Gene Literature Dashboard

<h4>Background</h4>The mitogen-activated protein kinase (MAPK) signaling pathway participates in several steps of tumour development and is considered a prominent therapeutic target for the design of chemotherapeutic agents. We evaluated the expressions of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MEK), an upstream regulator of ERK, and Raf kinase inhibitor protein (RKIP), and investigated correlations of these expressions with clinicopathological features and outcomes in gastric cancer.<h4>Methods</h4>Tumour samples were obtained from 105 patients with gastric adenocarcinomas who underwent radical gastrectomy. The expressions of phosphorylated ERK (p-ERK), phosphorylated MEK (p-MEK), and RKIP were analysed by immunohistochemical staining.<h4>Results</h4>Expression of RKIP, p-MEK, and p-ERK was found in 69 (66%), 54 (51%), and 64 (61%) of all tumours, respectively. RKIP expression negatively correlated with the depth of invasion (p < 0.001), lymph node involvement (p = 0.028), and Union for International Cancer Control (UICC) stage (p = 0.007). RKIP expression was associated with significantly longer relapse-free survival (RFS) (p = 0.0033), whereas p-MEK was not (p = 0.79). Patients with p-ERK expression had slightly, but not significantly shorter RFS than those without such expression (p = 0.054). Patients with positive p-ERK and negative RKIP expression had significantly shorter RFS than the other patients (p < 0.001). The combination of RKIP and p-ERK expression was an independent prognostic factor (hazard ratio, 2.4; 95% confidence interval, 1.3 - 4.6; p = 0.008).<h4>Conclusions</h4>Our results demonstrated that loss of RKIP was associated with tumour progression and poor survival. Negative RKIP expression combined with positive p-ERK expression was an independent predictor of poor outcomes in patients with gastric cancer.

<h4>Background</h4>Although several studies have shown that serum antithrombin III (ATIII) has anti-inflammatory effects, the prognostic value of ATIII in HCC is unknown. We investigated the influence of preoperative ATIII levels on the outcome of patients who underwent hepatectomy for hepatocellular carcinoma (HCC).<h4>Methods</h4>Data from 440 patients (314 patients with ATIII ≥ 70 % and 126 patients with ATIII <70 %) who underwent curative hepatectomy for HCC were retrospectively collected and analyzed. To overcome bias due to the different distribution of covariates for the 2 groups, propensity score matching was performed on the patients, and outcomes were compared.<h4>Results</h4>The propensity score analysis revealed that 65 patients with ATIII of ≥ 70 % (group 1) and 65 patients with ATIII of <70 % (group 2) had the same preoperative and operative characteristics (excluding the ATIII level). The overall survival rate and the disease-free survival rate was significantly higher in group 1 than in group 2 (P = 0.005 and 0.011, respectively). Multivariate analysis showed that ATIII was a significant favorable factor for overall survival and disease-free survival of patients with HCC after curative hepatectomy.<h4>Conclusions</h4>The prognosis of patients with HCC was found to be associated with preoperative antithrombin III levels. ATIII may be useful for predicting outcomes of patients with HCC after curative hepatectomy.

Pseudoxanthoma elasticum in humans and dystrophic cardiac calcification in mice are heritable disorders characterized by dystrophic calcification of soft connective tissues related to the defective function of the ABCC6 (human)/Abcc6 (mouse) transporter. Of particular interest is the finding of calcified vibrissae in Abcc6(-/-) mice, which facilitates the study of dystrophic calcification by histological techniques. We aimed to determine whether mice prone to dystrophic cardiac calcification (C3H/HeOuJ and DBA/2J strains) presented similar vibrissae changes and to evaluate the value of microcomputed tomography to quantify the extent of mystacial vibrissae calcifications. These calcifications were absent in DBA/2J and C57BL/6J control mice. In both Abcc6(-/-) and C3H/HeOuJ mice, calcifications progressed in a caudal-rostral direction with aging. However, the calcification process was delayed in C3H/HeOuJ mice, indicating an incomplete expression of the calcification phenotype. We also found that the calcification process in the cephalic region was not limited to mystacial vibrissae but was also present in other periorbital sensorial vibrissae. The vibrissae calcification was circular and encompassed the medial region of the vibrissae capsule, adjacent to the ring and cavernous sinuses (the areas adjacent to blood and lymphatic vessels). Collectively, our findings confirm that Abcc6 acts as an inhibitor of spontaneous chronic mineralization and that microcomputed tomography is a valuable noninvasive tool for the assessment of the calcification phenotype in Abcc6-deficient mice.

Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40 (SV-40). Subsequent investigations have revealed that it is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. Since then, NGAL expression has been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. Its expression is also dysregulated in several benign and malignant diseases. Its small size, secreted nature and relative stability have led to it being investigated as a diagnostic and prognostic biomarker in numerous diseases including inflammation and cancer. Functional studies, conducted primarily on lipocalin 2 (Lcn2), the mouse homologue of human NGAL have revealed that Lcn2 has a strong affinity for iron complexed to both bacterial siderophores (iron-binding proteins) and certain human proteins like norepinephrine. By sequestering iron-laden siderophores, Lcn2 deprives bacteria of a vital nutrient and thus inhibits their growth (bacteriostatic effect). In malignant cells, its proposed functions range from inhibiting apoptosis (in thyroid cancer cells), invasion and angiogenesis (in pancreatic cancer) to increasing proliferation and metastasis (in breast and colon cancer). Ectopic expression of Lcn2 also promotes BCR-ABL induced chronic myelogenous leukemia in murine models. By transporting iron into and out of the cell, NGAL also regulates iron responsive genes. Further, it stabilizes the proteolytic enzyme matrix metalloprotease-9 (MMP-9) by forming a complex with it, and thereby prevents its autodegradation. The factors regulating NGAL expression are numerous and range from pro-inflammatory cytokines like interleukins, tumor necrosis factor-α and interferons to vitamins like retinoic acid. The purpose of this review article is to examine the expression, structure, regulation and biological role of NGAL and critically assess its potential as a novel diagnostic and prognostic marker in both benign and malignant human diseases.

The major histocompatibility complex (MHC) glycoprotein family, also referred to as human leukocyte antigens, present endogenous and exogenous antigens to T lymphocytes for recognition and response. These molecules play a central role in enabling the immune system to distinguish self from non-self, which is the basis for protective immunity against pathogenic infections and disease while at the same time representing a serious obstacle for tissue transplantation. All known MHC family members, like the majority of secreted, cell surface, and other immune-related molecules, carry asparagine (N)-linked glycans. The immune system has evolved increasing complexity in higher-order organisms along with a more complex pattern of protein glycosylation, a relationship that may contribute to immune function beyond the early protein quality control events in the endoplasmic reticulum that are commonly known. The broad MHC family maintains peptide sequence motifs for glycosylation at sites that are highly conserved across evolution, suggesting importance, yet functional roles for these glycans remain largely elusive. In this review, we will summarize what is known about MHC glycosylation and provide new insight for additional functional roles for this glycoprotein modification in mediating immune responses.

<h4>Context</h4>The pathogenetic mechanisms of kidney stone formation are complex and involve both metabolic and environmental risk factors. Over the past decade, major advances have been made in the understanding of the pathogenesis, diagnosis, and treatment of kidney stone disease.<h4>Evidence acquisition and synthesis</h4>Both original and review articles were found via PubMed search reporting on pathophysiology, diagnosis, and management of kidney stones. These resources were integrated with the authors' knowledge of the field.<h4>Conclusion</h4>Nephrolithiasis remains a major economic and health burden worldwide. Nephrolithiasis is considered a systemic disorder associated with chronic kidney disease, bone loss and fractures, increased risk of coronary artery disease, hypertension, type 2 diabetes mellitus, and the metabolic syndrome. Further understanding of the pathophysiological link between nephrolithiasis and these systemic disorders is necessary for the development of new therapeutic options.

Synapse abnormalities in Huntington's disease (HD) patients can precede clinical diagnosis and neuron loss by decades. The polyglutamine expansion in the huntingtin (htt) protein that underlies this disorder leads to perturbations in many cellular pathways, including the disruption of Rab11-dependent endosomal recycling. Impairment of the small GTPase Rab11 leads to the defective formation of vesicles in HD models and may thus contribute to the early stages of the synaptic dysfunction in this disorder. Here, we employ transgenic Drosophila melanogaster models of HD to investigate anomalies at the synapse and the role of Rab11 in this pathology. We find that the expression of mutant htt in the larval neuromuscular junction decreases the presynaptic vesicle size, reduces quantal amplitudes and evoked synaptic transmission and alters larval crawling behaviour. Furthermore, these indicators of early synaptic dysfunction are reversed by the overexpression of Rab11. This work highlights a potential novel HD therapeutic strategy for early intervention, prior to neuronal loss and clinical manifestation of disease.

Gonadotropin Regulated Testicular RNA Helicase (GRTH/Ddx25) is a testis-specific multifunctional RNA helicase and an essential post-transcriptional regulator of spermatogenesis. GRTH transports relevant mRNAs from nucleus to cytoplasmic sites of meiotic and haploid germ cells and associates with actively translating polyribosomes. It is also a negative regulator of steroidogenesis in Leydig cells. To obtain a genome-wide perspective of GRTH regulated genes, in particularly those associated with polyribosomes, microarray differential gene expression analysis was performed using polysome-bound RNA isolated from testes of wild type (WT) and GRTH KO mice. 792 genes among the entire mouse genome were found to be polysomal GRTH-linked in WT. Among these 186 were down-regulated and 7 up-regulated genes in GRTH null mice. A similar analysis was performed using total RNA extracted from purified germ cell populations to address GRTH action in individual target cells. The down-regulation of known genes concerned with spermatogenesis at polysomal sites in GRTH KO and their association with GRTH in WT coupled with early findings of minor or unchanged total mRNAs and abolition of their protein expression in KO underscore the relevance of GRTH in translation. Ingenuity pathway analysis predicted association of GRTH bound polysome genes with the ubiquitin-proteasome-heat shock protein signaling network pathway and NFκB/TP53/TGFB1 signaling networks were derived from the differentially expressed gene analysis. This study has revealed known and unexplored factors in the genome and regulatory pathways underlying GRTH action in male reproduction.

Mutations in both the amyloid precursor protein (APP) and the presenilin (PSEN) genes cause familial Alzheimer's disease (FAD) with autosomal dominant inheritance and early onset of disease. The clinical course and neuropathology of FAD and sporadic Alzheimer's disease are highly similar, and patients with FAD constitute a unique population in which to conduct treatment and, in particular, prevention trials with novel pharmaceutical entities. It is critical, therefore, to exactly defi ne the molecular consequences of APP and PSEN FAD mutations. Both APP and PSEN mutations drive amyloidosis in FAD patients through changes in the brain metabolism of amyloid-β (Aβ) peptides that promote the formation of pathogenic aggregates. APP mutations do not seem to impair the physiological functions of APP. In contrast, it has been proposed that PSEN mutations compromise γ-secretase-dependent and -independent functions of PSEN. However, PSEN mutations have mostly been studied in model systems that do not accurately refl ect the genetic background in FAD patients. In this review, we discuss the reported cellular phenotypes of APP and PSEN mutations, the current understanding of their molecular mechanisms, the need to generate faithful models of PSEN mutations, and the potential bias of APP and PSEN mutations on therapeutic strategies that target Aβ.

The therapeutic response to a drug treatment is a mixture of direct pharmacological action and placebo effect. Therefore, harnessing the positive aspects of the placebo effect and reducing the negative ones could potentially benefit the patient. This article is aimed at providing an overview for clinicians of the importance of contextual psychosocial variables in determining treatment response, and the specific focus is on determinants of the placebo response. A better understanding of the physiological, psychological, and social mechanisms of placebo may aid in predicting which contexts have the greatest potential for inducing positive treatment responses. We examine the evidence for the role of psychological traits, including optimism, pessimism, and the effect of patient expectations on therapeutic outcome. We discuss the importance of the patient-practitioner relationship and how this can be used to enhance the placebo effect, and we consider the ethical challenges of using placebos in clinical practice.

Three new types of terminally connected ABA-heterotrimesogens and heterodimesogens, composed of a bent 3,5-diphenyl-1,2,4-oxadiazole central unit and one or two rod-shaped 4-cyanobiphenyl cores or one 2-phenyl-1,3,4-thiadiazole core, connected by flexible spacers, have been synthesized, and their mesomorphic behavior was studied by optical polarizing microscopy (PM), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). All dimesogens exhibit broad ranges of cybotactic nematic phases (N(cybA) and N(cybC)), in some cases accompanied by additional mesophases (CybA or SmC) at lower temperature. The combination of the 3,5-diphenyl-1,2,4-oxadiazole unit with one cyanobiphenyl core leads to the removal of tilted smectic and cybotactic nematic phases (SmC, N(cybC)), which are replaced by the nontilted CybA phases and nematic phases composed of SmA-type clusters (N(cybA)). The orthogonal cybotactic nematic phases of bent-core mesogens are of special interest for achieving biaxial nematic phases of the orthorhombic type. The orthogonal (N(cybA)) and skewed (N(cybC)) cybotactic nematic phases were distinguished by XRD and optical observations.

Neural crest cells are a population of multipotent stem cell-like progenitors that arise at the neural plate border in vertebrates, migrate extensively, and give rise to diverse derivatives such as melanocytes, craniofacial cartilage and bone, smooth muscle, peripheral and enteric neurons and glia. The neural crest gene regulatory network (NC-GRN) includes a number of key factors that are used reiteratively to control multiple steps in the development of neural crest cells, including the acquisition of stem cell attributes. It is therefore essential to understand the mechanisms that control the distinct functions of such reiteratively used factors in different cellular contexts. The context-dependent control of neural crest specification is achieved through combinatorial interaction with other factors, post-transcriptional and post-translational modifications, and the epigenetic status and chromatin state of target genes. Here we review the current understanding of the NC-GRN, including the role of the neural crest specifiers, their links to the control of "stemness," and their dynamic context-dependent regulation during the formation of neural crest progenitors.

Copper deficiency leads to anemia but the mechanism is unknown. Copper deficiency also leads to hypoferremia, which may limit erythropoiesis. The hypoferremia may be due to limited function of multicopper oxidases (MCO) hephaestin in enterocytes or GPI-ceruloplasmin in macrophages of liver and spleen whose function as a ferroxidase is thought essential for iron transfer out of cells. Iron release may also be limited by ferroportin (Fpn), the iron efflux transporter. Fpn may be lower following copper deficiency because of impaired ferroxidase activity of MCO. Fpn is also dependent on the liver hormone hepcidin as Fpn is degraded when hepcidin binds to Fpn. Anemia and hypoferremia both down regulate hepcidin by separate mechanisms. Current studies confirmed and extended earlier studies with copper-deficient (CuD) rats that suggested low hepicidin resulted in augmented Fpn. However, current studies in CuD dams failed to confirm a correlation that hepcidin expression was associated with low transferrin receptor 2 (TfR2) levels and also challenged the dogma that holotransferrin can explain the correlation with hepcidin. CuD dams exhibited hypoferremia, low liver TfR2, anemia in some rats, yet no depression in Hamp expression, the hepcidin gene. Normal levels of GDF-15, the putative erythroid cytokine that suppresses hepcidin, were detected in plasma of CuD and iron-deficient (FeD) dams. Importantly, FeD dams did display greatly lower Hamp expression. Normal hepcidin in these CuD dams is puzzling since these rats may need extra iron to meet needs of lactation and the impaired iron transfer noted previously.

<h4>Background</h4>Mice with peroxisome deficiency in neural cells (Nestin-Pex5-/-) develop a neurodegenerative phenotype leading to motor and cognitive disabilities and early death. Major pathologies at the end stage of disease include severe demyelination, axonal degeneration and neuroinflammation. We now investigated the onset and progression of these pathological processes, and their potential interrelationship. In addition, the putative role of oxidative stress, the impact of plasmalogen depletion on the neurodegenerative phenotype, and the consequences of peroxisome elimination in the postnatal period were studied.<h4>Methods</h4>Immunohistochemistry in association with gene expression analysis was performed on Nestin-Pex5-/- mice to document demyelination, axonal damage and neuroinflammation. Also Gnpat-/- mice, with selective plasmalogen deficiency and CMV-Tx-Pex5-/- mice, with tamoxifen induced generalized loss of peroxisomes were analysed.<h4>Results</h4>Activation of the innate immune system is a very early event in the pathological process in Nestin-Pex5-/- mice which evolves in chronic neuroinflammation. The complement factor C1q, one of the earliest up regulated transcripts, was expressed on neurons and oligodendrocytes but not on microglia. Transcripts of other pro- and anti-inflammatory genes and markers of phagocytotic activity were already significantly induced before detecting pathologies with immunofluorescent staining. Demyelination, macrophage activity and axonal loss co-occurred throughout the brain. As in patients with mild peroxisome biogenesis disorders who develop regressive changes, demyelination in cerebellum and brain stem preceded major myelin loss in corpus callosum of both Nestin-Pex5-/- and CMV-Tx-Pex5-/- mice. These lesions were not accompanied by generalized oxidative stress throughout the brain. Although Gnpat-/- mice displayed dysmyelination and Purkinje cell axon damage in cerebellum, confirming previous observations, no signs of inflammation or demyelination aggravating with age were observed.<h4>Conclusions</h4>Peroxisome inactivity triggers a fast neuroinflammatory reaction, which is not solely due to the depletion of plasmalogens. In association with myelin abnormalities this causes axon damage and loss.

Raf-1 kinase inhibitory protein (RKIP), a suppressor of metastasis, is associated inversely with the progression and metastasis of human malignancies. The present study evaluated relationships between RKIP expression and metastatic potential, clinicopathological characteristics and patient outcome in esophageal squamous cell carcinoma (ESCC). We examined tissue specimens from 138 patients with thoracic ESCC. Using immunohistochemistry, RKIP expression was detected in ESCC in situ, primary ESCC and nodal metastatic ESCC. RKIP expression was reduced in 28.9% (13/45) of ESCC in situ, in 50.0% (69/138) of primary ESCC and in 71.4% (65/91) of nodal metastatic ESCC. These levels of RKIP down-regulation differed significantly. RKIP expression was associated inversely with histological grade (P=0.008), pathological T stage (P=0.044), lymphatic invasion (P=0.019), regional lymph node metastasis (LNM; P=0.002) and stage (P=0.041). Pathological T stage (P=0.001), lymphatic invasion (P<0.001) and reduced RKIP expression (P=0.039) were independent predictors of regional LNM in ESCC. In addition, the postoperative survival of patients with RKIP-reduced ESCC was significantly shorter than for patients with RKIP-positive ESCC (P=0.004). Reduced RKIP expression in ESCC correlated with advanced disease, regional LNM and poor prognosis. RKIP expression may serve as a novel clinical biomarker in patients with ESCC.

Circulating levels of adiponectin, a hormone produced predominantly by adipocytes, are highly heritable and are inversely associated with type 2 diabetes mellitus (T2D) and other metabolic traits. We conducted a meta-analysis of genome-wide association studies in 39,883 individuals of European ancestry to identify genes associated with metabolic disease. We identified 8 novel loci associated with adiponectin levels and confirmed 2 previously reported loci (P = 4.5×10(-8)-1.2×10(-43)). Using a novel method to combine data across ethnicities (N = 4,232 African Americans, N = 1,776 Asians, and N = 29,347 Europeans), we identified two additional novel loci. Expression analyses of 436 human adipocyte samples revealed that mRNA levels of 18 genes at candidate regions were associated with adiponectin concentrations after accounting for multiple testing (p<3×10(-4)). We next developed a multi-SNP genotypic risk score to test the association of adiponectin decreasing risk alleles on metabolic traits and diseases using consortia-level meta-analytic data. This risk score was associated with increased risk of T2D (p = 4.3×10(-3), n = 22,044), increased triglycerides (p = 2.6×10(-14), n = 93,440), increased waist-to-hip ratio (p = 1.8×10(-5), n = 77,167), increased glucose two hours post oral glucose tolerance testing (p = 4.4×10(-3), n = 15,234), increased fasting insulin (p = 0.015, n = 48,238), but with lower in HDL-cholesterol concentrations (p = 4.5×10(-13), n = 96,748) and decreased BMI (p = 1.4×10(-4), n = 121,335). These findings identify novel genetic determinants of adiponectin levels, which, taken together, influence risk of T2D and markers of insulin resistance.

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.

Most clinical studies suggest that the prevalence and severity of liver steatosis are higher in patients infected with hepatitis C virus (HCV) genotype 3 than in patients infected with other genotypes. This may reflect the diversity and specific intrinsic properties of genotype 3 virus proteins. We analyzed the possible association of particular residues of the HCV core and NS5A proteins known to dysregulate lipid metabolism with steatosis severity in the livers of patients chronically infected with HCV. We used transmission electron microscopy to quantify liver steatosis precisely in a group of 27 patients, 12 of whom were infected with a genotype 3 virus, the other 15 being infected with viruses of other genotypes. We determined the area covered by lipid droplets in liver tissues and analyzed the diversity of the core and NS5A regions encoded by the viral variants circulating in these patients. The area covered by lipid droplets did not differ significantly between patients infected with genotype 3 viruses and those infected with other genotypes. The core and NS5A protein sequences of the viral variants circulating in patients with mild or severe steatosis were evenly distributed throughout the phylogenic trees established from all the collected sequences. Thus, individual host factors seem to play a much greater role than viral factors in the development of severe steatosis in patients chronically infected with HCV, including those infected with genotype 3 viruses.

Signal transduction and activator of transcription-3 (STAT3) plays an important role in neuronal survival, regeneration and repair after brain injury. We previously demonstrated that STAT3 is activated in brain after cerebral ischemia specifically in neurons. The effect was sex-specific and modulated by sex steroids, with higher activation in females than males. In the current study, we used a proteomics approach to identify downstream proteins affected by ischemia in male and female wild-type (WT) and neuron-specific STAT3 knockout (KO) mice. We established four comparison groups based on the transgenic condition and the hemisphere analyzed, respectively. Moreover, the sexual variable was taken into account and male and female animals were analyzed independently. Results support a role for STAT3 in metabolic, synaptic, structural and transcriptional responses to cerebral ischemia, indeed the adaptive response to ischemia/reperfusion injury is delayed in neuronal-specific STAT3 KO mice. The differences observed between males and females emphasize the importance of sex-specific neuronal survival and repair mechanisms, especially those involving antioxidant and energy-related activities, often caused by sex hormones.

Working memory deficits are found in different psychiatric populations and are most pronounced in schizophrenia. There is preliminary evidence from pharmacological studies that the verbal and visuospatial subcomponents of working memory are subject to differential neurotransmitter modulation. Here, we investigated the impact of well-known polymorphisms of the dopamine transporter gene (SLC6A3, DAT) and the catechol-O-methyl-transferase gene (COMT) as well as the serotonin transporter gene (SLC6A4, 5-HTT) on these specific working memory subcomponents in a mixed sample of patients and healthy individuals. Twenty healthy subjects and 80 patients diagnosed with schizophrenia, bipolar I disorder, or obsessive-compulsive disorder underwent genotyping for the DAT variable number of tandem repeats (VNTR), the COMT val/met-, and the 5-HTT promoter length polymorphism (5-HTTLPR) and neuropsychological testing using a battery of well-characterized, brain circuit-specific working memory tasks. DAT genotype revealed a significant and selective effect on visuospatial working memory, while there was no effect on verbal working memory functioning. 5-HTT genotype, by contrast, exerted a significant and selective effect on verbal working memory task performance. COMT genotype did not show any influence on either working memory domain. The results of the present study provide evidence for a differential impact of genetic polymorphisms of the dopaminergic and serotonergic systems on verbal and visuospatial working memory functioning. Together with prior evidence suggesting the existence of subgroups of schizophrenia patients exhibiting isolated deficits in only one working memory domain, this finding further supports the idea of endophenotypically and pathophysiologically distinct subgroups of schizophrenia with implications for personalized therapeutic approaches.

<h4>Background</h4>Although recent research evidence suggests an association between household air pollution from solid fuel use, such as coal or biomass, and cardiovascular events such as hypertension, little epidemiologic data are available concerning such exposure effects on cardiovascular endpoints other than hypertension. We explored the association between in-home solid fuel use and self-reported diagnoses of cardiovascular endpoints, such as hypertension, coronary heart disease (CHD), stroke, and diabetes.<h4>Methods</h4>We analyzed 14,068 Chinese adults, aged 18 years and older. Odds ratios (OR) and the corresponding 95% confidence intervals (CI) were estimated using logistic regression models for the risk of each outcome after adjusting for potential confounders.<h4>Results</h4>The use of solid fuel in home was significantly associated with an increased risk for hypertension (OR 1.70, 95% CI 1.40 to 2.07), CHD (OR 2.58, 95% CI 1.53 to 4.32), and diabetes (OR 2.48, 95% CI 1.59 to 3.86), after adjusting for potential confounders. Compared with individuals in the lowest tertile of the duration of solid fuel exposure, those in the highest tertile of the duration of solid fuel exposure had an increased odds of hypertension (OR 1.73, 95% CI 1.45 to 2.06), stroke (OR 1.87, 95% CI 1.03 to 3.38), and diabetes (OR 3.18, 95% CI 2.11 to 4.78).<h4>Conclusions</h4>Our data suggest that in-home solid fuel exposure maybe associated with increased risk for hypertension, CHD, stroke, and diabetes in the Chinese adult population. Further large-scale longitudinal studies are warranted to confirm these findings.

<h4>Background</h4>Longitudinal growth associations with genetic variants identified for adult BMI may provide insights into the timing of obesity susceptibility.<h4>Objective</h4>The objective was to explore associations of known BMI loci with measures of body size from birth to adulthood.<h4>Design</h4>A total of 2537 individuals from a longitudinal British birth cohort were genotyped for 11 genetic variants robustly associated with adult BMI (in/near FTO, MC4R, TMEM18, GNPDA2, KCTD15, NEGR1, BDNF, ETV5, SEC16B, SH2B1, and MTCH2). We derived an obesity-risk-allele score, comprising the sum of BMI-increasing alleles in each individual, and examined this for an association with birth weight and repeated measures of weight, height, and BMI SD scores (SDS) at 11 time points between ages 2 and 53 y.<h4>Results</h4>The obesity-risk-allele score showed borderline significant association with birth weight (0.019 SDS/allele; P = 0.05) and was more clearly associated with higher weight and BMI at all time points between ages 2 and 53 y; the strongest associations with weight occurred at ages 11 and 20 y (both 0.056 SDS/allele). In longitudinal analyses, the score was positively associated with weight gain only between birth and 11 y (0.003 SDS/allele per year; 95% CI: 0.001, 0.004; P = 0.001). The risk-allele score was associated with taller height at 7 y (0.031 SDS/allele; P = 0.002) and greater height gains between 2 and 7 y (0.007 SDS/allele per year; P < 0.001), but not with adult height (P = 0.5).<h4>Conclusions</h4>The combined effect of adult obesity susceptibility variants on weight gain was confined to childhood. These variants conferred a faster tempo of height growth that was evident before the pubertal years.

More than 76 million people world-wide are estimated to have diagnosable alcohol use disorders (AUDs) (alcohol abuse or dependence), making these disorders a major global health problem. Pharmacotherapy offers promising means for treating AUDs, and significant progress has been made in the past 20 years. The US Food and Drug Administration approved three of the four medications for alcoholism in the last two decades. Unfortunately, these medications do not work for everyone, prompting the need for a personalized approach to optimize clinical benefit or more efficacious medications that can treat a wider range of patients, or both. To promote global health, the potential reorganization of the National Institutes of Health (NIH) must continue to support the National Institute on Alcohol Abuse and Alcoholism's (NIAAA's) vision of ensuring the development and delivery of new and more efficacious medications to treat AUDs in the coming decade. To achieve this objective, the NIAAA Medications Development Team has identified three fundamental long-range goals: (1) to make the drug development process more efficient; (2) to identify more efficacious medications, personalize treatment approaches, or both; and (3) to facilitate the implementation and adaptation of medications in real-world treatment settings. These goals will be carried out through seven key objectives. This paper describes those objectives in terms of rationale and strategy. Successful implementation of these objectives will result in the development of more efficacious and safe medications, provide a greater selection of therapy options and ultimately lessen the impact of this devastating disorder.

<h4>Objectives</h4>To investigate the characteristics and prevalence of poststroke depression (PSD) and poststroke emotional incontinence (PSEI) and the factors related to these conditions at admission and 3 months after stroke.<h4>Methods</h4>We evaluated 508 consecutive patients with acute ischemic stroke for PSD and PSEI at admission and 3 months later. PSD was evaluated using the Beck Depression Inventory, and PSEI was evaluated using Kim's criteria. Blood samples were collected and genotyped for the promoter region of the serotonin transporter protein (5-HTTLPR) and the number of tandem repeats within intron 2 (STin2 VNTR). Perceived social support (the ENRICHD Social Support Inventory) was also measured.<h4>Results</h4>PSD and PSEI were present in 13.7% and 9.4% of patients, respectively, at admission and in 17.7% and 11.7%, respectively, at 3 months after stroke. Multivariate analyses showed that PSD at admission was associated with the NIH Stroke Scale score at admission (p < 0.001), whereas PSD at 3 months was associated with the presence of microbleeds (p < 0.01) and perceived low social support (p < 0.001). In contrast, only lesion location (p = 0.022) was associated with PSEI at admission, whereas modified Rankin Scale score (p = 0.019), STin2 VNTR (p = 0.040), and low social support (p = 0.042) were related to PSEI 3 months after stroke.<h4>Conclusions</h4>Diverse factors such as neurologic dysfunction, lesion location, microbleeds, genetic traits, and social support are differently related to acute and subacute emotional disturbances. Strategies to prevent or manage these problems should consider these differences.

Hereditary hemochomatosis (HH) is caused by mutations in several genes, including HFE and transferrin receptor-2 (TFR2). Loss of either protein decreases expression of the iron regulatory hormone hepcidin by the liver, leading to inappropriately high iron uptake from the diet, and resulting in systemic iron overload. In tissue culture, overexpressed HFE and TFR2 physically interact. Hepatocellular overexpression of Hfe in vivo increases hepcidin expression, despite an associated decrease in Tfr2. On this basis, we hypothesized that Tfr2 would not be required for Hfe-dependent up-regulation of hepcidin. We show that hepatocellular overexpression of Hfe in Tfr2(Y245X/Y245X) mice leads to hepcidin induction eventuating in iron deficiency and a hypochromic, microcytic anemia. Furthermore, coimmunoprecipitation studies using liver lysates did not provide evidence for physical interaction between Hfe and Tfr2 in vivo. In conclusion, we demonstrate that Tfr2 is not essential for Hfe-mediated induction of hepcidin expression, supporting the possibility that TFR2 may regulate iron metabolism in an HFE-independent manner.

The aim of this study was to identify genes that influence iron regulation under varying dietary iron availability. Male and female mice from 20+ BXD recombinant inbred strains were fed iron-poor or iron-adequate diets from weaning until 4 mo of age. At death, the spleen, liver, and blood were harvested for the measurement of hemoglobin, hematocrit, total iron binding capacity, transferrin saturation, and liver, spleen and plasma iron concentration. For each measure and diet, we found large, strain-related variability. A principal-components analysis (PCA) was performed on the strain means for the seven parameters under each dietary condition for each sex, followed by quantitative trait loci (QTL) analysis on the factors. Compared with the iron-adequate diet, iron deficiency altered the factor structure of the principal components. QTL analysis, combined with PosMed (a candidate gene searching system) published gene expression data and literature citations, identified seven candidate genes, Ptprd, Mdm1, Picalm, lip1, Tcerg1, Skp2, and Frzb based on PCA factor, diet, and sex. Expression of each of these is cis-regulated, significantly correlated with the corresponding PCA factor, and previously reported to regulate iron, directly or indirectly. We propose that polymorphisms in multiple genes underlie individual differences in iron regulation, especially in response to dietary iron challenge. This research shows that iron management is a highly complex trait, influenced by multiple genes. Systems genetics analysis of iron homeostasis holds promise for developing new methods for prevention and treatment of iron deficiency anemia and related diseases.

MicroRNAs (miRs) regulate diverse molecular and cellular processes including oligodendrocyte (OL) precursor cell (OPC) proliferation and differentiation in rodents. However, the role of miRs in human OPCs is poorly understood. To identify miRs that may regulate these processes in humans, we isolated OL lineage cells from human white matter and analyzed their miR profile. Using endpoint RT-PCR assays and quantitative real-time PCR, we demonstrate that miR-219, miR-338, and miR-17-92 are enriched in human white matter and expressed in acutely isolated human OLs. In addition, we report the expression of closely related miRs (miR-219-1-3p, miR-219-2-3p, miR-1250, miR-657, miR-3065-5p, miR-3065-3p) in both rodent and human OLs. Our findings demonstrate that miRs implicated in rodent OPC proliferation and differentiation are regulated in human OLs and may regulate myelination program in humans. Thus, these miRs should be recognized as potential therapeutic targets in demyelinating disorders.

Following reports of an increased incidence of amyotrophic lateral sclerosis (ALS) in U.S. veterans, we have conducted a high-density genome-wide association study (GWAS) of ALS outcome and survival time in a sample of U.S. veterans. We tested ∼1.3 million single nucleotide polymorphisms (SNPs) for association with ALS outcome in 442 incident Caucasian veteran cases diagnosed with definite or probable ALS and 348 Caucasian veteran controls. To increase power, we also included genotypes from 5909 publicly-available non-veteran controls in the analysis. In the survival analysis, we tested for association between SNPs and post-diagnosis survival time in 639 Caucasian veteran cases with definite or probable ALS. After this discovery phase, we performed follow-up genotyping of 299 SNPs in an independent replication sample of Caucasian veterans and non-veterans (ALS outcome: 183 cases and 961 controls; survival: 118 cases). Although no SNPs reached genome-wide significance in the discovery phase for either phenotype, three SNPs were statistically significant in the replication analysis of ALS outcome: rs6080539 (177 kb from PCSK2), rs7000234 (4 kb from ZNF704), and rs3113494 (13 kb from LOC100506746). Two SNPs located in genes that were implicated by previous GWA studies of ALS were marginally significant in the pooled analysis of discovery and replication samples: rs17174381 in DPP6 (p = 4.4×10(-4)) and rs6985069 near ELP3 (p = 4.8×10(-4)). Our results underscore the difficulty of identifying and convincingly replicating genetic associations with a rare and genetically heterogeneous disorder such as ALS, and suggest that common SNPs are unlikely to account for a substantial proportion of patients affected by this devastating disorder.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.

Serotonin and noradrenaline reuptake inhibitors have shown to produce antinociceptive effects in several animal models of neuropathic pain. In the present work, we have analyzed the density of brain and spinal serotonin and noradrenaline transporters (5-HTT and NAT) in a rat model of neuropathic pain, the spinal nerve ligation (SNL). Quantitative autoradiography revealed a significant decrease in the density of 5-HTT ([(3)H]citalopram binding) at the level of the lumbar spinal cord following 2 weeks of neuropathic surgery (lamina V, -40%: 6.01±0.64 nCi/mg tissue in sham-animals vs 3.59±1.56 in SNL-animals; lamina X, -30%: 9.10±2.00 vs 6.40±1.93 and lamina IX, -22%: 12.01±2.41 vs 9.42±1.58). By contrast, NAT density ([(3)H]nisoxetine binding) was significantly increased (lamina I-II, +34%: 2.20±0.45 vs 2.96±0.65; lamina V, +57%: 1.34±0.28 vs 2.11±0.66; and lamina IX, +58%: 2.39±0.71 vs 3.78±1.10). At supraspinal structures, SNL induced adaptive changes only in the density of 5-HTT (septal nuclei, +33%: 10.18±2.03 vs 13.53±1.14; CA3 field of hippocampus, +18%: 6.94±1.01 vs 8.21±0.81; paraventricular thalamic nucleus, +21%: 15.18±1.88 vs 18.35±2.08; lateral hypothalamic area, +40%: 12.68±1.90 vs 17.8±2.55; ventromedial hypothalamic nuclei, +19%: 7.16±0.92 vs 8.55±0.40; and dorsal raphe nucleus, +15%: 35.22±3.88 vs 40.68±3.11). Thus, we demonstrate, in the SNL model of neuropathic pain, the existence of opposite changes in the spinal expression of 5-HTT (down-regulation) and NAT (up-regulation), and the presence of supraspinal adaptive changes (up-regulation) only on 5-HTT density. These findings may help understanding the pathogeny of neuropathic pain and the differential analgesic action of antidepressants targeting 5-HTT and/or NAT transporters.

<h4>Background</h4>Tandem repeats are multiple duplications of substrings in the DNA that occur contiguously, or at a short distance, and may involve some mutations (such as substitutions, insertions, and deletions). Tandem repeats have been extensively studied also for their association with the class of repeat expansion diseases (mostly affecting the nervous system). Comparative studies on the output of different tools for finding tandem repeats highlighted significant differences among the sets of detected tandem repeats, while many authors pointed up how critical it is the right choice of parameters.<h4>Results</h4>In this paper we present TReaDS - Tandem Repeats Discovery Service, a tandem repeat meta search engine. TReaDS forwards user requests to several state of the art tools for finding tandem repeats and merges their outcome into a single report, providing a global, synthetic, and comparative view of the results. In particular, TReaDS allows the user to (i) simultaneously run different algorithms on the same data set, (ii) choose for each algorithm a different setting of parameters, and (iii) obtain a report that can be downloaded for further, off-line, investigations. We used TReaDS to investigate sequences associated with repeat expansion diseases.<h4>Conclusions</h4>By using the tool TReaDS we discover that, for 27 repeat expansion diseases out of a currently known set of 29, long fuzzy tandem repeats are covering the expansion loci. Tests with control sets confirm the specificity of this association. This finding suggests that long fuzzy tandem repeats can be a new class of cis-acting elements involved in the mechanisms leading to the expansion instability.We strongly believe that biologists can be interested in a tool that, not only gives them the possibility of using multiple search algorithm at the same time, with the same effort exerted in using just one of the systems, but also simplifies the burden of comparing and merging the results, thus expanding our capabilities in detecting important phenomena related to tandem repeats.

<h4>Background</h4>The prediction of fibrosis is an essential part of the assessment and management of patients with chronic liver disease. Non-invasive tests (NITs) have a number of advantages over the traditional standard of fibrosis assessment by liver biopsy, including safety, cost-effectiveness, and widespread accessibility.<h4>Objectives</h4>The aim of this study was to determine the accuracy of certain biomarkers and transient elastography (TE) alone or in combination to predict the stage of liver fibrosis in chronic hepatitis C (CHC). Also, we examined whether the combination of certain biomarkers and TE could increase the diagnostic accuracy of liver fibrosis assessment.<h4>Patients and method</h4>A total of 446 patients who were previously diagnosed with CHC were included in the study. In the study group, 6 blood-based scores (APRI, Forns, Fib-4, Hepascore, FibroTest, and Fibrometer) were calculated, and TE was performed to validate the stage of fibrosis, compared with liver biopsy (LB) as the standard.<h4>Results</h4>Significant fibrosis (F ≥ 2) was predicted with an AUROC of 0.727, 0.680, 0.714, 0.778, 0.688, 0.797, and 0.751 for the APRI, Forns, Fib-4, FibroTest, Hepascore, and Fibrometer scores and TE (Fibroscan), respectively. Severe fibrosis (F ≥ 3) was predicted, with AUROCs ranging between 0.705 and 0.811 for Hepascore and Fibrometer, respectively. Of the biomarkers, Fibrometer had the highest AUROC value in predicting both significant and severe fibrosis. The combination of APRI or FIB-4 with Fibrometer increased the diagnostic accuracy for significant fibrosis (from 69.07 to 82.27 for APRI, P = 0.001 and from 57.74 to 81.33, P = 0.001 for Fib-4). Combining APRI or Fib-4 with TE also increased the diagnostic accuracy (from 69.07 to 80.70%, P = 0.001 for APRI and from 57.74 to 81.33%, P = 0.001 for Fib-4) for significant fibrosis. The association that included Fibrotest was also reliable for the improvement of diagnostic accuracy. These combinations were more accurate or the assessment of severe fibrosis.<h4>Conclusions</h4>The synchronous association between a simple, inexpensive score and a complex but expensive score or TE increases the diagnostic accuracy of non-invasive methods for the assessment of liver fibrosis stage.

No abstract available.

No abstract available.

In 1987, Carney et al described a rare thyroid tumor termed hyalinizing trabecular adenoma presenting characteristics consisting of a trabecular growth pattern and hyalinizing stroma. In subsequent reports, the observed nuclear features and RET alterations led this tumor to be linked to papillary carcinoma. Subsequent reports concerning hyalinizing trabecular carcinoma further complicated its classification. To avoid uncertainties, the definition of hyalinizing trabecular tumor (HTT) is more widely used. Herein, a case of HTT is reported in detail, and the circumstances are also discussed. HTT is thought to be particularly differentiated from papillary carcinoma despite the identical high frequency of nuclear grooves and cytoplasmic inclusions, and MIB-1-positive staining is one of the most accurate diagnostic methods due to the distinct membrane-positive pattern noted in HTT. It is believed that most HTTs are benign and lobectomy is the standard treatment. Pathologists should offer surgeons information concerning diagnosis overlapping with effective treatment.

Hydroxyapatite (HA) doped with trace elements has attracted much attention recently owing to its excellent biological functions. Herein, we use a facile co-precipitation method to incorporate selenium into HA by adding sodium selenite during synthesis. The obtained selenium-substituted HA products are needle-like nanoparticles which have  size and crystallinity that are similar to those of the pure HA nanoparticles (HANs) when the selenium content is low. HANs are found to have the ability to induce the apoptosis of osteosarcoma cells, and the anti-tumour effects are enhanced after incorporation of selenium. Meanwhile, the nanoparticles can also support the growth of bone marrow stem cells. Furthermore, the flow cytometric results indicate that the apoptosis induction of osteosarcoma cells is caused by the increased reactive oxygen species and decreased mitochondrial membrane potential. These results show that the selenium-substituted HANs are potentially promising bone graft materials in osteosarcoma treatment due to their dual functions of supporting normal cell growth and inducing tumour cell apoptosis.

Glycopeptides are extremely useful for basic research and clinical applications, but access to structurally defined glycopeptides is limited by the difficulties in synthesizing this class of compounds. In this study, we demonstrate that many common peptide coupling conditions used to prepare O-linked glycopeptides result in substantial amounts of epimerization at the α position. In fact, epimerization resulted in up to 80% of the non-natural epimer, indicating that it can be the major product in some reactions. Through a series of mechanistic studies, we demonstrate that the enhanced epimerization relative to nonglycosylated amino acids is due to a combination of factors, including a faster rate of epimerization, an energetic preference for the unnatural epimer over the natural epimer, and a slower overall rate of peptide coupling. In addition, we demonstrate that use of 2,4,6-trimethylpyridine (TMP) as the base in peptide couplings produces glycopeptides with high efficiency and low epimerization. The information and improved reaction conditions will facilitate the preparation of glycopeptides as therapeutic compounds and vaccine antigens.

Rhes, the Ras Homolog Enriched in Striatum, is a GTP-binding protein whose gene was discovered during a screen for mRNAs preferentially expressed in rodent striatum. This 266 amino acid protein is intermediate in size between small Ras-like GTP-binding proteins and α-subunits of heterotrimeric G proteins. It is most closely related to another Ras-like GTP-binding protein termed Dexras1 or AGS1. Although subsequent studies have shown that the rhes gene is expressed in other brain areas in addition to striatum, the striatal expression level is relatively high, and Rhes protein is likely to play a vital role in striatal physiology and pathology. Indeed, it has recently been shown to interact with the Huntingtin protein and play a pivotal role in the selective vulnerability of striatum in Huntington's disease (HD). Not surprisingly, Rhes can interact with multiple proteins to affect striatal physiology at multiple levels. Functional studies have indicated that Rhes plays a role in signaling by striatal G protein-coupled receptors (GPCR), although the details of the mechanism remain to be determined. Rhes has been shown to bind to both α- and β-subunits of heterotrimeric G proteins and to affect signaling by both Gi/o- and Gs/olf-coupled receptors. In this context, Rhes can be classified as a member of the family of accessory proteins to GPCR signaling. With documented effects in dopamine- and opioid-mediated behaviors, an interaction with thyroid hormone systems and a role in HD pathology, Rhes is emerging as an important protein in striatal physiology and pathology.

Calcium is one of the most abundant minerals in the body and its metabolism is one of the basic biologic processes in humans. Although historically linked primarily to bone structural development and maintenance, calcium is now recognized as a key component of many physiologic pathways necessary for optimum health including cardiovascular, neurological, endocrine, renal, and gastrointestinal systems. A recent meta-analysis published in August 2011 showed a potential increase in cardiovascular events related to calcium supplementation. The possible mechanism of action of this correlation has not been well elucidated. This topic has generated intense interest due to the widespread use of calcium supplements, particularly among the middle aged and elderly who are at the most risk from cardiac events. Prior studies did not control for potential confounding factors such as the use of statins, aspirin or other medications. These controversial results warrant additional well-designed studies to investigate the relationship between calcium supplementation and cardiovascular outcomes. The purpose of this review is to highlight the current literature in regards to calcium supplementation and cardiovascular health; and to identify areas of future research.

<h4>Background</h4>Recently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation.<h4>Methods</h4>We analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions.<h4>Results</h4>We observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively).<h4>Conclusions</h4>We demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application.<h4>Trial registration</h4>The ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821.

<h4>Background</h4>It is supposed that fibroblasts present in tumour microenvironment increase cancer invasiveness and its ability to metastasize but the mechanisms have not been clearly defined yet. Thus, the current study was designed to assess changes in gene expression in five various cancer cell lines grown as a co-culture with the carcinoma-associated fibroblasts (CAFs) in vitro.<h4>Results</h4>A carcinoma-associated fibroblast cell line was isolated from a canine mammary cancer. Then, a co-culture of cancer cells with the CAFs was established and maintained for 72 hrs. Having sorted the cells, a global gene expression in cancer cells using DNA microarrays was examined. The analysis revealed an up-regulation of 100 genes and a down-regulation of 106 genes in the cancer cells grown as a co-culture with the CAFs in comparison to control conditions. The PANTHER binomial statistics tool was applied to determine statistically over-manifested pathways (p < 0.05). Bulk of the up-regulated genes are involved in the adhesion, the angiogenesis, the epithelial-mesenchymal transition (EMT) and generally take part in the developmental processes. These results were further confirmed using real-time qPCR. Moreover, a wound-healing assay and growth characteristics on Matrigel matrix showed that CAFs increase cancer cell migration and matrix invasion.<h4>Conclusion</h4>The results of the current study showed that the co-culturing of cancer cells and the CAFs caused significant changes to the cancer gene expression. The presence of the CAFs in a microenvironment of cancer cells promotes adhesion, angiogenesis and EMT.

Our goal was to study the effect of heat treatment temperature and heating rate on the microstructure and crystalline phases and assess the domain of existence of submicrometer fluorapatite crystals in niobium-doped fluorapatite glass-ceramics for biomedical applications. Glass-ceramic specimens were prepared by casting and heat treatment between 700 and 1200°C using a fast or a slow heating rate. The microstructure was characterized by atomic force microscopy and scanning electron microscopy. Crystalline phases were analyzed by x-ray diffraction. AFM of the as-cast glass revealed that amorphous phase separation occurred in this system. XRD confirmed the presence of fluorapatite in all specimens, together with forsterite and enstatite at higher temperatures. Both heating rate and heat treatment temperature strongly influenced microstructure and crystallinity. A dual microstructure with submicrometer fluorapatite crystals and polygonal forsterite crystals was obtained when slow heating rates and crystallization temperatures between 950 and 1100°C were used. Needle-shaped fluorapatite crystals appeared after heat treatment above 1100°C. Fast heating rates led to an increase in crystal size. Heat treatment temperatures should remain below 1100°C, together with slow heating rates, to prevent crystal dissolution, and preserve a dual microstructure of finely dispersed submicrometer crystals without growth of needle-shaped crystals.

Recent research has demonstrated that aberrant sphingolipid signaling is an important mechanism of chemoresistance in solid tumors. Sphingosine kinase (Sphk), the primary enzyme metabolizing the sphingolipid ceramide into sphingosine-1-phosphate (S1P), is a primary mediator of breast cancer promotion, survival and chemoresistance. However, to date the mechanism of Sphk-mediated drug resistance is poorly understood. Using the dual sphingosine kinase isozyme inhibitor, SKI-II (4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol), we explored the effects of sphingosine kinase inhibition on multi-drug-resistant breast cancer cells. We demonstrate that SKI-II alters endogenous sphingolipid signaling and decreases cancer proliferation, survival and viability. Furthermore, pharmacological inhibition of Sphk1/2 induced intrinsic apoptosis in these cells through modulation of the NF-κB pathway. SKI-II decreases NF-κB transcriptional activity through altered phosphorylation of the p65 subunit. Taken together, these results suggest that Sphk may be a promising therapeutic target in chemoresistant cancers.

We report the first genome-wide association study of a joint analysis using 795 Han Chinese individuals with treatment-refractory schizophrenia (TRS) and 806 controls. Three loci showed suggestive significant association with TRS were identified. These loci include: rs10218843 (P = 3.04 × 10(-7)) and rs11265461 (P = 1.94 × 10(-7)) are adjacent to signaling lymphocytic activation molecule family member 1 (SLAMF1); rs4699030 (P = 1.94 × 10(-6)) and rs230529 (P = 1.74 × 10(-7)) are located in the gene nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1); and rs13049286 (P = 3.05 × 10(-5)) and rs3827219 (P = 1.66 × 10(-5)) fall in receptor-interacting serine/threonine-protein kinase 4 (RIPK4). One isolated single nucleotide polymorphism (SNP), rs739617 (P = 3.87 × 10(-5)) was also identified to be associated with TRS. The -94delATTG allele (rs28362691) located in the promoter region of NFKB1 was identified by resequencing and was found to associate with TRS (P = 4.85 × 10(-6)). The promoter assay demonstrated that the -94delATTG allele had a significant lower promoter activity than the -94insATTG allele in the SH-SY5Y cells. This study suggests that rs28362691 in NFKB1 might be involved in the development of TRS.

<h4>Background</h4>Dopamine transporter (DAT) homozygous knockout (DAT(-/-)) mice have a 10-fold higher extracellular (DA) concentration in the caudate-putamen and nucleus accumbens than do wildtype (DAT(+/+)) mice, but show reduced presynaptic DA synthesis and fewer postsynaptic D(2) receptors. One aspect of neurotransmission involves DA binding to postsynaptic D(2)-like receptors coupled to cytosolic phospholipase A(2) (cPLA(2)), which releases the second messenger, arachidonic acid (AA), from synaptic membrane phospholipid. We hypothesized that tonic overactivation of D(2)-like receptors in DAT(-/-) mice due to the excess DA would not increase brain AA signaling, because of compensatory downregulation of postsynaptic DA signaling mechanisms.<h4>Methods</h4>[1-(14)C]AA was infused intravenously for 3 min in unanesthetized DAT(+/+), heterozygous (DAT(+/-)), and DAT(-/-) mice. AA incorporation coefficients k* and rates J(in), markers of AA metabolism and signaling, were imaged in 83 brain regions using quantitative autoradiography; brain cPLA(2)-IV activity also was measured.<h4>Results</h4>Neither k* nor J(in) for AA in any brain region, or brain cPLA(2)-IV activity, differed significantly among DAT(-/-), DAT(+/-), and DAT(+/+) mice.<h4>Conclusions</h4>These results differ from reported increases in k* and J(in) for AA, and in brain cPLA(2) expression, in serotonin reuptake transporter (5-HTT) knockout mice, and suggest that postsynaptic dopaminergic neurotransmission mechanisms involving AA are downregulated despite elevated DA in DAT(-/-) mice.

The invasion of squamous cell carcinoma (SCC) is a significant cause of morbidity and mortality. Here, we identify an E3 ligase, Traf6 and a de-ubiquitinating enzyme, Cezanne/ZA20D1, as important regulators of this process in organotypic models. Traf6 can promote the formation of Cdc42-dependent F-actin microspikes. Furthermore, Traf6 has a key role in autocrine interleukin-1β signalling in SCC cells, which in turn is required to drive the expression of tumour necrosis factor α (TNFα). TNFα acts in a paracrine manner to increase the invasion-promoting potential of carcinoma-associated fibroblasts (CAFs). Exogenous TNFα signalling can restore invasion in cells depleted of Traf6. In conclusion, Traf6 has two important roles in SCC invasion: it promotes cell intrinsic Cdc42-dependent regulation of the actin cytoskeleton and enables production of the paracrine signal, TNFα, that enhances the activity of CAFs.

Netrin is a chemotrophic factor known to regulate a number of neurodevelopmental processes, including cell migration, axon guidance, and synaptogenesis. Although the role of Netrin in synaptogenesis is conserved throughout evolution, the mechanisms by which it instructs synapse assembly are not understood. Here we identify a mechanism by which the Netrin receptor UNC-40/DCC instructs synaptic vesicle clustering in vivo. UNC-40 localized to presynaptic regions in response to Netrin. We show that UNC-40 interacted with CED-5/DOCK180 and instructed CED-5 presynaptic localization. CED-5 in turn signaled through CED-10/Rac1 and MIG-10/Lamellipodin to organize the actin cytoskeleton in presynaptic regions. Localization of this signaling pathway to presynaptic regions was necessary for synaptic vesicle clustering during synapse assembly but not for the subcellular localization of active zone proteins. Thus, vesicle clustering and localization of active zone proteins are instructed by separate pathways downstream of Netrin. Our data indicate that signaling modules known to organize the actin cytoskeleton during guidance can be co-opted to instruct synaptic vesicle clustering.

Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

With the completion of the human genome sequence, biomedical sciences have entered in the "omics" era, mainly due to high-throughput genomics techniques and the recent application of mass spectrometry to proteomics analyses. However, there is still a time lag between these technological advances and their application in the clinical setting. Our work is designed to build bridges between high-performance proteomics and clinical routine. Protein extracts were obtained from fresh frozen normal lung and non-small cell lung cancer samples. We applied a phosphopeptide enrichment followed by LC-MS/MS. Subsequent label-free quantification and bioinformatics analyses were performed. We assessed protein patterns on these samples, showing dozens of differential markers between normal and tumor tissue. Gene ontology and interactome analyses identified signaling pathways altered on tumor tissue. We have identified two proteins, PTRF/cavin-1 and MIF, which are differentially expressed between normal lung and non-small cell lung cancer. These potential biomarkers were validated using western blot and immunohistochemistry. The application of discovery-based proteomics analyses in clinical samples allowed us to identify new potential biomarkers and therapeutic targets in non-small cell lung cancer.

We report the clinical, neuropsychological, genetic, and radiological features of a large five-generation African-American kindred from the southern USA presenting with a progressive akinetic-rigid syndrome and severe dementia, but clinically insignificant chorea, due to mutations in junctophillin 3 (JPH3). Overt disease onset was in the mid-20s to late 30s with cognitive decline, REM sleep disturbance, or psychiatric features, followed by development of a levodopa-unresponsive akinetic-rigid motor syndrome. Dystonia and myoclonus were present in some subjects. A bedridden, nonverbal severely akinetic-rigid state developed within 10 to 15 years after onset. CTG repeat expansions ranged from 47 to 53. Imaging revealed generalized cerebral atrophy with severe striatal involvement and putaminal rim hyperintensity. Analysis of our kindred indicates that JPH3 mutations should be considered in the differential diagnosis of early-onset dementia and hypokinetic-rigid syndromes in individuals of African descent. Moreover, chorea may not be overtly manifest at presentation or during significant parts of the disease course.

Angelica sinensis (AS) is a traditional Chinese herbal medicine that has been formulated clinically to treat various form of skin trauma and to help wound healing. However, the mechanism by which it works remains a mystery. In this study we have established a new platform to evaluate the pharmacological effects of total AS herbal extracts as well as its major active component, ferulic acid (FA), using proteomic and biochemical analysis. Cytotoxic and proliferation-promoting concentrations of AS ethanol extracts (AS extract) and FA were tested, and then the cell extracts were subject to 2D PAGE analysis. We found 51 differentially expressed protein spots, and these were identified by mass spectrometry. Furthermore, biomolecular assays, involving collagen secretion, migration, and ROS measurements, gave results that are consistent with the proteomic analysis. In this work, we have demonstrated a whole range of pharmacological effects associated with Angelica sinensis that might be beneficial when developing a wound healing pharmaceutical formulation for the herbal medicine.

Background. Adult-Onset Still's Disease (AOSD) is an immune-mediated systemic disease with quotidian-spiking fever, rash, and inflammatory arthritis. Hyperferritinemia is a prominent feature, often used for screening. Methods. The key terms "ferritin" and "hyperferritinemia" were used to search PubMed and Medline and were cross-referenced with "Still's Disease." Results. Hyperferritinemia, although nonspecific, is particularly prevalent in AOSD. While most clinicians associate ferritin with iron metabolism, this is mostly true for the H isoform and not for the L isoform that tends to increase dramatically in hyperferritenemia. In these situations, hyperferritinemia is not associated with iron metabolism and may even mask an underlying iron deficiency. We review, in systematic fashion, the current basic science and clinical literature regarding the regulation of ferritin and its use in the diagnosis and management of AOSD. Conclusion. Serum hyperferritinemia in AOSD has been described for 2 decades, although its mechanism has not yet been completely elucidated. Regulation by proinflammatory cytokines such as interleukin (IL)-1b, IL-6, IL-18, MCSF, and INF-α provides a link to the disease pathogenesis and may explain rapid resolution of hyperferritinemia after targeted treatment and inhibition of key cytokines.

Synovial sarcoma (SS), an aggressive type of soft tissue tumor, occurs mostly in adolescents and young adults. The origin and molecular mechanism of the development of SS remain only partially known. Over 90% of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of SS18-SSX1 or SS18-SSX2 fusion genes. In recent years, several reports describing direct and indirect interactions of SS18-SSX1/SSX2 oncoproteins have been published. These reports suggest that the fusion proteins particularly affect the cell growth, cell proliferation, TP53 pathway, and chromatin remodeling mechanisms, contributing to SS oncogenesis. Additional research efforts are required to fully explore the protein-protein interactions of SS18-SSX oncoproteins and the pathways that are regulated by these partnerships for the development of effective targeted therapy.

This article reports the preparation of gold plasmonic transducers using a nanoparticle self-assembly/heating method and the characterization of the films using scattering-type scanning near-field optical microscopy (s-SNOM). Nanoparticle-polymer multilayer films were prepared by the layer-by-layer assembly on glass slides by alternating exposures to monodisperse Au(25) nanoparticles and ionic polymer linkers. Thermal evaporation of organic matters from the nanoparticle-polymer multilayer films at 600 °C allowed the nanoparticles to coalescence and form nanostructured films. Characterization of the nanostructured films generated from Au(25) nanoparticles using atomic force microscopy (AFM) showed that the films have rounded, small, island-like morphologies (d: 30-50 nm) with a pit in the center of many islands. However, further characterizations with s-SNOM revealed that the produced nanoislands contain a single gold cluster in a pit surrounded by donut-shaped dielectric species. Formation of such a structure is thought to be resulted from the embedding of gold clusters under the reorganized polysiloxane binder coatings and glass surfaces during heat treatment of the Au(25) nanoparticle multilayer films. The nanostructured films displayed strong surface plasmon resonance bands in UV-vis spectra with a peak absorbance occurring at ~545-550 nm. The optical sensing capability of the films was examined using D-glucose-functionalized gold island films with the interaction of Concanavalin A (ConA). The result showed that the adsorption of ConA on island films causes a large change in the LSPR band intensity.

Gap junctions (GJs) are expressed in most cell types of the nervous system, including neuronal stem cells, neurons, astrocytes, oligodendrocytes, cells of the blood brain barrier (endothelial cells and astrocytes) and under inflammatory conditions in microglia/macrophages. GJs connect cells by the docking of two hemichannels, one from each cell with each hemichannel being formed by 6 proteins named connexins (Cx). Unapposed hemichannels (uHC) also can be open on the surface of the cells allowing the release of different intracellular factors to the extracellular space. GJs provide a mechanism of cell-to-cell communication between adjacent cells that enables the direct exchange of intracellular messengers, such as calcium, nucleotides, IP(3), and diverse metabolites, as well as electrical signals that ultimately coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival and death. Despite their essential functions in physiological conditions, relatively little is known about the role of GJs and uHC in human diseases, especially within the nervous system. The focus of this review is to summarize recent findings related to the role of GJs and uHC in physiologic and pathologic conditions of the central nervous system.

<h4>Background</h4>Biomarkers released from the heart at early stage of ischemia are very important to diagnosis of ischemic heart disease and salvage myocytes from death. Known specific markers for blood tests including CK-MB, cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are released after the onset of significant necrosis instead of early ischemia. Thus, they are not good biomarkers to diagnose myocardial injury before necrosis happens. Therefore, in this study, we performed proteomic analysis on effluents from perfused human hearts of donors at different ischemic time.<h4>Results</h4>After global ischemia for 0 min, 30 min and 60 min at 4°C, effluents from five perfused hearts were analyzed respectively, by High performance liquid chromatography-Chip-Mass spectrometry (HPLC-Chip-MS) system. Total 196 highly reliable proteins were identified. 107 proteins were identified at the beginning of ischemia, 174 and 175 proteins at ischemic 30 min and ischemic 60 min, respectively. With the exception of cardiac troponin I and T, all known biomarkers for myocardial ischemia were detected in our study. However, there were four glycolytic enzymes and two targets of matrix metalloproteinase released significantly from the heart when ischemic time was increasing. These proteins were L-lactate dehydrogenase B(LDHB), glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate isomerase (GPI), phosphoglycerate mutase 2 (PGAM2), gelsolin and isoform 8 of titin. PGAM2, LDHB and titin were measured with enzyme-linked immunosorbent assays kits. The mean concentrations of LDHB and PGAM2 in samples showed an increasing trend when ischemic time was extending. In addition, 33% identified proteins are involved in metabolism. Protein to protein interaction network analysis showed glycolytic enzymes, such as isoform alpha-enolase of alpha-enolase, isoform 1 of triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, had more connections than other proteins in myocardial metabolism during ischemia.<h4>Conclusion</h4>It is the first time to use effluents of human perfused heart to study the proteins released during myocardial ischemia by HPLC-Chip-MS system. There might be many potential biomarkers for mild ischemic injury in myocardium, especially isoform 8 of titin and M-type of PGAM2 that are more specific in the cardiac tissue than in the others. Furthermore, glycolysis is one of the important conversions during early ischemia in myocardium. This finding may provide new insight into pathology and biology of myocardial ischemia, and potential diagnostic and therapeutic biomarkers.

Intense research on the pathogenesis of Huntington's disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, revealed multiple potential mechanisms, among which mitochondrial alterations had emerged as key determinants of the natural history of the disease. Pharmacological and genetic animal models of mitochondrial dysfunction in the striatum, which is mostly affected in HD corroborated a key role for these organelles in the pathogenesis of the disease. Here, we will give an account of the recent evidence indicating that the mitochondria-shaping machinery is altered in HD models and patients. Since its correction can counteract HD mitochondrial dysfunction and cellular damage, drugs impacting on mitochondrial shape are emerging as a new possibility of treatment for this devastating condition.

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials.

Although stress can alter the susceptibility of patients and animal models to convulsive epilepsy, little is known about the role of stress and glucocorticoid hormones in absence epilepsy. We measured the basal and acute stress-induced (foot-shocks: FS) concentrations of corticosterone in WAG/Rij rats, non-epileptic inbred ACI rats and outbred Wistar rats. The WAG/Rij strain is a genetic model for absence epilepsy and comorbidity for depression, which originates from the population of Wistar rats and, therefore, shares their genetic background. In a separate experiment, WAG/Rij rats were exposed to FS on three consecutive days. Electroencephalograms (EEGs) were recorded before and after FS, and the number of absence seizures (spike-wave-discharges, SWDs) was quantified. Both WAG/Rij rats and ACI rats exhibited elevated basal levels of corticosterone and a rapid corticosterone increase in response to acute stress. The WAG/Rij rats also displayed the most rapid normalization of corticosterone during the recovery phase compared to that of ACI and Wistar rats. FS had a biphasic effect on SWDs; an initial suppression was followed by an aggravation of the SWDs. By the third day, this aggravation of seizures was present in the hour preceding FS. This increase in SWDs may arise from anticipatory stress about the upcoming FS. Together, these results suggest that the distinct secretion profile of corticosterone found in WAG/Rij rats may contribute to the severity of the epileptic phenotype. Although the acute stressor results in an initial suppression of SWDs followed by an increase in SWDs, stress prior to a predictable negative event aggravates absences.

Neuroanatomists posit that the central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST) comprise two major nodes of a macrostructural forebrain entity termed the extended amygdala. The extended amygdala is thought to play a critical role in adaptive motivational behavior and is implicated in the pathophysiology of maladaptive fear and anxiety. Resting functional connectivity of the Ce was examined in 107 young anesthetized rhesus monkeys and 105 young humans using standard resting-state functional magnetic resonance imaging (fMRI) methods to assess temporal correlations across the brain. The data expand the neuroanatomical concept of the extended amygdala by finding, in both species, highly significant functional coupling between the Ce and the BST. These results support the use of in vivo functional imaging methods in nonhuman and human primates to probe the functional anatomy of major brain networks such as the extended amygdala.

In a previous study, an antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) and by gel filtration and HPLC. In this study, the functional peptide was characterized by partial acid digestion using HCl and HPLC. We obtained three peaks from the AFP-J, the first and third peaks were not active in the tested fungal strain. However, the second peak, which was named Potide-J, was active (MIC; 6.25 μg/mL) against Candida albicans. The amino acid sequences were analyzed by automated Edman degradation, and the amino acid sequence of Potide-J was determined to be Ala-Val-Cys-Glu-Asn-Asp-Leu-Asn-Cys-Cys. Mass spectrometry showed that its molecular mass was 1083.1 Da. Finally, we confirmed that a disulfide bond was present between Cys(3) and Cys(9) or Cys(10). Using this structure, Potide-J was synthesized via solid-phase methods. In these experiments, only the linear sequence was shown to display strong activity against Candida albicans. These results suggest that Potide-J may be an excellent candidate compound for the development of commercially applicable antibiotic agents.

Heat shock protein 60 (HSP60) is a highly conserved and multi-functional molecular chaperone that plays an essential role in both cellular metabolism and stress response. Portunus trituberculatus is an important marine fishery and aquaculture species, and water salinity condition influenced its artificial propagations significantly. In order to investigate the function of P. trituberculatus HSP60 against osmotic stress, P. trituberculatus HSP60 gene was firstly cloned. The full-length cDNA of PtHSP60 contains 1,743 nucleotides encoding 577 amino acids with a calculated molecular weight of 61.25 kDa. Multiple alignments indicated that the deduced amino acid sequences of PtHSP60 shared a high level of identity with invertebrate and vertebrate HSP60 sequence including shrimp, fruit fly, zebrafish, and human. The expression profiles of PtHSP60 at mRNA and protein levels under salinity treatment were investigated by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. It was found that the mRNA transcripts of PtHSP60 gene varied among different tissues under normal salinity conditions, and the antennal gland showed the highest expression level among the tissues tested. As for low salinity challenge, the mRNA expression of PtHSP60 gene was higher in the gill and appendicular muscle compared with other tissues, and gill and hypodermis represented the higher gene expressions during the hyperosmotic stress, which indicated that those tissues were salinity-sensitive tissues. In addition, salinity challenges significantly altered the expression of PtHSP60 at mRNA and protein level in a salinity- and time-dependent manner in P. trituberculatus gill tissue. The results indicate that PtHSP60 played important roles in mediating the salinity stress in P. trituberculatus.

No abstract available.

Panic disorder patients are vulnerable to recurrent panic attacks. Two neurochemical hypotheses have been proposed to explain this susceptibility. The first assumes that panic patients have deficient serotonergic inhibition of neurons localized in the dorsal periaqueductal gray matter of the midbrain that organize defensive reactions to cope with proximal threats and of sympathomotor control areas of the rostral ventrolateral medulla that generate most of the neurovegetative symptoms of the panic attack. The second suggests that endogenous opioids buffer normal subjects from the behavioral and physiological manifestations of the panic attack, and their deficit brings about heightened suffocation sensitivity and separation anxiety in panic patients, making them more vulnerable to panic attacks. Experimental results obtained in rats performing one-way escape in the elevated T-maze, an animal model of panic, indicate that the inhibitory action of serotonin on defense is connected with activation of endogenous opioids in the periaqueductal gray. This allows reconciliation of the serotonergic and opioidergic hypotheses of panic pathophysiology, the periaqueductal gray being the fulcrum of serotonin-opioid interaction.

Twenty years after gene therapy was introduced in the clinic, advances in the technique continue to garner headlines as successes pique the interest of clinicians, researchers, and the public. Gene therapy's appeal stems from its potential to revolutionize modern medical therapeutics by offering solutions to myriad diseases through treatments tailored to a specific individual's genetic code. Both viral and non-viral vectors have been used in the clinic, but the low transfection efficiencies when non-viral vectors are used have lead to an increased focus on engineering new gene delivery vectors. To address the challenges facing non-viral or synthetic vectors, specifically lipid-based carriers, we have focused on three main themes throughout our research: (1) The release of the nucleic acid from the carrier will increase gene transfection. (2) The use of biologically inspired designs, such as DNA binding proteins, to create lipids with peptide-based headgroups will improve delivery. (3) Mimicking the natural binding patterns observed within DNA, by using lipids having a nucleoside headgroup, will produce unique supramolecular assembles with high transfection efficiencies. The results presented in this Account demonstrate that engineering the chemical components of the lipid vectors to enhance nucleic acid binding and release kinetics can improve the cellular uptake and transfection efficacy of nucleic acids. Specifically, our research has shown that the incorporation of a charge-reversal moiety to initiate a shift of the lipid from positive to negative net charge improves transfection. In addition, by varying the composition of the spacer (rigid, flexible, short, long, or aromatic) between the cationic headgroup and the hydrophobic chains, we can tailor lipids to interact with different nucleic acids (DNA, RNA, siRNA) and accordingly affect delivery, uptake outcomes, and transfection efficiency. The introduction of a peptide headgroup into the lipid provides a mechanism to affect the binding of the lipid to the nucleic acid, to influence the supramolecular lipoplex structure, and to enhance gene transfection activity. Lastly, we discuss the in vitro successes that we have had when using lipids possessing a nucleoside headgroup to create unique self-assembled structures and to deliver DNA to cells. In this Account, we state our hypotheses and design elements as well as describe the techniques that we have used in our research to provide readers with the tools to characterize and engineer new vectors.

<h4>Background</h4>There is no standard serum biomarker used for diagnosis or early detection of recurrence for renal cell carcinoma (RCC) patients. MicroRNAs (miRNAs) are abundant and highly stable in blood serum, and have been recently described as powerful circulating biomarkers in a wide range of solid cancers. Our aim was to identify miRNA signature that can distinguish the blood serum of RCC patients and matched healthy controls and validate identified miRNAs as potential biomarkers for RCC.<h4>Methods</h4>In the screening phase of the study, blood serum of 15 RCC patients and 12 matched healthy controls were analyzed by use of the TaqMan Low-Density Arrays enabling parallel identification of expression levels of 667 miRNAs through qRT-PCR-based approach. In the validation phase, identified miRNAs were further evaluated on the independent group of 90 RCC patients and 35 matched healthy controls by use of individual qRT-PCR assays and statistically evaluated.<h4>Results</h4>We identified 30 miRNAs differentially expressed between serum of RCC patients and healthy controls: 19 miRNAs were up-regulated and 11 miRNAs were down-regulated in RCC patients. MiR-378, miR-451 and miR-150 were further evaluated in the independent group of patients, and two of them were successfully validated: levels of miR-378 were increased (p = 0.0003, AUC = 0.71), miR-451 levels were decreased (p < 0.0001, AUC = 0.77) in serum of RCC patients. Combination of miR-378 and miR-451 enable identification of RCC serum with the sensitivity of 81%, specificity 83% and AUC = 0.86.<h4>Conclusions</h4>Circulating miRNAs in serum are promising biomarkers in RCC.

<h4>Background</h4>Pneumococcal nasopharyngeal carriage precedes invasive infection and is the source for dissemination of the disease. Differences in sampling methodology, isolation or identification techniques, as well as the period (pre -or post-vaccination) when the study was performed, can influence the reported rates of colonization and the distribution of serotypes carried.<h4>Objectives</h4>To evaluate the prevalence and dynamics of pneumococcal nasopharyngeal colonization in healthy children aged 6-34 months attending a day care center with a high level of hygiene and no overcrowding. The study was performed 3-4 years after the 7-valent pneumococcal vaccine was introduced, using multiple methodologies to detect and characterize the isolates.<h4>Methods</h4>Over 12 months, 25 children were sampled three times, 53 children twice and 27 children once. Three Streptococcus pneumoniae typing techniques were used: Quellung, Pneumotest-Latex-kit and multiplex-polymerase chain reaction (PCR). The similarity of isolates of the same serotype was established by pulsed field gel electrophoresis (PFGE) and occasionally the multilocus sequence type (ST) was also determined.<h4>Results</h4>Overall pneumococcal carriage and multiple colonization rates were 89.5% (94/105) and 39%, respectively. Among 218 pneumococci detected, 21 different serotypes and 13 non-typeable isolates were found. The most prevalent serotypes were 19A, 16F and 15B. Serotypes 15B, 19A and 21 were mainly found as single carriage; in contrast serotypes 6B, 11A and 20, as well as infrequent serotypes, were isolated mainly as part of multiple carriage. Most 19A isolates were ST193 but most serotypes showed high genetic heterogeneity. Changes in the pneumococci colonizing each child were frequent and the same serotype detected on two occasions frequently showed a different genotype. By multiplex-PCR, 100% of pneumococci could be detected and 94% could be serotyped versus 80.3% by the Quellung reaction and Pneumotest-Latex in combination (p < 0.001).<h4>Conclusions</h4>Rates of S. pneumoniae carriage and multiple colonization were very high. Prevalent serotypes differed from those found in similar studies in the pre-vaccination period. In the same child, clearance of a pneumococcal strain and acquisition of a new one was frequent in a short period of time. The most effective technique for detecting pneumococcal nasopharyngeal carriers was multiplex-PCR.

<h4>Objective</h4>To understand the correlation of the acidic monomer/hydroxyapatite (HAp) reaction with the photopolymerization behaviour of self-etching adhesives with different aggressiveness.<h4>Methods</h4>Two commercial self-etching adhesives the strong Adper Prompt L-Pop (APLP, pH ~ 0.8) and the mild Adper Easy Bond (AEB, pH ~ 2.5) were used. HAp powders were incorporated into both adhesives to acquire solutions with concentrations of 0, 1, 3, 5, 7 wt%. The attenuated total reflectance Fourier transform infrared (ATR/FT-IR) technique was employed to collect the in situ spectra during light-curing, from which the degree of conversion (DC) and polymerization rate (PR) were calculated. The pH of each tested solution was also measured.<h4>Results</h4>Without HAp incorporation, the DC and PR of the strong APLP (7.8% and 0.12%/s, respectively) were much lower than those of the mild AEB (85.5% and 5.7%/s, respectively). The DC and PR of APLP displayed an apparent increasing trend with the HAp content. For example, the DC increased from 7.8% to 58.4% and the PR increased from 0.12 to 3.8%/s when the HAp content increased from 0 to 7 wt%. In contrast, the DC and PR of AEB were much less affected by the HAp content. The observations were correlated well with the spectral and pH changes, which indicated that APLP underwent a higher extent of chemical reaction with HAp than AEB.<h4>Conclusions</h4>The results disclosed the important role of the acidic monomer/HAp chemical reaction in improving the photopolymerization of the strong (low-pH) self-etching adhesives such as APLP. The phenomenon of polymerization improvement strongly depended on the adhesive aggressiveness.

Protein aggregation is a continuous process in our cells. Some proteins aggregate in a regulated manner required for different vital functional processes in the cells whereas other protein aggregates result from misfolding caused by various stressors. The decision to form an aggregate is largely made by chaperones and chaperone-assisted proteins. Proteins that are damaged beyond repair are degraded either by the proteasome or by the lysosome via autophagy. The aggregates can be degraded by the proteasome and by chaperone-mediated autophagy only after dissolution into soluble single peptide species. Hence, protein aggregates as such are degraded by macroautophagy. The selective degradation of protein aggregates by macroautophagy is called aggrephagy. Here we review the processes of aggregate formation, recognition, transport, and sequestration into autophagosomes by autophagy receptors and the role of aggrephagy in different protein aggregation diseases.

Lupus nephritis (LN) is one of the most serious complications of systemic lupus erythematosus (SLE) since it is the major predictor of poor prognosis. In susceptible individuals suffering of SLE, in situ formation and deposit of immune complexes (ICs) from apoptotic bodies occur in the kidneys as a result of an amplified epitope immunological response. IC glomerular deposits generate release of proinflammatory cytokines and cell adhesion molecules causing inflammation. This leads to monocytes and polymorphonuclear cells chemotaxis. Subsequent release of proteases generates endothelial injury and mesangial proliferation. Presence of ICs promotes adaptive immune response and causes dendritic cells to release type I interferon. This induces maturation and activation of infiltrating T cells, and amplification of Th2, Th1 and Th17 lymphocytes. Each of them, amplify B cells and activates macrophages to release more proinflammatory molecules, generating effector cells that cannot be modulated promoting kidney epithelial proliferation and fibrosis. Herein immunopathological findings of LN are reviewed.

The early developments of brain positron emission tomography (PET), including the methodological advances that have driven progress, are outlined. The considerable past achievements of brain PET have been summarized in collaboration with contributing experts in specific clinical applications including cerebrovascular disease, movement disorders, dementia, epilepsy, schizophrenia, addiction, depression and anxiety, brain tumors, drug development, and the normal healthy brain. Despite a history of improving methodology and considerable achievements, brain PET research activity is not growing and appears to have diminished. Assessments of the reasons for decline are presented and strategies proposed for reinvigorating brain PET research. Central to this is widening the access to advanced PET procedures through the introduction of lower cost cyclotron and radiochemistry technologies. The support and expertize of the existing major PET centers, and the recruitment of new biologists, bio-mathematicians and chemists to the field would be important for such a revival. New future applications need to be identified, the scope of targets imaged broadened, and the developed expertize exploited in other areas of medical research. Such reinvigoration of the field would enable PET to continue making significant contributions to advance the understanding of the normal and diseased brain and support the development of advanced treatments.

The common genetic variation of the serotonin transporter-linked polymorphic region (5-HTTLPR) has been related to depressive symptoms, in particular after stressful life events. Although it has been investigated in the past, results suggesting that the 5-HTTLPR genotype also affects hippocampal volume are often inconsistent and it remains unclear to what extent reduced hippocampal volume is influenced by the effect of stressful life events and 5-HTTLPR genotype. Moreover, sex, which is known to affect the prevalence of depression substantially, has not been taken into account when trying to disentangle the interactive effect of common genetic variation and environmental stressors on the hippocampus. We investigated this potentially relevant three-way interaction using an automatic magnetic resonance imaging (MRI)-based segmentation of the hippocampus in 357 healthy individuals. We determined the 5-HTTLPR genotype as a biallelic locus and childhood adversity (CA) using a standard questionnaire. An interaction for hippocampal volume was found between the factors sex, genotype, and severe CA (p=0.010) as well as an interaction between genotype and severe CA (p=0.007) in men only. Post hoc tests revealed that only male S'-allele carriers with severe CA had smaller hippocampi (p=0.002). Interestingly, there was no main effect of genotype in men, while female S'-allele carriers had smaller hippocampi than L'L' carriers (p=0.023). Our results indicate that sex modulates the interactive effect of the 5-HTTLPR genotype and CA on hippocampal volume. While the S'-allele is associated with hippocampal volume independent of CA in women, men only have smaller hippocampi if they carry the risk allele and experienced severe CA.

<h4>Background</h4>The role of serotonin transporter (SERT) gene polymorphism in irritable bowel syndrome (IBS) has been demonstrated. However, the expression of SERT mRNA and proteins in the colonic mucosa with different 5-HTT gene-linked polymorphic region (5-HTTLPR) genotypes remains unknown. We examined SERT mRNA and protein levels in colon biopsies from patients with different 5-HTTLPR genotypes and evaluated the links between the polymorphism and the expression levels.<h4>Methods</h4>Two hundred and fifty-four patients with IBS and 120 healthy subjects were studied. DNA samples were extracted from peripheral blood and genotyped by polymerase chain reaction (PCR). SERT mRNA and protein levels were evaluated by quantitative real time PCR and western blotting. The promoter efficiency of the serotonin transporter promoter (SERT-P) was evaluated with luciferase reporter system.<h4>Key results</h4>The frequency of the L/L genotype in C-IBS group was significantly higher than that in the control and D-IBS. However, the S/S genotype in D-IBS was significantly higher than that in C-IBS. The transcriptional efficiency of the L/L genotype was significantly higher than that in the L/S and S/S genotype. Patients with the L/L genotype demonstrated increased production of the SERT protein when compared with L/S and S/S patients. The l variant increased SERT promoter activity by 2.43-fold when compared with the s variant.<h4>Conclusions & inferences</h4>Polymorphism in the promoter region of the SERT gene can influence the expression of SERT mRNA and the levels of the SERT protein in the colonic mucosa, thereby playing a key role in motility-related symptoms of IBS patients.

Human rhinovirus (HRV) and other members of the enterovirus genus bind small-molecule antiviral compounds in a cavity buried within the viral capsid protein VP1. These compounds block the release of the viral protein VP4 and RNA from inside the capsid during the uncoating process. In addition, the antiviral compounds prevent "breathing" motions, the transient externalization of the N-terminal regions of VP1 and VP4 from the inside of intact viral capsid. The site for externalization of VP1/VP4 or release of RNA is likely between protomers, distant to the binding cavity for antiviral compounds. Molecular dynamics simulations were conducted to explore how the antiviral compound, WIN 52084, alters properties of the HRV 14 capsid through long-distance effect. We developed an approach to analyze capsid dynamics in terms of correlated radial motion and the shortest paths of correlated motions. In the absence of WIN, correlated radial motion is observed between residues separated by as much as 85 Å, a remarkably long distance. The most frequently populated path segments of the network were localized near the fivefold symmetry axis and included those connecting the N termini of VP1 and VP4 with other regions, in particular near twofold symmetry axes, of the capsid. The results provide evidence that the virus capsid exhibits concerted long-range dynamics, which have not been previously recognized. Moreover, the presence of WIN destroys this radial correlation network, suggesting that the underlying motions contribute to a mechanistic basis for the initial steps of VP1 and VP4 externalization and uncoating.

Gliomas are the most common and devastating central nervous system neoplasms. A gender bias exists in their development: females are at lower risk than males, implicating estrogen-mediated protective effects. Estrogen functions are mediated by two estrogen receptor (ER) subtypes: ERα, which functions as tumor promoter, and ERβ, which functions as tumor suppressor. We examined the potential use of ERβ agonists as a novel therapeutic to curb the growth of gliomas. Western analysis of six glioma model cells showed detectable expression of ERβ with little or no ERα. Treatment of glioma cells with ERβ agonists resulted in significant decrease in proliferation. Immunohistochemical analysis of tumor tissues revealed that ERβ expression is downregulated in high-grade gliomas. We found that ERβ agonists promote both expression and tumor-suppressive functions of ERβ in glioma cells. Liquiritigenin, a plant-derived ERβ agonist significantly reduced in vivo tumor growth in a xenograft model. Compared with control mice, animals treated with liquiritigenin had greater than 50% reduction in tumor volume and size. Immunohistochemical analysis of tumors revealed a significant increase in the nuclear ERβ expression with a concomitant decrease in cell proliferation in the liquiritigenin-treated group. Our results suggest that ERβ signaling has a tumor-suppressive function in gliomas. Because ERβ agonists are currently in clinical trials and are well tolerated with fewer side effects, identification of an ERβ agonist as a therapeutic agent can be readily extended to clinical use with current chemotherapies, providing an additional tool for enhancing survival in glioma patients.

Pectin is a normal constituent of cell walls of green plants. When supplied externally to live cells or walls isolated from the large-celled green alga Chara corallina, pectin removes calcium from load-bearing cross-links in the wall, loosening the structure and allowing it to deform more rapidly under the action of turgor pressure. New Ca(2+) enters the vacated positions in the wall and the externally supplied pectin binds to the wall, depositing new wall material that strengthens the wall. A calcium pectate cycle has been proposed for these sub-reactions. In the present work, the cycle was tested in C. corallina by depriving the wall of external Ca(2+) while allowing the cycle to run. The prediction is that growth would eventually be disrupted by a lack of adequate deposition of new wall. The test involved adding pectate or the calcium chelator EGTA to the Ca(2+)-containing culture medium to bind the calcium while the cycle ran in live cells. After growth accelerated, turgor and growth eventually decreased, followed by an abrupt turgor loss and growth cessation. The same experiment with isolated walls suggested the walls of live cells became unable to support the plasma membrane. If instead the pectate or EGTA was replaced with fresh Ca(2+)-containing culture medium during the initial acceleration in live cells, growth was not disrupted and returned to the original rates. The operation of the cycle was thus confirmed, providing further evidence that growth rates and wall biosynthesis are controlled by these sub-reactions in plant cell walls.

Genome-wide transcriptional profiling was used to characterize the molecular underpinnings of neocortical organization in rhesus macaque, including cortical areal specialization and laminar cell-type diversity. Microarray analysis of individual cortical layers across sensorimotor and association cortices identified robust and specific molecular signatures for individual cortical layers and areas, prominently involving genes associated with specialized neuronal function. Overall, transcriptome-based relationships were related to spatial proximity, being strongest between neighboring cortical areas and between proximal layers. Primary visual cortex (V1) displayed the most distinctive gene expression compared to other cortical regions in rhesus and human, both in the specialized layer 4 as well as other layers. Laminar patterns were more similar between macaque and human compared to mouse, as was the unique V1 profile that was not observed in mouse. These data provide a unique resource detailing neocortical transcription patterns in a nonhuman primate with great similarity in gene expression to human.

Retinoblastoma is the most common intraocular tumor in children. Malfunctioning of many signaling pathways regulating cell survival or apoptosis, make the disease more vulnerable. Notably, resistance to chemotherapy mediated by MRP-1, lung-resistance protein (LRP) is the most challenging aspect to treat this disease. Presently, much attention has been given to the recently developed anticancer drug nutlin-3a because of its non-genotoxic nature and potency to activate tumor suppressor protein p53. However, being a substrate of multidrug resistance protein MRP1 and Pgp its application has become limited. Currently, research has step towards reversing Multi drug resistance (MDR) by using curcumin, however its clinical relevance is restricted by plasma instability and poor bioavailability. In the present investigation we tried to encapsulate nutlin-3a and curcumin in PLGA nanoparticle (NPs) surface functionalized with folate to enhance therapeutic potential of nutlin-3a by modulating MDR. We document that curcumin can inhibit the expression of MRP-1 and LRP gene/protein in a concentration dependent manner in Y79 cells. In vitro cellular cytotoxicity, cell cycle analysis and apoptosis studies were done to compare the effectiveness of native drugs (single or combined) and single or dual drug loaded nanoparticles (unconjugated/folate conjugated). The result demonstrated an augmented therapeutic efficacy of targeted dual drug loaded NPs (Fol-Nut-Cur-NPs) over other formulation. Enhanced expression or down regulation of proapoptotic/antiapoptotic proteins respectively and down-regulation of bcl2 and NFκB gene/protein by Fol-Nut-Cur-NPs substantiate the above findings. This is the first investigation exploring the role of curcumin as MDR modulator to enhance the therapeutic potentiality of nutlin-3a, which may opens new direction for targeting cancer with multidrug resistance phenotype.

Aside from a decrease in the high-density lipoprotein (HDL) cholesterol levels, qualitative abnormalities of HDL can contribute to an increase in cardiovascular (CV) risk in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Dysfunctional HDL leads to an alteration of reverse cholesterol transport and the antioxidant and anti-inflammatory properties of HDL. In this study, a quantitative proteomics approach, based on iTRAQ labeling and nanoflow liquid chromatography mass spectrometry analysis, was used to generate detailed data on HDL-associated proteins. The HDL composition was compared between seven chronic HD patients and a pool of seven healthy controls. To confirm the proteomics results, specific biochemical assays were then performed in triplicate in the 14 samples as well as 46 sex-matched independent chronic HD patients and healthy volunteers. Of the 122 proteins identified in the HDL fraction, 40 were differentially expressed between the healthy volunteers and the HD patients. These proteins are involved in many HDL functions, including lipid metabolism, the acute inflammatory response, complement activation, the regulation of lipoprotein oxidation, and metal cation homeostasis. Among the identified proteins, apolipoprotein C-II and apolipoprotein C-III were significantly increased in the HDL fraction of HD patients whereas serotransferrin was decreased. In this study, we identified new markers of potential relevance to the pathways linked to HDL dysfunction in HD. Proteomic analysis of the HDL fraction provides an efficient method to identify new and uncharacterized candidate biomarkers of CV risk in HD patients.

No abstract available.

In polyglutamine (polyQ) containing fragments of the Huntington's disease protein huntingtin (htt), the N-terminal 17 amino acid htt(NT) segment serves as the core of α-helical oligomers whose reversible assembly locally concentrates the polyQ segments, thereby facilitating polyQ amyloid nucleation. A variety of aggregation inhibitors have been described that achieve their effects by neutralizing this concentrating function of the htt(NT) segment. In this paper we characterize the nature and limits of this inhibition for three means of suppressing htt(NT)-mediated aggregation. We show that the previously described action of htt(NT) peptide-based inhibitors is solely due to their ability to suppress the htt(NT)-mediated aggregation pathway. That is, under htt(NT) inhibition, nucleation of polyQ amyloid formation by a previously described alternative nucleation mechanism proceeds unabated and transiently dominates the aggregation process. Removal of the bulk of the htt(NT) segment by proteolysis or mutagenesis also blocks the htt(NT)-mediated pathway, allowing the alternative nucleation pathway to dominate. In contrast, the previously described immunoglobulin-based inhibitor, the antihtt(NT) V(L) 12.3 protein, effectively blocks both amyloid pathways, leading to stable accumulation of nonamyloid oligomers. These data show that the htt(NT)-dependent and -independent pathways of amyloid nucleation in polyQ-containing htt fragments are in direct kinetic competition. The results illustrate how amyloid polymorphism depends on assembly mechanism and kinetics and have implications for how the intracellular environment can influence aggregation pathways.

<h4>Introduction</h4>A younger age at menarche and an older age at menopause are well established risk factors for breast cancer. Recent genome-wide association studies have identified several novel genetic loci associated with these two traits. However, the association between these loci and breast cancer risk is unknown.<h4>Methods</h4>In this study, we investigated 19 and 17 newly identified single nucleotide polymorphisms (SNPs) from the ReproGen Consortium that have been associated with age at menarche and age at natural menopause, respectively, and assessed their associations with breast cancer risk in 6 population-based studies among up to 3,683 breast cancer cases and 34,174 controls in white women of European ancestry. In addition, we used these SNPs to calculate genetic risk scores (GRSs) based on their associations with each trait.<h4>Results</h4>After adjusting for age and potential population stratification, two age at menarche associated SNPs (rs1079866 and rs7821178) and one age at natural menopause associated SNP (rs2517388) were associated with breast cancer risk (p values, 0.003, 0.009 and 0.023, respectively). The odds ratios for breast cancer corresponding to per-risk-allele were 1.14 (95% CI, 1.05 to 1.24), 1.08 (95% CI, 1.02 to 1.15) and 1.10 (95% CI, 1.01 to 1.20), respectively, and were in the direction predicted by their associations with age at menarche or age at natural menopause. These associations did not appear to be attenuated by further controlling for self-reported age at menarche, age at natural menopause, or known breast cancer susceptibility loci. Although we did not observe a statistically significant association between any GRS for reproductive aging and breast cancer risk, the 4th and 5th highest quintiles of the younger age at menarche GRS had odds ratios of 1.14 (95% CI, 1.01 to 1.28) and 1.13 (95% CI, 1.00 to 1.27), respectively, compared to the lowest quintile.<h4>Conclusions</h4>Our study suggests that three genetic variants, independent of their associations with age at menarche or age at natural menopause, were associated with breast cancer risk and may contribute modestly to breast cancer risk prediction; however, the combination of the 19 age at menarche or the 17 age at natural menopause associated SNPs did not appear to be useful for identifying a high risk subgroup for breast cancer.

<h4>Background</h4>Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described.<h4>Results</h4>Our results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp.<h4>Conclusions</h4>EGF activates TTP expression via ELK-1 and EGR-1 transcription factors.

No abstract available.

The apoptotic effector caspase-6 (CASP6) has been clearly identified as a drug target due to its strong association with neurodegeneration and axonal pruning events as well as its crucial roles in Huntington disease and Alzheimer disease. CASP6 activity is suppressed by ARK5-mediated phosphorylation at Ser(257) with an unclear mechanism. In this work, we solved crystal structures of ΔproCASP6S257E and p20/p10S257E, which mimicked the phosphorylated CASP6 zymogen and activated CASP6, respectively. The structural investigation combined with extensive biochemical assay and molecular dynamics simulation studies revealed that phosphorylation on Ser(257) inhibited self-activation of CASP6 zymogen by "locking" the enzyme in the TEVD(193)-bound "inhibited state." The structural and biochemical results also showed that phosphorylation on Ser(257) inhibited the CASP6 activity by steric hindrance. These results disclosed the inhibition mechanism of CASP6 phosphorylation and laid the foundation for a new strategy of rational CASP6 drug design.

Oligodendrocyte precursor cells (OPCs) are a unique type of glia that are responsible for the myelination of the central nervous system. OPC migration is important for myelin formation during central nervous system development and repair. However, the precise extracellular and intracellular mechanisms that regulate OPC migration remain elusive. Slits were reported to regulate neurodevelopmental processes such as migration, adhesion, axon guidance, and elongation through binding to roundabout receptors (Robos). However, the potential roles of Slits/Robos in oligodendrocytes remain unknown. In this study, Slit2 was found to be involved in regulating the dispersal of OPCs through the association between Robo1 and Fyn. Initially, we examined the expression of Robos in OPCs both in vitro and in vivo. Subsequently, the Boyden chamber assay showed that Slit2 could inhibit OPC migration. RoboN, a specific inhibitor of Robos, could significantly attenuate this effect. The effects were confirmed through the explant migration assay. Furthermore, treating OPCs with Slit2 protein deactivated Fyn and increased the level of activated RhoA-GTP. Finally, Fyn was found to form complexes with Robo1, but this association was decreased after Slit2 stimulation. Thus, we demonstrate for the first time that Slit2 regulates the dispersal of oligodendrocyte precursor cells through Fyn and RhoA signaling.

Huntington disease is a genetic neurodegenerative disorder that arises from an expanded polyglutamine region in the N terminus of the HD gene product, huntingtin. Protein inclusions comprised of N-terminal fragments of mutant huntingtin are a characteristic feature of disease, though are likely to play a protective role rather than a causative one in neurodegeneration. Soluble oligomeric assemblies of huntingtin formed early in the aggregation process are candidate toxic species in HD. In the present study, we established an in vitro system to generate recombinant huntingtin in mammalian cells. Using both denaturing and native gel analysis, we have identified novel oligomeric forms of mammalian-derived expanded huntingtin exon-1 N-terminal fragment. These species are transient and were not previously detected using bacterially expressed exon-1 protein. Importantly, these species are recognized by 3B5H10, an antibody that recognizes a two-stranded hairpin conformation of expanded polyglutamine believed to be associated with a toxic form of huntingtin. Interestingly, comparable oligomeric species were not observed for expanded huntingtin shortstop, a 117-amino acid fragment of huntingtin shown previously in mammalian cell lines and transgenic mice, and here in primary cortical neurons, to be non-toxic. Further, we demonstrate that expanded huntingtin shortstop has a reduced ability to form amyloid-like fibrils characteristic of the aggregation pathway for toxic expanded polyglutamine proteins. Taken together, these data provide a possible candidate toxic species in HD. In addition, these studies demonstrate the fundamental differences in early aggregation events between mutant huntingtin exon-1 and shortstop proteins that may underlie the differences in toxicity.

<h4>Background</h4>Most malignancies identified by thyroid fine-needle aspiration (FNA) are papillary thyroid carcinoma (PTC). This study sought to determine if clinically adverse features of PTC correlate with the preceding cytologic diagnosis.<h4>Methods</h4>Thyroid FNA diagnoses were correlated with subsequent histopathologic findings.<h4>Results</h4>From 6175 thyroid FNAs, histologic follow-up confirmed PTC in 52 of 184 (28%) FNAs with atypia of undetermined significance (AUS), 52 of 190 (27%) FNAs suspicious for follicular neoplasm, 182 of 229 (79%) FNAs that were suspicious for malignancy, and 188 of 198 (95%) FNAs that were malignant (M). Sex, age, and disease multifocality did not differ among FNA diagnosis groups. However, PTCs following an M FNA were more likely to have a higher American Joint Committee on Cancer T and N stage, and have lymphovascular invasion and/or extrathyroidal extension. Two patients had distant metastasis at initial surgery, whereas 16 developed subsequent recurrence and/or metastasis; all had a preceding M FNA. High-risk histologic subtypes of PTC also stratify to the M category, accounting at least partly for the association of cytologic diagnosis with adverse pathological parameters. Conversely, follicular variants of PTC predominate in non-M categories.<h4>Conclusions</h4>The Bethesda System for Reporting Thyroid Cytopathology conveys malignancy risk, but also predicts the presence of pathological risk factors and disease progression when the malignancy is PTC. M diagnoses identify higher risk PTCs, whereas AUS diagnoses identify low-risk PTCs, mostly follicular variants. These findings support the concept of conservative clinical management for some patients with AUS, while suggesting that a central neck dissection may be routinely justified in some patients with a M FNA.

The Saccharomyces Genome Database (SGD) is compiling and annotating a comprehensive catalogue of functional sequence elements identified in the budding yeast genome. Recent advances in deep sequencing technologies have enabled for example, global analyses of transcription profiling and assembly of maps of transcription factor occupancy and higher order chromatin organization, at nucleotide level resolution. With this growing influx of published genome-scale data, come new challenges for their storage, display, analysis and integration. Here, we describe SGD's progress in the creation of a consolidated resource for genome sequence elements in the budding yeast, the considerations taken in its design and the lessons learned thus far. The data within this collection can be accessed at http://browse.yeastgenome.org and downloaded from http://downloads.yeastgenome.org. DATABASE URL: http://www.yeastgenome.org.

In the past few years, there have been significant advances in the research on cancer stem cells (CSCs). The emerging evidences have demonstrated that CSCs and epithelial-mesenchymal transition (EMT)-type cells, which share molecular characteristics with CSCs, play critical roles in drug resistance, invasion, and metastasis. Pancreatic cancer (PC) has a high mortality due to both intrinsic (de novo) and extrinsic (acquired) drug resistance, leading to increased invasive and metastatic potential of PC cells. Therefore, targeting pancreatic CSCs and EMT-type cells could be a novel therapeutic strategy for the treatment of PC. In this article, we will review the current state of our knowledge on the role of pancreatic CSCs and EMT-type cells, and summarize the novel therapeutic strategies that could target pancreatic CSCs and EMT-type cells, leading to the reversal of EMT phenotype, the induction of drug sensitivity, and the inhibition of invasion and metastasis of PC, which is expected to yield better treatment outcome.

An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.

<h4>Background</h4>In Drosophila melanogaster, the dosage-compensation system that equalizes X-linked gene expression between males and females, thereby assuring that an appropriate balance is maintained between the expression of genes on the X chromosome(s) and the autosomes, is at least partially mediated by the Male-Specific Lethal (MSL) complex. This complex binds to genes with a preference for exons on the male X chromosome with a 3' bias, and it targets most expressed genes on the X chromosome. However, a number of genes are expressed but not targeted by the complex. High affinity sites seem to be responsible for initial recruitment of the complex to the X chromosome, but the targeting to and within individual genes is poorly understood.<h4>Results</h4>We have extensively examined X chromosome sequence variation within five types of gene features (promoters, 5' UTRs, coding sequences, introns, 3' UTRs) and intergenic sequences, and assessed its potential involvement in dosage compensation. Presented results show that: the X chromosome has a distinct sequence composition within its gene features; some of the detected variation correlates with genes targeted by the MSL-complex; the insulator protein BEAF-32 preferentially binds upstream of MSL-bound genes; BEAF-32 and MOF co-localizes in promoters; and that bound genes have a distinct sequence composition that shows a 3' bias within coding sequence.<h4>Conclusions</h4>Although, many strongly bound genes are close to a high affinity site neither our promoter motif nor our coding sequence signatures show any correlation to HAS. Based on the results presented here, we believe that there are sequences in the promoters and coding sequences of targeted genes that have the potential to direct the secondary spreading of the MSL-complex to nearby genes.

<h4>Introduction</h4>In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1.<h4>Methods</h4>IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors.<h4>Results</h4>IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER+ breast cancer cells.<h4>Conclusion</h4>his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER+ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.

<h4>Background</h4>The prevalence of childhood obesity has increased worldwide, which is a serious concern as obesity is associated with many negative immediate and long-term health consequences. Therefore, the treatment of overweight and obesity in children and adolescents is strongly recommended. Inpatient weight-loss programs have shown to be effective particularly regarding short-term weight-loss, whilst little is known both on the long-term effects of this treatment and the determinants of successful weight-loss and subsequent weight maintenance.The purpose of this study is to evaluate the short, middle and long-term effects of an inpatient weight-loss program for children and adolescents and to investigate the likely determinants of weight changes, whereby the primary focus lies on the potential role of differences in polymorphisms of adiposity-relevant genes.<h4>Methods/design</h4>The study involves overweight and obese children and adolescents aged 6 to 19 years, who participate in an inpatient weight-loss program for 4 to 6 weeks. It started in 2006 and it is planned to include 1,500 participants by 2013. The intervention focuses on diet, physical activity and behavior therapy. Measurements are taken at the start and the end of the intervention and comprise blood analyses (DNA, lipid and glucose metabolism, adipokines and inflammatory markers), anthropometry (body weight, height and waist circumference), blood pressure, pubertal stage, and exercise capacity. Physical activity, dietary habits, quality of life, and family background are assessed by questionnaires. Follow-up assessments are performed 6 months, 1, 2, 5 and 10 years after the intervention: Children will complete the same questionnaires at all time points and visit their general practitioner for examination of anthropometric parameters, blood pressure and assessment of pubertal stage. At the 5 and 10 year follow-ups, blood parameters and exercise capacity will be additionally measured.<h4>Discussion</h4>Apart from illustrating the short, middle and long-term effects of an inpatient weight-loss program, this study will contribute to a better understanding of inter-individual differences in the regulation of body weight, taking into account the role of genetic predisposition and lifestyle factors.<h4>Trial registration</h4>NCT01067157.

From the 19th century to the present, the complex indole alkaloid strychnine has engaged the chemical community. In this Review, we examine why strychnine has been and remains today an important target for directed synthesis efforts. A selection of the diverse syntheses of strychnine is discussed with the aim of identifying their influence on the evolution of the strategy and tactics of organic synthesis.

Magnetic resonance imaging (MRI) of solids is rarely attempted. One of the main reasons is that the broader MR linewidths, compared to the narrow resonance of the hydrogen ((1)H) in free water, limit both the attainable spatial resolution and the signal-to-noise ratio. Basic physics research, stimulated by the quest to build a quantum computer, gave rise to a unique MR pulse sequence that offers a solution to this long-standing problem. The "quadratic echo" significantly narrows the broad MR spectrum of solids. Applying field gradients in sync with this line-narrowing sequence offers a fresh approach to carry out MRI of hard and soft solids with high spatial resolution and with a wide range of potential uses. Here we demonstrate that this method can be used to carry out three-dimensional MRI of the phosphorus ((31)P) in ex vivo bone and soft tissue samples.

Growing evidence suggests serotonin's role in anxiety and depression is mediated by its effects on learned fear associations. Pharmacological and genetic manipulations of serotonin signaling in mice alter the retention of fear extinction learning, which is inversely associated with anxious temperament in mice and humans. Here, we test whether genetic variation in serotonin signaling in the form of a common human serotonin transporter polyadenylation polymorphism (STPP/rs3813034) is associated with spontaneous fear recovery after extinction. We show that the risk allele of this polymorphism is associated with impaired retention of fear extinction memory and heightened anxiety and depressive symptoms. These STPP associations in humans mirror the phenotypic effects of serotonin transporter knockout in mice, highlighting the STPP as a potential genetic locus underlying interindividual differences in serotonin transporter function in humans. Furthermore, we show that the serotonin transporter polyadenylation profile associated with the STPP risk allele is altered through the chronic administration of fluoxetine, a treatment that also facilitates retention of extinction learning. The propensity to form persistent fear associations due to poor extinction recall may be an intermediate phenotype mediating the effects of genetic variation in serotonergic function on anxiety and depression. The consistency and specificity of these data across species provide robust support for this hypothesis and suggest that the little-studied STPP may be an important risk factor for mood and anxiety disorders in humans.

Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT(1A) and 5HT(2A) receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments.

The pore-forming Ca(2+) channel subunit Ca(V)3.2 mediates a low voltage-activated (T-type) Ca(2+) current (I(CaT)) that contributes pivotally to neuronal and cardiac pacemaker activity. Despite the importance of tightly regulated Ca(V)3.2 levels, the mechanisms regulating its transcriptional dynamics are not well understood. Here, we have identified two key factors that up- and down-regulate the expression of the gene encoding Ca(V)3.2 (Cacna1h). First, we determined the promoter region and observed several stimulatory and inhibitory clusters. Furthermore, we found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the Ca(V)3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the Ca(V)3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo. Subsequent chromatin immunoprecipitation assays in NG108-15 cells and mouse hippocampi confirmed specific Egr1 binding to the Ca(V)3.2 promoter. Congruently, whole-cell I(CaT) values were significantly larger after Egr1 overexpression. Intriguingly, Egr1-induced activation of the Ca(V)3.2 promoter was effectively counteracted by the repressor element 1-silencing transcription factor (REST). Thus, Egr1 and REST can bi-directionally regulate Ca(V)3.2 promoter activity and mRNA expression and, hence, the size of I(CaT). This mechanism has critical implications for the regulation of neuronal and cardiac Ca(2+) homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy.

<h4>Background and methods</h4>Familial aggregation of lung cancer exists after accounting for cigarette smoking. However, the extent to which family history affects risk by smoking status, histology, relative type and ethnicity is not well described. This pooled analysis included 24 case-control studies in the International Lung Cancer Consortium. Each study collected age of onset/interview, gender, race/ethnicity, cigarette smoking, histology and first-degree family history of lung cancer. Data from 24,380 lung cancer cases and 23,305 healthy controls were analysed. Unconditional logistic regression models and generalised estimating equations were used to estimate odds ratios and 95% confidence intervals.<h4>Results</h4>Individuals with a first-degree relative with lung cancer had a 1.51-fold increase in the risk of lung cancer, after adjustment for smoking and other potential confounders (95% CI: 1.39, 1.63). The association was strongest for those with a family history in a sibling, after adjustment (odds ratios (OR) = 1.82, 95% CI: 1.62, 2.05). No modifying effect by histologic type was found. Never smokers showed a lower association with positive familial history of lung cancer (OR = 1.25, 95% CI: 1.03, 1.52), slightly stronger for those with an affected sibling (OR = 1.44, 95% CI: 1.07, 1.93), after adjustment.<h4>Conclusions</h4>The occurrence of lung cancer among never smokers and similar magnitudes of the effect of family history on lung cancer risk across histological types suggests familial aggregation of lung cancer is independent of those risks associated with cigarette smoking. While the role of genetic variation in the aetiology of lung cancer remains to be fully characterised, family history assessment is immediately available and those with a positive history represent a higher risk group.

<h4>Background</h4>Necdin, a MAGE family protein expressed primarily in the nervous system, has been shown to interact with both nuclear and cytoplasmic proteins, but the mechanism of its nucleocytoplasmic transport are unknown.<h4>Methodology/principal findings</h4>We carried out a large-scale interaction screen using necdin as a bait in the yeast RRS system, and found a wide range of potential interactors with different subcellular localizations, including over 60 new candidates for direct binding to necdin. Integration of these interactions into a comprehensive network revealed a number of coherent interaction modules, including a cytoplasmic module connecting to necdin through huntingtin-associated protein 1 (Hap1), dynactin and hip-1 protein interactor (Hippi); a nuclear P53 and Creb-binding-protein (Crebbp) module, connecting through Crebbp and WW domain-containing transcription regulator protein 1 (Wwtr1); and a nucleocytoplasmic transport module, connecting through transportins 1 and 2. We validated the necdin-transportin1 interaction and characterized a sequence motif in necdin that modulates karyopherin interaction. Surprisingly, a D234P necdin mutant showed enhanced binding to both transportin1 and importin β1. Finally, exclusion of necdin from the nucleus triggered extensive cell death.<h4>Conclusions/significance</h4>These data suggest that necdin has multiple roles within protein complexes in different subcellular compartments, and indicate that it can utilize multiple karyopherin-dependent pathways to modulate its localization.

Epilepsy is characterized by spontaneous recurrent seizures and comprises a diverse group of syndromes with different etiologies. Epileptogenesis refers to the process whereby the brain becomes epileptic and can be related to several factors, such as acquired structural brain lesions, inborn brain malformations, alterations in neuronal signaling, and defects in maturation and plasticity of neuronal networks. In this review, we will focus on alterations of brain development that lead to an hyperexcitability phenotype in adulthood, providing examples from both animal and human studies. Malformations of cortical development (including focal cortical dysplasia, lissencephaly, heterotopia, and polymicrogyria) are frequently epileptogenic and result from defects in cell proliferation in the germinal zone and/or impaired neuronal migration and differentiation. Delayed or reduced arrival of inhibitory interneurons into the cortical plate is another possible cause of epileptogenesis. GABAergic neurons are generated during early development in the ganglionic eminences, and failure to pursue migration toward the cortex alters the excitatory/inhibitory balance resulting in aberrant network hyperexcitability. More subtle defects in the developmental assembly of excitatory and inhibitory synapses are also involved in epilepsy. For example, mutations in the presynaptic proteins synapsins and SNAP-25 cause derangements of synaptic transmission and plasticity which underlie appearance of an epileptic phenotype. Finally, there is evidence that defects in synapse elimination and remodeling during early "critical periods" can trigger hyperexcitability later in life. Further clarification of the developmental pathways to epilepsy has important implications for disease prevention and therapy.

<h4>Motivation</h4>This work constructs a closed loop Bayesian Network framework for predictive medicine via integrative analysis of publicly available gene expression findings pertaining to various diseases.<h4>Results</h4>An automated pipeline was successfully constructed. Integrative models were made based on gene expression data obtained from GEO experiments relating to four different diseases using Bayesian statistical methods. Many of these models demonstrated a high level of accuracy and predictive ability. The approach described in this paper can be applied to any complex disorder and can include any number and type of genome-scale studies.

<h4>Unlabelled</h4>Clinical application of the prognostic gene expression signature has been delayed due to the large number of genes and complexity of prediction algorithms. In the current study we aimed to develop an easy-to-use risk score with a limited number of genes that can robustly predict prognosis of patients with hepatocellular carcinoma (HCC). The risk score was developed using Cox coefficient values of 65 genes in the training set (n = 139) and its robustness was validated in test sets (n = 292). The risk score was a highly significant predictor of overall survival (OS) in the first test cohort (P = 5.6 × 10(-5), n = 100) and the second test cohort (P = 5.0 × 10(-5) , n = 192). In multivariate analysis, the risk score was a significant risk factor among clinical variables examined together (hazard ratio [HR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = 0.001 for OS).<h4>Conclusion</h4>The risk score classifier we have developed can identify two clinically distinct HCC subtypes at early and late stages of the disease in a simple and highly reproducible manner across multiple datasets.

Dendrites from a single neuron may be highly branched but typically do not overlap. Self-avoidance behavior has been shown to depend on cell-specific membrane proteins that trigger mutual repulsion. Here we report the unexpected discovery that a diffusible cue, the axon guidance protein UNC-6 (Netrin), is required for self-avoidance of sister dendrites from the PVD nociceptive neuron in Caenorhabditis elegans. We used time-lapse imaging to show that dendrites fail to withdraw upon mutual contact in the absence of UNC-6 signaling. We propose a model in which the UNC-40 (Deleted in Colorectal Cancer; DCC) receptor captures UNC-6 at the tips of growing dendrites for interaction with UNC-5 on the apposing branch to induce mutual repulsion. UNC-40 also responds to dendritic contact through another pathway that is independent of UNC-6. Our findings offer a new model for how an evolutionarily conserved morphogenic cue and its cognate receptors can pattern a fundamental feature of dendritic architecture.

We trained Segway, a dynamic Bayesian network method, simultaneously on chromatin data from multiple experiments, including positions of histone modifications, transcription-factor binding and open chromatin, all derived from a human chronic myeloid leukemia cell line. In an unsupervised fashion, we identified patterns associated with transcription start sites, gene ends, enhancers, transcriptional regulator CTCF-binding regions and repressed regions. Software and genome browser tracks are at http://noble.gs.washington.edu/proj/segway/.

Although the involvement of immune mechanisms in multiple sclerosis (MS) is undisputed, some argue that there is insufficient evidence to support the hypothesis that MS is an autoimmune disease, and that the difference between immune- and autoimmune disease mechanisms has yet to be clearly delineated. Uncertainties surrounding MS disease pathogenesis and the modest efficacy of currently used disease modifying treatments (DMTs) in the prevention of disability, warrant the need to explore other possibilities. It is evident from the literature that people diagnosed with MS differ widely in symptoms and clinical outcome--some patients have a benign disease course over many years without requiring any DMTs. Attempting to include all patients into a single entity is an oversimplification and may obscure important observations with therapeutic consequences. In this review we advocate an individualised approach named Pathology Supported Genetic Testing (PSGT), in which genetic tests are combined with biochemical measurements in order to identify subgroups of patients requiring different treatments. Iron dysregulation in MS is used as an example of how this approach may benefit patients. The theory that iron deposition in the brain contributes to MS pathogenesis has caused uncertainty among patients as to whether they should avoid iron. However, the fact that a subgroup of people diagnosed with MS show clinical improvement when they are on iron supplementation emphasises the importance of individualised therapy, based on genetic and biochemical determinations.

Mouse phenotype data represents a valuable resource for the identification of disease-associated genes, especially where the molecular basis is unknown and there is no clue to the candidate gene's function, pathway involvement or expression pattern. However, until recently these data have not been systematically used due to difficulties in mapping between clinical features observed in humans and mouse phenotype annotations. Here, we describe a semantic approach to solve this problem and demonstrate highly significant recall of known disease-gene associations and orthology relationships. A Web application (MouseFinder; www.mousemodels.org) has been developed to allow users to search the results of our whole-phenome comparison of human and mouse. We demonstrate its use in identifying ARTN as a strong candidate gene within the 1p34.1-p32 mapped locus for a hereditary form of ptosis.

Glucose uptake and metabolism are impaired in Alzheimer's disease (AD) brain, which appear to be a cause, rather than a consequence of neurodegeneration. Recently, the gene of the 14th isoform of subfamily A of solute carrier family 2 (SLC2A14), encoding glucose transporter 14 (GLUT14), was identified for the association in vivo with AD pathology of Tau, and rs10845990 within SLC2A14 showed association with AD in Caucasians. In order to evaluate the involvement of the SLC2A14 polymorphism in the risk of developing late-onset Alzheimer's disease (LOAD) in Chinese, we performed an independent case-control association study in a Han Chinese population (597 LOAD cases and 605 healthy controls). There were significant differences in genotype and allele frequencies between LOAD cases and controls (genotype P = 0.015, allele P = 0.039). The G-carrying genotype (GT + GG) individuals showed a 1.41-fold increased risk compared with the TT genotype carriers (odds ratio (OR) = 1.41, 95 % confidence interval (CI) = 1.11-1.79, P = 0.005, Power = 83.6 %). After stratification by ApoE ε4-carrying status, rs10845990 polymorphism was only significantly associated with LOAD in non-ApoE ε4 allele carriers (P < 0.001). Multivariate logistic regression analysis also conferred this positive association between the SNP rs10845990 and LOAD in the dominant and additive model after adjustment for age, gender, and the ApoE ε4 carrier status. These results suggested that SLC2A14 polymorphism has a possible role in changing the genetic susceptibility to LOAD in a Han Chinese population.

Huntington's disease (HD) is a genetically neurodegenerative disease, affecting the central nervous system and leading to mental and motor dysfunctions. To date, there is no cure for HD; as a result, HD patients gradually suffer devastating symptoms, such as chorea, weight loss, depression and mood swings, until death. According to previous studies, the exon 1 region of the huntingtin (HTT) gene with expanded CAG trinucleotide repeats plays a critical role in causing HD. In vitro studies using exon 1 of HTT fused with green fluorescent protein (GFP) gene have facilitated discovering several mechanisms of HD. However, whether this chimera construct exerts similar functions in vivo is still not clear. Here, we report the generation of transgenic mice carrying GFP fused with mutant HTT exon 1 containing 84 CAG trinucleotide repeats, and the evaluation of phenotypes via molecular, neuropathological and behavioral analyses. Results show that these transgenic mice not only displayed neuropathological characteristics, observed either by green fluorescent signals or by immunohistochemical staining, but also progressively developed pathological and behavioral symptoms of HD. Most interestingly, these transgenic mice showed significantly differential expression levels of nuclear aggregates between cortex and striatum regions, highly mimicking selective expression of mutant HTT in HD patients. To the best of our knowledge, this is the first report showing different nuclear diffusion profiling in mouse models with transgenic mice carrying the exon 1 region of mutant HTT. Our model will be beneficial for tracing the expression of mutant HTT and accelerating the understanding of selective pathological progression in HD.

To better characterize the interaction of protein-cysteines with sodium arsenite, arsenic-binding proteins were identified from the arsenic-resistant Chinese hamster ovary cell line SA7 using a p-aminophenylarsine oxide (PAO)-agarose matrix in combination with proteomic techniques. Twenty of the isolated arsenic-binding proteins were further peptide-mapped by MALDI-Q-TOF-MS. The binding capacity of PAO-agarose-retained proteins was then verified by re-applying Escherichia coli overexpressed recombinant proteins with various numbers of cysteine residues onto the PAO-agarose matrix. The results showed that recombinant heat shock protein 27 (HSP27, with one cysteine residue), reticulocalbin-3 (RCN3, with no cysteine residue), galectin-1 (GAL1, with six cysteine residues), but not peroxiredoxin 6 (Prdx6, with one cysteine residue but not retained by the PAO-agarose matrix), were bound to the PAO-agarose matrix. The six free cysteine residues in GAL1 were individually or double-mutated to alanine by means of site-directed mutagenesis and subjected to CD and ICP-MS analysis. The binding capacity of GAL1 for sodium arsenite was significantly attenuated in C16A, C88A and all double mutant clones. Taken together, our current data suggest that the cysteine residues in GAL1 may play a critical role in the binding of arsenic, but that in the case of RCN3 and Prdx6, this interaction may be mediated by other factors.

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.

Malformations of cerebral cortical development include a wide range of developmental disorders that are common causes of neurodevelopmental delay and epilepsy. In addition, study of these disorders contributes greatly to the understanding of normal brain development and its perturbations. The rapid recent evolution of molecular biology, genetics and imaging has resulted in an explosive increase in our knowledge of cerebral cortex development and in the number and types of malformations of cortical development that have been reported. These advances continue to modify our perception of these malformations. This review addresses recent changes in our perception of these disorders and proposes a modified classification based upon updates in our knowledge of cerebral cortical development.

Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.

<h4>Background</h4>Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. METHODOLGY: 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31.<h4>Principal findings</h4>In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31.<h4>Conclusions</h4>Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection without using an exfoliating brush may offer a cost effective, rapid, non-invasive, and reliable means for development of epigenetic salivary rinse biomarkers.

Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptation to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588(Q95)-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588(Q95)-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.

Messenger RNA decay is an essential step in gene expression to set mRNA abundance in the cytoplasm. The binding of proteins and/or noncoding RNAs to specific recognition sequences or secondary structures within mRNAs dictates mRNA decay rates by recruiting specific enzyme complexes that perform the destruction processes. Often, the cell coordinates the degradation or stabilization of functional subsets of mRNAs encoding proteins collectively required for a biological process. As well, extrinsic or intrinsic stimuli activate signal transduction pathways that modify the mRNA decay machinery with consequent effects on decay rates and mRNA abundance. This review is an update to our 2001 Gene review on mRNA stability in mammalian cells, and we survey the enormous progress made over the past decade.

Over the past several years, the success of genome-wide association studies (GWAS) and pharmacogenomics has gradually begun to enable personalized medicine in some fields. In the field of liver diseases, host genetic factors are now very useful in clinical practice for predicting treatment outcome and adverse reactions for pegylated interferon plus ribavirin combination therapy against chronic hepatitis C virus (HCV) infection. Recently, three virus-related hepatocellular carcinoma (HCC) GWAS were reported from Asia. One study examined hepatitis B virus-related HCC in China, where hepatitis B is very prevalent, and the other two examined HCV-related HCC in Japan. We identified a common variant in the DEPDC5 locus associated with HCV-related HCC, and another group identified an association involving the MICA locus. In this review, we compare the results of these GWAS and earlier candidate gene studies. Further research is needed to determine the role of these single nucleotide polymorphisms on HCC risk, but identification of these markers could make it possible to assess the magnitude of the risk of cancer based on each patient's genetic background. Consideration of the genetic background of the patients will likely play a role in personalized medicine for HCC, and understanding the mechanism underlying the association could suggest novel promising therapeutic targets in the future.

We have previously shown that adeno-associated virus type 2 (AAV2) undergoes anterograde axonal transport in rat and non-human primate brain. We screened other AAV serotypes for axonal transport and found that AAV6 is transported almost exclusively in a retrograde direction and, in the same way as AAV2, it is also neuron-specific in rat brain. Our findings show that axonal transport of AAV is serotype dependent and this has implications for gene therapy of neurological diseases such as Huntington's disease.

Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma.

<h4>Objective</h4>Independent replication with large cohorts and metaanalysis of genetic associations are necessary to validate genetic susceptibility factors. The known tumor necrosis factor (ligand) superfamily, member 4 gene (TNFSF4) systemic lupus erythematosus (SLE) risk locus has been found to be associated with systemic sclerosis (SSc) in 2 studies, but with discrepancies between them for genotype-phenotype correlation. Our objective was to validate TNFSF4 association with SSc and determine the subset with the higher risk.<h4>Methods</h4>Known SLE and SSc TNFSF4 susceptibility variants (rs2205960, rs1234317, rs12039904, rs10912580, and rs844648) were genotyped in 1031 patients with SSc and 1014 controls of French white ancestry. Genotype-phenotype association analysis and meta analysis of available data were performed, providing a population study of 4989 patients with SSc and 4661 controls, all of European white ancestry.<h4>Results</h4>Allelic and genotypic associations were observed for the 5 single-nucleotide polymorphisms (SNP) with the subset of patients with SSc who are positive for anticentromere antibodies (ACA) and only a trend for association with SSc and limited cutaneous SSc. Rs2205960 exhibited the strongest allelic association in ACA+ patients with SSc [p = 0.0015; OR 1.37 (1.12-1.66)], with significant intra-cohort association when compared to patients with SSc positive for ACA. Metaanalysis confirmed overall association with SSc but also raised preferential association with the ACA+ subset and strongest effect with rs2205960 [T allele p = 0.00013; OR 1.33 (1.15-1.54) and TT genotype p = 0.00046; OR 2.02 (1.36-2.98)].<h4>Conclusion</h4>We confirm TNFSF4 as an SSc susceptibility gene and rs2205960 as a putative causal variant with preferential association in the ACA+ SSc subphenotype.

The unusual cyclin-dependent protein kinase 5 (CDK5) was discovered based on its sequence homology to cell cycle regulating CDKs. CDK5 was found to be active in brain tissues, where it is not involved in cell cycle regulation but in the regulation of neuronal cell differentiation and neurocytoskeleton dynamics. An aberrant regulation of CDK5 leads to the development of various neurodegenerative diseases including Alzheimer's disease. Although CDK5 is not regulated by cyclins, its activity does depend on the association with a protein activator and the presence or absence of further inhibitory factors. Recently, CDK5RAP1 was discovered to inhibit the active CDK5 kinase. Here, we show that CDK5RAP1 is a radical SAM enzyme, which postsynthetically converts the RNA modification N6-isopentenyladenosine (i(6)A) into 2-methylthio-N6-isopentenyladenosine (ms(2)i(6)A). This conversion is surprisingly not limited to mitochondrial tRNA, where the modification was known to exist. Instead, CDK5RAP1 introduces the modification also into nuclear RNA species establishing a link between postsynthetic kinase-based protein modification and postsynthetic RNA modification.

While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.

The thalamus is essential for sensory perception. In mammals, work on the mouse has taught us most of what we know about how it develops and connects to the cortex. The mature thalamus of all mammalian species comprises numerous anatomically distinct collections of neurons called nuclei that differ in function, connectivity, and molecular constitution. At the time of its initial appearance as a distinct structure following neural tube closure, the thalamus is already patterned by the regional expression of numerous regulatory genes. This patterning, which lays down the blueprint for later development of thalamic nuclei, predates the development of thalamocortical projections. In this review we apply novel analytical methods to gene expression data available in the Allen Developing Mouse Brain Atlas to highlight the complex organized molecular heterogeneity already present among cells in the thalamus from the earliest stages at which it contains differentiating neurons. This early patterning is likely to invest in axons growing from different parts of the thalamus the ability to navigate in an ordered way to their appropriate area in the cerebral cortex. We review the mechanisms and cues that thalamic axons use, encounter, and interpret to attain the cortex. Mechanisms include guidance by previously generated guidepost cells, such as those in the subpallium that maintain thalamic axonal order and direction, and axons such as those of reciprocal projections from intermediate structures or from the cortex itself back toward the thalamus. We show how thalamocortical pathfinding involves numerous guidance cues operating at a series of steps along their route. We stress the importance of the combinatorial actions of multiple genes for the development of the numerous specific identities and functions of cells in this exquisitely complex system and their orderly innervation of the cortex.

More than 800 published genetic association studies have implicated dozens of potential risk loci in Parkinson's disease (PD). To facilitate the interpretation of these findings, we have created a dedicated online resource, PDGene, that comprehensively collects and meta-analyzes all published studies in the field. A systematic literature screen of -27,000 articles yielded 828 eligible articles from which relevant data were extracted. In addition, individual-level data from three publicly available genome-wide association studies (GWAS) were obtained and subjected to genotype imputation and analysis. Overall, we performed meta-analyses on more than seven million polymorphisms originating either from GWAS datasets and/or from smaller scale PD association studies. Meta-analyses on 147 SNPs were supplemented by unpublished GWAS data from up to 16,452 PD cases and 48,810 controls. Eleven loci showed genome-wide significant (P < 5 × 10(-8)) association with disease risk: BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT, MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25. In addition, we identified novel evidence for genome-wide significant association with a polymorphism in ITGA8 (rs7077361, OR 0.88, P  =  1.3 × 10(-8)). All meta-analysis results are freely available on a dedicated online database (www.pdgene.org), which is cross-linked with a customized track on the UCSC Genome Browser. Our study provides an exhaustive and up-to-date summary of the status of PD genetics research that can be readily scaled to include the results of future large-scale genetics projects, including next-generation sequencing studies.

Although genome-wide association studies (GWAS) have identified hundreds of complex trait loci, the pathomechanisms of most remain elusive. Studying the genetics of risk factors predisposing to disease is an attractive approach to identify targets for functional studies. Intracranial aneurysms (IA) are rupture-prone pouches at cerebral artery branching sites. IA is a complex disease for which GWAS have identified five loci with strong association and a further 14 loci with suggestive association. To decipher potential underlying disease mechanisms, we tested whether there are IA loci that convey their effect through elevating blood pressure (BP), a strong risk factor of IA. We performed a meta-analysis of four population-based Finnish cohorts (n(FIN)  =  11 266) not selected for IA, to assess the association of previously identified IA candidate loci (n  =  19) with BP. We defined systolic BP (SBP), diastolic BP, mean arterial pressure, and pulse pressure as quantitative outcome variables. The most significant result was further tested for association in the ICBP-GWAS cohort of 200 000 individuals. We found that the suggestive IA locus at 5q23.2 in PRDM6 was significantly associated with SBP in individuals of European descent (p(FIN)  =  3.01E-05, p(ICBP-GWAS)  =  0.0007, p(ALL)  =  8.13E-07). The risk allele of IA was associated with higher SBP. PRDM6 encodes a protein predominantly expressed in vascular smooth muscle cells. Our study connects a complex disease (IA) locus with a common risk factor for the disease (SBP). We hypothesize that common variants in PRDM6 can contribute to altered vascular wall structure, hence increasing SBP and predisposing to IA. True positive associations often fail to reach genome-wide significance in GWAS. Our findings show that analysis of traditional risk factors as intermediate phenotypes is an effective tool for deciphering hidden heritability. Further, we demonstrate that common disease loci identified in a population isolate may bear wider significance.

Staphylococcus aureus infection causes oxidative stress in neutrophils. The immune cells use reactive oxygen species (ROS) for carrying out their normal functions while an excess amount of ROS can attack cellular components that lead to cell damage. The present study was aimed to test the protective role of nanoconjugated vancomycin against vancomycin-sensitive Staphylococcus aureus (VSSA) and vancomycin-resistant Staphylococcus aureus (VRSA) infection induced oxidative stress in neutrophils. VSSA- and VRSA-infection were developed in Swiss mice by intraperitoneal injection of 5 × 10(6) CFU/mL bacterial solutions. Nanoconjugated vancomycin was treated to VSSA- and VRSA-infected mice at its effective dose for 10 days. Vancomycin was treated to VSSA and VRSA infected mice at similar dose, respectively, for 10 days. The result reveals that in vivo VSSA and VRSA infection significantly increases the level of lipid peroxidation, protein oxidation, oxidized glutathione level, and nitrite generation and decreases the level of reduced glutathione, antioxidant enzyme status, and glutathione-dependent enzymes as compared to control group; which were increased or decreased significantly near to normal in nanoconjugated vancomycin-treated group. These finding suggests the potential use and beneficial protective role of nanoconjugated vancomycin against VSSA and VRSA infection induced oxidative imbalance in neutrophils.

Cryptogenic cirrhosis (CC) is defined as cirrhosis occurring in an individual without an identifiable cause of liver disease, such as excessive alcohol consumption, viral hepatitis infection, hemochromatosis, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, chronic intake of medications that could induce cirrhosis, alpha-1-antitrypsin deficiency, Wilson's disease, or any other rare cause of cirrhosis according to the clinical context. Cryptogenic cirrhosis is a common cause of liver-related morbidity and mortality in the United States. Nonalcoholic fatty liver disease is now recognized as the most common cause of cryptogenic cirrhosis. A biopsy specimen is also important for detecting histological advanced disease, which may be clinically silent and undetected by liver-related tests or diagnostic imaging. We are presenting an unusual case of a patient with cryptogenic cirrhosis found to have Kayser-Fleischer-like rings without evidence of Wilson's disease.

An anti-fibrotic compound produced by Streptomycesn xiamenensis, found in mangrove sediments, was investigated for possible therapeutic effects against fibrosis. The compound, N-[[3,4-dihydro-3S-hydroxy-2S-methyl-2-(4'R-methyl-3'S-pentenyl)-2H-1-benzopyran-6-yl]carbonyl]-threonine (1), was isolated from crude extracts and its structure, including the absolute configuration was determined by extensive spectroscopic data analyses, Mosher's method, Marfey's reagent and quantum mechanical calculations. In terms of biological effects, this compound inhibits the proliferation of human lung fibroblasts (WI26), blocks adhesion of human acute monocytic leukemia cells (THP-1) to a monolayer of WI26 cells, and reduces the contractile capacity of WI26 cells in three-dimensional free-floating collagen gels. Altogether, these data indicate that we have identified a bioactive alkaloid (1) with multiple inhibitory biological effects on lung excessive fibrotic characteristics, that are likely involved in fibrosis, suggesting that this molecule might indeed have therapeutic potential against fibrosis.

Serotonin (5-hydroxytryptamine, 5-HT) signaling is thought to modulate nervous system development. Genetic and pharmacological studies support the idea that altered 5-HT signaling during development can have enduring consequences on brain function and behavior. Recently, we discovered that 5-HT can modulate thalamic axon guidance in vitro and in vivo. Embryonic thalamic axons transiently express the 5-HT transporter (SERT; Slc6a4) and accumulate 5-HT, suggesting that the SERT activity of these axons may regulate 5-HT-modulated guidance cues. We tested whether pharmacologically blocking SERT using selective 5-HT reuptake inhibitors (SSRIs) would impact the action of 5-HT on thalamic axon responses to netrin-1 in vitro. Surprisingly, we observed that two high-affinity SSRIs, racemic citalopram ((RS)-CIT) and paroxetine, affect the outgrowth of embryonic thalamic axons, but differ with respect to their dependence on SERT blockade. Using a recently developed 'citalopram insensitive' transgenic mouse line and in vitro pharmacology, we show that the effect of (RS)-CIT effect is SERT independent, but rather arises from R-CIT activation of the orphan sigma-1 receptor(σ1, Oprs1). Our results reveal a novel σ1 activity in modulating axon guidance and a 5-HT independent action of a widely prescribed SSRI. By extension, (RS)-CIT and possibly other structurally similar SSRIs may have other off-target actions that can impact neural development and contribute to therapeutic efficacy or side effects.

<h4>Introduction</h4>Acquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).<h4>Methods</h4>We cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.<h4>Results</h4>Quantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.<h4>Conclusions</h4>Our data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.

<h4>Background</h4>DNA methylation constitutes a key epigenetic mechanism by which cells regulate gene transcription. Among its roles are the dynamic regulation of gene expression, for example, as part of an evolving immune response, and cell differentiation in specialized tissues. Here our aim was to study the impact of differences in methylation patterns in the intestine with regard to inflammatory bowel disease (IBD) susceptibility and activity.<h4>Methods</h4>Having extracted DNA from rectal biopsies, we conducted genome-wide methylation profiling using the HumanMethylation27 BeadChip microarray to identify genes showing evidence of differential methylation between cases of ulcerative colitis and Crohn's disease and healthy controls. Selected methylation signals were validated in an independent replication panel by pyrosequencing. Correlation with gene expression was sought by quantitative real-time polymerase chain reaction (RT-PCR).<h4>Results</h4>Multiple genes showed significant evidence of differential methylation, several appearing in both ulcerative colitis and Crohn's disease comparisons including THRAP2, FANCC, GBGT1, DOK2, TNFSF4, TNFSF12, and FUT7. Many more than expected by chance overlapped with genes previously implicated as playing a role in IBD susceptibility in genome-wide association scans, including CARD9, ICAM3, and IL8RB (P < 0.001). Correlation between methylation and gene expression was identified for selected transcripts.<h4>Conclusions</h4>Consistent differences in DNA methylation between IBD cases and controls at regulatory sites within these genes suggest that their altered transcription contributes to IBD pathogenesis.

We performed a genome wide association analysis of maternally-mediated genetic effects and parent-of-origin (POO) effects on risk of orofacial clefting (OC) using over 2,000 case-parent triads collected through an international cleft consortium. We used log-linear regression models to test individual SNPs. For SNPs with a P-value <10(-5) for maternal genotypic effects, we also applied a haplotype-based method, TRIMM, to extract potential information from clusters of correlated SNPs. None of the SNPs were significant at the genome wide level. Our results suggest neither maternal genome nor POO effects play major roles in the etiology of OC in our sample. This finding is consistent with previous genetic studies and recent population-based cohort studies in Norway and Denmark, which showed no apparent difference between mother-to-offspring and father-to-offspring recurrence of clefting. We, however, cannot completely rule out maternal genome or POO effects as risk factors because very small effects might not be detectable with our sample size, they may influence risk through interactions with environmental exposures or may act through a more complex network of interacting genes. Thus, the most promising SNPs identified by this study may still be worth further investigation.

Our objective was to investigate whether the C282Y (p.Cys 282 Tyr) and H63D (p. His 63 Asp) HFE polymorphisms were associated with sporadic amyotrophic lateral sclerosis (SALS) in the French population. We searched for a relation of HFE polymorphisms with the clinical characteristics of the disease. The HFE polymorphisms were studied in 824 patients with SALS and 583 controls. We compared the frequency of the polymorphisms between SALS and controls groups by univariate and multivariate statistics, taking into account gender, site, age-at-onset and survival. We did not observe significant difference in the frequency of H63D polymorphism between SALS and control group. We observed a significant difference for C282Y between patients and controls with a low frequency of the Y allele in patients (3.2%) compared to our control group (5.9%). Disease duration, distribution of gender, site-of-onset, age-at-onset did not differ between groups taking into account genotypes of each polymorphism. Our results in this large cohort of ALS patients indicate that H63D polymorphism is not associated with SALS in the French population. This conclusion does not exclude a weak effect of the HFE gene polymorphisms in certain ALS populations, or an effect of other rare HFE gene variants.

<h4>Background</h4>Liver biopsy is considered as the gold standard for assessing non-alcoholic fatty liver disease (NAFLD) histologic lesions in patients with severe obesity. The aim of this study was to perform an overview of 3 studies which assessed the performance of non-invasive markers of fibrosis (FibroTest), steatosis (SteatoTest) and steato-hepatitis (NashTest, ActiTest) in these patients.<h4>Methods</h4>494 patients with interpretable biopsy and biomarkers using of three prospective cohorts of patients with severe obesity (BMI >35 kg/m2) were included. Histology (NAS score) and the biochemical measurements were blinded to any other characteristics. The area under the ROC curves (AUROC), sensitivity, specificity, positive and negative predictive values were assessed. Weighted AUROC (wAUROC Obuchowski method) was used to prevent multiple testing and spectrum effect. Two meta-analyses were performed; one used the individual patient, and the other a classical meta-analysis.<h4>Results</h4>Prevalence of advanced fibrosis (bridging) was 9.9%, advanced steatosis (>33%) 54.2%, and steato-hepatitis (NAS score >4) 17.2%. The mean wAUROCs were: FibroTest for advanced fibrosis (95%CI; significance)  =  0.85 (0.83-0.87; P<0.0001); SteatoTest for advanced steatosis = 0.80 (0.79-0.83); and ActiTest for steato-hepatitis = 0.84 (0.82-0.86; P<0.0001). Using the classical meta-analysis (random effect model) the mean AUROCs were: FibroTest = 0.72 (0.63-0.79; P<0.0001); SteatoTest = 0.71 (0.66-0.75; P<0.0001); and ActiTest = 0.74 (0.68-0.79; P<0.0001). Despite more metabolic risk factors in one cohort, results were similar according to gender, presence of diabetes and between the 3 cohorts.<h4>Conclusion</h4>In patients with severe obesity, a significant diagnostic performance of FibroTest, SteatoTest and ActiTest was observed for liver lesions.

Dementia is a major problem of health in developed societies. Alzheimer's disease (AD), vascular dementia, and mixed dementia account for over 90% of the most prevalent forms of dementia. Both genetic and environmental factors are determinant for the phenotypic expression of dementia. AD is a complex disorder in which many different gene clusters may be involved. Most genes screened to date belong to different proteomic and metabolomic pathways potentially affecting AD pathogenesis. The ε4 variant of the APOE gene seems to be a major risk factor for both degenerative and vascular dementia. Metabolic factors, cerebrovascular disorders, and epigenetic phenomena also contribute to neurodegeneration. Five categories of genes are mainly involved in pharmacogenomics: genes associated with disease pathogenesis, genes associated with the mechanism of action of a particular drug, genes associated with phase I and phase II metabolic reactions, genes associated with transporters, and pleiotropic genes and/or genes associated with concomitant pathologies. The APOE and CYP2D6 genes have been extensively studied in AD. The therapeutic response to conventional drugs in patients with AD is genotype specific, with CYP2D6-PMs, CYP2D6-UMs, and APOE-4/4 carriers acting as the worst responders. APOE and CYP2D6 may cooperate, as pleiotropic genes, in the metabolism of drugs and hepatic function. The introduction of pharmacogenetic procedures into AD pharmacological treatment may help to optimize therapeutics.

MicroRNA (miRNA) genes, located in intergenic or intragenic non-coding regions of the genome, are transcribed and processed to small non-protein-coding RNA of approximately 22 nucleotides negatively regulating gene expression. Some miRNA have already been reported for their genetic alterations, aberrant expression and oncogenic or tumor-suppressive functions. After 2008, there has been a striking increase in the number of publications reporting tumor-suppressive miRNA (TS-miRNA) silenced epigenetically in various types of cancers, suggesting important clinical applications for miRNA-based molecular diagnosis and therapy for cancers. Here, we introduce a correlation of the gene silencing of TS-miRNA through CpG island hypermethylation with the genomic distances between intergenic and intragenic miRNA genes or protein-coding host genes and CpG islands located around these genes. Furthermore, we also discuss the potential of miRNA replacement therapy for cancers using double-stranded RNA mimicking TS-miRNA.

A higher prevalence of intermediate ataxin-2 CAG repeats in amyotrophic lateral sclerosis (ALS) patients has raised the possibility that CAG expansions in other polyglutamine disease genes could contribute to ALS neurodegeneration. We sought to determine whether expansions of the CAG repeat of the HTT gene that causes Huntington's disease, are associated with ALS. We compared the HTT CAG repeat length on a total of 3144 chromosomes from 1572 sporadic ALS patients and 4007 control chromosomes, and also tested its possible effects on ALS-specific parameters, such as age and site of onset and survival rate. Our results show that the CAG repeat in the HTT gene is not a risk factor for ALS nor modifies its clinical presentation. These findings suggest that distinct neuronal degeneration processes are involved in these two different neurodegenerative disorders.

Gangliosides are sialic acid-containing glycosphingolipids that are most abundant in the nervous system. They are localized primarily in the outer leaflets of plasma membranes and participated in cell-cell recognition, adhesion, and signal transduction and are integral components of cell surface microdomains or lipid rafts along with proteins, sphingomyelin and cholesterol. Ganglioside-rich lipid rafts play an important role in signaling events affecting neural development and the pathogenesis of certain diseases. Disruption of gangloside synthase genes in mice induces developmental defects and neural degeneration. Targeting ganglioside metabolism may represent a novel therapeutic strategy for intervention in certain diseases. In this review, we focus on recent advances on metabolic and functional studies of gangliosides in normal brain development and in certain neurological disorders.

Dendritic spines are the elementary structural units of neural plasticity. In a model of hormone replacement therapy (HT), we sought to determine the effect of estradiol (E) and progesterone (P) on gene expression related to synapse assembly in a laser-captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n = 2 animals/treatment), and the results were confirmed for pivotal genes with qRT-PCR on additional laser-captured material (n = 3 animals/treatment). Ovariectomized rhesus macaques were treated with placebo, E, or E + P via Silastic implants for 1 month. The midbrain was obtained, sectioned, and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an arcturus laser dissection microscope (Pixel II). RNA from laser-captured serotonin neurons was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a twofold or greater change in the expression of 63 probe sets in the cell adhesion molecule (CAM) category, and 31 probe sets in the synapse assembly category were similarly altered in E- and E + P-treated animals. qRT-PCR assays showed that E treatment induced a significant increase in ephrin receptor A4 (EPHA4) and in integrin A8 (ITGA8) but not in ephrin receptor B4 (EPHB4) or integrin B8 (ITGB8) expression. E also increased expression of cadherin 11 (CDH11), neuroligin 3 (NLGN3), neurexin 3 (NRXN3), syndecan 2 (SCD2), and neural cell adhesion molecule (NCAM) compared with placebo. Supplemental P treatment suppressed E-induced gene expression. In summary, ovarian steroids target gene expression of adhesion molecules in serotonin neurons that are important for synapse assembly.

<h4>Background</h4>A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD.<h4>Results</h4>Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified.<h4>Conclusions</h4>Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD.

<h4>Objective</h4>Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use.<h4>Methods</h4>Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, β-thromboglobulin and terminal complement complex SC5b-9).<h4>Results</h4>No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05).<h4>Conclusions</h4>The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts.

<h4>Background</h4>Genomic stability is one of the crucial prognostic factors for patients with endometrioid endometrial cancer (EEC). The impact of genomic stability on the tumour tissue proteome of EEC is not yet well established.<h4>Methods</h4>Tissue lysates of EEC, squamous cervical cancer (SCC), normal endometrium and squamous cervical epithelium were subjected to two-dimensional (2D) gel electrophoresis and identification of proteins by MALDI TOF MS. Expression of selected proteins was analysed in independent samples by immunohistochemistry.<h4>Results</h4>Diploid and aneuploid genomically unstable EEC displayed similar patterns of protein expression. This was in contrast to diploid stable EEC, which displayed a protein expression profile similar to normal endometrium. Approximately 10% of the differentially expressed proteins in EEC were specific for this type of cancer with differential expression of other proteins observed in other types of malignancy (e.g., SCC). Selected proteins differentially expressed in 2D gels of EEC were further analysed in an EEC precursor lesion, that is, atypical hyperplasia of endometrium, and showed increased expression of CLIC1, EIF4A1 and PRDX6 and decreased expression of ENO1, ANXA4, EMD and Ku70.<h4>Conclusion</h4>Protein expression in diploid and aneuploid genomically unstable EEC is different from the expression profile of proteins in diploid genomically stable EEC. We showed that changes in expression of proteins typical for EEC could already be detected in precursor lesions, that is, atypical hyperplasia of endometrium, highlighting their clinical potential for improving early diagnostics of EEC.

Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio-temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross-correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio-temporal map of molecular interactions.

Insulin regulates many cellular processes, but the full impact of insulin deficiency on cellular functions remains to be defined. Applying a mass spectrometry-based nontargeted metabolomics approach, we report here alterations of 330 plasma metabolites representing 33 metabolic pathways during an 8-h insulin deprivation in type 1 diabetic individuals. These pathways included those known to be affected by insulin such as glucose, amino acid and lipid metabolism, Krebs cycle, and immune responses and those hitherto unknown to be altered including prostaglandin, arachidonic acid, leukotrienes, neurotransmitters, nucleotides, and anti-inflammatory responses. A significant concordance of metabolome and skeletal muscle transcriptome-based pathways supports an assumption that plasma metabolites are chemical fingerprints of cellular events. Although insulin treatment normalized plasma glucose and many other metabolites, there were 71 metabolites and 24 pathways that differed between nondiabetes and insulin-treated type 1 diabetes. Confirmation of many known pathways altered by insulin using a single blood test offers confidence in the current approach. Future research needs to be focused on newly discovered pathways affected by insulin deficiency and systemic insulin treatment to determine whether they contribute to the high morbidity and mortality in T1D despite insulin treatment.

To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

Schizophrenia is a highly heritable neurodevelopmental disorder associated with alterations in synaptic connectivity. Deleted in colorectal cancer (DCC), a receptor for the guidance cue netrin-1, plays a pivotal role in organizing neuronal circuitry by guiding growing axons and dendrites to their correct targets and by influencing synaptic connectivity. Results from experiments we previously conducted in dcc-heterozygous mice show that DCC plays a critical role in the developmental organization of the mesocorticolimbic dopamine (DA) circuitry. Furthermore we have shown that reduced expression of DCC during development and/or throughout life confers resilience to the development of schizophrenia-like DA and behavioural abnormalities. Importantly, this "protective" phenotype only emerges after puberty. Here we assess whether DCC may contribute to the risk of schizophrenia. We examined single nucleotide polymorphisms (SNPs) located in the DCC gene for association with schizophrenia using a case-control sample consisting of 556 unrelated schizophrenic patients and 208 healthy controls. We found one SNP, rs2270954, to be nominally associated with schizophrenia; patients were less likely to be heterozygous at this locus and more likely to be homozygous for the minor allele (χ(2)=9.84, df=2, nominal p=0.0071). Intriguingly, this SNP is located within the 3' untranslated region, an area known to contain a number of regulatory sequences that determine the stability and translation efficacy of mRNA. These results, together with our previous findings from studies in rodents, point at DCC as a promising novel candidate gene that may contribute to the genetic basis behind individual differences in susceptibility to schizophrenia.

The distinguishing feature between iron homeostasis in single versus multicellular organisms is the need for multicellular organisms to transfer iron from sites of absorption to sites of utilization and storage. Ferroportin is the only known iron exporter and ferroportin plays an essential role in the export of iron from cells to blood. Ferroportin can be regulated at many different levels including transcriptionally, post-transcriptionally, through mRNA stability and post-translationally, through protein turnover. Additionally, ferroportin may be regulated in both cell-dependent and cell-autonomous fashions. Regulation of ferroportin is critical for iron homeostasis as alterations in ferroportin may result in either iron deficiency or iron overload. This article is part of a Special Issue entitled: Cell Biology of Metals.

The objective of our study was to evaluate whether feeding pseudopurpurin affects bone mineral density and bone geometry architecture in rats. Pseudopurpurin was extracted, analyzed and purified using an HLPC-ESI-MS. Rats were given 0% and 0.5% pseudopurpurin powder in their diet. Femurs of rats were examined at 0.5, 1 and 2 months after pseudopurpurin feeding. Compared with rats in the group 0%, the bone mineral density, and the calcium, magnesium, zinc and manganese concentrations in the rats femur in the group 0.5% increased significantly at 1 month and 2 months after pseudopurpurin feeding. Analytical results of micro-computed tomography showed that the group 0.5% displayed an increase in the trabecular volume fraction, trabecular thickness and trabecular number of the distal femur at 1 and 2 months after pseudopurpurin feeding, and the mean thickness, inner perimeter, outer perimeter, and area of the femur diaphysis were significantly increased at 2 months after pseudopurpurin feeding compared with the group 0%. In parallel, the trabecular separation and structure model index of the distal femur were decreased, compared with the group 0% at 1 and 2 months after pseudopurpurin feeding. In the 0.5% and 0% groups, there was no damage to kidney and liver by histopathology analysis. The long-term feeding of pseudopurpurin is safe for rats. The feeding of 0.5% pseudopurpurin which has specific chemical affinities for calcium for bone improvement and level of bone mineral density, enhances the geometry architecture compared with the 0% group.

<h4>Background</h4>Using gene co-expression analysis, researchers were able to predict clusters of genes with consistent functions that are relevant to cancer development and prognosis. We applied a weighted gene co-expression network (WGCN) analysis algorithm on glioblastoma multiforme (GBM) data obtained from the TCGA project and predicted a set of gene co-expression networks which are related to GBM prognosis.<h4>Methods</h4>We modified the Quasi-Clique Merger algorithm (QCM algorithm) into edge-covering Quasi-Clique Merger algorithm (eQCM) for mining weighted sub-network in WGCN. Each sub-network is considered a set of features to separate patients into two groups using K-means algorithm. Survival times of the two groups are compared using log-rank test and Kaplan-Meier curves. Simulations using random sets of genes are carried out to determine the thresholds for log-rank test p-values for network selection. Sub-networks with p-values less than their corresponding thresholds were further merged into clusters based on overlap ratios (>50%). The functions for each cluster are analyzed using gene ontology enrichment analysis.<h4>Results</h4>Using the eQCM algorithm, we identified 8,124 sub-networks in the WGCN, out of which 170 sub-networks show p-values less than their corresponding thresholds. They were then merged into 16 clusters.<h4>Conclusions</h4>We identified 16 gene clusters associated with GBM prognosis using the eQCM algorithm. Our results not only confirmed previous findings including the importance of cell cycle and immune response in GBM, but also suggested important epigenetic events in GBM development and prognosis.

Major depressive disorder (MDD) is a leading cause of disability worldwide and results tragically in the loss of almost one million lives in Western societies every year. This is due to poor understanding of the disease pathophysiology and lack of empirical medical tests for accurate diagnosis or for guiding antidepressant treatment strategies. Here, we have used shotgun proteomics in the analysis of post-mortem dorsolateral prefrontal cortex brain tissue from 24 MDD patients and 12 matched controls. Brain proteomes were pre-fractionated by gel electrophoresis and further analyzed by shotgun data-independent label-free liquid chromatography-mass spectrometry. This led to identification of distinct proteome fingerprints between MDD and control subjects. Some of these differences were validated by Western blot or selected reaction monitoring mass spectrometry. This included proteins associated with energy metabolism and synaptic function and we also found changes in the histidine triad nucleotide-binding protein 1 (HINT1), which has been implicated recently in regulation of mood and behavior. We also found differential proteome profiles in MDD with (n=11) and without (n=12) psychosis. Interestingly, the psychosis fingerprint showed a marked overlap to changes seen in the brain proteome of schizophrenia patients. These findings suggest that it may be possible to contribute to the disease understanding by distinguishing different subtypes of MDD based on distinct brain proteomic profiles.

A mass spectrometry-based plasma biomarker discovery workflow was developed to facilitate biomarker discovery. Plasma from either healthy volunteers or patients with pancreatic cancer was 8-plex iTRAQ labeled, fractionated by 2-dimensional reversed phase chromatography and subjected to MALDI ToF/ToF mass spectrometry. Data were processed using a q-value based statistical approach to maximize protein quantification and identification. Technical (between duplicate samples) and biological variance (between and within individuals) were calculated and power analysis was thereby enabled. An a priori power analysis was carried out using samples from healthy volunteers to define sample sizes required for robust biomarker identification. The result was subsequently validated with a post hoc power analysis using a real clinical setting involving pancreatic cancer patients. This demonstrated that six samples per group (e.g., pre- vs post-treatment) may provide sufficient statistical power for most proteins with changes>2 fold. A reference standard allowed direct comparison of protein expression changes between multiple experiments. Analysis of patient plasma prior to treatment identified 29 proteins with significant changes within individual patient. Changes in Peroxiredoxin II levels were confirmed by Western blot. This q-value based statistical approach in combination with reference standard samples can be applied with confidence in the design and execution of clinical studies for predictive, prognostic, and/or pharmacodynamic biomarker discovery. The power analysis provides information required prior to study initiation.

<h4>Background</h4>Peripheral arterial disease (PAD) is a relatively common manifestation of systemic atherosclerosis that leads to progressive narrowing of the lumen of leg arteries. Circulating monocytes are in contact with the arterial wall and can serve as reporters of vascular pathology in the setting of PAD. We performed gene expression analysis of peripheral blood mononuclear cells (PBMC) in patients with PAD and controls without PAD to identify differentially regulated genes.<h4>Methods</h4>PAD was defined as an ankle brachial index (ABI) ≤0.9 (n = 19) while age and gender matched controls had an ABI > 1.0 (n = 18). Microarray analysis was performed using Affymetrix HG-U133 plus 2.0 gene chips and analyzed using GeneSpring GX 11.0. Gene expression data was normalized using Robust Multichip Analysis (RMA) normalization method, differential expression was defined as a fold change ≥1.5, followed by unpaired Mann-Whitney test (P < 0.05) and correction for multiple testing by Benjamini and Hochberg False Discovery Rate. Meta-analysis of differentially expressed genes was performed using an integrated bioinformatics pipeline with tools for enrichment analysis using Gene Ontology (GO) terms, pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG), molecular event enrichment using Reactome annotations and network analysis using Ingenuity Pathway Analysis suite. Extensive biocuration was also performed to understand the functional context of genes.<h4>Results</h4>We identified 87 genes differentially expressed in the setting of PAD; 40 genes were upregulated and 47 genes were downregulated. We employed an integrated bioinformatics pipeline coupled with literature curation to characterize the functional coherence of differentially regulated genes.<h4>Conclusion</h4>Notably, upregulated genes mediate immune response, inflammation, apoptosis, stress response, phosphorylation, hemostasis, platelet activation and platelet aggregation. Downregulated genes included several genes from the zinc finger family that are involved in transcriptional regulation. These results provide insights into molecular mechanisms relevant to the pathophysiology of PAD.

Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response.

Staphylococcus aureus spreads on the surface of soft agar, a phenomenon we termed "colony spreading." Here, we found that S. aureus culture supernatant inhibited colony spreading. We purified δ-hemolysin (Hld, δ-toxin), a major protein secreted from S. aureus, as a compound that inhibits colony spreading. The culture supernatants of hld-disrupted mutants had 30-fold lower colony-spreading inhibitory activity than those of the parent strain. Furthermore, hld-disrupted mutants had higher colony-spreading ability than the parent strain. These results suggest that S. aureus negatively regulates colony spreading by secreting δ-hemolysin.

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. Although much investigation has focused on understanding dt pathology, the diverse cellular functions of dystonin isoforms remain poorly characterized. In this paper, we highlight novel functions of the dystonin-a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform-specific loss-of-function analysis, we found dystonin-a2 bound to MT-associated protein 1B (MAP1B) in the centrosomal region, where it maintained MT acetylation. In dt neurons, absence of the MAP1B-dystonin-a2 interaction resulted in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MT accumulation resulted in Golgi fragmentation and prevented anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A administration or MAP1B overexpression mitigated the observed defect. These cellular aberrations are apparent in prephenotype dorsal root ganglia and primary sensory neurons from dt mice, suggesting they are causal in the disorder.

<h4>Background</h4>Data are limited on the impact of methicillin-resistant Staphylococcus aureus (MRSA) on morbidity and mortality among very low birth weight (VLBW) infants with S aureus (SA) bacteremia and/or meningitis (B/M).<h4>Methods</h4>Neonatal data for VLBW infants (birth weight 401-1500 g) born January 1, 2006, to December 31, 2008, who received care at centers of the Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network were collected prospectively. Early-onset (≤72 hours after birth) and late-onset (>72 hours) infections were defined by blood or cerebrospinal fluid cultures and antibiotic treatment of ≥5 days (or death <5 days with intent to treat). Outcomes were compared for infants with MRSA versus methicillin-susceptible S aureus (MSSA) B/M.<h4>Results</h4>Of 8444 infants who survived >3 days, 316 (3.7%) had SA B/M. Eighty-eight had MRSA (1% of all infants, 28% of infants with SA); 228 had MSSA (2.7% of all infants, 72% of infants with SA). No infant had both MRSA and MSSA B/M. Ninety-nine percent of MRSA infections were late-onset. The percent of infants with MRSA varied by center (P < .001) with 9 of 20 centers reporting no cases. Need for mechanical ventilation, diagnosis of respiratory distress syndrome, necrotizing enterocolitis, and other morbidities did not differ between infants with MRSA and MSSA. Mortality was high with both MRSA (23 of 88, 26%) and MSSA (55 of 228, 24%).<h4>Conclusions</h4>Few VLBW infants had SA B/M. The 1% with MRSA had morbidity and mortality rates similar to infants with MSSA. Practices should provide equal focus on prevention and management of both MRSA and MSSA infections among VLBW infants.

<h4>Background</h4>NF-κB/p65 has been reported to be involved in regulation of chondrogenic differentiation. However, its function in relation to key chondrogenic factor Sox9 and onset of chondrogenesis during endochondral ossification is poorly understood. We hypothesized that the early onset of chondrogenic differentiation is initiated by transient NF-κB/p65 signaling.<h4>Methodology/principal findings</h4>The role of NF-κB/p65 in early chondrogenesis was investigated in different in vitro, ex vivo and in vivo endochondral models: ATDC5 cells, hBMSCs, chicken periosteal explants and growth plates of 6 weeks old mice. NF-κB/p65 activation was manipulated using pharmacological inhibitors, RNAi and activating agents. Gene expression and protein expression analysis, and (immuno)histochemical stainings were employed to determine the role of NF-κB/p65 in the chondrogenic phase of endochondral development. Our data show that chondrogenic differentiation is facilitated by early transient activation of NF-κB/p65. NF-κB/p65-mediated signaling determines early expression of Sox9 and facilitates the subsequent chondrogenic differentiation programming by signaling through key chondrogenic pathways.<h4>Conclusions/significance</h4>The presented data demonstrate that NF-κB/p65 signaling, as well as its intensity and timing, represents one of the transcriptional regulatory mechanisms of the chondrogenic developmental program of chondroprogenitor cells during endochondral ossification. Importantly, these results provide novel possibilities to improve the success of cartilage and bone regenerative techniques.

Several studies investigated the potential role of imaging modalities in prostate cancer patients in case of biochemical recurrence. However, the role of molecular imaging has not been well established yet. Considering the results of the literature and of our own experience, we tried to summarize the potential applications of (11)C-choline PET/CT in prostate cancer patients in case of biochemical relapse for the detection of lymph node and distant recurrence.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is considered to be one of the major public health problems worldwide. The development of therapies that target tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and co-stimulatory pathways that regulate the immune system have revolutionized the care of patients with RA. Despite these advances, many patients continue to experience symptomatic and functional impairment. To address this issue, more recent therapies that have been developed are designed to target intracellular signaling pathways involved in immunoregulation. Though this approach has been encouraging, there have been major challenges with respect to off-target organ side effects and systemic toxicities related to the widespread distribution of these signaling pathways in multiple cell types and tissues. These limitations have led to an increasing interest in the development of strategies for the macromolecularization of anti-rheumatic drugs, which could target them to the inflamed joints. This approach enhances the efficacy of the therapeutic agent with respect to synovial inflammation, while markedly reducing non-target organ adverse side effects. In this manuscript, we provide a comprehensive overview of the rational design and optimization of macromolecular prodrugs for treatment of RA. The superior and the sustained efficacy of the prodrug may be partially attributed to their Extravasation through Leaky Vasculature and subsequent Inflammatory cell-mediated Sequestration (ELVIS) in the arthritic joints. This biologic process provides a plausible mechanism, by which macromolecular prodrugs preferentially target arthritic joints and illustrates the potential benefits of applying this therapeutic strategy to the treatment of other inflammatory diseases.

<h4>Purpose</h4>To investigate the effect of netrin-1 on alkali burn-induced corneal inflammation and neovascularization.<h4>Methods</h4>The expression of netrin-1 and its receptors UNC5A, UNC5B, UNC5C, UNC5D, adenosine 2b receptor (A2BAR), deleted in colorectal cancer (DCC), and neogenin in normal and alkali-burned rat cornea were determined by RT-PCR and/or Western blot analysis, or immunostaining. Topical netrin-1 protein was applied to treat rat corneal alkali-burn injury for 14 consecutive days, started right after the injury or 10 days postinjury. Corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Corneal inflammatory cell infiltration was evaluated by immunostaining of anti-PMN and anti-ED1 antibodies. The expression of epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), and pigment epithelium-derived factor (PEDF) in rat cornea was determined by Western blot analysis.<h4>Results</h4>Netrin-1 and its receptor UNC5B were expressed in normal rat corneal epithelium and stromal cells, and their expression decreased after corneal alkali burn. Exogenous netrin-1 administered on rat ocular surfaces resolved alkali burn-induced corneal inflammation, and also suppressed corneal neovascularization. Furthermore, netrin-1 could reverse neovascularization in alkali-burned cornea. The authors found that netrin-1 executed the functions through various mechanisms, including upregulating EGF expression, accelerating epithelial wound healing, inhibiting neutrophil and macrophage infiltration, reducing corneal cell apoptosis, and restoring the equilibrium of VEGF and PEDF in the wounded cornea.<h4>Conclusions</h4>Netrin-1 could dampen inflammation, inhibit, and reverse neovascularization in alkali-burned cornea.

The mammalian genome encodes thousands of noncoding RNAs. These noncoding transcripts are broadly categorized into short noncoding RNAs, such as microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) of greater than 200 nt. While the role of miRNAs in development and cancer biology has been extensively studied, much less is known about the vast majority of noncoding transcripts represented by lncRNAs. LncRNAs are emerging as key regulators of developmental processes and as such, their frequent misregulation in tumorigenesis and disease in not unexpected. The role of lncRNAs in mammary gland development and breast cancer is just beginning to be elucidated. This review will discuss the role of lncRNAs in mammalian and mammary gland development. In addition, we will review the contributions of lncRNAs to the stepwise progression of tumorigenesis, highlighting the role of lncRNAs in breast cancer.

Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis-antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of ∼800 lncRNAs and ∼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life < 2 h) intergenic, cis-antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life > 16 h). Analysis of lncRNA features revealed that intergenic and cis-antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1, suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource (http://stability.matticklab.com) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ~7200 mouse lncRNAs.

Systemic sclerosis (SSc) is complex autoimmune disease affecting the connective tissue; influenced by genetic and environmental components. Recently, we performed the first successful genome-wide association study (GWAS) of SSc. Here, we perform a large replication study to better dissect the genetic component of SSc. We selected 768 polymorphisms from the previous GWAS and genotyped them in seven replication cohorts from Europe. Overall significance was calculated for replicated significant SNPs by meta-analysis of the replication cohorts and replication-GWAS cohorts (3237 cases and 6097 controls). Six SNPs in regions not previously associated with SSc were selected for validation in another five independent cohorts, up to a total of 5270 SSc patients and 8326 controls. We found evidence for replication and overall genome-wide significance for one novel SSc genetic risk locus: CSK [P-value = 5.04 × 10(-12), odds ratio (OR) = 1.20]. Additionally, we found suggestive association in the loci PSD3 (P-value = 3.18 × 10(-7), OR = 1.36) and NFKB1 (P-value = 1.03 × 10(-6), OR = 1.14). Additionally, we strengthened the evidence for previously confirmed associations. This study significantly increases the number of known putative genetic risk factors for SSc, including the genes CSK, PSD3 and NFKB1, and further confirms six previously described ones.

In light of observed changes in connectivity in schizophrenia and the highly heritable nature of the disease, neural connectivity may serve as an important intermediate phenotype for schizophrenia. However, how individual variants confer altered connectivity and which measure of brain connectivity is more proximal to the underlying genetic architecture (i.e., functional or structural) has not been well delineated. In this review we consider these issues and the relative sensitivity of imaging methodologies to schizophrenia-related changes in connectivity. We searched PubMed for studies considering schizophrenia risk genes AND functional or structural connectivity. Where data was available, summary statistics were used to determine an estimate of effect size (i.e., Cohen's d). A random-effects meta-analysis was used to consider (1) the largest effect and (2) all significant effects between functional and structural studies. Schizophrenia risk variants involved in neurotransmission, neurodevelopment and myelin function were found to be associated with altered neural connectivity. On average, schizophrenia risk genes had a large effect on functional (mean d = 0.76) and structural connectivity (mean d = 1.04). The examination of the largest effect size indicated that the outcomes of functional and structural studies were comparable (Q = 2.17, p > 0.05). Conversely, consideration of effect size estimates for all significant effects suggest that reported effect sizes in structural connectivity studies were more variable than in functional connectivity studies, and that there was a significant lack of homogeneity across the modalities (Q = 6.928, p = 0.008). Given the more variable profile of effect sizes associated with structural connectivity, these data may suggest that structural imaging methods are more sensitive to a wider range of effects, as opposed to functional studies which may only be able to determine large effects. These conclusions are limited by methodological considerations, and require further investigation involving larger samples, multiple genes, and novel analysis techniques for confirmation.

<h4>Background</h4>Genetic variability of the major subunit (CACNA1E) of the voltage-dependent Ca(2+) channel Ca(V)2.3 is associated to risk of type 2 diabetes, insulin resistance and impaired insulin secretion in nondiabetic subjects. The aim of the study was to test whether CACNA1E common variability affects beta cell function and/or insulin sensitivity in patients with newly diagnosed type 2 diabetes.<h4>Methodology/principal findings</h4>In 595 GAD-negative, drug naïve patients (mean ± SD; age: 58.5 ± 10.2 yrs; BMI: 29.9 ± 5 kg/m(2), HbA1c: 7.0±1.3) with newly diagnosed type 2 diabetes we: 1. genotyped 10 tag SNPs in CACNA1E region reportedly covering ∼93% of CACNA1E common variability: rs558994, rs679931, rs2184945, rs10797728, rs3905011, rs12071300, rs175338, rs3753737, rs2253388 and rs4652679; 2. assessed clinical phenotypes, insulin sensitivity by the euglycemic insulin clamp and beta cell function by state-of-art modelling of glucose/C-peptide curves during OGTT. Five CACNA1E tag SNPs (rs10797728, rs175338, rs2184945, rs3905011 and rs4652679) were associated with specific aspects of beta cell function (p<0.05-0.01). Both major alleles of rs2184945 and rs3905011 were each (p<0.01 and p<0.005, respectively) associated to reduced proportional control with a demonstrable additive effect (p<0.005). In contrast, only the major allele of rs2253388 was related weakly to more severe insulin resistance (p<0.05).<h4>Conclusions/significance</h4>In patients with newly diagnosed type 2 diabetes CACNA1E common variability is strongly associated to beta cell function. Genotyping CACNA1E might be of help to infer the beta cell functional phenotype and to select a personalized treatment.

Nine neurodegenerative disorders, called polyglutamine (polyQ) diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be negligible when the latter contain particularly long polyQ tracts.

No abstract available.

Targeted molecular radiotherapy opens unprecedented opportunities to eradicate cancer cells with minimal irradiation of normal tissues. Described in this study are radioactive cyclosaligenyl monophosphates designed to deliver lethal doses of radiation to cancer cells. These compounds can be radiolabeled with SPECT- and PET-compatible radionuclides as well as radionuclides suitable for Auger electron therapies. This characteristic provides an avenue for the personalized and comprehensive treatment strategy that comprises diagnostic imaging to identify sites of disease, followed by the targeted molecular radiotherapy based on the imaging results. The developed radiosynthetic methods produce no-carrier-added products with high radiochemical yield and purity. The interaction of these compounds with their target, butyrylcholinesterase, depends on the stereochemistry around the P atom. IC(50) values are in the nanomolar range. In vitro studies indicate that radiation doses delivered to the cell nucleus are sufficient to kill cells of several difficult to treat malignancies including glioblastoma and ovarian and colorectal cancers.

The aim of this study was to identify determinants of functional disability, patient's quality of life (QoL) and caregivers' burden in Huntington's disease (HD). Eighty HD patients participated in the study. Motor and behavioral disturbances as well as cognitive impairment were assessed using motor, behavioral and cognitive parts of the Unified Huntington Disease Rating Scale (UHDRS); Hamilton Depression Rating Scale was used to assess depression. Disability, health-related QoL and the impact of the disease on the caregivers were assessed using the following methods: UHDRS Functional Assessment Score, SF-36 Scale and Caregiver Burden Inventory. Multiple regression analysis showed that motor disturbances, cognitive impairment, apathy and disease duration were the independent predictors of disability. Depression and cognitive disturbances were the determinants of patient's QoL, while motor disturbances and depression were the predictors of the caregiver burden. Patient's disability and QoL as well as caregivers' burden should be taken into consideration while planning treatment strategy and the results of the present study show that the predictors of those treatment targets are different.

We sequenced the SERPINC1 gene in 26 patients (11 males) with antithrombin (AT) deficiency (22 type I, 4 type II), belonging to 18 unrelated families from Southern Italy. Heterozygous mutations were identified in 15/18 (83.3%) families. Of them, eight were novel mutations, each being identified in one family. Seven clearly cause impaired protein synthesis (four frameshift, one non-stop, one splicing and one 21bp deletion). One, present in a single patient, is a missense mutation thought to be causative because: a) it is absent in 100 chromosomes from controls; b) it involves a highly conserved amino acid, whose change is predicted to impair AT activity; c) no other mutation is present in the propositus. Severe mutations (i.e. nonsense, frameshift, deletions) were invariably identified in type I patients. In type II patients, 3/4 were missense mutations; the fourth leads to a 19 nucleotides shift in the stop codon. In addition to the type of mutation, the co-existence of other predisposing factors in most patients helps explain the severity of the present type I cases (age at first event, recurrence during prophylaxis). In the five families in which there was more than one member affected, the same genotype and a concordant clinical expression of the disease were found. We conclude that the molecular bases of AT deficiency in Southern Italy are different as compared to other geographic areas, and that molecular analysis and the study of the effect of the mutation may help predict the clinical expression of the disease.

Huntington's disease (HD) is a hereditary neurodegenerative disorder resulting from the expansion of a polyglutamine tract in the huntingtin protein. The expansion of cytosine-adenine-guanine repeats results in neuronal loss in the striatum and cortex. Mutant huntingtin (HTT) may cause toxicity via a range of different mechanisms. Recent studies indicate that impairment of wild-type HTT function may also contribute to HD pathogenesis. However, the mechanisms regulating HTT expression have not been well defined. In this study, we cloned 1,795 bp of the 5' flanking region of the human huntingtin gene (htt) and identified a 106-bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis reveals several putative regulatory elements including Sp1, NF-κB, HIF, CREB, NRSF, P53, YY1, AP1, and STAT in the huntingtin promoter. We found functional Sp1 response elements in the huntingtin promoter region. The expression of Sp1 enhanced huntingtin gene transcription and the inhibition of Sp1-mediated transcriptional activation reduced huntingtin gene expression. These results suggest that Sp1 plays an important role in the regulation of the human huntingtin gene expression at the mRNA and protein levels. Our study suggests that the dysregulation of Sp1-mediated huntingtin transcription, combining with mutant huntingtin's detrimental effect on other Sp1-mediated downstream gene function, may contribute to the pathogenesis of HD.

<h4>Background</h4>Genome-wide association studies (GWAS) have identified loci associated with ischemic stroke (IS) and cardiovascular disease (CVD) in European-descent individuals, but their replication in different populations has been largely unexplored.<h4>Methods and results</h4>Nine single nucleotide polymorphisms (SNPs) selected from GWAS and meta-analyses of stroke, and 86 SNPs previously associated with myocardial infarction and CVD risk factors, including blood lipids (high density lipoprotein [HDL], low density lipoprotein [LDL], and triglycerides), type 2 diabetes, and body mass index (BMI), were investigated for associations with incident IS in European Americans (EA) N=26 276, African-Americans (AA) N=8970, and American Indians (AI) N=3570 from the Population Architecture using Genomics and Epidemiology Study. Ancestry-specific fixed effects meta-analysis with inverse variance weighting was used to combine study-specific log hazard ratios from Cox proportional hazards models. Two of 9 stroke SNPs (rs783396 and rs1804689) were significantly associated with [corrected] IS hazard in AA; none were significant in this large EA cohort. Of 73 CVD risk factor SNPs tested in EA, 2 (HDL and triglycerides SNPs) were associated with IS. In AA, SNPs associated with LDL, HDL, and BMI were significantly associated with IS (3 of 86 SNPs tested). Out of 58 SNPs tested in AI, 1 LDL SNP was significantly associated with IS.<h4>Conclusions</h4>Our analyses showing lack of replication in spite of reasonable power for many stroke SNPs and differing results by ancestry highlight the need to follow up on GWAS findings and conduct genetic association studies in diverse populations. We found modest IS associations with BMI and lipids SNPs, though these findings require confirmation.

Mobile DNAs, also known as transposons or 'jumping genes', are widespread in nature and comprise an estimated 45% of the human genome. Transposons are divided into two general classes based on their transposition intermediate (DNA or RNA). Only one subclass, the non-LTR retrotransposons, which includes the Long INterspersed Element-1 (LINE-1 or L1), is currently active in humans as indicated by 96 disease-causing insertions. The autonomous LINE-1 is capable of retrotransposing not only a copy of its own RNA in cis but also other RNAs (Alu, SINE-VNTR-Alu (SVA), U6) in trans to new genomic locations through an element encoded reverse transcriptase. L1 can also retrotranspose cellular mRNAs, resulting in processed pseudogene formation. Here, we highlight recent reports that update our understanding of human L1 retrotransposition and their role in disease. Finally we discuss studies that provide insights into the past and current activity of these retrotransposons, and shed light on not just when, but where, retrotransposition occurs and its part in genetic variation.

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.

Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N = 16,388) with p<5×10(-8) of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p = 9.1×10(-9)), 7q11 (rs13236689, CD36, p = 2.8×10(-9)), 10q21 (rs7896518, JMJD1C, p = 2.3×10(-12)), 11q13 (rs477895, BAD, p = 4.9×10(-8)), and 20q13 (rs151361, SLMO2, p = 9.4×10(-9)). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N = 14,909) and one (11q13) in Hispanic Americans (N = 3,462). For MPV (N = 4,531), genetic variants in 3 regions were significant at p<5×10(-8), two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis.

Expansion of polyglutamine repeats is the cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease (HD). It is widely accepted that deregulation of the transcriptional coactivator CBP by expanded huntingtin (htt) plays an important role in HD molecular pathogenesis. In this study, we report on a novel target of expanded polyglutamine stretches, the transcriptional coactivator Jun activation domain-binding protein 1 (Jab1), which shares DNA-sequence-specific transcription factor targets with CBP. Jab1 also plays a major role in the degradation of the cyclin-dependent-kinase inhibitor and putative transcription cofactor p27(Kip1). We found that Jab1 accumulates in aggregates when co-expressed with either expanded polyglutamine stretches or N-terminal fragments of mutant htt. In addition, the coactivator function of Jab1 was suppressed both by aggregated expanded polyglutamine solely and by mutant htt. Inhibition by mutant htt even preceded the appearance of microscopic aggregation. In an exon 1 HD cell model, we found that endogenous Jab1 could be recruited into aggregates and that this was accompanied by the accumulation of p27(Kip1). Accumulation of p27(Kip1) was also found in brains derived from HD patients. The repression of Jab1 by various mechanisms coupled with an increase of p27(Kip1) at late stages may have important transcriptional effects. In addition, the interference with the Jab1-p27(Kip1) pathway may contribute to the observed lower incidence of cancer in HD patients and may also be relevant for the understanding of the molecular pathogenesis of polyglutamine disorders in general.

<h4>Background and methods</h4>A nano calcium-deficient hydroxyapatite (n-CDHA)-multi(amino acid) copolymer (MAC) composite bone substitute biomaterial was prepared using an in situ polymerization method. The composition, structure, and compressive strength of the composite was characterized, and the in vitro degradability in phosphate-buffered solution and preliminary cell responses to the composite were investigated.<h4>Results</h4>The composite comprised n-CDHA and an amide linkage copolymer. The compressive strength of the composite was in the range of 88-129 MPa, varying with the amount of n-CDHA in the MAC (ranging from 10 wt% to 50 wt%). Weight loss from the composite increased (from 32.2 wt% to 44.3 wt%) with increasing n-CDHA content (from 10 wt% to 40 wt%) in the MAC after the composite was soaked in phosphate-buffered solution for 12 weeks. The pH of the soaking medium varied from 6.9 to 7.5. MG-63 cells with an osteogenic phenotype were well adhered and spread on the composite surface. Viability and differentiation increased with time, indicating that the composite had no negative effects on MG-63 cells.<h4>Conclusion</h4>The n-CDHA-MAC composite had good cytocompatibility and has potential to be used as a bone substitute.

The authors describe the case of fatal cumulative toxicities in a 58-year-old AIDS-free, HIV-infected patient, who successively developed under highly active antiretroviral therapy (HAART): severe lipodystrophy, complicated osteoporosis, complicated non-cirrhotic portal hypertension of the liver (with ascites, portal thrombosis, oesophageal varices and protein-losing enteropathy) due to nodular regenerative hyperplasia. These cumulative HAART-related toxicities led to death, despite symptomatic treatment and the switch of antiretrovirals (especially didanosine) putatively involved in the process in these drug-mediated diseases. As morbidity and mortality in HIV infection continue to improve, it appears important to recognise such rare HAART-associated toxicities. This case illustrates the absolute necessity of investigating the long-term side effects of HAART in HIV infection, particularly in patients treated with first generation molecules. The switch strategy (switching old molecules to newer ones) is crucial in case of severe suspected toxicity and has to be discussed in asymptomatic patients largely exposed to first generation molecules, in order to prevent long-term toxicity.

Non-invasive monitoring of living cells in vivo provides an important tool in the development of cell-based therapies in cartilage tissue engineering. High-resolution magnetic resonance imaging (MRI) has been used to monitor target cell populations in vivo. However, the side-effects on cell function of the labelling reagents, such as superparamagnetic iron oxide (SPIO), are still unclear. This study investigated the effect of SPIO particles on the chondrogenic differentiation of human bone marrow stromal cells (HBMSCs), neonatal and adult chondrocytes in vitro. Cells were labelled with SPIO for 24 h and chondrogenesis induced in serum-free medium including TGFβ3. For labelled/unlabelled cells, viability, morphology and proliferation were determined using CellTracker™ Green and PicoGreen dsDNA assays. The expression of SOX9, COL2A1 and ACAN was investigated using qRT-PCR after 2, 7 and 14 days. The results showed that viability was unaffected in all of the cells but cell morphology changed towards a 'stretched' phenotype following SPIO uptake. Cell proliferation was reduced only for labelled neonatal chondrocytes. SOX9 and COL2A1 expression decreased at day 2 but not at days 7 and 14 for labelled HBMSCs and adult chondrocytes; ACAN expression was unaffected. In contrast, SOX9 and COL2A1 expression were unaffected in labelled neonatal chondrocytes but a decrease in ACAN expression was seen at day 14. The results suggest that downregulation of chondrogenic genes associated with SPIO labelling is temporary and target cell-dependent. Resovist® can be used to label HBMSCs or mature chondrocytes for MR imaging of cells for cartilage tissue engineering.

<h4>Background</h4>RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.<h4>Results</h4>Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.<h4>Conclusions</h4>Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.

<h4>Background</h4>Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis.<h4>Methods</h4>The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed.<h4>Results</h4>We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury.<h4>Conclusion</h4>In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Epigenetic regulation of gene expression is commonly altered in human cancer. We have observed alterations of DNA methylation and microRNA expression that reflect the biology of bladder cancer. This common disease arises by distinct pathways with low and high-grade differentiation. We hypothesized that epigenetic gene regulation reflects an interaction between histone and DNA modifications, and differences between normal and malignant urothelial cells represent carcinogenic events within bladder cancer. To test this we profiled two repressive histone modifications (H3K9m3 and H3K27m3) using ChIP-Seq, cytosine methylation using MeDIP and mRNA expression in normal and malignant urothelial cell lines. In genes with low expression we identified H3K27m3 and DNA methylation each in 20-30% of genes and both marks in 5% of genes. H3K9m3 was detected in 5-10% of genes but was not associated with overall expression. DNA methylation was more closely related to gene expression in malignant than normal cells. H3K27m3 was the epigenetic mark most specifically correlated to gene silencing. Our data suggest that urothelial carcinogenesis is accompanied by a loss of control of both DNA methylation and H3k27 methylation. From our observations we identified a panel of genes with cancer specific-epigenetic mediated aberrant expression including those with reported carcinogenic functions and members potentially mediating a positive epigenetic feedback loop. Pathway enrichment analysis revealed genes marked by H3K9m3 were involved with cell homeostasis, those marked by H3K27m3 mediated pro-carcinogenic processes and those marked with cytosine methylation were mixed in function. In 150 normal and malignant urothelial samples, our gene panel correctly estimated expression in 65% of its members. Hierarchical clustering revealed that this gene panel stratified samples according to the presence and phenotype of bladder cancer.

<h4>Introduction</h4>Hydroxyapatite (HA) is the principal inorganic constituent of human bone. Due to its good biocompatibility and osteoconductivity, all kinds of HA particles were prepared by different methods. Numerous reports demonstrated that the properties of HA affected its biological effects.<h4>Methods</h4>Two kinds of nanohydroxyapatite with different sizes and crystallinities were obtained via a hydrothermal treatment method under different temperatures. It was found that at a temperature of 140°C, a rod-like crystal (n-HA1) with a diameter of 23 ± 5 nm, a length of 47 ± 14 nm, and crystallinity of 85% ± 5% was produced, while at a temperature of 80°C, a rod-like crystal (n-HA2) with a diameter of 16 ± 3 nm, a length of 40 ± 10 nm, and crystallinity of 65% ± 3% was produced. The influence of nanohydroxyapatite size and crystallinity on osteoblast viability was studied by MTT, scanning electron microscopy, and flow cytometry.<h4>Results</h4>n-HA1 gave a better biological response than n-HA2 in promoting cell growth and inhibiting cell apoptosis, and also exhibited much more active cell morphology. Alkaline phosphatase activity for both n-HA2 and n-HA1 was obviously higher than for the control, and no significant difference was found between n-HA1 and n-HA2. The same trend was observed on Western blotting for expression of type I collagen and osteopontin. In addition, it was found by transmission electron microscopy that large quantities of n-HA2 entered into the cell and damaged the cellular morphology. Release of tumor necrosis factor alpha from n-HA2 was markedly higher than from n-HA1, indicating that n-HA2 might trigger a severe inflammatory response.<h4>Conclusion</h4>This work indicates that not all nanohydroxyapatite should be considered a good biomaterial in future clinical applications.

Huntington's Disease (HD) is a neurodegenerative disease caused by a CAG triplet-repeat expansion-mutation in the Huntingtin gene. Subjects at risk for HD can be identified by genetic testing in the prodromal phase. Structural changes of basal-ganglia nuclei such as the caudate nucleus are well-replicated findings observable early in prodromal-HD subjects and may be preceded by distinct functional alterations of cortico-striatal circuits. This study aims to assess functional integrity of the motor system as a cortico-striatal circuit with particular clinical relevance in HD. Ten subjects in the prodromal phase of HD and ten matched controls were administered blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) at rest (3T). Functional connectivity was measured as synchrony of BOLD activity between the caudate nucleus and thirteen cortical brain regions (seeds). Basal-ganglia volumes were assessed as established markers of disease progression in prodromal-HD. Linear regression analysis was performed to test for a relationship between structural changes and group differences in functional connectivity. Prodromal-HD subjects showed reduced BOLD synchrony between two seeds in the premotor cortex (BA6) and the caudate nucleus. While similar effect sizes could be observed for reduced basal-ganglia volumes and differences in functional connectivity, coefficients of determination indicate a moderate relationship between functional connectivity and striatal atrophy. Our data show reduced cortico-striatal functional connectivity at rest in prodromal-HD and suggest a relation to early structural brain changes. Additional longitudinal studies are necessary to elucidate the temporal relationship between functional alterations and earliest structural brain changes in prodromal-HD.

The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (∼111 GBq/μmol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ∼ RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the α(v)β(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor α(v)β(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin α(v)β(3)-positive tumors.

Discoveries made over the past 20 years highlight the importance of mRNA decay as a means of modulating gene expression and thereby protein production. Up until recently, studies largely focused on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay and the ribonucleases that catalyse decay. Now, current studies have begun to elucidate how the decay process is regulated. This Review examines our current understanding of how mammalian cell mRNA decay is controlled by different signalling pathways and lays out a framework for future research.

Seromucinous hamartoma is a benign lesion of the sinonasal tract. Since its description in 1974, only a small number of additional cases have been reported. It is composed of a proliferation of seromucinous glands and ducts within a variable fibrous stroma. The serous component typically stains positively for S100 (at least focally) and lacks p63 positive abluminal cells. The lack of myoepithelial/basal cells is an important diagnostic feature of seromucinous hamartoma; their absence could lead to an incorrect diagnosis of low-grade sinonasal adenocarcinoma. We report the case of a polypoid mass resected from the posterior nasal cavity and nasopharynx of a 54-year-old woman. The lesion contained a population of small and large glands lined by cuboidal to flattened cells within a hypocellular stroma varying from dense and sclerotic to myxoid. Additionally, there was a superficial focus of ciliated invaginated surface epithelium and glands. Throughout the lesion there were no cytologic or architectural features of malignancy. The histologic features were diagnostic of seromucinous hamartoma. Immunohistochemistry showed focal S100 positivity of the serous glands. However, in contrast to previously reported cases, the glands focally showed an outer basal layer that was calponin, p63 and actin positive. Our case demonstrates two important points. First, complete absence of p63 staining should not necessarily be a required feature in the diagnosis of seromucinous hamartoma. Second, the ciliated larger glands--in keeping with respiratory epithelial adenomatoid hamartoma (REAH)--support the suggestion that seromucinous hamartoma and REAH are a spectrum of lesions, often seen together.

<h4>Background</h4>The p38α mitogen-activated protein kinase (MAPK) is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear.<h4>Methods</h4>To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms.<h4>Results</h4>Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation.<h4>Conclusion</h4>Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

Cell culture/xenograft and gene arrays of clinical material document that development of castration resistant prostate cancer (CRPC) cells involves acquisition of adaptive auto-regulation resulting in >25-fold increase in Androgen Receptor (AR) protein expression in a low androgen environment. Such adaptive AR increase paradoxically is a liability in castrated hosts, however, when supraphysiologic androgen is acutely replaced. Cell synchronization/anti-androgen response is due to AR binding to replication complexes (RC) at origin of replication sites (ORS) in early G1 associated with licensing/restricting DNA for single round of duplication during S-phase. When CRPC cells are acutely exposed to supraphysiologic androgen, adaptively increased nuclear AR is over-stabilized, preventing sufficient degradation in mitosis, inhibiting DNA re-licensing, and thus death in the subsequent cell cycle. These mechanistic results and the fact that AR/RC binding occurs in metastatic CRPCs directly from patients provides a paradigm shifting rationale for bipolar androgen therapy (BAT) in patient progressing on chronic androgen ablation. BAT involves giving sequential cycles alternating between periods of acute supraphysiologic androgen followed by acute ablation to take advantage of vulnerability produced by adaptive auto-regulation and binding of AR to RC in CRPC cells. BAT therapy is effective in xenografts and based upon positive results has entered clinical testing.

Protein misfolding and aggregation are associated with many neurodegenerative diseases, including Huntington's disease. The cellular machinery for maintaining proteostasis includes molecular chaperones that facilitate protein folding and reduce proteotoxicity. Increasing the protein folding capacity of cells through manipulation of DNAJ chaperones has been shown to suppress aggregation and ameliorate polyglutamine toxicity in cells and flies. However, to date these promising findings have not been translated to mammalian models of disease. To address this issue, we developed transgenic mice that over-express the neuronal chaperone HSJ1a (DNAJB2a) and crossed them with the R6/2 mouse model of Huntington's disease. Over-expression of HSJ1a significantly reduced mutant huntingtin aggregation and enhanced solubility. Surprisingly, this was mediated through specific association with K63 ubiquitylated, detergent insoluble, higher order mutant huntingtin assemblies that decreased their ability to nucleate further aggregation. This was dependent on HSJ1a client binding ability, ubiquitin interaction and functional co-operation with HSP70. Importantly, these changes in mutant huntingtin solubility and aggregation led to improved neurological performance in R6/2 mice. These data reveal that prevention of further aggregation of detergent insoluble mutant huntingtin is an additional level of quality control for late stage chaperone-mediated neuroprotection. Furthermore, our findings represent an important proof of principle that DNAJ manipulation is a valid therapeutic approach for intervention in Huntington's disease.

It is now established that, as compared to normal cells, the cancer cell genome has an overall inverse distribution of DNA methylation ("methylome"), i.e., predominant hypomethylation and localized hypermethylation, within "CpG islands" (CGIs). Moreover, although cancer cells have reduced methylation "fidelity" and genomic instability, accurate maintenance of aberrant methylomes that underlie malignant phenotypes remains necessary. However, the mechanism(s) of cancer methylome maintenance remains largely unknown. Here, we assessed CGI methylation patterns propagated over 1, 3, and 5 divisions of A2780 ovarian cancer cells, concurrent with exposure to the DNA cross-linking chemotherapeutic cisplatin, and observed cell generation-successive increases in total hyper- and hypo-methylated CGIs. Empirical bayesian modeling revealed five distinct modes of methylation propagation: (1) heritable (i.e., unchanged) high-methylation (1186 probe loci in CGI microarray); (2) heritable (i.e., unchanged) low-methylation (286 loci); (3) stochastic hypermethylation (i.e., progressively increased, 243 loci); (4) stochastic hypomethylation (i.e., progressively decreased, 247 loci); and (5) considerable "random" methylation (582 loci). These results support a "stochastic model" of DNA methylation equilibrium deriving from the efficiency of two distinct processes, methylation maintenance and de novo methylation. A role for cis-regulatory elements in methylation fidelity was also demonstrated by highly significant (p<2.2×10(-5)) enrichment of transcription factor binding sites in CGI probe loci showing heritably high (118 elements) and low (47 elements) methylation, and also in loci demonstrating stochastic hyper-(30 elements) and hypo-(31 elements) methylation. Notably, loci having "random" methylation heritability displayed nearly no enrichment. These results demonstrate an influence of cis-regulatory elements on the nonrandom propagation of both strictly heritable and stochastically heritable CGIs.

MicroRNAs are gaining importance as regulators of gene expression with the capability to fine-tune and modulate cellular events. The complex network with their selective targets (mRNAs/genes) pave way for regulation of many physiological processes. Dysregulation of normal neuronal activities could result in accumulation of substances that are detrimental to neuronal functions and subsequently result in neurotoxicity. Neurotoxicity-mediated pathophysiological conditions could then manifest as diseases or disabilities like Parkinson's and Alzheimer's which have debilitating implications. Such toxicity can be a result of individuals predisposed due to genetic inheritance or from other sources such as brain tumours. Neurotoxicity can also be brought about by external agents like drugs and alcohol as well as brain injury with miRNAs playing a pivotal role in diseases. It is therefore vital to understand the expression of these microRNAs and their impact on neuronal activities. In this paper, we discuss some of the neuronal pathophysiological conditions that could be caused by dysregulated microRNAs.

RAF kinase inhibitor protein (RKIP) is a negative regulator of the RAS-mitogen-activated protein kinase/extracellular signal-regulated kinase signaling cascade. We investigated its role in acute myeloid leukemia (AML), an aggressive malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Western blot analysis revealed loss of RKIP expression in 19/103 (18%) primary AML samples and 4/17 (24%) AML cell lines but not in 10 CD34+ HSPC specimens. In in-vitro experiments with myeloid cell lines, RKIP overexpression inhibited cellular proliferation and colony formation in soft agar. Analysis of two cohorts with 103 and 285 AML patients, respectively, established a correlation of decreased RKIP expression with monocytic phenotypes. RKIP loss was associated with RAS mutations and in transformation assays, RKIP decreased the oncogenic potential of mutant RAS. Loss of RKIP further related to a significantly longer relapse-free survival and overall survival in uni- and multivariate analyses. Our data show that RKIP is frequently lost in AML and correlates with monocytic phenotypes and mutations in RAS. RKIP inhibits proliferation and transformation of myeloid cells and decreases transformation induced by mutant RAS. Finally, loss of RKIP seems to be a favorable prognostic parameter in patients with AML.

Caspi et al. (2003) found an interaction between the serotonin transporter polymorphism gene (5-HTTLPR) and stressful life events on depression. Subsequent attempts to replicate have been inconsistent. The present research included long allele variants modified by SNP rs25531 and tested the interaction on adolescents' trajectories of anxious/depressed symptoms, with consideration of possible age effects. Adolescents (N = 574), of whom 436 were genotyped, were followed from ages 12 to 17. Analyses demonstrated a G × E interaction in predicting the development of anxious/depressed symptoms. Specifically, adolescents with lower serotonin transcriptional efficiency (TE) genotypes whose mothers reported more stressful events were reported to show more anxious/depressed symptoms and greater increases in the development of symptoms of anxiety and depression than were higher TE adolescents, particularly at ages 16 and 17. Interactions did not differ by gender. Findings demonstrate that stress may affect adolescents' likelihood of experiencing anxious/depressed symptoms when they have a low serotonin TE (A/G-modified 5-HTTLPR) genotype and suggest that the vulnerability may be stronger in late than early adolescence.

<h4>Background</h4>Diabetes can lead to serious microvascular complications such as proliferative diabetic retinopathy (PDR), which results in severe vision loss. The diabetes-induced alterations in the vitreous protein composition in diabetic patients with PDR may be responsible for the presence of PDR. The vitreous humour can be utilised in a variety of studies aimed toward the discovery of new targets for the treatment or prevention of PDR and the identification of novel disease mechanisms. The aim of this study was to compare the protein profile of vitreous humour from diabetic patients with PDR with that of vitreous humour from normal human eyes donated for corneal transplant.<h4>Results</h4>Vitreous humour from type 2 diabetic patients with PDR (n = 10) and from normal human eyes donated for corneal transplant (n = 10) were studied. The comparative proteomic analysis was performed using two-dimensional fluorescence difference gel electrophoresis (2-D DIGE). Differentially produced proteins (abundance ratio > 2 or < -2, p < 0.01) were identified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF tandem mass spectrometry. A total of 1242 protein spots were detected on the 2-D master gel of the samples, and 57 spots that exhibited statistically significant variations were successfully identified. The spots corresponded to peptide fragments of 29 proteins, including 8 proteins that increased and 21 proteins that decreased in PDR. Excluding the serum proteins from minor vitreous haemorrhage, 19 proteins were found to be differentially produced in PDR patients compared with normal subjects; 6 of these proteins have never been reported to be differentially expressed in PDR vitreous: N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 (DDAH 1), tubulin alpha-1B chain, gamma-enolase, cytosolic acyl coenzyme A thioester hydrolase, malate dehydrogenase and phosphatidylethanolamine-binding protein 1 (PEBP 1). The differential production of pigment epithelium-derived factor (PEDF) and clusterin was confirmed by Western blot analysis.<h4>Conclusions</h4>These data provide an in-depth analysis of the human vitreous proteome and reveal protein alterations that are possibly involved in the pathogenesis of PDR. Further investigation of these special proteins may provide potential new targets for the treatment or the prevention of PDR.

Dosage compensation equalizes X-linked gene expression between the sexes. This process is achieved in Caenorhabditis elegans by hermaphrodite-specific, dosage compensation complex (DCC)-mediated, 2-fold X chromosome downregulation. How the DCC downregulates gene expression is not known. By analyzing the distribution of histone modifications in nuclei using quantitative fluorescence microscopy, we found that H4K16 acetylation (H4K16ac) is underrepresented and H4K20 monomethylation (H4K20me1) is enriched on hermaphrodite X chromosomes in a DCC-dependent manner. Depletion of H4K16ac also requires the conserved histone deacetylase SIR-2.1, while enrichment of H4K20me1 requires the activities of the histone methyltransferases SET-1 and SET-4. Our data suggest that the mechanism of dosage compensation in C. elegans involves redistribution of chromatin-modifying activities, leading to a depletion of H4K16ac and an enrichment of H4K20me1 on the X chromosomes. These results support conserved roles for histone H4 chromatin modification in worm dosage compensation analogous to those seen in flies, using similar elements and opposing strategies to achieve differential 2-fold changes in X-linked gene expression.

Throughout life, intestinal Lgr5+ stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1, and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1-/-, Tcf3(flox), and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.

Antidepressant usage in prodromal Huntington Disease (HD) remains uncharacterized, despite its relevance in designing experiments, studying outcomes of HD, and evaluating the efficacy of therapeutic interventions. We searched baseline medication logs of 787 prodromal HD and 215 healthy comparison (HC) participants for antidepressant use. Descriptive and mixed-effects logistic regression modeling characterized usage across participants. At baseline, approximately one in five prodromal HD participants took antidepressants. Of those, the vast majority took serotonergic antidepressants (selective serotonin reuptake inhibitor (SSRI) or serotonin/norepinephrine reuptake inhibitor (SNRI)). Significantly more prodromal HD participants used serotonergic antidepressants than their HC counterparts. Because of the prevalence of these medications, further analyses focused on this group alone. Mixed-effects logistic regression modeling revealed significant relationships of both closer proximity to diagnosis and female sex with greater likelihood to be prescribed a serotonergic antidepressant. More prodromal HD participants took antidepressants in general and specifically the subclass of serotonergic antidepressants than their at-risk counterparts, particularly when they were closer to predicted time of conversion to manifest HD. These propensities must be considered in studies of prodromal HD participants.

Axon pathfinding is essential for the establishment of proper neuronal connections during development. Advances in neuroimaging and genomic technologies, coupled with animal modeling, are leading to the identification of an increasing number of human disorders that result from aberrant axonal wiring. In this review, we summarize the recent clinical, genetic and molecular advances with regard to three human disorders of axon guidance: Horizontal gaze palsy with progressive scoliosis, Congenital mirror movements, and Congenital fibrosis of the extraocular muscles, Type III.

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.

<h4>Background</h4>Iron isomaltoside 1000 is a novel injectable iron compound which offers potential advantages in the treatment of subjects with iron-deficiency anemia. We studied the pharmacokinetics (PK) of this novel compound in subjects with mild-to-moderate inflammatory bowel disease (IBD).<h4>Methods</h4>This open-label, crossover, single-center trial was conducted in 12 subjects with IBD who were allocated to one of the two single intravenous (IV) bolus sequences of iron isomaltoside 1000: 100 mg followed by 200 mg, or vice-versa. PK variables were analyzed according to a single-compartment model.<h4>Results</h4>The concentration-versus-time relationship for isomaltoside-bound iron (IBI) and total iron (TI) showed first-order kinetics with small deviations from dose-linearity. The area under the concentration-time curve (AUC) values in h * μg/mL for IBI following 100 mg and 200 mg doses were 888 and 2141 respectively, and for TI following 100 mg and 200 mg doses, the AUC values were 1010 and 2319 respectively. The corresponding maximum serum concentration (C(max)) values in μg/mL were 35.6 and 68.6 for IBI, and 37.3 and 71.1 for TI. The half-life (T(½)) values for IBI and TI were between 20.8-23.5 hours. The apparent volume of distribution (V(D)) ranged from 3.0-3.5 L. Only approximately 1% of the doses administered were excreted in the urine. No serious adverse event (SAE) was reported. One subject was withdrawn after the 100 mg dose due to abdominal pain and flushing.<h4>Conclusion</h4>At the administered doses, iron isomaltoside 1000 showed first-order PK, and did not raise safety concerns in patients with IBD. The PK parameters for IBI were close to those of TI.

The prevalence of obesity, which is a heritable trait that arises from the interactions of multiple genes and lifestyle factors, continues to increase worldwide, causing serious health problems and imposing a substantial economic burden on societies. For the past several years, various genetic epidemiological approaches have been utilized to identify genetic loci for obesity. Recent evidence suggests that development of obesity involves hormones and neurotransmitters (such as leptin, cocaine- and amphetamine-regulated transcript (CART), and ghrelin) that regulate appetite and energy expenditure. These hormones act on specific centers in the brain that regulate the sensations of satiety. Mutations in these hormones or their receptors can lead to obesity. Aberrant circadian rhythms and biochemical pathways in peripheral organs or tissues have also been implicated in the pathology of obesity. More interestingly, increasing evidence indicates a potential relation between obesity and central nervous system disorders (such as cognitive deficits). This paper discusses recent advances in the field of genetics of obesity with an emphasis on several established loci that influence obesity. These recently identified loci may hold the promise to substantially improve our insights into the pathophysiology of obesity and open up new therapeutic strategies to combat growing obesity epidemic facing the human population today.

Hydroxyapatite (HA) is a precursor of bone and has been studied as a biomaterial. We attempted HA to apply to protein delivery systems. In this study, the association and dissociation properties of two types of bioactive proteins, cytochrom c and insulin, to HA were investigated. Cytochrom c was less associated with HA than insulin, which was easily released from it. However, the release of insulin from HA was slow. Insulin was released from HA at pH 7.4 more rapidly than at pH 3. The association and dissociation properties might be influenced by the size, solubility and net charge of protein. HA is a potential protein carrier with controlled release.

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically interpreted translocations and inversions. We confirm that the recently described phenomenon of 'chromothripsis' (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline, where it can resolve to a relatively balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign copy-number variants (CNVs). We compared these results to experimentally generated DNA breakage-repair by sequencing seven transgenic animals, revealing extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion was the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations.

Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.

In addition to its central roles in protein quality control, regulation of cell cycle, intracellular signaling, DNA damage response and transcription regulation, the ubiquitin-proteasome system (UPS) plays specific roles in the nervous system, where it contributes to precise connectivity through development, and later assures functionality by regulating a wide spectrum of neuron-specific cellular processes. Aberrations in this system have been implicated in the etiology of neurodevelopmental and neurodegenerative diseases. In this review, we provide an updated view on the UPS and highlight recent findings concerning its role in normal and diseased nervous systems. We discuss the advantages of the model organism Caenorhabditis elegans as a tool to unravel the major unsolved questions concerning this biochemical pathway and its involvement in nervous system function and dysfunction, and expose the new possibilities, using state-of-the-art techniques, to assess UPS function using this model system.

Poly(lactide-co-glycolide) (PLGA) has most often been employed for the controlled release of protein formulations because of its safety profile with non-toxic degradation products. Nevertheless, such formulations have been plagued by a local acidic microenvironment and protein-polymer interactions, which result in chemical and physical denaturation of loaded proteins and often unfavorable release profiles. This study investigated the pH change of inner PLGA microsphere (MS) using charged (PLGA)(n)-b-branched polyethyleneimine (bPEI) micelles. The designed micelles can be transformed into either micelle or reverse micelle (RM) depending on the solvent and RM can form microspheres. In addition, (PLGA)(n)-b-bPEI can be modified into (PLGA)(n)-b-(carboxylated bPEI) via carboxylation of the primary amines. Cationic micelle (CM) or anionic micelle (AM) was complexed with counter-charged proteins leading to nanosized particles (approximately 100nm). In the micelle/protein complexes, the micelles mostly maintained their proton buffering capacity, and consequently, prevented or delayed the typical decrease in pH caused by degradation of PLGA in aqueous solution. Reconstitutable micelle/protein complexes allowed for increased and fine-tuned protein loading (~20wt.% when using CM1 (CM prepared from PLGA(36kDa)-b-bPEI(25kDa))/insulin complexes) in PLGA MS. In CM2 (CM prepared from (PLGA(36kDa))(2)-b-bPEI(25kDa))/insulin (4 of weight ratio (WR) of micelle to protein; WR4)-loaded PLGA MS, CM2 strongly prevented the micellar nanoenvironmental pH (pH 6.6 within 5days and then approximately pH 8.5) to be acidified in PLGA MS for 9weeks, unlike CM2-free PLGA MS. In conclusion, our findings propose that the proton buffering capacity and protein loading in PLGA MS can be tuned by controlling the complexation ratios of micelles and proteins, polymeric architectures of (PLGA)(n)-b-bPEI copolymers and WR of micelle/protein complexes and PLGA (or RM).

The development of tasks measuring behaviors specific to the three major symptom categories for autism makes it possible to differentiate mouse models of autism spectrum disorders (ASD) in terms of changes in these specific categories. Prior studies indicate that BTBR T+tf/J mice, the strain that has been evaluated most extensively, show autism-relevant changes in all three symptom categories; reciprocal social interactions; communication; and repetitive, ritualized behaviors. This report reviews the behaviors of oxytocin receptor (Oxtr) and Mecp2(308/Y) wild-type (WT) and knockout (KO) mice, in a number of tests specifically designed to provide information on behaviors that may show functional parallels to the core symptoms of ASD. Oxtr KO mice show robust decreases in reciprocal social interactions, and reduced levels of communication, but no changes in repetitive, ritualized behaviors; whereas Mecp2(308/Y) KO mice show a slight but consistent enhancement of social behavior and communication, and no changes in repetitive, ritualized behaviors. This data base, although small, strongly indicates that mouse models can sort the diagnostic symptoms of autism, and suggests that biological and physiological analyses of these strains may be capable of providing differential information on the brain systems involved in particular symptoms of this disorder. Profiles of behavioral changes in other mouse models of ASD should provide additional specificity in the search for biomarkers associated with particular ASD symptoms and symptom clusters.

The discovery and development of small molecule cancer drugs has been revolutionised over the last decade. Most notably, we have moved from a one-size-fits-all approach that emphasized cytotoxic chemotherapy to a personalised medicine strategy that focuses on the discovery and development of molecularly targeted drugs that exploit the particular genetic addictions, dependencies and vulnerabilities of cancer cells. These exploitable characteristics are increasingly being revealed by our expanding understanding of the abnormal biology and genetics of cancer cells, accelerated by cancer genome sequencing and other high-throughput genome-wide campaigns, including functional screens using RNA interference. In this review we provide an overview of contemporary approaches to the discovery of small molecule cancer drugs, highlighting successes, current challenges and future opportunities. We focus in particular on four key steps: Target validation and selection; chemical hit and lead generation; lead optimization to identify a clinical drug candidate; and finally hypothesis-driven, biomarker-led clinical trials. Although all of these steps are critical, we view target validation and selection and the conduct of biology-directed clinical trials as especially important areas upon which to focus to speed progress from gene to drug and to reduce the unacceptably high attrition rate during clinical development. Other challenges include expanding the envelope of druggability for less tractable targets, understanding and overcoming drug resistance, and designing intelligent and effective drug combinations. We discuss not only scientific and technical challenges, but also the assessment and mitigation of risks as well as organizational, cultural and funding problems for cancer drug discovery and development, together with solutions to overcome the 'Valley of Death' between basic research and approved medicines. We envisage a future in which addressing these challenges will enhance our rapid progress towards truly personalised medicine for cancer patients.

The proteasome is a multi-catalytic protein complex whose primary function is the degradation of abnormal or foreign proteins. Upon exposure of cells to interferons (IFNs), the β1i/LMP2, β2i/MECL-1, and β5i/LMP7 subunits are induced and incorporated into newly synthesized immunoproteasomes (IP), which are thought to function solely as critical players in the optimization of the CD8(+) T-cell response. However, the observation that IP are present in several non-immune tissues under normal conditions and/or following pathological events militates against the view that its role is limited to MHC class I presentation. In support of this concept, the recent use of genetic models deficient for β1i/LMP2, β2i/MECL-1, or β5i/LMP7 has uncovered unanticipated functions for IP in innate immunity and non-immune processes. Herein, we review recent data in an attempt to clarify the role of IP beyond MHC class I epitope presentation with emphasis on its involvement in the regulation of protein homeostasis, cell proliferation, and cytokine gene expression.

<h4>Introduction</h4>The capacity of bone marrow mesenchymal stromal cells (BMSCs) to be induced into chondrocytes has drawn much attention for cell-based cartilage repair. BMSCs represent a small proportion of cells of the bone marrow stromal compartment and, thus, culture expansion is a necessity for therapeutic use. However, there is no consensus on how BMSCs should be isolated nor expanded to maximize their chondrogenic potential. During embryonic development pluripotent stem cells differentiate into chondrocytes and form cartilage in a hypoxic microenvironment.<h4>Methods</h4>Freshly harvested human BMSCs were isolated and expanded from the aspirates of six donors, under either hypoxic conditions (3% O2) or normoxic conditions (21% O2). A colony-forming unit fibroblastic (Cfu-f) assay was used to determine the number of cell colonies developed from each donor. BMSCs at passage 2 (P2) were characterized by flow cytometry for the phenotypic expression of cell surface markers on mesenchymal stem cells. BMSCs at P2 were subsequently cultured in vitro as three-dimensional cell pellets in a defined serum-free chondrogenic medium under normoxic and hypoxic conditions. Chondrogenic differentiation of the BMSCs was characterized by biochemical and histological methods and by quantitative gene-expression analysis.<h4>Results</h4>After 14 days of culture, the number of BMSC colonies developed under hypoxia was generally higher (8% to 38% depending on donor) than under normoxia. BMSCs were positive for the cell surface markers CD13, CD29, CD44, CD73, CD90, CD105 and CD151, and negative for CD34. Regardless of the oxygen tension during pellet culture, hypoxia-expanded BMSC pellets underwent a more robust chondrogenesis than normoxia-expanded BMSC pellets after three weeks of culture, as judged by increased glycosaminoglycan synthesis and Safranin O staining, along with increased mRNA expression of aggrecan, collagen II and Sox9. Hypoxic conditions enhanced the mRNA expression of hypoxia inducible factor-2 alpha (HIF-2α) but suppressed the mRNA expression of collagen X in BMSC pellet cultures regardless of the oxygen tension during BMSC isolation and propagation.<h4>Conclusions</h4>Taken together, our data demonstrate that isolation and expansion of BMSCs under hypoxic conditions augments the chondrogenic potential of BMSCs. This suggests that hypoxia-mediated isolation and expansion of BMSCs may improve clinical applications of BMSCs for cartilage repair.

The genetics of skeletal muscle lineage commitment are deceptively complicated. MyoD overexpression is sufficient to convert fibroblasts into skeletal muscle myotubes. In vivo, there are a number of different steps of differentiation that require a large network of transcription factors that control differentiation and homeostasis of skeletal muscle progenitors. Each transcription factor has been shown to have the ability to promote the next factor in the cascade, but the mechanisms regulating the transitions remain incomplete. Recently, microRNAs have been shown to be important for a large number of developmental and oncogenic processes. In this review, we will discuss recent advances in the understanding of how microRNA is critical for skeletal muscle development by interacting with protein-coding genes that had previously been shown to be important for myogenesis.

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin-α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down-modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in >90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras-association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.

In this paper, we present the design and implementation of the integrated proactive surveillance system for prostate cancer (PASS-PC). The integrated PASS-PC is a multi-institutional web-based system aimed at collecting a variety of data on prostate cancer patients in a standardized and efficient way. The integrated PASS-PC was commissioned by the Prostate Cancer Foundation (PCF) and built through the joint of efforts by a group of experts in medical oncology, genetics, pathology, nutrition, and cancer research informatics. Their main goal is facilitating the efficient and uniform collection of critical demographic, lifestyle, nutritional, dietary and clinical information to be used in developing new strategies in diagnosing, preventing and treating prostate cancer.The integrated PASS-PC is designed based on common industry standards - a three tiered architecture and a Service- Oriented Architecture (SOA). It utilizes open source software and programming languages such as HTML, PHP, CSS, JQuery, Drupal and MySQL. We also use a commercial database management system - Oracle 11g. The integrated PASS-PC project uses a "confederation model" that encourages participation of any interested center, irrespective of its size or location. The integrated PASS-PC utilizes a standardized approach to data collection and reporting, and uses extensive validation procedures to prevent entering erroneous data. The integrated PASS-PC controlled vocabulary is harmonized with the National Cancer Institute (NCI) Thesaurus. Currently, two cancer centers in the USA are participating in the integrated PASS-PC project.THE FINAL SYSTEM HAS THREE MAIN COMPONENTS: 1. National Prostate Surveillance Network (NPSN) website; 2. NPSN myConnect portal; 3. Proactive Surveillance System for Prostate Cancer (PASS-PC). PASS-PC is a cancer Biomedical Informatics Grid (caBIG) compatible product. The integrated PASS-PC provides a foundation for collaborative prostate cancer research. It has been built to meet the short term goal of gathering prostate cancer related data, but also with the prerequisites in place for future evolution into a cancer research informatics platform. In the future this will be vital for successful prostate cancer studies, care and treatment.

<h4>Abstract</h4>Two series of novel 4-chloro-2-(benzylthio)-5-(1,3,4-oxadiazol-2-yl)benzenesulfonamides and their <i>N</i>-aroyl derivatives have been synthesized and evaluated for in vitro anticancer activity against the full NCI-60 cell line panel. Most of the compounds exhibited antiproliferative activity. Among them a compound bearing an <i>N</i>-(thien-2-ylcarbonyl) moiety showed broad-spectrum activity with 50% growth inhibition (GI₅₀) values in the range of 2.02-7.82 μM over 50 cell lines.<h4>Graphical abstract</h4>.