first-line drug in myotonic syndromes, a group of muscle disorders characterized by membrane over-excitability. We previously showed that the β-adrenoceptor modulators, clenbuterol and propranolol, block voltage-gated sodium channels in a manner reminiscent to mexiletine, whereas salbutamol and nadolol do not. We now developed a pharmacological rat model of myotonia congenita to perform in vivo preclinical test of antimyotonic drugs. Myotonia was induced by i.p. injection of 30 mg/kg of anthracene-9-carboxylic acid (9-AC), a muscle chloride channel blocker, and evaluated by measuring the time of righting reflex (TRR). The TRR was prolonged from <0.5 s in control conditions to a maximum of ∼4 s, thirty minutes after 9-AC injection, then gradually recovered in a few hours. Oral administration of mexiletine twenty minutes after 9-AC injection significantly hampered the TRR prolongation, with an half-maximum efficient dose (ED(50)) of 12 mg/kg. Both propranolol and clenbuterol produced a dose-dependent antimyotonic effect similar to mexiletine, with ED(50) values close to 20 mg/kg. Antimyotonic effects of 40 mg/kg mexiletine and propranolol lasted for 2 h. We also demonstrated, using patch-clamp methods, that both propranolol enantiomers exerted a similar block of skeletal muscle hNav1.4 channels expressed in HEK293 cells. The two enantiomers (15 mg/kg) also showed a similar antimyotonic activity in vivo in the myotonic rat. Among the drugs tested, the R(+)-enantiomer of propranolol may merit further investigation in humans, because it exerts antimyotonic effect in the rat model, while lacking of significant activity on the β-adrenergic pathway. This study provides a new and useful in vivo preclinical model of myotonia congenita in order to individuate the most promising antimyotonic drugs to be tested in humans.

Numerous genes/SNPs in autoimmune diseases (ADs) are identified through genome-wide association studies (GWAS) and likely to contribute in developing autoimmune phenotypes. Constructions of biologically meaningful pathways are necessary to determine how these genes interact with each other and with other small molecules to develop various complex AD phenotypes prior to beginning time-consuming rigorous experimentation. We have constructed biological pathways with genetically identified genes leading to shared AD phenotypes. Various environmental and endogenous factors interact with these AD associated genes suggesting their critical role in developing diseases and further association studies could be designed for assessing the role of these factors with risk allele in a specific gene. Additionally, existing drugs that have been used long before the identification of these genetically associated genes also interact with these newly associated genes. Thus advanced therapeutic strategies could be designed by grouping patients with risk allele(s) in particular genes that directly or closely interact with the specified drugs. This drug-susceptible gene network will not only increase our understanding about the additional molecular basis for effectiveness against these diseases but also indicate which drug could be more effective for those patients carrying risk allele(s) in that gene. Additionally, we have also identified several interlinking genes in the pathways that could be used for designing future association studies.

Numerous studies have focused on the transcriptional signatures that underlie the maintenance of embryonic stem cell (ESC) pluripotency. However, it remains unclear whether ESC retain transcriptional aberrations seen in in vitro cultured embryos. Here we report the first global transcriptional profile comparison between ESC generated from either in vitro cultured or in vivo derived primate embryos by microarray analysis. Genes involved in pluripotency, oxygen regulation and the cell cycle were downregulated in rhesus ESC generated from in vitro cultured embryos (in vitro ESC). Significantly, several gene differences are similarly downregulated in preimplantation embryos cultured in vitro, which have been associated with long term developmental consequences and disease predisposition. This data indicates that prior to derivation, embryo quality may influence the molecular signature of ESC lines, and may differentially impact the physiology of cells prior to or following differentiation.

Protein kinase D (PKD) has emerged as a potential therapeutic target in multiple pathological conditions, including cancer and heart diseases. Potent and selective small molecule inhibitors of PKD are valuable for dissecting PKD-mediated cellular signaling pathways and for therapeutic application. In this study, we evaluated a targeted library of 235 small organic kinase inhibitors for PKD1 inhibitory activity at a single concentration. Twenty-eight PKD inhibitory chemotypes were identified and six exhibited excellent PKD1 selectivity. Five of the six lead structures share a common scaffold, with compound 139 being the most potent and selective for PKD vs PKC and CAMK. Compound 139 was an ATP-competitive PKD1 inhibitor with a low double-digit nanomolar potency and was also cell-active. Kinase profiling analysis identified this class of small molecules as pan-PKD inhibitors, confirmed their selectivity again PKC and CAMK, and demonstrated an overall favorable selectivity profile that could be further enhanced through structural modification. Furthermore, using a PKD homology model based on similar protein kinase structures, docking modes for compound 139 were explored and compared to literature examples of PKD inhibition. Modeling of these compounds at the ATP-binding site of PKD was used to rationalize its high potency and provide the foundation for future further optimization. Accordingly, using biochemical screening of a small number of privileged scaffolds and computational modeling, we have identified a new core structure for highly potent PKD inhibition with promising selectivity against closely related kinases. These lead structures represent an excellent starting point for the further optimization and the design of selective and therapeutically effective small molecule inhibitors of PKD.

Diabetic nephropathy (DN), the leading cause of end-stage renal failure, is clinically manifested by albuminuria and a progressive decline in glomerular filtration rate. The risk factors and mechanisms that contribute to the development and progression of DN are still incompletely defined. To address the involvement of bradykinin B(2)-receptors (B(2)R) in DN, we used a genome wide approach to study the effects of diabetes on differential renal gene expression profile in wild type and B(2)R knockout (B(2)R(-/-)) mice. Diabetes was induced with streptozotocin and plasma glucose levels and albumin excretion rate (AER) were measured at predetermined times throughout the 23 week study period. Longitudinal analysis of AER indicated that diabetic B(2)R(-/-)D null mice had a significantly decreased AER levels compared to wild type B(2)R(+/+)D mice (P = 0.0005). Results from the global microarray study comparing gene expression profiles among four groups of mice respectively: (B(2)R(+/+)C, B(2)R(+/+)D, B(2)R(-/-)C and B(2)R(-/-)D) highlighted the role of several altered pathological pathways in response to disruption of B(2)R and to the diabetic state that included: endothelial injury, oxidative stress, insulin and lipid metabolism and inflammatory process with a marked alteration in the pro-apoptotic genes. The findings of the present study provide a global genomics view of biomarkers that highlight the mechanisms and putative pathways involved in DN.

<h4>Aim</h4>To evaluate the effects of tanshinone IIA (Tan IIA), a lipophilic diterpene from the Chinese herb Salvia miltiorrhiza, on lipopolysaccharide (LPS)-induced disseminated intravascular coagulation (DIC) in rabbits.<h4>Methods</h4>LPS-induced DIC model was made in adult male New Zealand rabbits by continuous intravenous infusion of LPS (0.5 mg/kg) via marginal ear vein for 6 h. The animals were simultaneously administered with Tan IIA (1, 3 and 10 mg/kg) or heparin (500 000 IU/kg) through continuous infusion via the contralateral marginal ear vein for 6 h. Before and 2 and 6 h after the start of LPS infusion, blood samples were taken for biochemical analyses.<h4>Results</h4>Continuous infusion of LPS into the rabbits gradually impaired the hemostatic parameters, damaged renal and liver functions, increased the plasma TNF-α level, and led to a high mortality rate (80%). Treatment of the rabbits with Tan IIA dose-dependently attenuated the increase in activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrin-fibrinogen degradation products (FDP); ameliorated the decrease in plasma levels of fibrinogen and platelets; and reversed the decline in activity of protein C and antithrombin III. Meanwhile, the treatment significantly suppressed the increase in the plasma levels of aminotransferase, creatinine and TNF-α, and led to much lower mortality (46.7% and 26.7% for the medium- and high-dose groups). Treatment of the rabbits with the high dose of heparin also effectively improved the hemostatic parameters, ameliorated liver and renal injuries, and reduced the plasma level of TNF-α, and significantly reduced the mortality (33.3%).<h4>Conclusion</h4>Tan IIA exerts a protective effect against DIC in rabbits.

Psychopathy is a multidimensional construct characterized by an interpersonally manipulative and emotionally detached personality profile that differentiates it from other antisocial syndromes. Previous research with youth has linked the long allele of the serotonin transporter gene in the presence of environmental stress with the interpersonal and affective traits of psychopathy, but these relationships have yet to be examined in relation to adult psychopathy. Consequently, we examined how serotonin transporter (5-HTTLPR) polymorphisms, monoamine oxidase-A (MAO-A) variants, and childhood abuse measured with the Childhood Trauma Questionnaire relate to dimensions of psychopathy in a forensic sample of 237 men with elevated levels of environmental adversity. We found that the emotional deficits characterizing the affective factor of psychopathy, as measured by the Psychopathy Checklist: Screening Version, were highest among carriers of the 5-HTT long allele. Furthermore, the impulsive and irresponsible lifestyle features of psychopathy were higher among low-activity than high-activity MAO-A carriers. These genetic effects were unexpectedly not moderated by a history of childhood abuse. Results provide evidence on the molecular genetics correlates of psychopathic traits in adulthood, relationships that should be investigated further in future research.

Deregulation of transcription arising from mutations in key signaling pathways is a hallmark of cancer. In melanoma, the most aggressive and lethal form of skin cancer, the Brn-2 transcription factor (POU3F2) regulates proliferation and invasiveness and lies downstream from mitogen-activated protein kinase (MAPK) and Wnt/β-catenin, two melanoma-associated signaling pathways. In vivo Brn-2 represses expression of the microphthalmia-associated transcription factor, MITF, to drive cells to a more stem cell-like and invasive phenotype. Given the key role of Brn-2 in regulating melanoma biology, understanding the signaling pathways that drive Brn-2 expression is an important issue. Here, we show that inhibition of phosphatidylinositol 3-kinase (PI3K) signaling reduces invasiveness of melanoma cells in culture and strongly inhibits Brn-2 expression. Pax3, a transcription factor regulating melanocyte lineage-specific genes, directly binds and regulates the Brn-2 promoter, and Pax3 expression is also decreased upon PI3K inhibition. Collectively, our results highlight a crucial role for PI3K in regulating Brn-2 and Pax3 expression, reveal a mechanism by which PI3K can regulate invasiveness, and imply that PI3K signaling is a key determinant of melanoma subpopulation diversity. Together with our previous work, the results presented here now place Brn-2 downstream of three melanoma-associated signaling pathways.

This report describes one in a series of National Institute of Health (NIH) supported conferences aimed at enhancing the ability of leaders of psychiatry residency training to teach research literacy and produce both clinician-scholars and physician-scientists in their home programs. Most psychiatry training directors would not consider themselves research scholars or even well-schooled in evidence based practice. Yet they are the front line educators to prepare tomorrow's psychiatrists to keep up with, critically evaluate, and in some cases actually participate in the discovery of new and emerging psychiatric knowledge. This annual conference is meant to help psychiatry training directors become more enthusiastic, knowledgeable and pedagogically prepared to create research-friendly environments at their home institutions, so that more trainees will, in turn, become research literate, practice evidence-based psychiatry, and enter research fellowships and careers. The overall design of each year's meeting is a series of plenary sessions introducing participants to new information pertaining to the core theme of that year's meeting, integrated with highly interactive small group teaching sessions designed to consolidate knowledge and provide pragmatic teaching tools appropriate for residents at various levels of training. The theme of each meeting, selected to be a compelling and contemporary clinical problem, serves as a vehicle to capture training directors' attention while teaching relevant brain science, research literacy and effective pedagogy. This report describes the content and assessment of the 2011 annual pre-meeting, "Evidence-based Approaches to Suicide Risk Assessment and Prevention: Insights from the Neurosciences and Behavioral Sciences for use in Psychiatry Residency Training."

Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by an expanded CAG repeat in the gene encoding huntingtin (HTT). Therapeutic approaches to lower mutant HTT (mHTT) levels are expected to proceed to human trials, but noninvasive quantification of mHTT is not currently possible. The importance of the peripheral immune system in neurodegenerative disease is becoming increasingly recognized. Peripheral immune cells have been implicated in HD pathogenesis, but HTT levels in these cells have not been quantified before. A recently described time-resolved Förster resonance energy transfer (TR-FRET) immunoassay was used to quantify mutant and total HTT protein levels in leukocytes from patients with HD. Mean mHTT levels in monocytes, T cells, and B cells differed significantly between patients with HD and controls and between pre-manifest mutation carriers and those with clinical onset. Monocyte and T cell mHTT levels were significantly associated with disease burden scores and caudate atrophy rates in patients with HD. mHTT N-terminal fragments detected in HD PBMCs may explain the progressive increase in mHTT levels in these cells. These findings indicate that quantification of mHTT in peripheral immune cells by TR-FRET holds significant promise as a noninvasive disease biomarker.

Astragaloside IV (AGS-IV) is a main active ingredient of Astragalus membranaceus Bunge, a medicinal herb used for cardiovascular diseases (CVD). In this work, we investigated the therapeutic mechanisms of AGS-IV at a network level by computer-assisted target identification with the in silico inverse docking program (INVDOCK). Targets included in the analysis covered all signaling pathways thought to be implicated in the therapeutic actions of all CVD drugs approved by US FDA. A total of 39 putative targets were identified. Three of these targets, calcineurin (CN), angiotensin-converting enzyme (ACE), and c-Jun N-terminal kinase (JNK), were experimentally validated at a molecular level. Protective effects of AGS-IV were also compared with the CN inhibitor cyclosporin A (CsA) in cultured cardiomyocytes exposed to adriamycin. Network analysis of protein-protein interactions (PPI) was carried out with reference to the therapeutic profiles of approved CVD drugs. The results suggested that the therapeutic effects of AGS-IV are based upon a combination of blocking calcium influx, vasodilation, anti-thrombosis, anti-oxidation, anti-inflammation and immune regulation.

<h4>Background</h4>A peptide mimetic of a ligand for the galactose/N-acetylgalactosamine-specific C-type lectin receptors (GCLR) exhibited monocyte-stimulating activity, but did not extend survival when applied alone against a syngeneic murine malignant glioma. In this study, the combined effect of GCLRP with radiation was investigated.<h4>Methods</h4>C57BL/6 mice underwent stereotactic intracranial implantation of GL261 glioma cells. Animals were grouped based on randomized tumor size by magnetic resonance imaging on day seven. One group that received cranial radiation (4 Gy on days seven and nine) only were compared with animals treated with radiation and GCLRP (4 Gy on days seven and nine combined with subcutaneous injection of 1 nmol/g on alternative days beginning on day seven). Magnetic resonance imaging was used to assess tumor growth and correlated with survival rate. Blood and brain tissues were analyzed with regard to tumor and contralateral hemisphere using fluorescence-activated cell sorting analysis, histology, and enzyme-linked immunosorbent assay.<h4>Results</h4>GCLRP activated peripheral monocytes and was associated with increased blood precursors of dendritic cells. Mean survival increased (P < 0.001) and tumor size was smaller (P < 0.02) in the GCLRP + radiation group compared to the radiation-only group. Accumulation of dendritic cells in both the tumoral hemisphere (P < 0.005) and contralateral tumor-free hemisphere (P < 0.01) was associated with treatment.<h4>Conclusion</h4>Specific populations of monocyte-derived brain cells develop critical relationships with malignant gliomas. The biological effect of GCLRP in combination with radiation may be more successful because of the damage incurred by tumor cells by radiation and the enhanced or preserved presentation of tumor cell antigens by GCLRP-activated immune cells. Monocyte-derived brain cells may be important targets for creating effective immunological modalities such as employing the receptor system described in this study.

Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-l-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251.

During erythropoiesis, erythropoietin stimulates induction of erythroid transcription factors that activate expression of erythroid genes including the erythropoietin receptor (EPO-R) that results in increased sensitivity to erythropoietin. DNA binding of the basic helix-loop-helix transcription factor, TAL1/SCL, is required for normal erythropoiesis. A link between elevated TAL1 and excessive erythrocytosis is suggested by erythroid progenitor cells from a patient that exhibits unusually high sensitivity to erythropoietin with concomitantly elevated TAL1 and EPO-R expression. We found that TAL1 regulates EPO-R expression mediated via three conserved E-box binding motifs (CAGCTG) in the EPO-R 5' untranslated transcribed region. TAL1 increases association of the GATA-1·TAL1·LMO2·LDB1 transcription activation complex to the region that includes the transcription start site and the 5' GATA and 3' E-box motifs flanking the EPO-R transcription start site suggesting that TAL1 promotes accessibility of this region. Nucleosome shifting has been demonstrated to facilitate TAL1 but not GATA-1 binding to regulate target gene expression. Accordingly, we observed that with induced expression of EPO-R in hemotopoietic progenitor cells, nucleosome phasing shifts to increase the linker region containing the EPO-R transcription start site and TAL1 binds to the flanking 5' GATA and 3' E-box regions of the promoter. These data suggest that TAL1 binds to the EPO-R promoter to activate EPO-R expression and provides a potential link to elevated EPO-R expression leading to hypersensitivity to erythropoietin and the resultant excessive erythrocytosis.

Raf kinase inhibitory protein (RKIP) has been identified as a member of a novel class of molecules which implicated in cancer progression and suppress the metastatic spread of tumors. The aim of this study was to investigate the promoter methylation and expression of RKIP, determine the prognostic significance of RKIP in gastric cardia adenocarcinoma (GCA). MSP approach and immunohistochemistry methods were used respectively to examine methylation status and protein expression of RKIP in GCA tissues. The frequency of RKIP methylation in GCA tumor tissues (62.1 %) was significantly higher than that in corresponding normal tissues (4.1 %) and was associated with TNM stage, histological differentiation, depth of invasion, LN metastasis, distant metastasis or recurrence, and upper gastrointestinal cancers (UGIC) family history. Positive staining of RKIP in GCA tumor tissues (34.5 %) was significantly decreased than that in corresponding normal tissues (84.1 %) and was associated with RKIP methylation. RKIP may act as a tumor suppressor gene in GCA by regulation of the Raf-1/MEK/ERK signaling pathway. GCA patients in stage III and IV, with positive UGIC family history, and hypermethylation and down-expression of RKIP were most likely to develop metastatic disease and also showed the worse survival. RKIP methylation in GCA was an independent prognostic marker for survival using multivariate Cox regression analysis (P = 0.04). In all, aberrant hypermethylation of RKIP may be one of the mechanisms that lead to loss or down expression of the gene in GCA especially in individuals with UGIC family history. Additionally, hypermethylation and loss of RKIP expression may be used as a marker to predict clinical outcome of GCA.

Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. There are two forms of sideroblastic anemia, i.e., congenital sideroblastic anemia (CSA) and acquired sideroblastic anemia. In order to clarify the pathophysiology of sideroblastic anemia, a nationwide survey consisting of clinical and molecular genetic analysis was performed in Japan. As of January 31, 2012, data of 137 cases of sideroblastic anemia, including 72 cases of myelodysplastic syndrome (MDS)-refractory cytopenia with multilineage dysplasia (RCMD), 47 cases of MDS-refractory anemia with ring sideroblasts (RARS), and 18 cases of CSA, have been collected. Hemoglobin and MCV level in CSA are significantly lower than those of MDS, whereas serum iron level in CSA is significantly higher than those of MDS. Of 14 CSA for which DNA was available for genetic analysis, 10 cases were diagnosed as X-linked sideroblastic anemia due to ALAS2 gene mutation. The mutation of SF3B1 gene, which was frequently mutated in MDS-RS, was not detected in CSA patients. Together with the difference of clinical data, it is suggested that genetic background, which is responsible for the development of CSA, is different from that of MDS-RS.

Co-existence of multiple causes of liver injury increases the risk of hepatocellular carcinoma (HCC) development. HCC usually develops in patients with cirrhosis although it may also occur in individuals with no or mild liver disease, in particular in cases with hepatitis B virus (HBV) infection. Here we report the case of a 43year-old man with HFE-haemochromatosis, seronegative for hepatitis B and C infections, who developed HCC in the absence of severe liver damage. Both tumoural and non-tumoural liver DNA extracts were tested by nested-PCR and primers specific for four different HBV genomic regions in order to evaluate the presence of occult HBV infection. Only X gene sequences were detected in tumour (but not in non-tumour) DNA extracts. HBV-Alu PCR showed a HBV integration involving a 5'-deleted X gene with an intact enhancer-II/basal-core promoter region. The viral-host junction sequencing revealed that this integrant was located upstream of the partitioning-defective-6-homolog-gamma gene (PARD6G) and real time-PCR quantification demonstrated that PARD6G was overexpressed in tumour compared to non-tumour liver tissues. In conclusion, the combination of HFE-haemochromatosis and occult HBV infection in this patient might have led to a sequel of cellular events that determined the development of HCC even in the absence of cirrhosis.

<h4>Background</h4>We initiated this preclinical study in order to analyze the impact of sorafenib single treatment versus combination treatment in human colorectal cancer.<h4>Methods</h4>The effect of increasing sorafenib doses on proliferation, apoptosis, migration, and activation of signal cascades was analyzed in vitro. The effect of sorafenib single treatment versus 5-fluorouracil (5-FU) single treatment and combination therapy on in vivo proliferation and target cytokine receptor/ligand expression was analyzed in a human colon cancer xenograft mouse model using HT29 tumor cells.<h4>Results</h4>In vitro, SW480 and HT29 cell lines were sensitive to sorafenib, as compared to Caco2 and SW620 cell lines, independent of the mutation status of K-ras, Raf, PTEN, or PI3K. The effect on migration was marginal, but distinct differences in caspases activation were seen. Combination strategies were beneficial in some settings (sorafenib + 5-FU; irinotecan) and disadvantageous in others (sorafenib + oxaliplatin), depending on the chemotherapeutic drug and cell line chosen. Sensitive cell lines revealed a downregulation of AKT and had a weak expression level of GADD45β. In resistant cell lines, pp53 and GADD45β levels decreased upon sorafenib exposure. In vivo, the combination treatment of sorafenib and 5-FU was equally effective as the respective monotherapy concerning tumor proliferation. Interestingly, treatment with either sorafenib or 5-FU resulted in a significant decrease of VEGFR1 and PDGFRβ expression intensity.<h4>Conclusions</h4>In colorectal cancer, a sensitivity towards sorafenib exists, which seems similarly effective as a 5-FU monotherapy. A combination therapy, in contrast, does not show any additional effect.

<h4>Background</h4>Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2-15% of patients. We hypothesized that sensitization/challenge of CFTR(-/-) mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF.<h4>Methods</h4>FABP-hCFTR(+/-)-CFTR(-/-) mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines.<h4>Results</h4>Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR(-/-) mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells.<h4>Conclusions</h4>The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.

Mimicking the structural nanomolecular extracellular matrix with synthetically designed nanosized materials is a relatively new approach, which can be applied in the field of bone tissue engineering. Likewise, bone tissue-engineered constructs can be aided in their development by the use of several types of mechanical stimuli. In this study, we wanted to combine nanotextured biomaterials and centrifugation in one multifactorial system. Mesenchymal stem cells were isolated from rat bone marrow, and cultured on a nanogrooved polystyrene substrate (200-nm-wide pitch with a depth of 50 nm). Constant centrifugation of 10 g was applied to cells up to 7 days. Results showed that on a nanogrooved substrate osteoblast-like cells align parallel to the groove direction. Centrifugation of 10 g also affected cell morphology on a smooth surface. Moreover, cell alignment was significantly reduced for cells grown on nanogrooved substrates, which were subsequently subjected to centrifugation. Independently, both stimuli increased the number of cells after 7 days of culture. However, when both stimuli were combined, an additive effect on cell number was observed, followed by an enhanced effect on osteocalcin mRNA expression and matrix mineralization. In conclusion, biomaterial surface modification as well as centrifugation are effective means to enhance bone cell behavior, moreover, readily available to many tissue engineers.

Pancreatic cancer is among the most lethal malignancies worldwide. We aimed to identify novel prognostic markers by applying mass spectrometry (MS)-based proteomic analysis to formalin-fixed paraffin-embedded (FFPE) tissues. Resectable, node positive pancreatic ductal adenocarcinoma (PDAC) with poor (n = 4) and better (n = 4) outcomes, based on survival duration, with essentially the same clinicopathological backgrounds, and noncancerous pancreatic ducts (n = 5) were analyzed. Cancerous and noncancerous cells collected from FFPE tissue sections by laser microdissection (LMD) were processed for liquid chromatography (LC)-tandem MS (MS/MS). Candidate proteins were identified by semiquantitative comparison and then analyzed quantitatively using selected reaction monitoring (SRM)-based MS. To confirm the associations between candidate proteins and outcomes, we immunohistochemically analyzed a cohort of 87 cases. In result, totally 1,229 proteins were identified and 170 were selected as candidate proteins for SRM-based targeted proteomics. Fourteen proteins overexpressed in cancerous as compared to noncancerous tissue showed different expressions in the poor and better outcome groups. Among these proteins, we found that three novel proteins ECH1, OLFM4 and STML2 were overexpressed in poor group than in better group, and that one known protein GTR1 was expressed reciprocally. Kaplan-Meier analysis showed high expressions of all four proteins to correlate with significantly worse overall survival (p < 0.05). In conclusion, we identified four proteins as candidates of prognostic marker of PDAC. The combination of shotgun proteomics verified by SRM and validated by immunohistochemistry resulted in the prognostic marker discovery that will contribute the understanding of PDAC biology and therapeutic development.

<h4>Background</h4>Genome-wide association (GWA) studies have led to a paradigm shift in how researchers study the genetics underlying disease. Many GWA studies are now publicly available and can be used to examine whether or not previously proposed candidate genes are supported by GWA data. This approach is particularly important for the field of alcoholism because the contribution of many candidate genes remains controversial.<h4>Methods</h4>Using the Human Genome Epidemiology (HuGE) Navigator, we selected candidate genes for alcoholism that have been frequently examined in scientific articles in the past decade. Specific candidate loci as well as all the reported single nucleotide polymorphisms (SNPs) in candidate genes were examined in the Study of Addiction: Genetics and Environment (SAGE), a GWA study comparing alcohol-dependent and nondependent subjects.<h4>Results</h4>Several commonly reported candidate loci, including rs1800497 in DRD2, rs698 in ADH1C, rs1799971 in OPRM1, and rs4680 in COMT, are not replicated in SAGE (p > 0.05). Among candidate loci available for analysis, only rs279858 in GABRA2 (p = 0.0052, OR = 1.16) demonstrated a modest association. Examination of all SNPs reported in SAGE in over 50 candidate genes revealed no SNPs with large frequency differences between cases and controls, and the lowest p-value of any SNP was 0.0006.<h4>Conclusions</h4>We provide evidence that several extensively studied candidate loci do not have a strong contribution to risk of developing alcohol dependence in European and African ancestry populations. Owing to the lack of coverage, we were unable to rule out the contribution of other variants, and these genes and particular loci warrant further investigation. Our analysis demonstrates that publicly available GWA results can be used to better understand which if any of previously proposed candidate genes contribute to disease. Furthermore, we illustrate how examining the convergence of candidate gene and GWA studies can help elucidate the genetic architecture of alcoholism and more generally complex diseases.

<h4>Background</h4>Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Several recent transcriptomic studies in GBM have identified different signatures involving immune genes associated with GBM pathology, overall survival (OS) or response to treatment.<h4>Methods</h4>In order to clarify the immune signatures found in GBM, we performed a co-expression network analysis that grouped 791 immune-associated genes (IA genes) in large clusters using a combined dataset of 161 GBM specimens from published databases. We next studied IA genes associated with patient survival using 3 different statistical methods. We then developed a 6-IA gene risk predictor which stratified patients into two groups with statistically significantly different survivals. We validated this risk predictor on two other Affymetrix data series, on a local Agilent data series, and using RT-Q-PCR on a local series of GBM patients treated by standard chemo-radiation therapy.<h4>Results</h4>The co-expression network analysis of the immune genes disclosed 6 powerful modules identifying innate immune system and natural killer cells, myeloid cells and cytokine signatures. Two of these modules were significantly enriched in genes associated with OS. We also found 108 IA genes linked to the immune system significantly associated with OS in GBM patients. The 6-IA gene risk predictor successfully distinguished two groups of GBM patients with significantly different survival (OS low risk: 22.3 months versus high risk: 7.3 months; p < 0.001). Patients with significantly different OS could even be identified among those with known good prognosis (methylated MGMT promoter-bearing tumor) using Agilent (OS 25 versus 8.1 months; p < 0.01) and RT-PCR (OS 21.8 versus 13.9 months; p < 0.05) technologies. Interestingly, the 6-IA gene risk could also distinguish proneural GBM subtypes.<h4>Conclusions</h4>This study demonstrates the immune signatures found in previous GBM genomic analyses and suggests the involvement of immune cells in GBM biology. The robust 6-IA gene risk predictor should be helpful in establishing prognosis in GBM patients, in particular in those with a proneural GBM subtype, and even in the well-known good prognosis group of patients with methylated MGMT promoter-bearing tumors.

<h4>Purpose</h4>The outcomes of adjuvant chemoradiation for locally advanced rectal cancer are nonuniform among patients with matching prognostic factors. We explored the role of molecular markers for predicting the outcome of adjuvant chemoradiation for rectal cancer patients.<h4>Methods and materials</h4>The study included 68 patients with stages II to III rectal adenocarcinoma who were treated with total mesorectal excision and adjuvant chemoradiation. Chemotherapy based on 5-fluorouracil and leucovorin was intravenously administered each month for 6-12 cycles. Radiation therapy consisted of 54 Gy delivered in 30 fractions. Immunostaining of surgical specimens for COX-2, EGFR, VEGF, thymidine synthase (TS), and Raf kinase inhibitor protein (RKIP) was performed.<h4>Results</h4>The median follow-up was 65 months. Eight locoregional (11.8%) and 13 distant (19.1%) recurrences occurred. Five-year locoregional failure-free survival (LRFFS), distant metastasis-free survival (DMFS), disease-free survival (DFS), and overall survival (OS) rates for all patients were 83.9%, 78.7%, 66.7%, and 73.8%, respectively. LRFFS was not correlated with TNM stage, surgical margin, or any of the molecular markers. VEGF overexpression was significantly correlated with decreased DMFS (P=.045), while RKIP-positive results were correlated with increased DMFS (P=.025). In multivariate analyses, positive findings for COX-2 (COX-2+) and VEGF (VEGF+) and negative findings for RKIP (RKIP-) were independent prognostic factors for DMFS, DFS, and OS (P=.035, .014, and .007 for DMFS; .021, .010, and <.0001 for DFS; and .004, .012, and .001 for OS). The combination of both COX-2+ and VEGF+ (COX-2+/VEGF+) showed a strong correlation with decreased DFS (P=.007), and the combinations of RKIP+/COX-2- and RKIP+/VEGF- showed strong correlations with improved DFS compared with the rest of the patients (P=.001 and <.0001, respectively).<h4>Conclusions</h4>Molecular markers can be valuable in predicting treatment outcome of adjuvant chemoradiation for rectal cancer patients.

Huntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and γ-glutamyl transpeptidase (γ-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

Speech disturbances (e.g., altered prosody) have been described in symptomatic Huntington's Disease (HD) individuals, however, the extent to which speech changes in gene positive pre-manifest (PreHD) individuals is largely unknown. The speech of individuals carrying the mutant HTT gene is a behavioural/motor/cognitive marker demonstrating some potential as an objective indicator of early HD onset and disease progression. Speech samples were acquired from 30 individuals carrying the mutant HTT gene (13 PreHD, 17 early stage HD) and 15 matched controls. Participants read a passage, produced a monologue and said the days of the week. Data were analysed acoustically for measures of timing, frequency and intensity. There was a clear effect of group across most acoustic measures, so that speech performance differed in-line with disease progression. Comparisons across groups revealed significant differences between the control and the early stage HD group on measures of timing (e.g., speech rate). Participants carrying the mutant HTT gene presented with slower rates of speech, took longer to say words and produced greater silences between and within words compared to healthy controls. Importantly, speech rate showed a significant correlation to burden of disease scores. The speech of early stage HD differed significantly from controls. The speech of PreHD, although not reaching significance, tended to lie between the performance of controls and early stage HD. This suggests that changes in speech production appear to be developing prior to diagnosis.

A range of crystalline and nano-sized carbonate- and silicate-substituted hydroxyapatite has been successfully produced by using continuous hydrothermal flow synthesis technology. Ion-substituted calcium phosphates are better candidates for bone replacement applications (due to improved bioactivity) as compared to phase-pure hydroxyapatite. Urea was used as a carbonate source for synthesising phase pure carbonated hydroxyapatite (CO₃-HA) with ≈5 wt% substituted carbonate content (sample 7.5CO₃-HA) and it was found that a further increase in urea concentration in solution resulted in biphasic mixtures of carbonate-substituted hydroxyapatite and calcium carbonate. Transmission electron microscopy images revealed that the particle size of hydroxyapatite decreased with increasing urea concentration. Energy-dispersive X-ray spectroscopy result revealed a calcium deficient apatite with Ca:P molar ratio of 1.45 (±0.04) in sample 7.5CO₃-HA. For silicate-substituted hydroxyapatite (SiO₄-HA) silicon acetate was used as a silicate ion source. It was observed that a substitution threshold of ∼1.1 wt% exists for synthesis of SiO₄-HA in the continuous hydrothermal flow synthesis system, which could be due to the decreasing yields with progressive increase in silicon acetate concentration. All the as-precipitated powders (without any additional heat treatments) were analysed using techniques including Transmission electron microscopy, X-ray powder diffraction, Differential scanning calorimetry, Thermogravimetric analysis, Raman spectroscopy and Fourier transform infrared spectroscopy.

Cholangiocarcinoma is notoriously difficult to diagnose, and the mortality rate is high due to late clinical presentation. CpG island promoter methylation is frequently seen in cancer development. In the present study, we aimed at identifying novel epigenetic biomarkers with the potential to improve the diagnostic accuracy of cholangiocarcinoma. Microarray data analyses of cholangiocarcinoma cell lines treated with epigenetic drugs and their untreated counterparts were compared with previously published gene expression profiles of primary tumors and with non-malignant controls. Genes responding to the epigenetic treatment that were simultaneously downregulated in primary cholangiocarcinoma compared with controls (n = 43) were investigated for their promoter methylation status in cancer cell lines from the gastrointestinal tract. Genes commonly methylated in cholangiocarcinoma cell lines were subjected to quantitative methylation-specific polymerase chain reaction in a total of 93 clinical samples (cholangiocarcinomas and non-malignant controls). CDO1, DCLK1, SFRP1 and ZSCAN18, displayed high methylation frequencies in primary tumors and were unmethylated in controls. At least one of these four biomarkers was positive in 87% of the tumor samples, with a specificity of 100%. In conclusion, the novel methylation-based biomarker panel showed high sensitivity and specificity for cholangiocarcinoma. The potential of these markers in early diagnosis of this cancer type should be further explored.

The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.

Stroke is the leading cause of adult disability, worldwide. Metalloproteins and metals play key roles in epigenetic events in living organisms, including hypertension, the most important modifiable risk factor for stroke. Thus, metalloproteins may be important target biomarkers for disease diagnosis. The primary goal of this study was to assess metal containing proteins in blood plasma, detected by ICP-MS, followed by ESIMS for peptide/protein identification. We then compared the relative concentration differences between samples from patients with ischemic stroke, hemorrhagic stroke and stroke mimics. In 29 plasma samples (10 stroke mimics, 10 ischemic stroke and 9 hemorrhagic stroke patients) previously collected from patients who presented to the University of Cincinnati Emergency Department within 12 hours of symptom onset for a plasma banking project. For the metal associated protein study, Mg, Mn, Cu, Se concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and ischemic stroke patients vs. hemorrhagic stroke patients. Pb concentrations were statistically different when compared between stroke mimics vs. ischemic stroke patients and Mo levels were statistically the same among the three groups. In addition, we also report concentration levels and preliminary correlation studies for total elemental analysis among the three sets of patients. This pilot study demonstrates that mass spectrometry methods may be highly valuable in detecting novel stroke biomarkers in blood plasma. Expanded studies are warranted to confirm these findings.

Toll-like receptors (TLRs) are a group of pattern recognition receptors that play a crucial role in the induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral double-stranded RNA. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Limited studies have applied proteomics toward understanding the dynamics of TLR signaling. Herein, a proteomics approach identified 14-3-3ε and 14-3-3σ proteins as new members of the TLR signaling complex. Toward the functional characterization of 14-3-3ε and 14-3-3σ in TLR signaling, we have shown that both of these proteins impair TLR2, TLR3, TLR4, TLR7/8, and TLR9 ligand-induced IL-6, TNFα, and IFN-β production. We also show that 14-3-3ε and 14-3-3σ impair TLR2-, TLR3-, TLR4-, TLR7/8-, and TLR9-mediated NF-κB and IFN-β reporter gene activity. Interestingly, although the 14-3-3 proteins inhibit poly(I:C)-mediated RANTES production, 14-3-3 proteins augment Pam(3)CSK(4), LPS, R848, and CpG-mediated production of RANTES (regulated on activation normal T cell expressed and secreted) in a Mal (MyD88 adaptor-like)/MyD88-dependent manner. 14-3-3ε and 14-3-3σ also bind to the TLR adaptors and to both TRAF3 and TRAF6. Our study conclusively shows that 14-3-3ε and 14-3-3σ play a major regulatory role in balancing the host inflammatory response to viral and bacterial infections through modulation of the TLR signaling pathway. Thus, manipulation of 14-3-3 proteins may represent novel therapeutic targets for inflammatory conditions and infections.

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

<h4>Background & aims</h4>Porphyria cutanea tarda (PCT) is an iron-related disorder caused by reduced activity of hepatic uroporphyrinogen decarboxylase; it can be treated by phlebotomy or low doses of hydroxychloroquine. We performed a prospective pilot study to compare the efficacy and safety of these therapies.<h4>Methods</h4>We analyzed data from 48 consecutive patients with well-documented PCT to characterize susceptibility factors; patients were treated with phlebotomy (450 mL, every 2 weeks until they had serum ferritin levels of 20 ng/mL) or low-dose hydroxychloroquine (100 mg orally, twice weekly, until at least 1 month after they had normal plasma levels of porphyrin). We compared the time required to achieve a normal plasma porphyrin concentration (remission, the primary outcome) for 17 patients treated with phlebotomy and 13 treated with hydroxychloroquine.<h4>Results</h4>The time to remission was a median 6.9 months for patients who received phlebotomy and 6.1 months for patients treated with hydroxychloroquine treatment (6.7 and 6.5 mo for randomized patients), a difference that was not significant (log-rank, P = .06 and P = .95, respectively). The sample size was insufficient to confirm noninferiority of hydroxychloroquine treatment (hazard ratio, 2.19; 95% confidence interval, 0.95-5.06) for all patients. Patients who received hydroxychloroquine had substantially better compliance. There were no significant side effects of either treatment.<h4>Conclusions</h4>Hydroxychloroquine, 100 mg twice weekly, is as effective and safe as phlebotomy in patients with PCT, although noninferiority was not established. Given these results, higher-dose regimens of hydroxychloroquine, which have more side effects, do not seem justified. Compliance was better and projected costs were lower for hydroxychloroquine than phlebotomy treatment. Long-term studies are needed to compare durability of response. ClinicalTrials.gov number, NCT01573754.

Twin and adoption studies have demonstrated a significant contribution of both genetic and environmental factors to antisocial and delinquent behavior. Associations have been reported between the serotonin transporter (5-HTT) and aggression, and between socioeconomic status (SES), aggression, and serotonergic functions of the brain. We aimed to investigate associations between the 5-HTTLPR genotype and family SES in relation to delinquent behavior among adolescents. A total of 1,467 17- to 18-year-old students in the county of Västmanland, Sweden, anonymously completed a questionnaire and gave a saliva sample. Family SES had a U-shaped relation to delinquency, where adolescents with low and high family SES were the most delinquent. There were curvilinear interactions between the 5-HTTLPR genotype and family SES in relation to delinquency. Among individuals having high family SES, boys with the LL (homozygous for the long allele) or LS (heterozygous) genotypes and girls with the SS (homozygous for the short allele) or LS (heterozygous) genotypes showed the highest delinquency scores. Among individuals having low family SES, boys with the LL (homozygous for the long allele) genotype and girls with the LS (heterozygous) genotype showed the highest delinquency scores. The present study suggests evidence for an interaction between family SES and the 5-HTTLPR genotype in relation to juvenile delinquency.

<h4>Background</h4>Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by the loss of motor neurons. Its etiology remains unknown, but several hypothesis have been raised to explain motor neuron death, including oxidative stress. Dysregulation of cellular iron metabolism can lead to increased oxidative stress, and existing data argue for a role of iron metabolism in ALS pathophysiology.<h4>Methods</h4>We performed a retrospective analysis of iron metabolism (IM) variables (serum levels of iron, transferrin, ferritin, and TSC for Transferrin Saturation Coefficient) in a cohort of 694 ALS patients and 297 healthy controls.<h4>Results</h4>Serum ferritin levels and TSC were higher, whereas serum transferrin levels were lower in ALS patients than controls. In addition, patients with a high level serum ferritin had a shorter survival time compared to those with low level serum ferritin (618 days versus 921 days for men subgroup; p = .007). Site of onset and ALS-FRS score were not associated with IM variables.<h4>Conclusion</h4>This study suggests that ALS patients may have increased iron storage, as measured by increased serum ferritin and TSC. Elevated serum ferritin may also have a deleterious impact on survival in ALS.

Huntington disease (HD) phenotypes without a HTT mutation are known as HD-like (HDL) syndromes and are caused by mutations in other loci. HDL2, almost indistinguishable from HD, is due to expansions in the Junctophilin 3 locus (JPH3) with a worldwide Sub-Saharan ethnic origin. Sixteen independent patients with involuntary movements, psychiatric disturbances and ataxia not having a HTT mutation were searched for loci PRNP (prion protein, HDL1), JPH3 (HDL2), ATN1 (dentatorubral-pallidoluysian atrophy), ATX2 (spinocerebellar ataxia 2) ATXN3 (spinocerebellar ataxia 3), and TBP (spinocerebellar ataxia 17=HDL4). Markers Duffy, Kell, Diego, D9S1120, plus six JPH3 intragenic single-nucleotide polymorphisms were tested to ascertain ethnic origin. Four unrelated choreic patients had an expanded allele at JPH3. Three of them carried the African marker Duffy null. All four families carried with the mutation the same haplotype most frequent in African populations; Amerindian alleles D9D1120*9 and Diego A; or Kell allele K were absent. HDL2 in Venezuela had a low, but higher relative frequency (2.6%) than that in other Caucasoid populations. It should be searched first in choreic patients not having HTT mutations. The most likely remote ethnic origin for all detected families was African.

Preterm birth (PTB) is the leading cause of infant mortality. PTB pathophysiology overlaps with those of adult cardiovascular, immune and metabolic disorders (CIMD), with mechanisms including inflammation, immunotolerance, thrombosis, and nutrient metabolism. Whereas many genetic factors for CIMD have been identified, progress in PTB has lagged. We hypothesized that highly validated genetic risk factors for CIMD may also be associated with PTB. We conducted case-control study of four female cohorts with spontaneous PTB (n = 673) versus term (n = 1119). Of 35 SNPs genotyped, there were 13 statistically significant associations (P < 0.05), which were more than expected (binomial test; P = 0.02). In US White (307 cases/342 controls), the G allele of HLA-DQA1 (A/G) rs9272346 was protective for PTB in the initial discovery cohort (P = 0.02; OR = 0.65; 95 % CI 0.46, 0.94). This protective association replicated (P = 0.02; OR = 0.85; 95 % CI 0.75, 0.97) nominally in the Danish Cohort (883 cases, 959 controls), but lost significance upon multiple testing correction. We observed more statistically significant associations than expected, suggesting that chance is an unlikely explanation for one or more of the associations. Particularly, a protective association of the G allele of HLA-DQA1 was found in two independent cohorts, and in previous studies, this same allele was found to protect against type-1-diabetes (meta-analysis P value 5.52 × 10(-219)). Previous investigations have implicated HLA phenotypic variation in recurrent fetal loss and in chronic chorioamnionitis. Given the limited sample size in his study, we suggest larger studies to further investigate possible HLA genetic involvement in PTB.

<h4>Purpose</h4>Recent developments in genomics have led to expanded carrier screening panels capable of assessing hundreds of causal mutations for genetic disease. This new technology enables simultaneous measurement of carrier frequencies for many diseases. As the resultant rank-ordering of carrier frequencies impacts the design and prioritization of screening programs, the accuracy of this ranking is a public health concern.<h4>Methods</h4>A total of 23,453 individuals from many obstetric, genetics, and infertility clinics were referred for routine recessive disease carrier screening. Multiplex carrier screening was performed and results were aggregated for this study.<h4>Results</h4>Twenty-four percent of individuals were identified as carriers for at least one of 108 disorders, and 5.2% were carriers for multiple disorders. We report tabulations of carrier frequency by self-identified ethnicity and disease.<h4>Conclusion</h4>To our knowledge, this study of a large, ethnically diverse clinical sample provides the most accurate measurements to date of carrier frequencies for hundreds of recessive alleles. The study also yields information on the clinical considerations associated with routine use of expanded panels and provides support for a pan-ethnic screening paradigm that minimizes the use of "racial" categories by the physician, as recommended by recent guidelines.

Huntington's disease (HD) is characterized clinically by chorea, motor impairment, psychiatric manifestations, and dementia. Atrophy of the striatum is the neuropathological hallmark of HD, and previous studies have suggested that striatal atrophy correlates more closely with motor impairment than with chorea. Motor impairment, as measured by motor impairment score, correlates with functional disability in HD patients, but chorea does not. In this study, we investigated the relation between neuronal loss and these motor features. We conducted neuropathological and stereologic assessments of neurons in putamen and subthalamic nuclei in HD patients and age-matched controls. In putamen, we estimated the total number and volume of medium spiny neurons labeled with dopamine- and cAMP-regulated phosphoprotein 32 kDa (DARPP-32). In subthalamic nuclei, we estimated the total number of neurons on hematoxylin & eosin/luxol fast blue stains. In putamen of HD, immunohistochemistry showed DARPP-32 neuronal atrophy with extensive disruption of neurites and neuropil; stereologic studies found significant decreases in both the number and size of DARPP-32 neurons; we also detected a significant reduction of overall putamen volume in HD patients, compared to controls. In subthalamic nuclei, there was a mild, but significant, neuronal loss in the HD group. The loss of neurons in putamen and subthalamic nuclei as well as putaminal atrophy were significantly correlated with severity of motor impairment, but not with chorea. Our findings suggest that neuronal loss and atrophy in striatum and neuronal loss in subthalamic nuclei contribute specifically to the motor impairment of HD, but not to chorea.

Peptide Nucleic Acids (PNAs) linked to high molecular weight (MW) poly(ethylene oxide) (PEO) derivatives could be useful conjugates for the direct functionalisation of gold surfaces dedicated to Surface Plasmon Resonance (SPR)-based DNA sensing. However their use is hampered by the difficulty to obtain them through a convenient and economical route. In this work we compared three synthetic strategies to obtain PNA-high MW PEO conjugates composed of (a) a 15-mer PNA sequence as the probe complementary to genomic DNA of ]Mycobacterium tuberculosis, (b) a PEO moiety (2 or 5 KDa MW) and (c) a terminal trityl-protected thiol necessary (after acidic deprotection) for grafting to gold surfaces. The 15-mer PNA was obtained by solid-phase synthesis. Its amino terminal group was later condensed to bi-functional PEO derivatives (2 and 5 KDa MW) carrying a Trt-cysteine at one end and a carboxyl group at the other end. The reaction was carried out either in solution, using HATU or PyOxim as coupling agents, or through the solid-phase approach, with 49.6%, 100% and 5.2% yield, respectively. A differential solvent extraction strategy for product purification without the need for chromatography is described. The ability of the 5 KDa PEO conjugate to function as a probe for complementary DNA detection was demonstrated using a Grating-Coupling Surface Plasmon Resonance (GC-SPR) system. The optimized PEO conjugation and purification protocols are economical and simple enough to be reproduced also within laboratories that are not highly equipped for chemical synthesis.

<h4>Objective</h4>Genetic variation in coagulation and fibrinolysis may affect the development of subclinical atherosclerosis modifying the risk of stroke and cardiovascular disease. However, data on the relationship between subclinical atherosclerosis and genes involved in the coagulation system are sparse. The objective of this study is to examine the association between single nucleotide polymorphisms (SNPs) in coagulation system genes and subclinical carotid plaque phenotypes.<h4>Methods</h4>From the Genetic Determinants of Subclinical Carotid Disease Study, 287 Dominicans were examined for carotid plaque presence, thickness, and surface irregularity by high-resolution B-mode carotid ultrasound. Logistic regression was used to test for association between 101 SNPs in 23 coagulation system genes and plaque phenotypes while controlling for age, sex, smoking, hypertension, dyslipidemia, and diabetes. Within gene haplotypes and interactions between genes were examined. A follow-up of SNPs in moderate to high (r(2)>0.25) linkage disequilibrium (LD) with those implicated in the discovery analysis (p ≤ 0.01) was performed in an independent sample of 301 Dominicans.<h4>Results</h4>The prevalence of carotid plaque (47% discovery; 46% follow-up) as well as the mean age (65 ± 8 discovery; 65 ± 9 follow-up) of the participants was similar in both datasets. Two genes (vWF and THBS1) were associated (p ≤ 0.01) with plaque size and surface irregularity. In follow-up, 5 SNPs in vWF were associated (p ≤ 0.05) with plaque size. SERPINE1 was an additional gene of interest in the haplotype and interaction analyses.<h4>Conclusions</h4>Variation in the vWF, THBS1, and SERPINE1 gene may play an important role in the pathogenesis of atherosclerotic plaque.

The Caenorhabditis elegans dosage compensation complex (DCC) equalizes X-chromosome gene dosage between XO males and XX hermaphrodites by two-fold repression of X-linked gene expression in hermaphrodites. The DCC localizes to the X chromosomes in hermaphrodites but not in males, and some subunits form a complex homologous to condensin. The mechanism by which the DCC downregulates gene expression remains unclear. Here we show that the DCC controls the methylation state of lysine 20 of histone H4, leading to higher H4K20me1 and lower H4K20me3 levels on the X chromosomes of XX hermaphrodites relative to autosomes. We identify the PR-SET7 ortholog SET-1 and the Suv4-20 ortholog SET-4 as the major histone methyltransferases for monomethylation and di/trimethylation of H4K20, respectively, and provide evidence that X-chromosome enrichment of H4K20me1 involves inhibition of SET-4 activity on the X. RNAi knockdown of set-1 results in synthetic lethality with dosage compensation mutants and upregulation of X-linked gene expression, supporting a model whereby H4K20me1 functions with the condensin-like C. elegans DCC to repress transcription of X-linked genes. H4K20me1 is important for mitotic chromosome condensation in mammals, suggesting that increased H4K20me1 on the X may restrict access of the transcription machinery to X-linked genes via chromatin compaction.

We screened 46 novel anilinoquinazoline derivatives for activity to inhibit proliferation of a panel of human cancer cell lines. Among them, Q15 showed potent in vitro growth-inhibitory activity towards cancer cell lines derived from colorectal cancer, lung cancer and multiple myeloma. It also showed antitumor activity towards multiple myeloma KMS34 tumor xenografts in lcr/scid mice in vivo. Unlike the known anilinoquinazoline derivative gefitinib, Q15 did not inhibit cytokine-mediated intracellular tyrosine phosphorylation. Using our mRNA display technology, we identified hCAP-G2, a subunit of condensin II complex, which is regarded as a key player in mitotic chromosome condensation, as a Q15 binding partner. Immunofluorescence study indicated that Q15 compromises normal segregation of chromosomes, and therefore might induce apoptosis. Thus, our results indicate that hCAP-G2 is a novel therapeutic target for development of drugs active against currently intractable neoplasms.

GWAS has facilitated greatly the discovery of risk SNPs associated with complex diseases. Traditional methods analyze SNP individually and are limited by low power and reproducibility since correction for multiple comparisons is necessary. Several methods have been proposed based on grouping SNPs into SNP sets using biological knowledge and/or genomic features. In this article, we compare the linear kernel machine based test (LKM) and principal components analysis based approach (PCA) using simulated datasets under the scenarios of 0 to 3 causal SNPs, as well as simple and complex linkage disequilibrium (LD) structures of the simulated regions. Our simulation study demonstrates that both LKM and PCA can control the type I error at the significance level of 0.05. If the causal SNP is in strong LD with the genotyped SNPs, both the PCA with a small number of principal components (PCs) and the LKM with kernel of linear or identical-by-state function are valid tests. However, if the LD structure is complex, such as several LD blocks in the SNP set, or when the causal SNP is not in the LD block in which most of the genotyped SNPs reside, more PCs should be included to capture the information of the causal SNP. Simulation studies also demonstrate the ability of LKM and PCA to combine information from multiple causal SNPs and to provide increased power over individual SNP analysis. We also apply LKM and PCA to analyze two SNP sets extracted from an actual GWAS dataset on non-small cell lung cancer.

<h4>Objective</h4>To study the incidence, clinical presentation and outcome of intracranial hemorrhagic complications in adult patients with community associated bacterial meningitis.<h4>Methods</h4>Nationwide prospective cohort study from all hospitals in the Netherlands, from 1 March 2006, through 31 December 2010.<h4>Results</h4>Of the 860 episodes of bacterial meningitis that were included, 24 were diagnosed with intracranial hemorrhagic complications: 8 upon presentation and 16 during clinical course. Clinical presentation between patients with or without intracranial hemorrhage was similar. Causative bacteria were Streptococcus pneumoniae in 16 patients (67%), Staphylococcus aureus in 5 (21%), Pseudomonas aeruginosa and Listeria monocytogenes both in 1 patient (4%). Occurrence of intracranial hemorrhage was associated with death (63% vs. 15%, P<0.001) and unfavorable outcome (94% vs. 34%, P<0.001). The use of anticoagulants on admission was associated with a higher incidence of intracranial hemorrhages (odds ratio 5.84, 95% confidence interval 2.17-15.76).<h4>Conclusion</h4>Intracranial hemorrhage is a rare but devastating complication in patients with community-associated bacterial meningitis. Since anticoagulant therapy use is associated with increased risk for intracranial hemorrhage, physicians may consider reversing or temporarily discontinuing anticoagulation in patients with bacterial meningitis.

Although acute myeloid leukaemia (AML) has long been recognized for its morphological and cytogenetic heterogeneity, recent high-resolution genomic profiling has demonstrated a complexity even greater than previously imagined. This complexity can be seen in the number and diversity of genetic alterations, epigenetic modifications, and characteristics of the leukaemic stem cells. The broad range of abnormalities across different AML subtypes suggests that improvements in clinical outcome will require the development of targeted therapies for each subtype of disease and the design of novel clinical trials to test these strategies. It is highly unlikely that further gains in long-term survival rates will be possible by mere intensification of conventional chemotherapy. In this review, we summarize recent studies that provide new insight into the genetics and biology of AML, discuss risk stratification and therapy for this disease, and profile some of the therapeutic agents currently under investigation.

No abstract available.

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe²⁺ on permeation and gating of Ca(V)3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe²⁺ state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe²⁺ rapidly blocked currents with 2 mM extracellular Ca²⁺ in a voltage-dependent manner, as described by a Woodhull model with K(D) = 2.5 mM at 0 mV and apparent electrical distance δ = 0.17. Extracellular Fe²⁺ also shifted activation to more-depolarized voltages (by ∼10 mV with 1.8 mM extracellular Fe²⁺) somewhat more strongly than did extracellular Ca²⁺ or Mg²⁺, which is consistent with a Gouy-Chapman-Stern model with surface charge density σ = 1 e(-)/98 Ų and K(Fe) = 4.5 M⁻¹ for extracellular Fe²⁺. In the absence of extracellular Ca²⁺ (and with extracellular Na⁺ replaced by TEA), Fe²⁺ carried detectable, whole-cell, inward currents at millimolar concentrations (73 ± 7 pA at -60 mV with 10 mM extracellular Fe²⁺). With a two-site/three-barrier Eyring model for permeation of Ca(V)3.1 channels, we estimated a transport rate for Fe²⁺ of ∼20 ions/s for each open channel at -60 mV and pH 7.2, with 1 μM extracellular Fe²⁺ (with 2 mM extracellular Ca²⁺). Because Ca(V)3.1 channels exhibit a significant "window current" at that voltage (open probability, ∼1%), Ca(V)3.1 channels represent a likely pathway for Fe²⁺ entry into cells with clinically relevant concentrations of extracellular Fe²⁺.

Male-biased genes-those expressed at higher levels in males than in females-are underrepresented on the X chromosome of Drosophila melanogaster. Several evolutionary models have been posited to explain this so-called demasculinization of the X. Here, we show that the apparent paucity of male-biased genes on the X chromosome is attributable to global X-autosome differences in expression in Drosophila testes, owing to a lack of sex chromosome dosage compensation in the male germline, but not to any difference in the density of testis-specific or testis-biased genes on the X chromosome. First, using genome-wide gene expression data from 20 tissues, we find no evidence that genes with testis-specific expression are underrepresented on the X chromosome. Second, using contrasts in gene expression profiles among pairs of tissues, we recover a statistical underrepresentation of testis-biased genes on the X but find that the pattern largely disappears once we account for the lack of dosage compensation in the Drosophila male germline. Third, we find that computationally "demasculinizing" the autosomes is not sufficient to produce an expression profile similar to that of the X chromosome in the testes. Our findings thus show that the lack of sex chromosome dosage compensation in Drosophila testes can explain the apparent signal of demasculinization on the X, whereas evolutionary demasculinization of the X cannot explain its overall reduced expression in the testes.

A major event in mammalian male sex determination is the induction of the testis determining factor Sry and its downstream gene Sox9. The current study provides one of the first genome wide analyses of the downstream gene binding targets for SRY and SOX9 to help elucidate the molecular control of Sertoli cell differentiation and testis development. A modified ChIP-Chip analysis using a comparative hybridization was used to identify 71 direct downstream binding targets for SRY and 109 binding targets for SOX9. Interestingly, only 5 gene targets overlapped between SRY and SOX9. In addition to the direct response element binding gene targets, a large number of atypical binding gene targets were identified for both SRY and SOX9. Bioinformatic analysis of the downstream binding targets identified gene networks and cellular pathways potentially involved in the induction of Sertoli cell differentiation and testis development. The specific DNA sequence binding site motifs for both SRY and SOX9 were identified. Observations provide insights into the molecular control of male gonadal sex determination.

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington's Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.

Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis.

Hepatitis C virus (HCV) exhibits a narrow host range and a specific tissue tropism. Mice expressing major entry receptors for HCV permit viral entry, and therefore the species tropism of HCV infection is considered to be reliant on the expression of the entry receptors. However, HCV receptor candidates are expressed and replication of HCV-RNA can be detected in several nonhepatic cell lines, suggesting that nonhepatic cells are also susceptible to HCV infection. Recently it was shown that the exogenous expression of a liver-specific microRNA, miR-122, facilitated the efficient replication of HCV not only in hepatic cell lines, including Hep3B and HepG2 cells, but also in nonhepatic cell lines, including Hec1B and HEK-293T cells, suggesting that miR-122 is required for the efficient replication of HCV in cultured cells. However, no infectious particle was detected in the nonhepatic cell lines, in spite of the efficient replication of HCV-RNA. In the nonhepatic cells, only small numbers of lipid droplets and low levels of very-low-density lipoprotein-associated proteins were observed compared with findings in the hepatic cell lines, suggesting that functional lipid metabolism participates in the assembly of HCV. Taken together, these findings indicate that miR-122 and functional lipid metabolism are involved in the tissue tropism of HCV infection. In this review, we would like to focus on the role of miR-122 and lipid metabolism in the cell tropism of HCV.

<h4>Background</h4>Studies conducted with gilthead sea bream (Sparus aurata L.) have determined the maximum dietary replacement of fish meal and oil without compromising growth or product quality. The present study aimed to analyze the effect of the nutritional background on fish health and fish fed plant protein-based diets with fish oil (FO diet) or a blend of vegetable oils (66VO diet) were exposed for 102 days to the intestinal myxosporean parasite Enteromyxum leei, and the intestine transcriptome was analyzed with a customized oligo-microarray of 7,500 annotated genes.<h4>Results</h4>Infection prevalence was high and similar in the two diet groups, but the outcome of the disease was more pronounced in fish fed the 66VO diet. No differences were found in the transcriptome of both diet control groups, whereas the number of differentially expressed genes in infected groups was considerable. K-means clustering of these differentially expressed genes identified four expression patterns that reflected the progression of the disease with the magnitude of the fold-change being higher in infected 66VO fish. A positive correlation was found between the time of infection and the magnitude of the transcriptional change within the 66VO group, being higher in early infected animals. Within this diet group, a strong up-regulation of many components of the immune specific response was evidenced, whereas other genes related to complement response and xenobiotic metabolism were down-regulated.<h4>Conclusions</h4>The high replacement of fish oil by vegetable oils in practical fish feeds did not modify the intestine transcriptome of gilthead sea bream, but important changes were apparent when fish were exposed to the myxosporean E. leei. The detected changes were mostly a consequence rather than a cause of the different disease progression in the two diet groups. Hence, the developed microarray constitutes an excellent diagnostic tool to address changes associated with the action of intestinal pathogens, but lacks a prognostic value to predict in advance the different susceptibility of growing fish to the current pathogen.

Neurodegenerative diseases constitute a class of illnesses marked by pathological protein aggregation in the brains of affected individuals. Although these disorders are invariably characterized by the degeneration of highly specific subpopulations of neurons, protein aggregation occurs in all cells, which indicates that toxicity arises only in particular cell biological contexts. Aggregation-associated disorders are unified by a common cell biological feature: the deposition of the culprit proteins in inclusion bodies. The precise function of these inclusions remains unclear. The starting point for uncovering the origins of disease pathology must therefore be a thorough understanding of the general cell biological function of inclusions and their potential role in modulating the consequences of aggregation. Here, we show that in human cells certain aggregate inclusions are active compartments. We find that toxic aggregates localize to one of these compartments, the juxtanuclear quality control compartment (JUNQ), and interfere with its quality control function. The accumulation of SOD1G93A aggregates sequesters Hsp70, preventing the delivery of misfolded proteins to the proteasome. Preventing the accumulation of SOD1G93A in the JUNQ by enhancing its sequestration in an insoluble inclusion reduces the harmful effects of aggregation on cell viability.

<h4>Background</h4>Many methods have been developed to identify disease genes and further module biomarkers of complex diseases based on gene expression data. It is generally difficult to distinguish whether the variations in gene expression are causative or merely the effect of a disease. The limitation of relying on gene expression data alone highlights the need to develop new approaches that can explore various data to reflect the casual relationship between network modules and disease traits.<h4>Methods</h4>In this work, we developed a novel network-based approach to identify putative causal module biomarkers of complex diseases by integrating heterogeneous information, for example, epigenomic data, gene expression data, and protein-protein interaction network. We first formulated the identification of modules as a mathematical programming problem, which can be solved efficiently and effectively in an accurate manner. Then, we applied our approach to colorectal cancer (CRC) and identified several network modules that can serve as potential module biomarkers for characterizing CRC. Further validations using three additional gene expression datasets verified their candidate biomarker properties and the effectiveness of the method. Functional enrichment analysis also revealed that the identified modules are strongly related to hallmarks of cancer, and the enriched functions, such as inflammatory response, receptor and signaling pathways, are specific to CRC.<h4>Results</h4>Through constructing a transcription factor (TF)-module network, we found that aberrant DNA methylation of genes encoding TF considerably contributes to the activity change of some genes, which may function as causal genes of CRC, and that can also be exploited to develop efficient therapies or effective drugs.<h4>Conclusion</h4>Our method can potentially be extended to the study of other complex diseases and the multiclassification problem.

<h4>Background</h4>Aggressive mature B-cell non-Hodgkin's lymphomas (BCL) sharing features of Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) (intermediate BL/DLBCL) but deviating with respect to one or more characteristics are increasingly recognized. The limited knowledge about these biologically heterogeneous lymphomas hampers their assignment to a known entity, raising incertitude about optimal treatment approaches. We therefore searched for discriminative, prognostic, and predictive factors for their better characterization.<h4>Patients and methods</h4>We analyzed 242 cytogenetically defined aggressive mature BCL for differential protein expression. Marker selection was based on recent gene-expression profile studies. Predictive models for diagnosis were established and validated by a different set of lymphomas.<h4>Results</h4>CSE1L- and inhibitor of DNA binding-3 (ID3)-overexpression was associated with the diagnosis of BL and signal transduction and transcription-3 (STAT3) with DLBCL (P<0.001 for all markers). All three markers were associated with patient outcome in DLBCL. A new algorithm discriminating BL from DLBCL emerged, including the expression of CSE1L, STAT3, and MYC translocation. This 'new classifier' enabled the identification of patients with intermediate BL/DLBCL who benefited from intensive chemotherapy regimens.<h4>Conclusion</h4>The proposed algorithm, which is based on markers with reliable staining properties for routine diagnostics, represents a novel valid tool in separating BL from DLBCL. Most interestingly, it allows segregating intermediate BL/DLBCL into groups with different treatment requirements.

Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA₂ G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA₂ receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA(1&2) double-knockout mice reconstituted with the LPA₂ receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA₂, Na⁺ - H⁺ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA₂-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.

Liquid crystal displays (LCD) are currently replacing the previously dominant cathode ray tubes (CRT) in most vision science applications. While the properties of the CRT technology are widely known among vision scientists, the photometric and temporal properties of LCDs are unfamiliar to many practitioners. We provide the essential theory, present measurements to assess the temporal properties of different LCD panel types, and identify the main determinants of the photometric output. Our measurements demonstrate that the specifications of the manufacturers are insufficient for proper display selection and control for most purposes. Furthermore, we show how several novel display technologies developed to improve fast transitions or the appearance of moving objects may be accompanied by side-effects in some areas of vision research. Finally, we unveil a number of surprising technical deficiencies. The use of LCDs may cause problems in several areas in vision science. Aside from the well-known issue of motion blur, the main problems are the lack of reliable and precise onsets and offsets of displayed stimuli, several undesirable and uncontrolled components of the photometric output, and input lags which make LCDs problematic for real-time applications. As a result, LCDs require extensive individual measurements prior to applications in vision science.

A better understanding of the aging process is necessary to ensure that the healthcare needs of an aging population are met. With the trend toward increased human life expectancies, identification of candidate genes affecting the regulation of lifespan and its relationship to environmental factors is essential. Through misexpression screening of EP mutant lines, we previously isolated several genes extending lifespan when ubiquitously overexpressed, including the two genes encoding the fatty-acid-binding protein and dodecenoyl-CoA delta-isomerase involved in fatty-acid β-oxidation, which is the main energy resource pathway in eukaryotic cells. In this study, we analyzed flies overexpressing the two main components of fatty-acid β-oxidation, and found that overexpression of fatty-acid-β-oxidation-related genes extended the Drosophila lifespan. Furthermore, we found that the ability of dietary restriction to extend lifespan was reduced by the overexpression of fatty-acid-β-oxidation-related genes. Moreover, the overexpression of fatty-acid-β-oxidation-related genes enhanced stress tolerance to oxidative and starvation stresses and activated the dFOXO signal, indicating translocation to the nucleus and transcriptional activation of the dFOXO target genes. Overall, the results of this study suggest that overexpression of fatty-acid-β-oxidation-related genes extends lifespan in a dietary-restriction-related manner, and that the mechanism of this process may be related to FOXO activation.

Celiac disease (CD) is an immune-mediated enteropathy, triggered by dietary wheat gluten and similar proteins of barley and rye in genetically susceptible individuals. The etiology of this disorder is complex, involving both environmental and genetic factors. The major genetic risk factor for CD is represented by HLA-DQ genes, which account for approximately 40% of the genetic risk; however, only a small percentage of carriers develop the disease. Gluten is the main environmental factor responsible for the signs and symptoms of the disease, but exposure to gluten does not fully explain the manifestation of CD. Epidemiological and clinical data suggest that environmental factors other than gluten might play a role in disease development, including early feeding practices (e.g., breast milk versus formula and duration of breastfeeding), infections, and alterations in the intestinal microbiota composition. Herein, we review what is known about the influence of dietary factors, exposure to infectious agents, and intestinal microbiota composition, particularly in early life, on the risk of developing CD, as well as the possible dietary strategies to induce or increase gluten tolerance.

Bioelectrical signals generated by ion channels play crucial roles in many cellular processes in both excitable and nonexcitable cells. Some ion channels are directly implemented in chemical signaling pathways, the others are involved in regulation of cytoplasmic or vesicular ion concentrations, pH, cell volume, and membrane potentials. Together with ion transporters and gap junction complexes, ion channels form steady-state voltage gradients across the cell membranes in nonexcitable cells. These membrane potentials are involved in regulation of such processes as migration guidance, cell proliferation, and body axis patterning during development and regeneration. While the importance of membrane potential in stem cell maintenance, proliferation, and differentiation is evident, the mechanisms of this bioelectric control of stem cell activity are still not well understood, and the role of specific ion channels in these processes remains unclear. Here we introduce the flatworm Macrostomum lignano as a versatile model organism for addressing these topics. We discuss biological and experimental properties of M. lignano, provide an overview of the recently developed experimental tools for this animal model, and demonstrate how manipulation of membrane potential influences regeneration in M. lignano.

Neurospheres are widely used to propagate and investigate neural stem cells (NSCs) and neural progenitors (NPs). However, the exact cell types present within neurospheres are still unknown. To identify cell types, we used single-cell mRNA profiling of 48 genes in 187 neurosphere cells. Using a clustering algorithm, we identified 3 discrete cell populations within neurospheres. One cell population [cluster unsorted (US) 1] expresses high Bmi1 and Hes5 and low Myc and Klf12. Cluster US2 shows intermediate expression of most of the genes analyzed. Cluster US3 expresses low Bmi1 and Hes5 and high Myc and Klf12. The mRNA profiles of these 3 cell populations correlate with a developmental timeline of early, intermediate, and late NPs, as seen in vivo from the mouse brain. We enriched the cell population for neurosphere-forming cells (NFCs) using morphological criteria of forward scatter (FSC) and side scatter (SSC). FSC/SSC(high) cells generated 2.29-fold more neurospheres than FSC/SSC(low) cells at clonal density. FSC/SSC(high) cells were enriched for NSCs and Lewis-X(+ve) cells, possessed higher phosphacan levels, and were of a larger cell size. Clustering of both FSC/SSC(high) and FSC/SSC(low) cells identified an NFC cluster. Significantly, the mRNA profile of the NFC cluster drew close resemblance to that of early NPs. Taken together, data suggest that the neurosphere culture system can be used to model central nervous system development, and that early NPs are the cell population that gives rise to neurospheres. In future work, it may be possible to further dissect the NFCs and reveal the molecular signature for NSCs.

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expansion of CAG trinucleotide repeats encoding for polyglutamine (polyQ) in the huntingtin (Htt) gene. Despite considerable effort, the mechanisms underlying the toxicity of the mutated Htt protein remains largely uncertain. To identify novel therapeutic targets, we recently employed the approach of tandem affinity purification and discovered that calretinin (Cr), a member of the EF-hand family of calcium-binding proteins, is preferentially associated with mHtt, although it also interacts with wild-type Htt. These observations were supported by coimmunoprecipitation and by colocalization of Cr with mHtt in neuronal cultures. Over- expression of Cr reduced mHtt-caused cytotoxicity in both non-neuronal and neuronal cell models of HD, whereas knockdown of Cr expression in the cells enhanced mHtt-caused neuronal cell death. In addition, over-expression of Cr was also associated with reduction of intracellular free calcium and activation of Akt. These results suggest that Cr may be a potential therapeutic target for treatment of HD.

The phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway is constitutively activated in a substantial proportion of prostate tumors and is considered a key mechanism supporting progression toward an androgen-independent status, for which no effective therapy is available. Therefore, PI3K inhibitors, alone or in combination with other cytotoxic drugs, could potentially be used to treat cancer with a constitutive activated PI3K/Akt pathway. To selectively target advanced prostate tumors with a constitutive activated PI3K/Akt pathway, a prostate cancer-specific PI3K inhibitor was generated by coupling the chemically modified form of the quercetin analogue LY294002 (HO-CH(2)-LY294002, compound 8) with the peptide Mu-LEHSSKLQL, in which the internal sequence HSSKLQ is a substrate for the prostate-specific antigen (PSA) protease. The result is a water-soluble and latent PI3K inhibitor prodrug (compound 11), its activation being dependent on PSA cleavage. Once activated, the L-O-CH(2)-LY294002 (compound 10) can specifically inhibit PI3K in PSA-secreting prostate cancer cells and induce apoptosis with a potency comparable to that of the original LY294002 compound.

We previously demonstrated that the histone deacetylase (HDAC) inhibitor, 4b, which preferentially targets HDAC1 and HDAC3, ameliorates Huntington's disease (HD)-related phenotypes in different HD model systems. In the current study, we investigated extensive behavioral and biological effects of 4b in N171-82Q transgenic mice and further explored potential molecular mechanisms of 4b action. We found that 4b significantly prevented body weight loss, improved several parameters of motor function and ameliorated Huntingtin (Htt)-elicited cognitive decline in N171-82Q transgenic mice. Pathways analysis of microarray data from the mouse brain revealed gene networks involving post-translational modification, including protein phosphorylation and ubiquitination pathways, associated with 4b drug treatment. Using real-time qPCR analysis, we validated differential regulation of several genes in these pathways by 4b, including Ube2K, Ubqln, Ube2e3, Usp28 and Sumo2, as well as several other related genes. Additionally, 4b elicited increases in the expression of genes encoding components of the inhibitor of kappaB kinase (IKK) complex. IKK activation has been linked to phosphorylation, acetylation and clearance of the Htt protein by the proteasome and the lysosome, and accordingly, we found elevated levels of phosphorylated endogenous wild-type (wt) Htt protein at serine 16 and threonine 3, and increased AcK9/pS13/pS16 immunoreactivity in cortical samples from 4b-treated mice. We further show that HDAC inhibitors prevent the formation of nuclear Htt aggregates in the brains of N171-82Q mice. Our findings suggest that one mechanism of 4b action is associated with the modulation of the ubiquitin-proteasomal and autophagy pathways, which could affect accumulation, stability and/or clearance of important disease-related proteins, such as Htt.

<h4>Context</h4>The primary goals of genome-wide association studies (GWAS) are to discover new molecular and biological pathways involved in the regulation of bone metabolism that can be leveraged for drug development. In addition, the identified genetic determinants may be used to enhance current risk factor profiles.<h4>Evidence acquisition</h4>There have been more than 40 published GWAS on skeletal phenotypes, predominantly focused on dual-energy x-ray absorptiometry-derived bone mineral density (BMD) of the hip and spine.<h4>Evidence synthesis</h4>Sixty-six BMD loci have been replicated across all the published GWAS, confirming the highly polygenic nature of BMD variation. Only seven of the 66 previously reported genes (LRP5, SOST, ESR1, TNFRSF11B, TNFRSF11A, TNFSF11, PTH) from candidate gene association studies have been confirmed by GWAS. Among 59 novel BMD GWAS loci that have not been reported by previous candidate gene association studies, some have been shown to be involved in key biological pathways involving the skeleton, particularly Wnt signaling (AXIN1, LRP5, CTNNB1, DKK1, FOXC2, HOXC6, LRP4, MEF2C, PTHLH, RSPO3, SFRP4, TGFBR3, WLS, WNT3, WNT4, WNT5B, WNT16), bone development: ossification (CLCN7, CSF1, MEF2C, MEPE, PKDCC, PTHLH, RUNX2, SOX6, SOX9, SPP1, SP7), mesenchymal-stem-cell differentiation (FAM3C, MEF2C, RUNX2, SOX4, SOX9, SP7), osteoclast differentiation (JAG1, RUNX2), and TGF-signaling (FOXL1, SPTBN1, TGFBR3). There are still 30 BMD GWAS loci without prior molecular or biological evidence of their involvement in skeletal phenotypes. Other skeletal phenotypes that either have been or are being studied include hip geometry, bone ultrasound, quantitative computed tomography, high-resolution peripheral quantitative computed tomography, biochemical markers, and fractures such as vertebral, nonvertebral, hip, and forearm.<h4>Conclusions</h4>Although several challenges lie ahead as GWAS moves into the next generation, there are prospects of new discoveries in skeletal biology. This review integrates findings from previous GWAS and provides a roadmap for future directions building on current GWAS successes.

Munc13s are presynaptic proteins that mediate synaptic vesicle priming and thereby control the size of the readily releasable pool of vesicles. During high synaptic activity, Munc13-1 and its closely related homolog, ubMunc13-2, bind Ca(2+)/calmodulin, resulting in enhanced priming activity and in changes of short-term synaptic plasticity characteristics. Here, we studied whether bMunc13-2 and Munc13-3, two remote isoforms of Munc13-1 with a neuronal subtype-specific expression pattern, mediate synaptic vesicle priming and regulate short-term synaptic plasticity in a Ca(2+)/calmodulin-dependent manner. We identified a single functional Ca(2+)/calmodulin binding site in these isoforms and provide structural evidence that all Munc13s employ a common mode of interaction with calmodulin despite the lack of sequence homology between their Ca(2+)/calmodulin binding sites. Electrophysiological analysis showed that, during high-frequency activity, Ca(2+)/calmodulin binding positively regulates the priming activity of bMunc13-2 and Munc13-3, resulting in an increase in the size of the readily releasable pool of vesicles and subsequently in strong short-term synaptic enhancement of neurotransmission. We conclude that Ca(2+)/calmodulin-dependent regulation of priming activity is structurally and functionally conserved in all Munc13 proteins, and that the composition of Munc13 isoforms in a neuron differentially controls its short-term synaptic plasticity characteristics.

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.

Huntingtin (htt) is a scaffold protein localized at the subcellular level and is involved in coordinating the activity of several protein for signaling and intracellular transport. The emerging properties of htt in intracellular trafficking prompted us to study the role of mutant htt (polyQ-htt) in the intracellular fate of epidermal growth factor receptor (EGFR), whose activity seems to be strictly regulated by htt. In particular, to evaluate whether protein trafficking dysfunction occurs in non-neuronal cells in the absence of functional htt, we monitored the EGFR protein in fibroblasts from homozygotic HD patients and their healthy counterpart. We found that polyQ-htt controls EGFR degradation and recycling. Lack of wild-type htt caused alteration of the ubiquitination cycle, formation of EGFR-incorporating high-molecular weight protein aggregates and abnormal EGFR distribution in endosomes of the degradation and recycling pathways after EGF stimulation. PolyQ-htt-induced alteration of EGFR trafficking affected cell migration and proliferation, at least in part, through inhibition of ERK signaling. To our knowledge the data here reported represent the first signaling and phenotypic characterization of polyQ-htt involvement in the modulation of growth factor stimulation in non-neuronal cells.

Huntington disease (HD) is a neurodegenerative disease caused by expansion of CAG repeats in the huntingtin (Htt) gene. The expression of hMTH1, the human hydrolase that degrades oxidized purine nucleoside triphosphates, grants protection in a chemical HD mouse model in which HD-like features are induced by the mitochondrial toxin 3-nitropropionic acid (3-NP). To further examine the relationship between oxidized dNTPs and HD-like neurodegeneration, we studied the effects of hMTH1 expression in a genetic cellular model for HD, such as striatal cells expressing mutant htt (Hdh(Q111)). hMTH1 expression protected these cells from 3-NP and H2O2-induced killing, by counteracting the mutant htt-dependent increased vulnerability and accumulation of nuclear and mitochondrial DNA 8-hydroxyguanine levels. hMTH1 expression reverted the decreased mitochondrial membrane potential characteristic of Hdh(Q111) cells and delayed the increase in mitochondrial reactive oxygen species associated with 3-NP treatment. Further indications of hMTH1-mediated mitochondrial protection are the partial reversion of 3-NP-induced alterations in mitochondrial morphology and the modulation of DRP1 and MFN1 proteins, which control fusion/fission rates of mitochondria. Finally, in line with the in vitro findings, upon 3-NP in vivo treatment, 8-hydroxyguanine levels in mitochondrial DNA from heart, muscle and brain are significantly lower in transgenic hMTH1-expressing mice than in wild-type animals.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, α,ω-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of α-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The α,ω-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-∈-amino caproic acid, by DCC coupling, resulting in the formation of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, (1)H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores.

Background. Iron deficiency anemia (IDA) is a common hematological complication with potentially serious clinical consequences that may require intravenous iron therapy. Ferric carboxymaltose (FCM) is a stable, nondextran iron formulation administered intravenously in large single doses to treat IDA. Objective. Two open-label, randomized, placebo-controlled trials evaluated safety of multiple or single 750 mg FCM doses compared to standard medical care (SMC) in IDA patients. Secondary endpoints were improvements in hemoglobin and iron indices. Design and Patients. Adults with hemoglobin ≤12 g/dL, ferritin ≤100 or ≤300 ng/mL with transferrin saturation ≤30% were randomized to receive single (n = 366) or weekly (n = 343) FCM or SMC (n = 360 and n = 366). Results. Significantly greater (P ≤ 0.001) increases in hemoglobin and iron indices occurred in FCM groups versus SMC. In the multidose study, up to two infusions of FCM were needed to reach target iron levels versus 3-5 of intravenous iron comparators. FCM and SMC groups had similar incidences and types of adverse events and serious adverse events. Transient hypophosphatemia not associated with adverse events or clinical sequelae occurred in the FCM groups. Conclusion. Intravenous FCM is safe, well tolerated, and associated with improvements in hemoglobin and iron indices comparable to SMC when administered in single doses of up to 750 mg at a rate of 100 mg/min. Fewer FCM infusions were required to reach target iron levels compared to other intravenous iron preparations.

We address leadership emergence and the possibility that there is a partially innate predisposition to occupy a leadership role. Employing twin design methods on data from the National Longitudinal Study of Adolescent Health, we estimate the heritability of leadership role occupancy at 24%. Twin studies do not point to specific genes or neurological processes that might be involved. We therefore also conduct association analysis on the available genetic markers. The results show that leadership role occupancy is associated with rs4950, a single nucleotide polymorphism (SNP) residing on a neuronal acetylcholine receptor gene (<i>CHRNB3</i>). We replicate this family-based genetic association result on an independent sample in the Framingham Heart Study. This is the first study to identify a specific genotype associated with the tendency to occupy a leadership position. The results suggest that what determines whether an individual occupies a leadership position is the complex product of genetic and environmental influences; with a particular role for rs4950.

We examined mouse models with altered adipocyte expression of mitoNEET, a protein residing in the mitochondrial outer membrane, to probe its impact on mitochondrial function and subsequent cellular responses. We found that overexpression of mitoNEET enhances lipid uptake and storage, leading to an expansion of the mass of adipose tissue. Despite the resulting massive obesity, benign aspects of adipose tissue expansion prevail, and insulin sensitivity is preserved. Mechanistically, we also found that mitoNEET inhibits mitochondrial iron transport into the matrix and, because iron is a rate-limiting component for electron transport, lowers the rate of β-oxidation. This effect is associated with a lower mitochondrial membrane potential and lower levels of reactive oxygen species-induced damage, along with increased production of adiponectin. Conversely, a reduction in mitoNEET expression enhances mitochondrial respiratory capacity through enhanced iron content in the matrix, ultimately corresponding to less weight gain on a high-fat diet. However, this reduction in mitoNEET expression also causes heightened oxidative stress and glucose intolerance. Thus, manipulation of mitochondrial function by varying mitoNEET expression markedly affects the dynamics of cellular and whole-body lipid homeostasis.

The development of personalised therapy and mechanism-targeted agents in oncology mandates the identification of the patient populations most likely to benefit from therapy. This paper discusses the increasing evidence as to the heterogeneity of the group of diseases called colorectal cancer. Differences in the aetiology and epidemiology of proximal and distal cancers are reflected in different clinical behaviour, histopathology, and molecular characteristics of these tumours. This may impact response both to standard cytotoxic therapies and mechanism-targeted agents. This disease heterogeneity leads to challenges in the design of clinical trials to assess novel therapies in the treatment of "colorectal cancer."

<h4>Unlabelled</h4><h4>Background</h4>Alzheimer's disease (AD) is a neurologically degenerative disorder that affects more than 20 million people worldwide. The selective butyrylcholinesterase (BChE) inhibitors and bivalent cholinesterase (ChE) inhibitors represent new treatments for AD.<h4>Findings</h4>A series of lycorine derivatives (1-10) were synthesized and evaluated for anti-cholinesterase activity. Result showed that the novel compound 2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7) was a dual inhibitor of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) with IC50 values of 11.40 ± 0.66 μM and 4.17 ± 0.29 μM, respectively. The structure-activity relationships indicated that (i) the 1-O-(methylthio)methyl substituent in lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii) the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChE than hAChE; and (iii) the oxidation of lycorine at C-2 decreases the activity.<h4>Conclusion</h4>Acylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE. Hence, further study on the modification of lycorine for ChE inhibition is necessary.

Cannabidiol (CBD), a major non-psychotropic constituent of fiber-type cannabis plant, has been reported to possess diverse biological activities, including anti-proliferative effect on cancer cells. Although CBD is obtained from non-enzymatic decarboxylation of its parent molecule, cannabidiolic acid (CBDA), few studies have investigated whether CBDA itself is biologically active. Results of the current investigation revealed that CBDA inhibits migration of the highly invasive MDA-MB-231 human breast cancer cells, apparently through a mechanism involving inhibition of cAMP-dependent protein kinase A, coupled with an activation of the small GTPase, RhoA. It is established that activation of the RhoA signaling pathway leads to inhibition of the mobility of various cancer cells, including MDA-MB-231 cells. The data presented in this report suggest for the first time that as an active component in the cannabis plant, CBDA offers potential therapeutic modality in the abrogation of cancer cell migration, including aggressive breast cancers.

Polyglutamine (polyQ) disorders share many similarities, such as a common mutation type in unrelated human causative genes, neurological character, and certain aspects of pathogenesis, including morphological and physiological neuronal alterations. The similarities in pathogenesis have been confirmed by findings that some experimental in vivo therapy approaches are effective in multiple models of polyQ disorders. Additionally, mouse models of polyQ diseases are often highly similar between diseases with respect to behavior and the features of the disease. The common features shared by polyQ mouse models may facilitate the investigation of polyQ disorders and may help researchers explore the mechanisms of these diseases in a broader context. To provide this context and to promote the understanding of polyQ disorders, we have collected and analyzed research data about the characterization and treatment of mouse models of polyQ diseases and organized them into two complementary Excel data tables. The data table that is presented in this review (Part I) covers the behavioral, molecular, cellular, and anatomic characteristics of polyQ mice and contains the most current knowledge about polyQ mouse models. The structure of this data table is designed in such a way that it can be filtered to allow for the immediate retrieval of the data corresponding to a single mouse model or to compare the shared and unique aspects of many polyQ models. The second data table, which is presented in another publication (Part II), covers therapeutic research in mouse models by summarizing all of the therapeutic strategies employed in the treatment of polyQ disorders, phenotypes that are used to examine the effects of the therapy, and therapeutic outcomes.

<h4>Objective</h4>We aimed to audit the regional management of central nervous system (CNS) infection in children.<h4>Methods</h4>The study was undertaken in five district general hospitals and one tertiary paediatric hospital in the Mersey region of the UK. Children admitted to hospital with a suspected CNS infection over a three month period were identified. Children were aged between 4 weeks and 16 years old. Details were recorded from the case notes and electronic records. We measured the appropriateness of management pathways as outlined by national and local guidelines.<h4>Results</h4>Sixty-five children were identified with a median age of 6 months (range 1 month to 15 years). Ten had a CNS infection: 4 aseptic meningitis, 3 purulent meningitis, 3 encephalitis [2 with herpes simplex virus (HSV) type 1]. A lumbar puncture (LP) was attempted in 50 (77%) cases but only 43 had cerebrospinal fluid (CSF) available for analysis. Of these 24 (57%) had a complete standard set of tests performed. Fifty eight (89%) received a third generation cephalosporin. Seventeen (26%) also received aciclovir with no obvious indication in 9 (53%). Only 11 (65%) of those receiving aciclovir had CSF herpes virus PCR. Seventeen had cranial imaging and it was the first management step in 14. Treatment lengths of both antibiotics and aciclovir were highly variable: one child with HSV encephalitis was only treated with aciclovir for 7 days.<h4>Conclusions</h4>The clinical management of children with suspected CNS infections across the Mersey region is heterogeneous and often sub-optimal, particularly for the investigation and treatment of viral encephalitis. National guidelines for the management of viral encephalitis are needed.

While hundreds of loci have been identified as reflecting strong-positive selection in human populations, connections between candidate loci and specific selective pressures often remain obscure. This study investigates broader patterns of selection in African populations, which are underrepresented despite their potential to offer key insights into human adaptation. We scan for hard selective sweeps using several haplotype and allele-frequency statistics with a data set of nearly 500,000 genome-wide single-nucleotide polymorphisms in 12 highly diverged African populations that span a range of environments and subsistence strategies. We find that positive selection does not appear to be a strong determinant of allele-frequency differentiation among these African populations. Haplotype statistics do identify putatively selected regions that are shared across African populations. However, as assessed by extensive simulations, patterns of haplotype sharing between African populations follow neutral expectations and suggest that tails of the empirical distributions contain false-positive signals. After highlighting several genomic regions where positive selection can be inferred with higher confidence, we use a novel method to identify biological functions enriched among populations' empirical tail genomic windows, such as immune response in agricultural groups. In general, however, it seems that current methods for selection scans are poorly suited to populations that, like the African populations in this study, are affected by ascertainment bias and have low levels of linkage disequilibrium, possibly old selective sweeps, and potentially reduced phasing accuracy. Additionally, population history can confound the interpretation of selection statistics, suggesting that greater care is needed in attributing broad genetic patterns to human adaptation.

Waldenström's macroglobulinemia (WM) is a clonal B-cell lymphoproliferative disorder (LPD) of post-germinal center nature. Despite the fact that the precise molecular pathway(s) leading to WM remain(s) to be elucidated, a hallmark of the disease is the absence of the immunoglobulin heavy chain class switch recombination. Using two-dimensional gel electrophoresis, we compared proteomic profiles of WM cells with that of other LPDs. We were able to demonstrate that WM constitutes a unique proteomic entity as compared with chronic lymphocytic leukemia and marginal zone lymphoma. Statistical comparisons of protein expression levels revealed that a few proteins are distinctly expressed in WM in comparison with other LPDs. In particular we observed a major downregulation of the double strand repair protein Ku70 (XRCC6); confirmed at both the protein and RNA levels in an independent cohort of patients. Hence, we define a distinctive proteomic profile for WM where the downregulation of Ku70-a component of the non homologous end-joining pathway-might be relevant in disease pathophysiology.

Certain oncolytic viruses exploit activated Ras signaling in order to replicate in cancer cells. Constitutive activation of the Ras/MEK pathway is known to suppress the effectiveness of the interferon (IFN) antiviral response, which may contribute to Ras-dependent viral oncolysis. Here, we identified 10 human cancer cell lines (out of 16) with increased sensitivity to the anti-viral effects of IFN-α after treatment with the MEK inhibitor U0126, suggesting that the Ras/MEK pathway underlies their reduced sensitivity to IFN. To determine how Ras/MEK suppresses the IFN response in these cells, we used DNA microarrays to compare IFN-induced transcription in IFN-sensitive SKOV3 cells, moderately resistant HT1080 cells, and HT1080 cells treated with U0126. We found that 267 genes were induced by IFN in SKOV3 cells, while only 98 genes were induced in HT1080 cells at the same time point. Furthermore, the expression of a distinct subset of IFN inducible genes, that included RIGI, GBP2, IFIT2, BTN3A3, MAP2, MMP7 and STAT2, was restored or increased in HT1080 cells when the cells were co-treated with U0126 and IFN. Bioinformatic analysis of the biological processes represented by these genes revealed increased representation of genes involved in the anti-viral response, regulation of apoptosis, cell differentiation and metabolism. Furthermore, introduction of constitutively active Ras into IFN sensitive SKOV3 cells reduced their IFN sensitivity and ability to activate IFN-induced transcription. This work demonstrates for the first time that activated Ras/MEK in human cancer cells induces downregulation of a specific subset of IFN-inducible genes.

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit.

Gastric cancer is a malignant disease that arises from the gastric epithelium. A potential biomarker for gastric cancer is the protein annexin A4 (ANXA4), an intracellular Ca(2+) sensor. ANXA4 is primarily found in epithelial cells, and is known to be involved in various biological processes, including apoptosis, cell cycling and anticoagulation. In respect to cancer, ANXA4-overexpression has been observed in cancers of various origins, including gastric tumors associated with Helicobacter pylori infection. H. pylori induces ANXA4 expression and intracellular [Ca(2+)](i) elevation, and is an important risk factor for carcinogenesis that results in gastric cancer. Despite this correlation, the role of ANXA4 in the progression of gastric tumors remains unclear. In this study, we have investigated whether ANXA4 can mediate the rate of cell growth and whether ANXA4 downstream signals are involved in tumorigenesis. After observing the rate of cell growth in real-time, we determined that ANXA4 promotes cell proliferation. The transcription gene profile of ANXA4-overexpressing cells was measured and analyzed by human exon arrays. From this transcriptional gene data, we show that overexpression of ANXA4 regulates genes that are known to be related to cancer, for example the activation of hyaluronan mediated motility receptor (RHAMM), AKT, and cyclin-dependent kinase 1 (CDK1) as well as the suppression of p21. The regulation of these genes further induces cancer cell proliferation. We also found Ca(2+) could regulate the transmission of downstream signals by ANXA4. We suggest that ANXA4 triggers a signaling cascade, leading to increased epithelial cell proliferation, ultimately promoting carcinogenesis. These results might therefore provide a new insight for gastric cancer therapy, specifically through the modification of ANXA4 activity.

The central serotonergic system has been implicated in the pathophysiology of panic disorder (PD) by evidence of abnormally elevated serotonin-turnover, reduced pre- and post-synaptic 5-HT(1A)-receptor sensitivity and binding and clinical improvement during administration of agents that enhance serotonergic transmission. Polymorphisms in genes that putatively influence serotonergic neurotransmission increase the vulnerability for developing PD specifically in males. We tested the hypotheses that serotonin transporter (5-HTT) binding is elevated in PD subjects vs. healthy controls in regions where in vivo evidence exists for both elevated 5-HTT and 5-HT(1A) receptor levels in PD and investigated whether the extent of this difference depends upon gender. Volunteers were out-patients with current PD (n=24) and healthy controls (n=24). The non-displaceable component of 5-HTT binding-potential (BP(ND)) was measured using positron emission tomography and the 5-HTT selective radioligand, [(11)C]DASB. PD severity was assessed using the PD Severity Scale. The 5-HTT-BP(ND) was increased in males with PD relative to male controls in the anterior cingulate cortex (F=8.96, p(FDR)=0.01) and midbrain (F=5.09, p(FDR)=0.03). In contrast, BP(ND) did not differ between females with PD and female controls in any region examined. The finding that 5-HTT-binding is elevated in males but not in females with PD converges with other evidence suggesting that dysfunction within the central serotonergic system exists in PD, and also indicates that such abnormalities are influenced by gender. These findings conceivably may reflect a sexual dimorphism that underlies the greater efficacy of serotonin reuptake inhibitor treatment in females vs. males with PD.

Afferent output in type II taste cells is mediated by ATP liberated through ion channels. It is widely accepted that pannexin 1 (Panx1) channels are responsible for ATP release in diverse cell types, including taste cells. While biophysical evidence implicates slow deactivation of ion channels following ATP release in taste cells, recombinant Panx1 activates and deactivates rapidly. This inconsistency could indicate that the cellular context specifies Panx1 functioning. We cloned Panx1 from murine taste tissue, and heterologously expressed it in three different cell lines: HEK-293, CHO and neuroblastoma SK-N-SH cells. In all three cell lines, Panx1 transfection yielded outwardly rectifying anion channels that exhibited fast gating and negligible permeability to anions exceeding 250 Da. Despite expression of Panx1, the host cells did not liberate ATP upon stimulation, making it unclear whether Panx1 is involved in taste-related ATP secretion. This issue was addressed using mice with genetic ablation of the Panx1 gene. The ATP-biosensor assay revealed that, in taste cells devoid of Panx1, ATP secretion was robust and apparently unchanged compared with the control. Our data suggest that Panx1 alone forms a channel that has insufficient permeability to ATP. Perhaps, a distinct subunit and/or a regulatory circuit that is absent in taste cells is required to enable a high ATP-permeability mode of a native Panx1-based channel.

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

We previously reported that nanoceria can slow retinal degeneration in the tubby mouse for two weeks by multiple systemic injections. However, the long-term protection of retinal structure and function by directly deliver of nanoceria to the eye had not been explored. In this study, 172 ng of nanoceria in 1 μl saline (1 mm) were intravitreally injected into tubby P7 pups and assays were performed at P28, P49, P80 and P120. The expression of antioxidant associated genes and photoreceptor-specific genes was significantly up regulated, the mislocalization of rod and cone opsins was decreased, and retinal structure and function were protected. These findings demonstrate that nanoceria can function as catalytic antioxidants in vivo and may be broad spectrum therapeutic agents for multiple types of ocular diseases.

<h4>Background & aims</h4>Hepatitis B virus (HBV) modulates the immune system to escape clearance. Plasmacytoid dendritic cells (pDCs) initiate antiviral immunity and might determine outcomes of HBV infections. Functional defects in pDCs and natural killer (NK) cells have been reported in patients with chronic HBV infection. However, the mechanisms of these immune dysfunctions and the interactions between pDCs and NK cells have not been determined. We investigated features of pDCs from patients with chronic HBV infection and their interactions with NK cells.<h4>Methods</h4>We used flow cytometry and cytokine assays to analyze pDCs from patients with chronic HBV infection (118 aviremic and 67 viremic) and compared them with pDCs from uninfected individuals (controls). We performed coculture assays to analyze the ability of pDCs to activate heterologous NK cells.<h4>Results</h4>Circulating and hepatic pDCs from patients with chronic HBV infection had higher levels of activation than pDCs from controls and defective responses to stimulation with Toll-like receptor 9 ligand (TLR9-L), regardless of the patient's viral load. TLR9-L-activated pDCs from viremic patients with HBV did not induce cytolytic activity of NK cells. This altered function of pDCs was associated with reduced expression of OX40L and could be reproduced by incubating control pDCs with plasma from viremic patients with HBV. A high level of interferon-induced protein 10 (IP-10 or CXCL10) and hepatitis B surface and e antigens might induce these defective pDC functions.<h4>Conclusions</h4>HBV escapes antiviral immunity by altering pDC functions, to disrupt interactions between pDC and NK cells. This could reduce immune control of HBV and lead to chronic infection.

<h4>Objective</h4>Adipocyte insulin resistance (IR) is a key feature early in the pathogenesis of type 2 diabetes mellitus (T2DM), and although scarce, data in the literature suggest a direct role for iron and iron metabolism-related factors in adipose tissue function and metabolism. Serum ferritin and transferrin were shown to be associated with muscle insulin resistance (IR) and T2DM, but little is known about the role of iron metabolism on adipose tissue. We therefore investigated whether markers of iron metabolism were associated with adipocyte IR and plasma adiponectin.<h4>Research design and methods</h4>Serum ferritin, transferrin, total iron, non-transferrin-bound iron (NTBI), transferrin saturation, and plasma adiponectin were determined in 492 individuals. Adipocyte IR was defined by the product of fasting insulin and nonesterified fatty acids (NEFAs). Using linear regression analyses, we investigated the difference in adipocyte IR or adiponectin (in %) according to differences in iron metabolism markers.<h4>Results</h4>Serum ferritin (β = 1.00% increase in adipocyte IR per 10 μg/L [95% CI 0.66-1.34]), transferrin (4.18% per 0.1 g/L [2.88-5.50]), total iron (1.36% per μmol/L [0.61-2.12]), and NTBI (5.14% per μmol/L [1.88-8.52]) were associated with adipocyte IR after adjustment for several covariates, including inflammatory markers. All markers of iron metabolism were also associated with NEFAs (all P < 0.01). In addition, ferritin and transferrin were inversely associated with adiponectin (both P < 0.01).<h4>Conclusions</h4>The observed associations of several markers of iron metabolism with adipocyte IR and adiponectin suggest that factors related to iron and iron metabolism may contribute to adipocyte IR early in the pathogenesis of T2DM.

Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

<h4>Objective</h4>The purpose of this study was to investigate the association between the T102C polymorphism in the serotonin 2A receptor gene and attention-deficit/hyperactivity disorder (ADHD) in Korean patients.<h4>Methods</h4>A total of 189 Korean children with ADHD as well as both parents of the ADHD children and 150 normal children participated in this study. DNA was extracted from blood samples from all of the subjects, and genotyping was conducted. Based on the allele and genotype information obtained, case-control analyses were performed to compare the ADHD and normal children, and Transmission disequilibrium tests (TDTs) were used for family-based association testing (number of trios=113). Finally, according to the significant finding which was showed in the case-control analyses, the results of behavioral characterastics and neuropsychological test were compared between ADHD children with and without the C allele.<h4>Results</h4>In the case-control analyses, statistically significant differences were detected in the frequencies of genotypes containing the C allele (χ(2)=4.73, p=0.030). In the family-based association study, TDTs failed to detect linkage disequilibrium of the T102C polymorphism associated with ADHD children. In the ADHD children, both the mean reaction time and the standard deviation of the reaction time in the auditory continuous performance test were longer in the group with the C allele compared to the group without the C allele.<h4>Conclusion</h4>The results of this study suggest that there is a significant genetic association between the T102C polymorphism in the serotonin 2A receptor gene and ADHD in Korean children.

<h4>Background</h4>Remipedia, a group of homonomously segmented, cave-dwelling, eyeless arthropods have been regarded as basal crustaceans in most early morphological and taxonomic studies. However, molecular sequence information together with the discovery of a highly differentiated brain led to a reconsideration of their phylogenetic position. Various conflicting hypotheses have been proposed including the claim for a basal position of Remipedia up to a close relationship with Malacostraca or Hexapoda. To provide new morphological characters that may allow phylogenetic insights, we have analyzed the architecture of the remipede brain in more detail using immunocytochemistry (serotonin, acetylated α-tubulin, synapsin) combined with confocal laser-scanning microscopy and image reconstruction techniques. This approach allows for a comprehensive neuroanatomical comparison with other crustacean and hexapod taxa.<h4>Results</h4>The dominant structures of the brain are the deutocerebral olfactory neuropils, which are linked by the olfactory globular tracts to the protocerebral hemiellipsoid bodies. The olfactory globular tracts form a characteristic chiasm in the center of the brain. In Speleonectes tulumensis, each brain hemisphere contains about 120 serotonin immunoreactive neurons, which are distributed in distinct cell groups supplying fine, profusely branching neurites to 16 neuropilar domains. The olfactory neuropil comprises more than 300 spherical olfactory glomeruli arranged in sublobes. Eight serotonin immunoreactive neurons homogeneously innervate the olfactory glomeruli. In the protocerebrum, serotonin immunoreactivity revealed several structures, which, based on their position and connectivity resemble a central complex comprising a central body, a protocerebral bridge, W-, X-, Y-, Z-tracts, and lateral accessory lobes.<h4>Conclusions</h4>The brain of Remipedia shows several plesiomorphic features shared with other Mandibulata, such as deutocerebral olfactory neuropils with a glomerular organization, innervations by serotonin immunoreactive interneurons, and connections to protocerebral neuropils. Also, we provided tentative evidence for W-, X-, Y-, Z-tracts in the remipedian central complex like in the brain of Malacostraca, and Hexapoda. Furthermore, Remipedia display several synapomorphies with Malacostraca supporting a sister group relationship between both taxa. These homologies include a chiasm of the olfactory globular tract, which connects the olfactory neuropils with the lateral protocerebrum and the presence of hemiellipsoid bodies. Even though a growing number of molecular investigations unites Remipedia and Cephalocarida, our neuroanatomical comparison does not provide support for such a sister group relationship.

Prostate cancer (PrCa) is the most common male cancer in developed countries and the second most common cause of cancer death after lung cancer. We recently reported a genome-wide linkage scan in 69 Finnish hereditary PrCa (HPC) families, which replicated the HPC9 locus on 17q21-q22 and identified a locus on 2q37. The aim of this study was to identify and to detect other loci linked to HPC. Here we used ordered subset analysis (OSA), conditioned on nonparametric linkage to these loci to detect other loci linked to HPC in subsets of families, but not the overall sample. We analyzed the families based on their evidence for linkage to chromosome 2, chromosome 17 and a maximum score using the strongest evidence of linkage from either of the two loci. Significant linkage to a 5-cM linkage interval with a peak OSA nonparametric allele-sharing LOD score of 4.876 on Xq26.3-q27 (ΔLOD=3.193, empirical P=0.009) was observed in a subset of 41 families weakly linked to 2q37, overlapping the HPCX1 locus. Two peaks that were novel to the analysis combining linkage evidence from both primary loci were identified; 18q12.1-q12.2 (OSA LOD=2.541, ΔLOD=1.651, P=0.03) and 22q11.1-q11.21 (OSA LOD=2.395, ΔLOD=2.36, P=0.006), which is close to HPC6. Using OSA allows us to find additional loci linked to HPC in subsets of families, and underlines the complex genetic heterogeneity of HPC even in highly aggregated families.

<h4>Introduction</h4>Primary lymphoma of the liver is an extremely rare finding, with the few such cases reported in the literature to date describing indeterminate imaging findings, being focused more on computed tomography. To the best of our knowledge, there is no prior report describing magnetic resonance imaging scan findings with such a lesion. In the case reported here, magnetic resonance imaging gave us the opportunity to ascertain the correct diagnosis, confirmed by histopathology, thus avoiding unnecessary surgery or other treatments. Although this condition is rare, knowledge of magnetic resonance imaging findings will be invaluable for radiologists and other medical subspecialties that may face such cases in the future in helping to provide adequate management for affected patients.<h4>Case presentation</h4>A focal lesion was incidentally detected by ultrasound in a 75-year-old asymptomatic Albanian man being treated for benign hypertrophy of prostate. Chest and abdomen computed tomography scans did not reveal any abnormal findings besides a solid focal lesion on the right lobe of the liver and a mild homogenous enlargement of the prostate gland. Subsequently, magnetic resonance imaging of the upper abdomen was performed for better characterization of this lesion. Our patient was free of symptoms and his laboratory test results were normal.<h4>Conclusions</h4>The magnetic resonance imaging scan results showed some distinctive features that helped us to make the correct diagnosis, and were thus very important in helping us provide the correct treatment for our patient.

DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs.

Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

Input from various signaling pathways in conjunction with specific transcription factors (TFs), noncoding RNAs, and epigenetic modifiers governs the maintenance of cellular identity. Endogenous or exogenous TFs operate within certain boundaries, which are set, in part, by the cell type-specific epigenetic landscape. Ectopic expression of selected TFs can override the cellular identity and induce reprogramming to alternative fates. In this minireview, we summarize many of the classic examples and a large number of recent studies that have taken advantage of TF-mediated reprogramming to produce cell types of biomedical relevance.

TNFR superfamily (TNFRSF)4 (OX40, CD134) and TNFRSF25 are costimulatory receptors that influence CD4(+) and CD8(+) T cell responses to cognate Ag. Independently, these receptors have been described to stimulate overlapping functions, including enhanced proliferation and activation for both regulatory T cells (CD4(+)Foxp3(+); Tregs) and conventional T cells (CD4(+)Foxp3(-) or CD8(+)Foxp3(-); Tconvs). To determine the relative functionality of TNFRSF4 and TNFRSF25 in T cell immunity, the activity of TNFRSF4 and TNFRS25 agonistic Abs was compared in the context of both traditional protein/adjuvant (OVA/aluminum hydroxide) and CD8(+)-specific heat shock protein-based (gp96-Ig) vaccine approaches. These studies demonstrate that both TNFRSF4 and TNFRSF25 independently and additively costimulate vaccine-induced CD8(+) T cell proliferation following both primary and secondary Ag challenge. In contrast, the activities of TNFRSF4 and TNFRSF25 were observed to be divergent in the costimulation of CD4(+) T cell immunity. TNFRSF4 agonists were potent costimulators of OVA/aluminum hydroxide-induced CD4(+) Tconv proliferation, but they only weakly costimulated Treg proliferation and IgG2a production, whereas TNFRSF25 agonists were strong costimulators of Treg proliferation, producers of IgG1, IgG2a, and IgG2b, and weak costimulators of CD4(+) Tconv proliferation. Interestingly, Ag-specific cellular and humoral responses were uncoupled upon secondary immunization, which was dramatically affected by the presence of TNFRSF4 or TNFRSF25 costimulation. These studies highlight the overlapping but nonredundant activities of TNFRSF4 and TNFRSF25 in T cell immunity, which may guide the application of receptor agonistic agents as vaccine adjuvants for infectious disease and tumor immunity.