Gene Literature Dashboard

<h4>Background</h4>Bovine mastitis is one of the most costly and prevalent diseases affecting dairy cows worldwide. In order to develop new strategies to prevent Escherichia coli-induced mastitis, a detailed understanding of the molecular mechanisms underlying the host immune response to an E. coli infection is necessary. To this end, we performed a global gene-expression analysis of mammary gland tissue collected from dairy cows that had been exposed to a controlled E. coli infection. Biopsy samples of healthy and infected utter tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR) and chronic stage, respectively. Differentially expressed (DE) genes for each stage were analyzed and the DE genes detected at T = 24 h were also compared to data collected from two previous E. coli mastitis studies that were carried out on post mortem tissue.<h4>Results</h4>Nine-hundred-eighty-two transcripts were found to be differentially expressed in infected tissue at T = 24 (P < 0.05). Up-regulated transcripts (699) were largely associated with immune response functions, while the down-regulated transcripts (229) were principally involved in fat metabolism. At T = 192 h, all of the up-regulated transcripts were associated with tissue healing processes. Comparison of T = 24 h DE genes detected in the three E. coli mastitis studies revealed 248 were common and mainly involved immune response functions. KEGG pathway analysis indicated that these genes were involved in 12 pathways related to the pro-inflammatory response and APR, but also identified significant representation of two unexpected pathways: natural killer cell-mediated cytotoxicity pathway (KEGG04650) and the Rig-I-like receptor signalling pathway (KEGG04622).<h4>Conclusions</h4>In E. coli-induced mastitis, infected mammary gland tissue was found to significantly up-regulate expression of genes related to the immune response and down-regulate genes related to fat metabolism. Up to 25% of the DE immune response genes common to the three E. coli mastitis studies at T = 24 h were independent of E. coli strain and dose, cow lactation stage and number, tissue collection method and gene analysis method used. Hence, these DE genes likely represent important mediators of the local APR against E. coli in the mammary gland.

Nuage (or commonly known as chromatoid body in mammals) is a conserved germline-specific organelle that has been linked to the Piwi-interacting RNA (piRNA) pathway. piRNAs are a class of gonadal-specific RNAs that are ~23-29 nucleotides in length and protect genome stability by repressing the expression of deleterious retrotransposons. More recent studies in Drosophila have implicated the piRNA pathway in other functions including canalization of embryonic development, regulation of maternal gene expression and telomere protection. We have recently shown that Vasa (known as Mouse Vasa Homolog in mouse), a nuage component, plays a mitotic role in promoting chromosome condensation and segregation by facilitating robust chromosomal localization of condensin I in the Drosophila germline. Vasa functions together with Aubergine (a PIWI family protein) and Spindle-E/mouse TDRD-9, two other nuage components that are involved in the piRNA pathway, therefore providing a link between the piRNA pathway and mitotic chromosome condensation. Here, we propose and discuss possible models for the role of Vasa and the piRNA pathway during mitosis. We also highlight relevant studies implicating mitotic roles for RNAs and/or nuage in other model systems and their implications for cancer development.

<h4>Background</h4>Haploinsufficient (HI) genes are those for which a reduction in copy number in a diploid from two to one results in significantly reduced fitness. Haploinsufficiency is increasingly implicated in human disease, and so predicting this phenotype could provide insights into the genetic mechanisms behind many human diseases, including some cancers.<h4>Results</h4>In the present work we show that orthologues of Saccharomyces cerevisiae HI genes are preferentially retained across the kingdom Fungi, and that the HI genes of S. cerevisiae can be used to predict haploinsufficiency in humans. Our HI gene predictions confirm known associations between haploinsufficiency and genetic disease, and predict several further disorders in which the phenotype may be relevant. Haploinsufficiency is also clearly relevant to the gene-dosage imbalances inherent in eukaryotic sex-determination systems. In S. cerevisiae, HI genes are over-represented on chromosome III, the chromosome that determines yeast's mating type. This may be a device to select against the loss of one copy of chromosome III from a diploid. We found that orthologues of S. cerevisiae HI genes are also over-represented on the mating-type chromosomes of other yeasts and filamentous fungi. In animals with heterogametic sex determination, accumulation of HI genes on the sex chromosomes would compromise fitness in both sexes, given X chromosome inactivation in females. We found that orthologues of S. cerevisiae HI genes are significantly under-represented on the X chromosomes of mammals and of Caenorhabditis elegans. There is no X inactivation in Drosophila melanogaster (increased expression of X in the male is used instead) and, in this species, we found no depletion of orthologues to yeast HI genes on the sex chromosomes.<h4>Conclusion</h4>A special relationship between HI genes and the sex/mating-type chromosome extends from S. cerevisiae to Homo sapiens, with the microbe being a useful model for species throughout the evolutionary range. Furthermore, haploinsufficiency in yeast can predict the phenotype in higher organisms.

<h4>Background</h4>The vascular disease in-stent restenosis (ISR) is characterized by formation of neointima and adverse inward remodeling of the artery after injury by coronary stent implantation. We hypothesized that the analysis of gene expression in peripheral blood mononuclear cells (PBMCs) would demonstrate differences in transcript expression between individuals who develop ISR and those who do not.<h4>Methods and results</h4>We determined and investigated PBMC gene expression of 358 patients undergoing an index procedure to treat in de novo coronary artery lesions with bare metallic stents, using a novel time-varying intercept model to optimally assess the time course of gene expression across a time course of blood samples. Validation analyses were conducted in an independent sample of 97 patients with similar time-course blood sampling and gene expression data. We identified 47 probesets with differential expression, of which 36 were validated upon independent replication testing. The genes identified have varied functions, including some related to cellular growth and metabolism, such as the NAB2 and LAMP genes.<h4>Conclusions</h4>In a study of patients undergoing bare metallic stent implantation, we have identified and replicated differential gene expression in peripheral blood mononuclear cells, studied across a time series of blood samples. The genes identified suggest alterations in cellular growth and metabolism pathways, and these results provide the basis for further specific functional hypothesis generation and testing of the mechanisms of ISR.

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.

proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and co-immunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371-445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1(-/-) neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1(-/-) neurons. Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.

Although local regulation of T-cell responses by epithelial cells is increasingly viewed as important, few molecules mediating such regulation have been identified. Skint1, a recently identified member of the Ig-supergene family expressed by thymic epithelial cells and keratinocytes, specifies the murine epidermal intraepithelial lymphocyte (IEL) repertoire. Investigating whether Skint1-related molecules might regulate IEL in other compartments, this study focuses on buytrophilin-like 1 (Btnl1), which is conspicuously similar to Skint1 and primarily restricted to small intestinal epithelium. Btnl1 protein is mostly cytoplasmic, but surface expression can be induced, and in vivo Btnl1 can be detected adjacent to the IEL. In a newly developed culture system, enforced epithelial cell expression of Btnl1 attenuated the cells' response to activated IEL, as evidenced by suppression of IL-6 and other inflammatory mediators. These findings offer a unique perspective on emerging genetic data that Btnl genes may comprise novel and important local regulators of gut inflammation.

Huntington's disease (HD) is a debilitating autosomal dominant, neurodegenerative disease with a fatal prognosis. Classical features include motor disturbances, dementia and psychiatric problems but are not restricted to this triad as patients often experience other abnormalities such as unintended weight loss, the exact cause of which is unknown. We studied the body composition of 25 premanifest HD and compared it to 25 control subjects using a dual energy x-ray absorptiometer (DEXA) scan. Like the R6/2 transgenic mouse model, we identified significantly lower bone mineral density z-scores in premanifest individuals, that was not related to any difference in testosterone, cortisol, leptin or Vitamin D levels. These results identify an early gene-related change that occurs in HD which not only could lead to a potential biomarker for the disease, but given it is also seen in other manifest neurodegenerative diseases, could also reveal a common disease related process.

<h4>Background</h4>Impulsivity has been associated with serotonergic system functions. However, few researchers have investigated the relationship between a polymorphism in the promoter of the serotonin transporter gene (5-HTTLPR) and the different components of impulsivity in a non-clinical population. The aim of this study was to investigate the relationship between a polymorphism in the promoter region of the serotonin transporter gene (5-HTTLPR) and the different components of impulsivity in a non-clinical population.<h4>Methodology/principal findings</h4>We administered two neuropsychological tests, the Continuous Performance Task and the Iowa Gambling Task, to 127 healthy participants to measure their levels of motor, attentional and non-planning impulsivity. Then, these participants were grouped by genotype and gender, and their scores on impulsivity measures were compared. There were no significant differences between group scores on attentional, motor and non-planning impulsivity.<h4>Conclusions/significance</h4>Our results suggest that 5-HTTLPR genotype is not significantly associated with subsets of impulsive behavior in a non-clinical sample when measured by neuropsychological tests. These findings are discussed in terms of the sensitivity of neuropsychological tests to detect impulsivity in a non-clinical population and the role of gender and race in the relationship between the 5-HTTLPR and impulsivity.

Since their arrival in the Tibetan Plateau during the Neolithic Age, Tibetans have been well-adapted to extreme environmental conditions and possess genetic variation that reflect their living environment and migratory history. To investigate the origin of Tibetans and the genetic basis of adaptation in a rigorous environment, we genotyped 30 Tibetan individuals with more than one million SNP markers. Our findings suggested that Tibetans, together with the Yi people, were descendants of Tibeto-Burmans who diverged from ancient settlers of East Asia. The valleys of the Hengduan Mountain range may be a major migration route. We also identified a set of positively-selected genes that belong to functional classes of the embryonic, female gonad, and blood vessel developments, as well as response to hypoxia. Most of these genes were highly correlated with population-specific and beneficial phenotypes, such as high infant survival rate and the absence of chronic mountain sickness.

Human VRK1 induces a stabilization and accumulation of p53 by specific phosphorylation in Thr18. This p53 accumulation is reversed by its downregulation mediated by Hdm2, requiring a dephosphorylated p53 and therefore also needs the removal of VRK1 as stabilizer. This process requires export of VRK1 to the cytosol and is inhibited by leptomycin B. We have identified that downregulation of VRK1 protein levels requires DRAM expression, a p53-induced gene. DRAM is located in the endosomal-lysosomal compartment. Induction of DNA damage by UV, IR, etoposide and doxorubicin stabilizes p53 and induces DRAM expression, followed by VRK1 downregulation and a reduction in p53 Thr18 phosphorylation. DRAM expression is induced by wild-type p53, but not by common human p53 mutants, R175H, R248W and R273H. Overexpression of DRAM induces VRK1 downregulation and the opposite effect was observed by its knockdown. LC3 and p62 were also downregulated, like VRK1, in response to UV-induced DNA damage. The implication of the autophagic pathway was confirmed by its requirement for Beclin1. We propose a model with a double regulatory loop in response to DNA damage, the accumulated p53 is removed by induction of Hdm2 and degradation in the proteasome, and the p53-stabilizer VRK1 is eliminated by the induction of DRAM that leads to its lysosomal degradation in the autophagic pathway, and thus permitting p53 degradation by Hdm2. This VRK1 downregulation is necessary to modulate the block in cell cycle progression induced by p53 as part of its DNA damage response.

<h4>Purpose</h4>Under different culture conditions, periodontal ligament (PDL) stem cells are capable of differentiating into cementoblast-like cells, adipocytes, and collagen-forming cells. Several previous studies reported that because of the stem cells in the PDL, the PDL have a regenerative capacity which, when appropriately triggered, participates in restoring connective tissues and mineralized tissues. Therefore, this study analyzed the genes involved in mineralization during differentiation of human PDL (hPDL) cells, and searched for candidate genes possibly associated with the mineralization of hPDL cells.<h4>Methods</h4>To analyze the gene expression pattern of hPDL cells during differentiation, the hPDL cells were cultured in two conditions, with or without osteogenic cocktails (β-glycerophosphate, ascorbic acid and dexamethasone), and a DNA microarray analysis of the cells cultured on days 7 and 14 was performed. Reverse transcription-polymerase chain reaction was performed to validate the DNA microarray data.<h4>Results</h4>The up-regulated genes on day 7 by hPDL cells cultured in osteogenic medium were thought to be associated with calcium/iron/metal ion binding or homeostasis (PDE1A, HFE and PCDH9) and cell viability (PCDH9), and the down-regulated genes were thought to be associated with proliferation (PHGDH and PSAT1). Also, the up-regulated genes on day 14 by hPDL cells cultured in osteogenic medium were thought to be associated with apoptosis, angiogenesis (ANGPTL4 and FOXO1A), and adipogenesis (ANGPTL4 and SEC14L2), and the down-regulated genes were thought to be associated with cell migration (SLC16A4).<h4>Conclusions</h4>This study suggests that when appropriately triggered, the stem cells in the hPDL differentiate into osteoblasts/cementoblasts, and the genes related to calcium binding (PDE1A and PCDH9), which were strongly expressed at the stage of matrix maturation, may be associated with differentiation of the hPDL cells into osteoblasts/cementoblasts.

Trafficking of transcription factors between the cytoplasm and the nucleus is an essential aspect of signal transduction, which is particularly challenging in neurons due to their highly polarized structure. Disruption in the subcellular localization of many proteins, including transcription factors, is observed in affected neurons of human neurodegenerative diseases. In these diseases, there is also growing evidence supporting alterations in nuclear transport as potential mechanisms underlying the observed mislocalization of proteins. Oxidative stress, which plays a key pathogenic role in these diseases, has also been associated with significant alterations in nuclear transport. After providing an overview of the major nuclear import and export pathways and discussing the impact of oxidative injury on nuclear trafficking of proteins, this review synthesizes emerging evidence for altered nuclear transport as a possible mechanism in the pathogenesis of neurodegenerative diseases. Potential strategies to overcome such deficits are also discussed.

In the nervous system, several key steps in cellular complexity and development are regulated by non-coding RNAs (ncRNAs) and the repressor element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF). REST recruits gene regulatory complexes to regulatory sequences, among them the repressor element-1/neuron-restrictive silencer element, and mediates developmental stage-specific gene expression or repression, chromatin (re-)organization or silencing for protein-coding genes as well as for several ncRNAs like microRNAs, short interfering RNAs or long ncRNAs. NcRNAs are far from being just transcriptional noise and are involved in chromatin accessibility, transcription and post-transcriptional processing, trafficking, or RNA editing. REST and its cofactor CoREST are both highly regulated through various ncRNAs. The importance of the correct regulation within the ncRNA network, the ncRNAome, is demonstrated when it comes to a deregulation of REST and/or ncRNAs associated with molecular pathophysiology underlying diverse disorders including neurodegenerative diseases or brain tumors.

<h4>Background</h4>Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes, but little is known about somatic alterations of mitotic spindle checkpoint genes in breast cancer.<h4>Methods</h4>To obtain further insight into the molecular mechanisms underlying aneuploidy in breast cancer, we used real-time quantitative RT-PCR to quantify the mRNA expression of 76 selected mitotic spindle checkpoint genes in a large panel of breast tumor samples.<h4>Results</h4>The expression of 49 (64.5%) of the 76 genes was significantly dysregulated in breast tumors compared to normal breast tissues: 40 genes were upregulated and 9 were downregulated. Most of these changes in gene expression during malignant transformation were observed in epithelial cells.Alterations of nine of these genes, and particularly NDC80, were also detected in benign breast tumors, indicating that they may be involved in pre-neoplastic processes.We also identified a two-gene expression signature (PLK1 + AURKA) which discriminated between DNA aneuploid and DNA diploid breast tumor samples. Interestingly, some DNA tetraploid tumor samples failed to cluster with DNA aneuploid breast tumors.<h4>Conclusion</h4>This study confirms the importance of previously characterized genes and identifies novel candidate genes that could be activated for aneuploidy to occur. Further functional analyses are required to clearly confirm the role of these new identified genes in the molecular mechanisms involved in breast cancer aneuploidy. The novel genes identified here, and/or the two-gene expression signature, might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies.

TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.

The polygenic nature of essential hypertension and its dependence on environmental factors pose a challenge for biomedical research. We hypothesized that the analysis of gene expression profiles from peripheral blood cells would distinguish patients with hypertension from normotensives. In order to test this, total RNA from peripheral blood cells was isolated. RNA was reversed-transcribed and labeled and gene expression analyzed using significance Analysis Microarrays (Stanford University, CA, USA). Briefly, Significance Analysis Microarrays (SAM) thresholding identified 31 up-regulated and 18 down-regulated genes with fold changes of ≥2 or ≤0.5 and q-value≤5% in expression. Statistically significantly gene ontology (GO) function and biological process differentially expressed in essential hypertension were MHC class II receptor activity and immune response respectively. Biological pathway analysis identified several related pathways which are associated with immune/inflammatory responses. Quantitative Real-Time RT-PCR results were consistent with the microarray results. The levels of C-reactive protein were higher in hypertensive patients than normotensives and inflammation-related genes were increased as well. In conclusion, genes enriched for "immune/inflammatory responses" may be associated with essential hypertension. In addition, there is a correlation between systemic inflammation and hypertension. It is anticipated that these findings may provide accurate and efficient strategies for prevention, diagnosis and control of this disorder.

This study investigated the potential of creating a charged polymeric micelle-based nucleic acid delivery system that could easily be reconstituted by the addition of water. (PLGA(36kDa))(2)-b-bPEI(25kDa) (PLGA MW 36 kDa, bPEI M(w) 25 kDa, PLGA:bPEI block ratio = 2) was synthesized and used to prepare cationic micelles. The copolymer retained proton-buffering capability from the bPEI block within the endosomal pH range. Micelle/pDNA complexes retained their particle size (100-150 nm) and surface charge (30-40 mV) following reconstitution. It was found that adding a small amount of low molecular weight bPEI (1.8 kDa) completely shielded pDNA in the micelle/pDNA complexes and enhanced transfection efficiency 50-100 fold for both fresh and reconstituted complexes without affecting complex size. Transfection efficiency for "reconstituted" micelle/pDNA/bPEI(1.8kDa) (WR 1) complexes was 16-fold higher than its "fresh" counterpart. Although transfection levels achieved using "reconstituted" micelle/pDNA/bPEI(1.8kDa) complexes were 3.6-fold lower than control "fresh" bPEI(25kDa)/pDNA (N/P 5) complexes, transfection levels were 39-fold higher than "reconstituted" bPEI(25kDa)/pDNA (N/P 5) complexes. The micelle/pDNA/bPEI(1.8kDa) system showed very low cytotoxicity in MCF7 cells even with pDNA doses up to 20 μg, and transfection levels increased linearly with increasing pDNA dose. These results indicate that this PLGA-b-bPEI polymeric micelle-based system is well suited as a reconstitutable gene delivery system, and has high potential for use as a delivery system for gene therapy applications.

A close relationship between tumor angiogenesis, growth, and carcinomatosis has been observed. Netrin-4 (NT-4) has been shown to regulate angiogenic responses. We aimed to examine the effects of NT-4 on colon tumor angiogenesis, growth, and carcinomatosis. We showed that NT-4 was expressed in human colon cancer cells (LS174). A 20-fold increase in NT-4 expression was stably induced by NT-4 pcDNA in LS174 cells. In vivo, a Matrigel angiogenesis assay showed that NT-4 overexpression altered vascular endothelial growth factor (VEGF)/basic fibroblast growth factor-induced angiogenesis. In nude mice with LS174 xenografts, NT-4 overexpression inhibited tumor angiogenesis and growth. In addition, these NT-4-involved inhibitory effects were associated with decreased tumor cell proliferation and increased tumor cell apoptosis. Using an orthotopic peritoneal carcinomatosis model, we demonstrated that NT-4 overexpression decreased colorectal cancer carcinomatosis. Moreover, carcinomatosis-related ascites formation was significantly decreased in mice transplanted with NT-4 LS174 cells versus control LS174 cells. The antiangiogenic activity of NT-4 was probably mediated by binding to its receptor neogenin. Netrin-4 had a direct effect on neither in vitro apoptosis and proliferation of cultured LS174 cells nor the VEGF-induced acute increase in vascular permeability in vivo. We propose that NT-4 overexpression decreases tumor growth and carcinomatosis, probably via an antiangiogenic effect, underlying the potential therapeutic interest in NT-4 in the treatment of colorectal cancer growth and carcinomatosis.

Neurodegenerative diseases result from the gradual and progressive loss of neural cells and lead to nervous system dysfunction. The rapidly advancing stem cell field is providing attractive alternative options for fighting these diseases. Results have provided proof of principle that cell replacement can work in humans with Parkinson's disease (PD). However, three clinical studies of cell transplantation were published that found no net benefit, while patients in two of the studies developed dyskinesias that persisted despite reductions in treatment. Induced pluripotent stem cells (iPSC) have major potential advantages because patient-specific neuroblasts are suitable for transplantation, avoid immune reactions, and can be produced without the use of human ES cells (hESC). Although iPSCs have not been successfully used in clinical trials for PD, patients with amyotrophic lateral sclerosis (ALS) were treated with autologous stem cells and, though they had some degree of decline one year after treatment, they were still improved compared with the preoperative period or without any drug therapy. In addition, neural stem cells (NSCs), via brain-derived neurotrophic factor (BDNF), have been shown to ameliorate complex behavioral deficits associated with widespread Alzheimer's disease (AD) pathology in a transgenic mouse model of AD. So far, the FDA lists 18 clinical trials treating multiple sclerosis (MS), but most are in preliminary stages. This article serves as an overview of recent studies in stem cell and regenerative approaches to the above chronic neurodegenerative disorders. There are still many obstacles to the use of stem cells as a cure for neurodegenerative disease, especially because we still don't fully understand the true mechanisms of these diseases. However, there is hope in the potential of stem cells to help us learn and understand a great deal more about the mechanisms underlying these devastating neurodegenerative diseases.

<h4>Aim</h4> To compare risk-adjusted outcomes at 18- to 22-month-corrected age for extremely low birth weight (ELBW) infants who never received phototherapy (NoPTx) to those who received any phototherapy (PTx) in the NICHD Neonatal Research Network randomized trial of Aggressive vs. Conservative Phototherapy.<h4>Methods</h4>Outcomes at 18 to 22-month-corrected age included death, neurodevelopmental impairment (NDI) and Bayley Scales Mental Developmental Index (MDI). Regression models evaluated the independent association of PTx with adverse outcomes controlling for centre and other potentially confounding variables.<h4>Results</h4>Of 1972 infants, 216 were NoPTx and 1756 were PTx. For the entire 501- to 1000-g-BW cohort, PTx was not independently associated with death or NDI (OR 0.85, 95% CI: 0.60-1.20), death or adverse neurodevelopmental endpoints. However, among infants 501-750 g BW, the rate of significant developmental impairment with MDI < 50 was significantly higher for NoPTx (29%) than PTx (12%) (p = 0.004).<h4>Conclusions</h4>Phototherapy did not appear to be independently associated with death or NDI for the overall ELBW group. Whether PTx increases mortality could not be excluded because of bias from deaths before reaching conservative treatment threshold. The higher rate of MDI < 50 in the 501- to 750-g-BW NoPTx group is concerning and consistent with NRN Trial results.

Glutathione S-transferase P1-1 (GSTP) is one member of the family of GSTs and is ubiquitously expressed in human tissues. The literature is replete with reports of high levels of GSTP linked either with cancer incidence or drug resistance, and yet no entirely cogent explanation for these correlations exists. The catalytic detoxification properties of the GST isozyme family have been a primary research focus for the last four decades. However, it has become apparent that they have undergone structural and functional convergence where evolutionary selective pressures have favored the emergence of noncatalytic properties of GSTP that has imbued this isozyme with expanded biological importance. For example, GSTP has now been linked with two cell-signaling functions that are critical to survival. Through protein:protein interactions, GSTP can sequester c-jun N-terminal kinase (JNK) and act as a negative regulator of this stress kinase. Pharmacologically, this activity has been linked with the activity of GSTP inhibitors in stimulating myeloproliferation. In addition, GSTP is linked with the forward S-glutathionylation reaction, a post-translational modification that impacts the function/activity of a number of proteins. Catalytic reversal of S-glutathionylation is well characterized, but the role of GSTP in catalyzing the forward reaction contributes to the "glutathionylation cycle." Moreover, GSTP is itself susceptible to S-glutathionylation, providing an autoregulatory loop for the cycle. Because oxidative stress regulates both S-glutathionylation and JNK-signaling pathways, such links may help to explain the aberrant patterns of GSTP expression in the cancer phenotype. As such, there is an ongoing preclinical and clinical platform of drug discovery and development around GSTP.

Huntington's disease (HD) is caused by an expanded CAG tract in the Interesting transcript 15 (IT15) gene encoding the 350 kDa huntingtin protein. Cellular stresses can trigger the release of huntingtin from the endoplasmic reticulum, allowing huntingtin nuclear entry. Here, we show that endogenous, full-length huntingtin localizes to nuclear cofilin-actin rods during stress and is required for the proper stress response involving actin remodeling. Mutant huntingtin induces a dominant, persistent nuclear rod phenotype similar to that described in Alzheimer's disease for cytoplasmic cofilin-actin rods. Using live cell temporal studies, we show that this stress response is similarly impaired when mutant huntingtin is present, or when normal huntingtin levels are reduced. In clinical lymphocyte samples from HD patients, we have quantitatively detected cross-linked complexes of actin and cofilin with complex formation varying in correlation with disease progression. By live cell fluorescence lifetime imaging measurement-Förster resonant energy transfer studies and western blot assays, we quantitatively observed that stress-activated tissue transglutaminase 2 (TG2) is responsible for the actin-cofilin covalent cross-linking observed in HD. These data support a direct role for huntingtin in nuclear actin re-organization, and describe a new pathogenic mechanism for aberrant TG2 enzymatic hyperactivity in neurodegenerative diseases.

The hereditary hemochromatosis protein HFE promotes the expression of hepcidin, a circulating hormone produced by the liver that inhibits dietary iron absorption and macrophage iron release. HFE mutations are associated with impaired hepatic bone morphogenetic protein (BMP)/SMAD signaling for hepcidin production. TMPRSS6, a transmembrane serine protease mutated in iron-refractory iron deficiency anemia, inhibits hepcidin expression by dampening BMP/SMAD signaling. In the present study, we used genetic approaches in mice to examine the relationship between Hfe and Tmprss6 in the regulation of systemic iron homeostasis. Heterozygous loss of Tmprss6 in Hfe(-/-) mice reduced systemic iron overload, whereas homozygous loss caused systemic iron deficiency and elevated hepatic expression of hepcidin and other Bmp/Smad target genes. In contrast, neither genetic loss of Hfe nor hepatic Hfe overexpression modulated the hepcidin elevation and systemic iron deficiency of Tmprss6(-/-) mice. These results indicate that genetic loss of Tmprss6 increases Bmp/Smad signaling in an Hfe-independent manner that can restore Bmp/Smad signaling in Hfe(-/-) mice. Furthermore, these results suggest that natural genetic variation in the human ortholog TMPRSS6 might modify the clinical penetrance of HFE-associated hereditary hemochromatosis, raising the possibility that pharmacologic inhibition of TMPRSS6 could attenuate iron loading in this disorder.

Romidepsin (depsipeptide or FK228) is a histone deacetylase inhibitor, one of a new class of agents active in T-cell lymphoma. A phase 2 trial was conducted in cutaneous (CTCL) and peripheral (PTCL) T-cell lymphoma. Major and durable responses in CTCL supported the approval of romidepsin for CTCL. Forty-seven patients with PTCL of various subtypes including PTCL NOS, angioimmunoblastic, ALK-negative anaplastic large cell lymphoma, and enteropathy-associated T-cell lymphoma were enrolled. All patients had received prior therapy with a median of 3 previous treatments (range 1-11); 18 (38%) had undergone stem-cell transplant. All patients were evaluated for toxicity; 2 patients discovered to be ineligible were excluded from response assessment. Common toxicities were nausea, fatigue, and transient thrombocytopenia and granulocytopenia. Complete responses were observed in 8 and partial responses in 9 of 45 patients, for an overall response rate of 38% (95% confidence interval 24%-53%). The median duration of overall response was 8.9 months (range 2-74). Responses were observed in various subtypes, with 6 responses among the 18 patients with prior stem-cell transplant. The histone deacetylase inhibitor romidepsin has single agent clinical activity associated with durable responses in patients with relapsed PTCL.

Microbial interactions in natural microbiota are, in many cases, crucial for the sustenance of the communities, but the precise nature of these interactions remain largely unknown because of the inherent complexity and difficulties in laboratory cultivation. Conventional pure culture-oriented cultivation does not account for these interactions mediated by small molecules, which severely limits its utility in cultivating and studying "unculturable" microorganisms from synergistic communities. In this study, we developed a simple microfluidic device for highly parallel co-cultivation of symbiotic microbial communities and demonstrated its effectiveness in discovering synergistic interactions among microbes. Using aqueous micro-droplets dispersed in a continuous oil phase, the device could readily encapsulate and co-cultivate subsets of a community. A large number of droplets, up to ∼1,400 in a 10 mm × 5 mm chamber, were generated with a frequency of 500 droplets/sec. A synthetic model system consisting of cross-feeding E. coli mutants was used to mimic compositions of symbionts and other microbes in natural microbial communities. Our device was able to detect a pair-wise symbiotic relationship when one partner accounted for as low as 1% of the total population or each symbiont was about 3% of the artificial community.

Epidemiological studies have suggested that there are many risk factors associated with breast cancer. Silencing tumor suppressor genes through epigenetic alterations play critical roles in breast cancer initiation, promotion and progression. As a growth promoter, Zeranol (Z) has been approved by the FDA and is widely used to enhance the growth of beef cattle in the United States. However, the safety of Z use as a growth promoter is still under debate. In order to provide more evidence to clarify this critical health issue, the current study investigated the effect of Z on the proliferation of primary cultured human normal and cancerous breast epithelial cells (PCHNBECs and PCHBCECs, respectively) isolated from the same patient using MTS assay, RT-PCR and Western blot analysis. We also conducted an investigation regarding the mechanisms that might be involved. Our results show that Z is more potent to stimulate PCHBCEC growth than PCHNBEC growth. The stimulatory effects of Z on PCHBCECs and PCHBCECs may be mediated by its down-regulating expression of the tumor suppressor gene p53 at the mRNA and protein levels. Further investigation showed that the expression of DNA methylatransferase 1 mRNA and protein levels is up-regulated by treatment with Z in PCHBCECs as compared to PCHNBECs, which suggests a role of Z in epigenetic modification involved in the regulation of p53 gene expression in PCHBCECs. Our experimental results imply the potentially adverse health effect of Z in breast cancer development. Further study is continuing in our laboratory.

If properly translated to clinical use, our knowledge about biomarkers may lead to a more effective way of combating colorectal cancer (CRC). Biomarkers are biomolecular, genetic, or cytogenetic attributes indicative of the disease's progression, predisposition, prognosis, or therapeutic options. For CRC, these include chromosomal instability, mutations in KRAS and TP53, loss of 18q, and elevated level of carcinoembryonic antigen (CEA), which are all associated with poor prognosis. The prognostic significance of 18q loss can be attributed to reduced expression of SMAD4, or DCC, although the chromosomal arm is actually heavily populated by genes whose downregulation correlate to worse survival. Potentially, identification of prognostic biomarkers can help the oncologist decide whether adjuvant chemotherapy is necessary after surgery. Testing for therapeutic biomarkers can be necessary if targeted therapeutics are being considered. The identification of highly penetrant predisposition markers (such as mutations in APC and MLH1) can be a lifesaver for carrier individuals, who would then have to undergo colonoscopy at an earlier age. Even sporadic CRCs may have some hereditary components, according to recent studies. Genome-wide association studies (using SNP arrays) showed that polymorphisms of certain genes can have subtle influence on CRC predisposition. Our own SNP array-based analysis suggested that long stretches of germline homozygosity (autozygosity), indicative of consanguinity, may also factor in CRC predisposition.

A series of fluorine-containing N-(2-methoxyphenyl)piperazine and N-(2-fluoroethoxy)piperazine analogues were synthesized, and their affinities for human dopamine D(2), D(3), and D(4) receptors were determined. Radioligand binding studies identified five compounds, 18a, 20a, 20c, 20e, and 21e, which bind with high affinity at D(3) (K(i) = 0.17-5 nM) and moderate to high selectivity for D(3) vs D(2) receptors (ranging from ∼25- to 163-fold). These compounds were also evaluated for intrinsic activity at D(2) and D(3) receptors using a forskolin-dependent adenylyl cyclase assay. This panel of compounds exhibits varying receptor subtype binding selectivity and intrinsic activity at D(2) vs D(3) receptors. These compounds may be useful for behavioral pharmacology studies on the role of D(2)-like dopamine receptors in neuropsychiatric and neurological disorders. Furthermore, compound 20e, which has the highest binding affinity and selectivity for the D(3) receptor (K(i) = 0.17 nM for D(3), 163-fold selectivity for D(3) vs D(2) receptors), represents a candidate fluorine-18 radiotracer for in vivo PET imaging studies on the regulation of D(3) receptor expression.

Reactive oxygen and nitrogen species, generated by neutrophils and macrophages in chronically inflamed tissues, readily damage DNA, producing a variety of potentially genotoxic etheno base lesions; such inflammation-related DNA damage is now known to contribute to carcinogenesis. Although the human alkyladenine DNA glycosylase (AAG) can specifically bind DNA containing either 1,N(6)-ethenoadenine (εA) lesions or 3,N(4)-ethenocytosine (εC) lesions, it can only excise εA lesions. AAG binds very tightly to DNA containing εC lesions, forming an abortive protein-DNA complex; such binding not only shields εC from repair by other enzymes but also inhibits AAG from acting on other DNA lesions. To understand the structural basis for inhibition, we have characterized the binding of AAG to DNA containing εC lesions and have solved a crystal structure of AAG bound to a DNA duplex containing the εC lesion. This study provides the first structure of a DNA glycosylase in complex with an inhibitory base lesion that is induced endogenously and that is also induced upon exposure to environmental agents such as vinyl chloride. We identify the primary cause of inhibition as a failure to activate the nucleotide base as an efficient leaving group and demonstrate that the higher binding affinity of AAG for εC versus εA is achieved through formation of an additional hydrogen bond between Asn-169 in the active site pocket and the O(2) of εC. This structure provides the basis for the design of AAG inhibitors currently being sought as an adjuvant for cancer chemotherapy.

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

<h4>Background</h4>There is a paucity of information on the clinical presentation and outcome of elderly hepatocellular carcinoma (HCC) patients. We performed a multicenter retrospective comparative study to assess the impact of age on potential differences in clinical characteristics, treatment patterns, and outcome in HCC patients.<h4>Methods</h4>We retrospectively analyzed HCC patients treated at two U.S. tertiary institutions from 1998 to 2008. Demographics, tumor parameters, etiology and severity of cirrhosis, treatment, and survival from diagnosis were collected and analyzed. After exclusion of transplanted patients, survival analyses were performed using the Kaplan-Meier method with log-rank tests and Cox proportional hazards models.<h4>Results</h4>Three hundred thirty-five HCC patients were divided into two groups: "elderly" (95 patients, age ≥ 70 years) and "younger" (240 patients, aged <70 years). The male/female (M/F) ratio was 5.8:1 and 1.7:1 in the younger and elderly groups, respectively (p < .0001). Hepatitis C virus (HCV) infection rate was 48.3% in younger and 21.1% in elderly patients (p < .0001); Child class B and C cirrhosis accounted for 35.8% in younger and 25.3% in elderly patients (p = .063). Compared with younger patients, the elderly received transplant less frequently (19.6% versus 5.3%, p = .0002) and were more likely to receive supportive care only (22.9% versus 36.8%, p = .01). No significant differences between the two age groups were seen in tumor parameters or other treatments received. Overall (p = .47) and HCC-specific survival rates (p = .38) were similar in both age groups.<h4>Conclusions</h4>Characteristics that distinguish elderly from younger HCC patients include lower M/F ratio, worse performance status, lower rate of HCV infection, and less advanced underlying cirrhosis. Elderly patients were less likely to have a liver transplant and more likely to receive supportive care only. However, overall and HCC-specific survival were similar between the two groups.

Human cartilage is a complex tissue of matrix proteins that vary in amount and orientation from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue engineering strategies is the inability of the resident chondrocytes to lay down new matrix with the same structural and resilient properties that it had upon its original formation. This is particularly true of the collagen network, which is susceptible to cleavage once proteoglycans are depleted. Thus, a thorough understanding of the similarities and particularly the marked differences in mechanisms of cartilage remodeling during development, osteoarthritis, and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. To identify and characterize effectors or regulators of cartilage remodeling in these processes, we are using culture models of primary human and mouse chondrocytes and cell lines and mouse genetic models to manipulate gene expression programs leading to matrix remodeling and subsequent chondrocyte hypertrophic differentiation, pivotal processes which both go astray in OA disease. Matrix metalloproteinases (MMP)-13, the major type II collagen-degrading collagenase, is regulated by stress-, inflammation-, and differentiation-induced signals that not only contribute to irreversible joint damage (progression) in OA, but importantly, also to the initiation/onset phase, wherein chondrocytes in articular cartilage leave their natural growth- and differentiation-arrested state. Our work points to common mediators of these processes in human OA cartilage and in early through late stages of OA in surgical and genetic mouse models.

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens.

Elevated serum ferritin concentrations are common in clinical practice. In this review, we provide an approach to interpreting the serum ferritin elevation in relationship to other clinical parameters including the patient history, transferrin saturation, serum concentrations of alanine, and aspartate aminotransferases (ALT, AST), testing for HFE mutations, liver imaging, liver biopsy, and liver iron concentration. We used observations from a large series of patients with hepatic iron overload documented by liver iron concentration measurement from two referral practices as a gold standard to guide the interpretation of the predictive values of non-invasive iron tests. Three case studies illustrate common problems in interpreting iron blood tests.

<h4>Background & aims</h4>Previous studies examining the relationship between the C282Y and H63D HFE mutations and presence of nonalcoholic fatty liver disease (NAFLD) have yielded conflicting results. The goal of this study was to systematically evaluate and summarize data on the association between these two variants and the presence of NAFLD.<h4>Methods</h4>The authors searched EMBASE and PUBMED from August 1, 1996 to August 12, 2010. Two investigators independently conducted data abstraction. Ethnic specific weighted prevalence was calculated and pooled odds ratios were estimated using the random effects model.<h4>Results</h4>From 2542 references, the authors included 16 case-control studies and 14 case-only studies, or 2610 cases and 7298 controls. The majority of the studies came from Caucasian populations (2287 cases and 4275 controls). The weighted prevalence of HFE mutations in cases was comparable to controls. The meta-analysis was restricted to Caucasians only because of the small sample size of non Caucasian participants. The pooled odds ratio for the presence of any HFE genetic variant in cases was 1.03 (95%CI: 0.90, 1.17; I(2): 65.8%, 95%CI: 38.5, 81.0). The presence of other genotypes and secondary analyses yielded similar non significant findings.<h4>Conclusions</h4>Our systematic review does not support an association between the HFE genetic variants and the presence of NAFLD.

Integrative genomics approaches associating DNA structure and transcriptomic analysis should allow the identification of cascades of events relating to tumor aggressiveness. While different genome alterations have been identified in pituitary tumors, none have ever been correlated with the aggressiveness. This study focused on one subtype of pituitary tumor, the prolactin (PRL) pituitary tumors, to identify molecular events associated with the aggressive and malignant phenotypes. We combined a comparative genomic hybridization and transcriptomic analysis of 13 PRL tumors classified as nonaggressive or aggressive. Allelic loss within the p arm region of chromosome 11 was detected in five of the aggressive tumors. Allelic loss in the 11q arm was observed in three of these five tumors, all three of which were considered as malignant based on the occurrence of metastases. Comparison of genomic and transcriptomic data showed that allelic loss impacted upon the expression of genes located in the imbalanced region. Data filtering allowed us to highlight five deregulated genes (DGKZ, CD44, TSG101, GTF2H1, HTATIP2), within the missing 11p region, potentially responsible for triggering the aggressive and malignant phenotypes of PRL tumors. Our combined genomic and transcriptomic analysis underlines the importance of chromosome allelic loss in determining the aggressiveness and malignancy of tumors.

<h4>Background</h4>Myelodysplastic Syndromes (MDSS) are pre-leukemic disorders with increasing incident rates worldwide, but very limited treatment options. Little is known about small regulatory RNAs and how they contribute to pathogenesis, progression and transcriptome changes in MDS.<h4>Methods</h4>Patients' primary marrow cells were screened for short RNAs (RNA-seq) using next generation sequencing. Exon arrays from the same cells were used to profile gene expression and additional measures on 98 patients obtained. Integrative bioinformatics algorithms were proposed, and pathway and ontology analysis performed.<h4>Results</h4>In low-grade MDS, observations implied extensive post-transcriptional regulation via microRNAs (miRNA) and the recently discovered Piwi interacting RNAs (piRNA). Large expression differences were found for MDS-associated and novel miRNAs, including 48 sequences matching to miRNA star (miRNA*) motifs. The detected species were predicted to regulate disease stage specific molecular functions and pathways, including apoptosis and response to DNA damage. In high-grade MDS, results suggested extensive post-translation editing via transfer RNAs (tRNAs), providing a potential link for reduced apoptosis, a hallmark for this disease stage. Bioinformatics analysis confirmed important regulatory roles for MDS linked miRNAs and TFs, and strengthened the biological significance of miRNA*. The "RNA polymerase II promoters" were identified as the tightest controlled biological function. We suggest their control by a miRNA dominated feedback loop, which might be linked to the dramatically different miRNA amounts seen between low and high-grade MDS.<h4>Discussion</h4>The presented results provide novel findings that build a basis of further investigations of diagnostic biomarkers, targeted therapies and studies on MDS pathogenesis.

<h4>Background</h4>Nonalcoholic fatty liver disease (NAFLD) is the most frequent disease associated with abnormal liver tests that is characterized by a wide spectrum of liver damage, ranging from simple macro vesicular steatosis to steatohepatitis (NASH), cirrhosis or liver carcinoma. Liver biopsy is the most precise test to differentiate NASH from other stages of NAFLD, but it is an invasive and expensive method. This study aimed to create a clinical laboratory score capable of identify individual with NASH in severely obese patients submitted to bariatric surgery.<h4>Methods</h4>The medical records from 66 patients submitted to gastroplasty were reviewed. Their chemistry profile, abdominal ultrasound (US) and liver biopsy done during the surgical procedure were analyzed. Patients were classified into 2 groups according to liver biopsy: Non-NASH group - those patients without NAFLD or with grade I, II or III steatosis; and NASH group - those with steatohepatitis or fibrosis. The t-test was used to compare each variable with normal distribution between NASH and Non-NASH groups. When comparing proportions of categorical variables, we used chi-square or z-test, where appropriate. A p-value < 0.05 was considered statistically significant.<h4>Results</h4>83% of patients with obesity grades II or III showed NAFLD, and the majority was asymptomatic. Total Cholesterol (TC)≥200 mg/dL, alanine aminotransferase (ALT) ≥30, AST/ALT ratio (AAR)≤ 1, gammaglutaril-transferase (γGT)≥30 U/L and abdominal US, compatible with steatosis, showed association with NASH group. We proposed 2 scores: Complete score (TC, ALT, AAR, γGT and US) and the simplified score, where US was not included. The combination of biochemical and imaging results improved accuracy to 84.4% the recognition of NASH (sensitivity 70%, specificity 88.6%, NPV 91.2%, PPV 63. 6%).<h4>Conclusion</h4>Alterations in TC, ALT, AAR, γGT and US are related to the most risk for NASH. The combination of biochemical and imaging results improved accuracy to 84.4% the recognition of NASH. Additionally, negative final scores exclude the presence of an advanced illness. Using this score, the severity of fatty liver infiltration would be predicted without the risks associated with hepatic biopsy.

<h4>Context</h4>A strong genetic influence on bone mineral density has been long established, and modern genotyping technologies have generated a flurry of new discoveries about the genetic determinants of bone mineral density (BMD) measured at a single time point. However, much less is known about the genetics of age-related bone loss. Identifying bone loss-related genes may provide new routes for therapeutic intervention and osteoporosis prevention.<h4>Evidence acquisition</h4>A review of published peer-reviewed literature on the genetics of bone loss was performed. Relevant studies were summarized, most of which were drawn from the period 1990-2010.<h4>Evidence synthesis</h4>Although bone loss is a challenging phenotype, available evidence supports a substantial genetic contribution. Some of the genes identified from recent genome-wide association studies of cross-sectional BMD are attractive candidate genes for bone loss, most notably genes in the nuclear factor κB and estrogen endocrine pathways. New insights into the biology of skeletal development and regulation of bone turnover have inspired new hypotheses about genetic regulation of bone loss and may provide new directions for identifying genes associated with bone loss.<h4>Conclusions</h4>Although recent genome-wide association and candidate gene studies have begun to identify genes that influence BMD, efforts to identify susceptibility genes specific for bone loss have proceeded more slowly. Nevertheless, clues are beginning to emerge on where to look, and as population studies accumulate, there is hope that important bone loss susceptibility genes will soon be identified.

Iron accumulation in the brain and increased oxidative stress are consistent observations in many neurodegenerative diseases. Thus, we have begun examination into gene mutations or allelic variants that could be associated with loss of iron homeostasis. One of the mechanisms leading to iron overload is a mutation in the HFE gene, which is involved in iron metabolism. The 2 most common HFE gene variants are C282Y (1.9%) and H63D (8.9%). The C282Y HFE variant is more commonly associated with hereditary hemochromatosis, which is an autosomal recessive disorder, characterized by iron overload in a number of systemic organs. The H63D HFE variant appears less frequently associated with hemochromatosis, but its role in the neurodegenerative diseases has received more attention. At the cellular level, the HFE mutant protein resulting from the H63D HFE gene variant is associated with iron dyshomeostasis, increased oxidative stress, glutamate release, tau phosphorylation, and alteration in inflammatory response, each of which is under investigation as a contributing factor to neurodegenerative diseases. Therefore, the HFE gene variants are proposed to be genetic modifiers or a risk factor for neurodegenerative diseases by establishing an enabling milieu for pathogenic agents. This review will discuss the current knowledge of the association of the HFE gene variants with neurodegenerative diseases: amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, and ischemic stroke. Importantly, the data herein also begin to dispel the long-held view that the brain is protected from iron accumulation associated with the HFE mutations.

Nonheme food ferritin (FTN) iron minerals, nonheme iron complexes, and heme iron contribute to the balance between food iron absorption and body iron homeostasis. Iron absorption depends on membrane transporter proteins DMT1, PCP/HCP1, ferroportin (FPN), TRF2, and matriptase 2. Mutations in DMT1 and matriptase-2 cause iron deficiency; mutations in FPN, HFE, and TRF2 cause iron excess. Intracellular iron homeostasis depends on coordinated regulation of iron trafficking and storage proteins encoded in iron responsive element (IRE)-mRNA. The noncoding IRE-mRNA structures bind protein repressors, IRP1 or 2, during iron deficiency. Integration of the IRE-RNA in translation regulators (near the cap) or turnover elements (after the coding region) increases iron uptake (DMT1/TRF1) or decreases iron storage/efflux (FTN/FPN) when IRP binds. An antioxidant response element in FTN DNA binds Bach1, a heme-sensitive transcription factor that coordinates expression among antioxidant response proteins like FTN, thioredoxin reductase, and quinone reductase. FTN, an antioxidant because Fe(2+) and O(2) (reactive oxygen species generators) are consumed to make iron mineral, is also a nutritional iron concentrate that is an efficiently absorbed, nonheme source of iron from whole legumes. FTN protein cages contain thousands of mineralized iron atoms and enter cells by receptor-mediated endocytosis, an absorption mechanism distinct from transport of nonheme iron salts (ferrous sulfate), iron chelators (ferric-EDTA), or heme. Recognition of 2 nutritional nonheme iron sources, small and large (FTN), will aid the solution of iron deficiency, a major public health problem, and the development of new policies on iron nutrition.

<h4>Purpose</h4>Although most patients with estrogen receptor α (ER)-positive breast cancer initially respond to endocrine therapy, many ultimately develop resistance to antiestrogens. However, mechanisms of antiestrogen resistance and biomarkers predictive of such resistance are underdeveloped.<h4>Experimental design</h4>We adapted four ER(+) human breast cancer cell lines to grow in an estrogen-depleted medium. A gene signature of estrogen independence was developed by comparing expression profiles of long-term estrogen-deprived (LTED) cells to their parental counterparts. We evaluated the ability of the LTED signature to predict tumor response to neoadjuvant therapy with an aromatase inhibitor and disease outcome following adjuvant tamoxifen. We utilized Gene Set Analysis (GSA) of LTED cell gene expression profiles and a loss-of-function approach to identify pathways causally associated with resistance to endocrine therapy.<h4>Results</h4>The LTED gene expression signature was predictive of high tumor cell proliferation following neoadjuvant therapy with anastrozole and letrozole, each in different patient cohorts. This signature was also predictive of poor recurrence-free survival in two studies of patients treated with adjuvant tamoxifen. Bioinformatic interrogation of expression profiles in LTED cells revealed a signature of MYC activation. The MYC activation signature and high MYC protein levels were both predictive of poor outcome following tamoxifen therapy. Finally, knockdown of MYC inhibited LTED cell growth.<h4>Conclusions</h4>A gene expression signature derived from ER(+) breast cancer cells with acquired hormone independence predicted tumor response to aromatase inhibitors and associated with clinical markers of resistance to tamoxifen. Activation of the MYC pathway was associated with this resistance.

Peroxiredoxin 6 (Prdx6), a bifunctional protein with GSH peroxidase and lysosomal-type phospholipase A(2) activities, has been localized to both cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization affects the balance between the two activities. Immunostaining localized Prdx6 to lysosome-related organelles in the MLE12 and A549 alveolar epithelial cell lines. Inhibition of trafficking by brefeldin A indicated processing of the protein through the vesicular pathway. Trafficking of Prdx6 was decreased by inhibitors of PKC, ERK, and p38 MAPK. Immunocytochemistry, immunoprecipitation, and an in situ proximity ligation assay (Duolink) showed that binding of the lysosomal targeting sequence of Prdx6 (amino acids 31-40) to 14-3-3ε was dependent on activity of PKC, ERK, and p38 MAPK. Knockdown of 14-3-3ε with siRNA inhibited the lysosomal targeting of Prdx6. In vitro study with recombinant proteins by pull-down assay and surface plasmon resonance confirmed the interaction of Prdx6 and 14-3-3ε. These findings suggest that ERK and p38 MAPK regulate subcellular localization of Prdx6 by activation of 14-3-3ε as a chaperone protein, resulting in its translocation to acidic organelles.

SRY-box-containing gene 9 (Sox9) is an essential transcription factor in chondrocyte lineage determination and differentiation. Recent studies demonstrated that Sox9 controls the transcription of chondrocyte-specific genes in association with several other transcriptional regulators. To further understand the molecular mechanisms by which Sox9 influences transcriptional events during chondrocyte differentiation, we attempted to identify transcriptional partners of Sox9 and to examine their roles in chondrocyte differentiation. We isolated AT-rich interactive domain-containing protein 5a (Arid5a; also known as Mrf1) as an activator of the Col2a1 gene promoter from an ATDC5 cDNA library. Arid5a was highly expressed in cartilage and induced during chondrocyte differentiation. Furthermore, Arid5a physically interacted with Sox9 in nuclei and up-regulated the chondrocyte-specific action of Sox9. Overexpression of Arid5a stimulated chondrocyte differentiation in vitro and in an organ culture system. In contrast, Arid5a knockdown inhibited Col2a1 expression in chondrocytes. In addition, Arid5a binds directly to the promoter region of the Col2a1 gene and stimulates acetylation of histone 3 in the region. Our results suggest that Arid5a may directly interact with Sox9 and thereby enhance its chondrocyte-specific action.

Under evolutionary pressure to counter the toxicity of iron and to maintain adequate iron supply for hemoglobin synthesis and essential metabolic functions, humans and other vertebrates have effective mechanisms to conserve iron and to regulate its concentration, storage, and distribution in tissues. The iron-regulatory hormone hepcidin, first described 10 years ago, and its receptor and iron channel ferroportin control the dietary absorption, storage, and tissue distribution of iron. Hepcidin causes ferroportin internalization and degradation, thereby decreasing iron transfer into blood plasma from the duodenum, from macrophages involved in recycling senescent erythrocytes, and from iron-storing hepatocytes. Hepcidin is feedback regulated by iron concentrations in plasma and the liver and by erythropoietic demand for iron. Genetic malfunctions affecting the hepcidin-ferroportin axis are a main cause of iron overload disorders but can also cause iron-restricted anemias. Modulation of hepcidin and ferroportin expression during infection and inflammation couples iron metabolism to host defense and decreases iron availability to invading pathogens. This response also restricts the iron supply to erythropoietic precursors and may cause or contribute to the anemia associated with infections and inflammatory disorders.

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML.

MicroRNAs (miRNAs) are a class of small (approx. 22 nt) noncoding RNAs that are capable of post-transcriptionally silencing mRNAs that contain sequences complementary to the miRNAs' 7- to 8-bp 'seed' sequence. As single miRNAs are often predicted to target up to hundreds of individual transcripts, miRNAs are able to broadly affect the overall protein expression state of the cell. This can translate into global effects on cellular health and differentiation state. Recently, several reports have identified crucial roles for miRNAs in controlling the production, differentiation, and health of myelinating cells of the mammalian nervous system. In this review, we will discuss how individual miRNAs regulate these various processes, and also how miRNA production in general is required for several stages of myelin generation and maintenance.

Recent evidence suggests that genetic variants that confer susceptibility to myocardial infarction (MI) may differ between men and women or between individuals with or without conventional risk factors for MI. We previously showed that rs6929846 of BTN2A1 and rs2569512 of ILF3 were significantly associated with MI in Japanese individuals. In the present study, we examined the associations of rs6929846 of BTN2A1 or rs2569512 of ILF3 to MI among individuals stratified by the absence or presence of hypertension, diabetes mellitus (DM) and chronic kidney disease (CKD). The study population was comprised of 5689 unrelated Japanese individuals, including 1626 subjects with MI and 4063 controls with or without hypertension, DM or CKD. Multivariable logistic regression analyses with adjustment for covariates revealed that rs6929846 of BTN2A1 was significantly associated with MI in individuals with (P=0.0001; odds ratio, 1.49) or without (P=1.6x10-7; odds ratio, 2.32) hypertension; in individuals with (P=0.0002; odds ratio, 1.65) or without (P=8.1x10-7; odds ratio, 1.76) DM; and in individuals without CKD (P=6.0x10-11; odds ratio, 2.03), but not in those with CKD. Similar analyses revealed that rs2569512 of ILF3 was significantly associated with MI in individuals with (P=0.0041; odds ratio, 1.26) or without (P=0.0051; odds ratio, 1.78) hypertension; in individuals with (P=0.0200; odds ratio, 1.46) or without (P=0.0174; odds ratio, 1.43) DM; and in individuals with (P=0.0011, odds ratio, 1.47) or without (P=0.0237; odds ratio, 1.34) CKD. Results suggested that the association of rs6929846 in BTN2A1 with MI was more apparent in low-risk individuals than in high-risk individuals, whereas the association of rs2569512 in ILF3 with MI was not influenced by the absence or presence of hypertension, DM or CKD. Stratification of subjects based on hypertension, DM or CKD may thus be informative in order to achieve personalized prevention of MI with the use of genetic information.

<h4>Background</h4>A debilitating anemia associated with low serum iron often accompanies inflammatory bowel disease (IBD). Increased production of the iron regulatory hormone hepcidin is implicated in its pathogenesis and may also contribute to the inflammatory process itself. Hepcidin expression is dependent on bone morphogenetic proteins (BMPs) like BMP6, but the mechanisms that increase hepcidin levels during intestinal inflammation are not clear. Here we test the hypothesis that inhibiting hepcidin expression may have beneficial effects in IBD, and also shed light on the mechanism of colitis-induced hepcidin upregulation.<h4>Methods</h4>Mice with T cell transfer colitis were treated with vehicle or one of three anti-BMP reagents: HJV.Fc, a recombinant protein that prevents binding of BMPs to their receptor, LDN-193189, a small molecule inhibitor of BMP signal transduction, and an anti-BMP6 antibody. The effects of these reagents on colitis severity, liver hepcidin mRNA, and serum iron were determined. The mechanism of hepcidin upregulation was investigated by examining BMP6 expression and activity and the effects of IL-6 deficiency.<h4>Results</h4>All the anti-BMP reagents inhibited hepcidin expression and increased serum iron levels in the colitic mice. They also produced modest reductions in colon inflammatory cytokine expression. Although hepcidin upregulation during colitis was dependent on BMP6, it was not associated with increased BMP6 expression or activity. IL-6 was required for increased hepcidin expression during colitis.<h4>Conclusions</h4>Inhibiting hepcidin expression may help to correct the anemia of IBD and may also attenuate intestinal inflammation. The mechanism of colitis-induced hepcidin upregulation involves both BMP6 and IL-6.

Epigenetics can be loosely defined as the study of cellular "traits" that influence biological phenotype in a fashion that is not dependent on the underlying primary DNA sequence. One setting in which epigenetics is likely to have a profound influence on biological phenotype is during intrauterine development. In this context there is a defined and critical window during which balanced homeostasis is essential for normal fetal growth and development. We have carried out a detailed structural and functional analysis of the placental epigenome at its maternal interface. Specifically, we performed genome wide analysis of DNA methylation in samples of chorionic villus (CVS) and maternal blood cells (MBC) using both commercially available and custom designed microarrays. We then compared these data with genome wide transcription data for the same tissues. In addition to the discovery that CVS genomes are significantly more hypomethylated than their MBC counterparts, we identified numerous tissue-specific differentially methylated regions (T-DMRs). We further discovered that these T-DMRs are clustered spatially along the genome and are enriched for genes with tissue-specific biological functions. We identified unique patterns of DNA methylation associated with distinct genomic structures such as gene bodies, promoters and CpG islands and identified both direct and inverse relationships between DNA methylation levels and gene expression levels in gene bodies and promoters respectively. Furthermore, we found that these relationships were significantly associated with CpG content. We conclude that the early gestational placental DNA methylome is highly organized and is significantly and globally associated with transcription. These data provide a unique insight into the structural and regulatory characteristics of the placental epigenome at its maternal interface and will drive future analyses of the role of placental dysfunction in gestational disease.

Physiological, hormonal, and genetic differences between males and females affect the prevalence, incidence, and severity of diseases and responses to therapy. Understanding these differences is important for designing safe and effective treatments. This paper summarizes sex differences that impact drug disposition and includes a general comparison of clinical pharmacology as it applies to men and women.

No abstract available.

Whether the presence of steroid receptors in luminal breast cancers renders them resistant to taxanes remains uncertain. Here we assess the role of progesterone receptors (PR) on taxane-induced cell death. We previously showed that estrogen receptor (ER)-positive human breast cancer cells that inducibly express PR-A or PR-B isoforms were protected from taxane-stimulated apoptosis when compared to the identical cells lacking PR. Surprisingly, PR-dependent protection occurred in the absence of progesterone, demonstrating that the unliganded receptors were biologically active. The present studies demonstrate that unliganded PR, focused on PR-A, protect breast cancer cells from taxane-stimulated apoptosis. The studies identify genes regulated by taxanes in isogenic ER-positive cells that either lack or express PR-A. We show that unliganded PR-A alters the gene expression pattern controlled by taxanes, especially multiple genes involved in the spindle assembly checkpoint, a group of proteins that insure proper attachment of microtubules to kinetochores during mitosis. Importantly, taxanes and unliganded PR regulate many of these genes in opposite directions. As a result, mitotic slippage is exacerbated by the presence of PR, leading to an increase in the number of multinucleated cells both in vitro and in xenograft tumors. We describe a simple new assay for assessing multinucleation in paraffin sections. We speculate that rather than inducing cell death, unliganded PR exploits multinucleation to promote cell survival from taxane therapy. This can be prevented with antiprogestin.

To compare and summarize the mechanisms, frequencies of occurrence, and classification schemes of spontaneous, experimental, and genetically engineered mouse skeletal neoplasms, the literature was reviewed, and archived case material at The Jackson Laboratory was examined. The frequency of occurrence of spontaneous bone neoplasms was less than 1% for most strains, with the exceptions of osteomas in CF-1 (5.5% and 10% in two studies) and OF-1 outbred strains (35%), and osteosarcomas in NOD/ShiLtJ (11.5%) and NOD-derived (7.1%) mice. The frequency was 100% for osteochondromas induced by conditional inactivation of exostoses (multiple) 1 (Ext1) in chondrocytes, osteosarcomas induced by tibial intramedullary inoculation of Moloney murine sarcoma virus, and osteosarcomas induced by conditional inactivation of Trp53-with or without inactivation of Rb1-in osteoblast precursors. Spontaneous osteogenic neoplasms were more frequent than spontaneous cartilaginous and vascular types. Malignant neoplasms were more frequent than benign ones. The age of occurrence for spontaneous neoplasms ranged from 37 to 720 days (M = 316.35) for benign neoplasms and 35 to 990 (M = 299.28) days for malignant. In genetically engineered mice, the average age of occurrence ranged from 28 to 70 days for benign and from 35 to 690 days for malignant. Histologically, nonosteogenic neoplasms were similar across strains and mutant stocks; osteogenic neoplasms exhibited greater diversity. This comparison and summarization of mouse bone neoplasms provides valuable information for the selection of strains to create, compare, and validate models of bone neoplasms.

Nucleosomes impede access to DNA. Therefore, nucleosome positioning is fundamental to genome regulation. Nevertheless, the molecular nucleosome positioning mechanisms are poorly understood. This is partly because in vitro reconstitution of in vivo-like nucleosome positions from purified components is mostly lacking, barring biochemical studies. Using a yeast extract in vitro reconstitution system that generates in vivo-like nucleosome patterns at S. cerevisiae loci, we find that the RSC chromatin remodelling enzyme is necessary for nucleosome positioning. This was previously suggested by genome-wide in vivo studies and is confirmed here in vivo for individual loci. Beyond the limitations of conditional mutants, we show biochemically that RSC functions directly, can be sufficient, but mostly relies on other factors to properly position nucleosomes. Strikingly, RSC could not be replaced by either the closely related SWI/SNF or the Isw2 remodelling enzyme. Thus, we pinpoint that nucleosome positioning specifically depends on the unique properties of the RSC complex.

<h4>Background</h4>Skeletal muscle is a complex, versatile tissue composed of a variety of functionally diverse fiber types. Although the biochemical, structural and functional properties of myofibers have been the subject of intense investigation for the last decades, understanding molecular processes regulating fiber type diversity is still complicated by the heterogeneity of cell types present in the whole muscle organ.<h4>Methodology/principal findings</h4>We have produced a first catalogue of genes expressed in mouse slow-oxidative (type 1) and fast-glycolytic (type 2B) fibers through transcriptome analysis at the single fiber level (microgenomics). Individual fibers were obtained from murine soleus and EDL muscles and initially classified by myosin heavy chain isoform content. Gene expression profiling on high density DNA oligonucleotide microarrays showed that both qualitative and quantitative improvements were achieved, compared to results with standard muscle homogenate. First, myofiber profiles were virtually free from non-muscle transcriptional activity. Second, thousands of muscle-specific genes were identified, leading to a better definition of gene signatures in the two fiber types as well as the detection of metabolic and signaling pathways that are differentially activated in specific fiber types. Several regulatory proteins showed preferential expression in slow myofibers. Discriminant analysis revealed novel genes that could be useful for fiber type functional classification.<h4>Conclusions/significance</h4>As gene expression analyses at the single fiber level significantly increased the resolution power, this innovative approach would allow a better understanding of the adaptive transcriptomic transitions occurring in myofibers under physiological and pathological conditions.

Fetal exposure to environmental insults increases the susceptibility to late-onset neuropsychiatric disorders. Alcohol is listed as one of such prenatal environmental risk factors and known to exert devastating teratogenetic effects on the developing brain, leading to complex neurological and psychiatric symptoms observed in fetal alcohol spectrum disorder (FASD). Here, we performed a coordinated transcriptome analysis of human and mouse fetal cerebral cortices exposed to ethanol in vitro and in vivo, respectively. Up- and down-regulated genes conserved in the human and mouse models and the biological annotation of their expression profiles included many genes/terms related to neural development, such as cell proliferation, neuronal migration and differentiation, providing a reliable connection between the two species. Our data indicate that use of the combined rodent and human model systems provides an effective strategy to reveal and analyze gene expression changes inflicted by various physical and chemical environmental exposures during prenatal development. It also can potentially provide insight into the pathogenesis of environmentally caused brain disorders in humans.

Acute myocardial infarction (AMI) is rarely associated with antiphospholipid syndrome. The treatment of these patients is a clinical challenge. We report the observations of 2 young adults (1 woman and 1 man), admitted in our acute care unit for acute myocardial infarction (AMI). A coagulopathy work-up concludes the existence of antiphospholipid syndrome (APS) in the 2 cases. APS syndrome was considered primary in 2 cases. All patients presented an intense inflammatory syndrome (high level of CRP). Anticardiolipine was present in the 2 cases. However, anti B2 glycoprotein I antibodies were detected in only one case. Emergency percutaneous transluminal coronary angioplasty (PTCA) with direct stenting had been performed successfully only in the first case, and the follow-up was uncomplicated. Thereafter, long-term oral anticoagulant appeared to be effective. The last patient was admitted because of peripheral acute ischemia of legs. Standard electrocardiogram showed signs of previous silent anteroseptal wall myocardial infarction confirmed by echocardiography. The latter revealed an apical thrombus and a very low left ventricular ejection fraction. Amputation of the right leg was necessary because of consultation occurred too late. However, he died four weeks later. Primary antiphospholipid syndrome should be considered as a cause of acute myocardial infarction in young adults, and PTCA with anticoagulant treatment is effective for initial treatment of this complication.

Incidental white matter lesions (WML) are a common neuroradiological finding in elderly people and have been linked to dementia and depression. Various mechanisms including hypoxia and increased production of reactive oxygen species (ROS) are implicated in the etiology of WML. The hemochromatosis (HFE) gene p.H63D and p.C282Y polymorphisms have been linked to dysregulation of iron metabolism and increased levels of ROS, whereas Angiotensin II receptor 1 (AGTR1) c.1166A → C polymorphism is known as a vascular risk factor. These genetic polymorphisms were characterized in brains donated to the UK MRC Cognitive Function and Ageing Study (CFAS) to assess their potential role in the risk for development of age-related WML. The study cohort comprised 258 brain donated to CFAS. WML severity was assessed in the postmortem brain donations using magnetic resonance imaging (MRI) scans and scored using the Scheltens' scale. Polymerase chain reaction (PCR) amplification of extracted DNA followed by restriction enzyme digestion was used to genotype the samples. Genotypes were validated using direct sequencing in a smaller sample. The results show that HFE p.H63D polymorphism is not associated with WML severity in the whole cohort. However, there is a significant association of the D allele with severity of WML in noncarriers of the APOE ε4 allele. No association is demonstrated between the HFE p.C282Y nor the AGTR1 c.1166A → C polymorphisms and WML severity. The HFE gene appears to be a genetic risk factor for severe aging WML independently of the APOE ε4 genotype. This would support the role of iron-related oxidative stress, in addition to previously studied factors, e.g., hypoxia as potential risk factors for developing prominent aging WML.

The human breast tumor microenvironment can display features of T helper type 2 (Th2) inflammation, and Th2 inflammation can promote tumor development. However, the molecular and cellular mechanisms contributing to Th2 inflammation in breast tumors remain unclear. Here, we show that human breast cancer cells produce thymic stromal lymphopoietin (TSLP). Breast tumor supernatants, in a TSLP-dependent manner, induce expression of OX40L on dendritic cells (DCs). OX40L(+) DCs are found in primary breast tumor infiltrates. OX40L(+) DCs drive development of inflammatory Th2 cells producing interleukin-13 and tumor necrosis factor in vitro. Antibodies neutralizing TSLP or OX40L inhibit breast tumor growth and interleukin-13 production in a xenograft model. Thus, breast cancer cell-derived TSLP contributes to the inflammatory Th2 microenvironment conducive to breast tumor development by inducing OX40L expression on DCs.

Huntington's disease is an inherited and incurable neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in huntingtin (encoded by HTT). PolyQ length determines disease onset and severity, with a longer expansion causing earlier onset. The mechanisms of mutant huntingtin-mediated neurotoxicity remain unclear; however, mitochondrial dysfunction is a key event in Huntington's disease pathogenesis. Here we tested whether mutant huntingtin impairs the mitochondrial fission-fusion balance and thereby causes neuronal injury. We show that mutant huntingtin triggers mitochondrial fragmentation in rat neurons and fibroblasts of individuals with Huntington's disease in vitro and in a mouse model of Huntington's disease in vivo before the presence of neurological deficits and huntingtin aggregates. Mutant huntingtin abnormally interacts with the mitochondrial fission GTPase dynamin-related protein-1 (DRP1) in mice and humans with Huntington's disease, which, in turn, stimulates its enzymatic activity. Mutant huntingtin-mediated mitochondrial fragmentation, defects in anterograde and retrograde mitochondrial transport and neuronal cell death are all rescued by reducing DRP1 GTPase activity with the dominant-negative DRP1 K38A mutant. Thus, DRP1 might represent a new therapeutic target to combat neurodegeneration in Huntington's disease.

The establishment of bacterial infections at mucosal epithelial surfaces is determined by the balance of virulence attributes of the pathogen with the activity of innate host defenses. Polymorphonuclear leukocytes (PMN) are key responders in many bacterial infections, but the mechanisms by which pathogens subvert these early responses to establish infection are largely undefined. Here, we model early interactions between human PMN and the primary cause of urinary tract infections, namely uropathogenic Escherichia coli (UPEC). Our objective was to define virulence phenotypes of uropathogens that permit evasion of PMN activity. We show that UPEC strains, as compared with laboratory and commensal E. coli, resist phagocytic killing and dampen the production of antimicrobial reactive oxygen species by PMN. Analysis of the transcriptional responses of PMN to E. coli strains revealed that UPEC exposure downregulates the expression of PMN genes that direct pro-inflammatory signaling and PMN chemotaxis, adhesion, and migration. Consistent with these data, UPEC attenuated transepithelial neutrophil recruitment in an in vitro model of acute infection and in a murine model of bacterial cystitis. We propose that these UPEC strategies are important in the establishment of epithelial infection, and that the findings are germane to bacterial infections at other epithelial surfaces.

Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.

More than a decade has passed since Zic family zinc finger proteins were discovered to be transcription factors controlling neuroectodermal differentiation (neural induction) in Xenopus laevis embryos. Although BMP-signal blocking has been shown to be a major upregulator of Zic genes in neuroectodermal differentiation, recent studies have revealed that FGF signaling and intracellular calcium elevation are also involved in regulating the expression of Zic genes. Different regulatory mechanisms have been found for the Zic1 and Zic3 genes, raising the possibility that functional synergism between them partly accounts for the integration of BMP-signal blocking and FGF signaling in neuroectodermal differentiation. Furthermore, mammalian Zic1 and Zic3 have been found to be neural-cell-fate-inducing and pluripotency-maintaining factors, respectively, leading us to the intriguing question of whether the mechanism underlying amphibian neuroectodermal differentiation is applicable to mammals. Comprehensive understanding of the Zic family genes is therefore essential for the study of the neuroectodermal differentiation and stem cell biology.

Behavioural assays represent sensitive methods for detecting neuronal dysfunction in model organisms. A number of manual methods have been established for Drosophila, however these are time-consuming and generate parameter-poor phenotype descriptors. Here, we have developed an automated computer vision system to monitor accurately the three-dimensional locomotor trajectories of flies. This approach allows the quantitative description of fly trajectories, using small fly cohorts and short acquisition times. The application of this approach to a Drosophila model of Alzheimer's disease enables the early detection of progressive locomotor deficits and the quantitative assessment of phenotype severity. The approach can be widely applied to different disease models in a number of model organisms.

Brain white matter hyperintensities are more prevalent in migraine patients than in the general population, but the pathogenesis and the risk factors of these hyperintensities are not fully elucidated. The authors analyzed the routine clinical data of 186 migraine patients who were referred to the Outpatient Headache Department of the Department of Neurology, Medical School, University of Pécs, Hungary between 2007 and 2009: 58 patients with white matter hyperintensities and 128 patients without white matter hyperintensities on 3 T MRI. Significant associations between the presence of white matter hyperintensities and longer disease duration (14.4 vs. 19.9 years, p = 0.004), higher headache frequency (4.1 vs. 5.5 attacks/month, p = 0.017), hyperhomocysteinemia (incidence of hyperintensity is 9/9 = 100%, p = 0.009) and thyroid gland dysfunction (incidence of hyperintensity is 8/14 = 57.1%, p = 0.038) were found. These data support the theory that both the disease duration and the attack frequency have a key role in the formation of migraine-related brain white matter hyperintensities, but the effects of comorbid diseases may also contribute to the development of the hyperintensities.

<h4>Background</h4>Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input.<h4>Results</h4>TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified.<h4>Conclusions</h4>TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes.

When assessing liver iron content using relaxometry, an average relaxation rate (R1, R2 or R2*) is usually determined from a region of interest or the entire liver. This is commonly performed by fitting the signal decay in individual voxels to an appropriate relaxation function. The voxel-level parameters resulting from the fits are combined to determine the average relaxation rate, and an empirically derived calibration curve is used to convert this single value to iron content. The goal of this study was to compare the precision and accuracy of this voxel-wise fitting to an alternative method that relies on first averaging the signals from all voxels within the region of interest and then determining the relaxation rate from a single fit. Systematic differences were observed when both methods were applied to clinical images. Mathematical simulations were employed to determine which method provided more robust estimates of the true relaxation rate. The mathematical simulations were then expanded to include a range of conditions expected in typical relaxometry images. The results show that voxel-wise fitting skews the relaxation rate estimates and increases variance, particularly when the true relaxation rate is moderate to fast, as it would be in liver with high iron content. The potential impact of these results on clinical decisions is discussed.

Iron storage diseases are rare conditions of dysregulated iron metabolism in man and animals. A genetic basis has been confirmed only for human haemochromatosis. Iron storage disease was diagnosed in six related, 2-year-old male red deer of the same herd. These animals presented with weight loss and rough hair coats. Haematological examination was unremarkable. At necropsy examination, gross lesions were restricted to cachexia. Microscopical examination revealed severe, diffuse hepatocellular necrosis and iron accumulation in hepatocytes, Kupffer cells, cardiac myocytes and renal tubular cells in all affected animals. Four animals also had moderate bridging fibrosis in the liver. Hepatic iron concentrations were increased (1108-2275 mg/kg wet weight; reference range 100-200 mg/kg). Drinking water in rusty iron tubs in the deer park contained eight times more iron than the accepted level for human drinking water. To test for a possible genetic basis of increased iron uptake and storage in red deer, the cervid haemochromatosis gene (HFE) was identified. Sequence comparisons between the six diseased animals and three healthy free-ranging unrelated animals failed to identify differences in the HFE sequences. Furthermore, the disease was not associated with common amino acid substitutions reported in human patients with haemochromatosis, including C282Y and H63D. Polymorphisms in other non-HFE genes involved in iron metabolism may have led to a higher sensitivity to iron and this, together with the high iron content of the drinking water, may have been the cause of the observed iron storage in these red deer.

R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice. We suggest that the prolonged onset and disease course observed in R6/2 mice with greatly expanded CAG repeats might result from differential upregulation of genes related to protein localization and clearance. Such genes may represent novel therapeutic avenues to decrease htt aggregate toxicity and cell death in HD patients, with Lrsam1 being a promising, novel candidate disease modifier.

<h4>Background</h4>CD20 is a cell surface protein exclusively expressed on B cells. It is a clinically validated target for Non-Hodgkin's lymphomas (NHL) and autoimmune diseases. The B cell receptor (BCR) plays an important role for development and proliferation of pre-B and B cells. Physical interaction of CD20 with BCR and components of the BCR signaling cascade has been reported but the consequences are not fully understood.<h4>Methodology</h4>In this study we employed antibodies against CD20 and against the BCR to trigger the respective signaling. These antibodies induced very similar expression patterns of up- and down-regulated genes in NHL cell lines indicating that CD20 may play a role in BCR signaling and vice versa. Two of the genes that were rapidly and transiently induced by both stimuli are CCL3 and CCL4. 4 hours after stimulation the concentration of these chemokines in culture medium reaches a maximum. Spleen tyrosine kinase Syk is a cytoplasmic tyrosine kinase and a key component of BCR signaling. Both siRNA mediated silencing of Syk and inhibition by selective small molecule inhibitors impaired CCL3/CCL4 protein induction after treatment with either anti-CD20 or anti-BCR antibodies.<h4>Conclusion</h4>Our results suggest that treatment with anti-CD20 antibodies triggers at least partially a BCR activation-like response in NHL cell lines.

<h4>Background</h4>We initiated a prospective trial to identify transcriptional alterations associated with acquired chemotherapy resistance from pre- and post-biopsy samples from the same patient and uncover potential molecular pathways involved in treatment failure to help guide therapeutic alternatives.<h4>Methodology/principal findings</h4>A prospective, high-throughput transcriptional profiling study was performed using endoscopic biopsy samples from 123 metastatic gastric cancer patients prior to cisplatin and fluorouracil (CF) combination chemotherapy. 22 patients who initially responded to CF were re-biopsied after they developed resistance to CF. An acquired chemotherapy resistance signature was identified by analyzing the gene expression profiles from the matched pre- and post-CF treated samples. The acquired resistance signature was able to segregate a separate cohort of 101 newly-diagnosed gastric cancer patients according to the time to progression after CF. Hierarchical clustering using a 633-gene acquired resistance signature (feature selection at P<0.01) separated the 101 pretreatment patient samples into two groups with significantly different times to progression (2.5 vs. 4.7 months). This 633-gene signature included the upregulation of AKT1, EIF4B, and RPS6 (mTOR pathway), DNA repair and drug metabolism genes, and was enriched for genes overexpressed in embryonic stem cell signatures. A 72-gene acquired resistance signature (a subset of the 633 gene signature also identified in ES cell-related gene sets) was an independent predictor for time to progression (adjusted P = 0.011) and survival (adjusted P = 0.034) of these 101 patients.<h4>Conclusion/significance</h4>This signature may offer new insights into identifying new targets and therapies required to overcome the acquired resistance of gastric cancer to CF.

Mesenchymal progenitor cells, a multipotent adult stem cell population, have the ability to differentiate into cells of connective tissue lineages, including fat, cartilage, bone and muscle, and therefore generate a great deal of interest for their potential use in regenerative medicine. During development, endochondral bone is formed from a template of cartilage that transforms into bone; however, mature articular cartilage remains in the articulating joints, where its principal role is reducing friction and dispersing mechanical load. Articular cartilage is prone to damage from sports injuries or ageing, which regularly progresses to more serious joint disorders, such as osteoarthritis. Osteoarthritis is a degenerative joint disease characterized by the thinning and eventual wearing of articular cartilage, and affects millions of people worldwide. Due to low chondrocyte motility and proliferative rates, and complicated by the absence of blood vessels, cartilage has a limited ability to self-repair. Current pharmaceutical and surgical interventions fail to generate repair tissue with the mechanical and cellular properties of native host cartilage. The long-term success of cartilage repair will therefore depend on regenerative methodologies resulting in the restoration of articular cartilage that closely duplicates the native tissue. For cell-based therapies, the optimal cell source must be readily accessible with easily isolated, abundant cells capable of collagen type II and sulfated proteoglycan production in appropriate proportions. Although a cell source with these therapeutic properties remains elusive, mesenchymal chondroprogenitors retain their expansion capacity with the promise of reproducing the structural or biomechanical properties of healthy articular cartilage. As current knowledge regarding chondroprogenitors is relatively limited, this review will focus on their origin and therapeutic application.

Synthesis of 1α,25-dihydroxyvitamin D(3)-3β-bromoacetate (1,25(OH)(2)D(3)-3-BE), a potential anti-cancer agent is presented. We also report that mechanism of action of 1,25(OH)(2)D(3)-3-BE may involve reduction of its catabolism, as evidenced by the reduced and delayed expression of 1α,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24) gene in cellular assays.

Dyslipidemia is an important risk factor for myocardial infarction (MI). We previously showed that gene polymorphisms associated with MI differed among individuals with different lipid profiles. We also showed that rs6929846 of BTN2A1 and rs2569512 of ILF3 were significantly associated with MI in Japanese individuals. In the present study, we examined the relationship between rs6929846 of BTN2A1 or rs2569512 of ILF3 and MI in individuals with low or high serum concentrations of triglycerides, high density lipoprotein (HDL) cholesterol, or low density lipoprotein (LDL) cholesterol, respectively. The study population comprised 5513 unrelated Japanese individuals, including 1537 subjects with MI and 3976 controls. Multivariable logistic regression analyses with adjustment for covariates revealed that rs6929846 of BTN2A1 was significantly associated with MI in individuals with low (P=3.1 x 10-5; odds ratio, OR=1.66) or high (P = 1.1 x 10⁻⁶; OR = 2.09) triglycerides; in individuals with low (P = 0.0082; OR = 1.75) or high (P = 2.0 x 10⁻⁹; OR = 1.85) HDL cholesterol; and in individuals with low (P = 3.2 x 10⁻⁷; OR = 1.75) or high (P = 2.8 x 10⁻⁵; OR =2.18) LDL cholesterol. Similar analyses revealed that rs2569512 of ILF3 was significantly associated with MI in individuals with low (P = 0.0066; OR = 1.47) or high (P = 0.0013; OR = 1.88) triglycerides; in individuals with low (P = 0.0059; OR = 1.96) or high (P = 0.0020; OR = 1.51) HDL cholesterol; and in individuals with low (P = 0.0004, OR = 1.62) LDL cholesterol, but not in those with high LDL cholesterol. The results suggest that the relationship between rs6929846 of BTN2A1 or rs2569512 of ILF3 and MI is influenced by the serum concentrations of HDL and LDL cholesterol, respectively. Stratification of subjects according to lipid profiles may thus be useful for the personalized prevention of MI based on genetic information.

Huntington's disease (HD) is a debilitating neurodegenerative condition with significant burdens on both patient and healthcare costs. Despite extensive research, treatment options for patients with this condition remain limited. Aberrant post-translational modification (PTM) of proteins is emerging as an important element in the pathogenesis of HD. These PTMs include acetylation, phosphorylation, methylation, sumoylation and ubiquitination. Several families of proteins are involved with the regulation of these PTMs. In this review, I discuss the current evidence linking aberrant PTMs and/or aberrant regulation of the cellular machinery regulating these PTMs to HD pathogenesis. Finally, I discuss the evidence suggesting that pharmacologically targeting one of these protein families the histone deacetylases may be of potential therapeutic benefit in the treatment of HD.

As is generally true with other age-related diseases, Alzheimer's disease (AD) involves oxidative damage to cellular components in the affected tissue, in this case the brain. The causes and consequences of oxidative stress in neurons in AD are not fully understood, but considerable evidence points to important roles for accumulation of amyloid β-peptide upstream of oxidative stress and perturbed cellular Ca(2+) homeostasis and energy metabolism downstream of oxidative stress. The identification of mutations in the β-amyloid precursor protein and presenilin-1 as causes of some cases of early onset inherited AD, and the development of cell culture and animal models based on these mutations has greatly enhanced our understanding of the AD process, and has greatly expanded opportunities for preclinical testing of potential therapeutic interventions. In this regard, and of particular interest to us, is the elucidation of adaptive cellular stress response pathways (ACSRP) that can counteract multiple steps in the AD neurodegenerative cascades, thereby limiting oxidative damage and preserving cognitive function. ACSRP can be activated by factors ranging from exercise and dietary energy restriction, to drugs and phytochemicals. In this article we provide an overview of oxidative stress and AD, with a focus on ACSRP and their potential for preventing and treating AD.

Over the past few years, several genes, proteins and signalling pathways that are required for embryogenesis have been shown to regulate tumour development and progression by playing a major part in overriding antitumour safeguard mechanisms. These include axon guidance cues, such as Netrins and Slits. Netrin 1 and members of the Slit family are secreted extracellular matrix proteins that bind to deleted in colorectal cancer (DCC) and UNC5 receptors, and roundabout receptors (Robos), respectively. Their expression is deregulated in a large proportion of human cancers, suggesting that they could be tumour suppressor genes or oncogenes. Moreover, recent data suggest that these ligand-receptor pairs could be promising targets for personalized anticancer therapies.

<h4>Background</h4>Regular colonoscopic surveillance for detection of dysplasia is recommended in longstanding inflammatory bowel disease (IBD), however, its sensitivity is disputed. Screening accuracy may increase by using a biomarker-based surveillance strategy.<h4>Methods</h4>A case-control study was performed to determine the prognostic value of DNA ploidy and p53 in IBD-related neoplasia. Cases with IBD-related colorectal cancer (CRC), detected in our surveillance program between 1985-2008, were selected and matched with two controls, for age, gender, disease characteristics, interval of follow-up, PSC, and previous surgery. Biopsies were assessed for DNA ploidy, p53, grade of inflammation and neoplasia. Progression to neoplasia was analyzed with Cox regression analysis, adjusting for potentially confounding variables.<h4>Results</h4>Adjusting for age, we found statistically significant Hazard ratios (HR) between development of CRC, and low grade dysplasia (HR5.5; 95%CI 2.6-11.5), abnormal DNA ploidy (DNA index (DI) 1.06-1.34, HR4.7; 95%CI 2.9-7.8 and DI>1.34, HR6.6; 95%CI 3.7-11.7) and p53 immunopositivity (HR3.0; 95%CI 1.9-4.7) over time. When adjusting for all confounders, abnormal DNA ploidy (DI 1.06-1.34, HR4.7; 95%CI 2.7-7.9 and DI>1.34, HR5.0; 95%CI 2.5-10.0) and p53 immunopositivity (HR1.7; 95%CI 1.0-3.1) remained statistically significant predictive of neoplasia.<h4>Conclusion</h4>In longstanding IBD, abnormal DNA ploidy and p53 immunopositivity are important risk factors of developing CRC. The yield of surveillance may potentially increase by adding these biomarkers to the routine assessment of biopsies.

Cyclin dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) has gained attention in the last years following the discovery, in 2005, that recessive mutations cause primary autosomal recessive microcephaly. This disease is seen as an isolated developmental defect of the brain, particularly of the cerebral cortex, and was thus historically also referred to as microcephalia vera. Unraveling the pathomechanisms leading to this human disease is fascinating scientists because it can convey insight into basic mechanisms of physiologic brain development (particularly of cortex formation). It also finds itself in the spotlight because of its implication in trends in mammalian evolution with a massive increase in the size of the cerebral cortex in primates. Here, we provide a timely overview of the current knowledge on the function of CDK5RAP2 and mechanisms that might lead to disease in humans when the function of this protein is disturbed.

<h4>Rationale</h4>Chromosome 12p has been linked to chronic obstructive pulmonary disease (COPD) in the Boston Early-Onset COPD Study (BEOCOPD), but a susceptibility gene in that region has not been identified.<h4>Objectives</h4>We used high-density single-nucleotide polymorphism (SNP) mapping to implicate a COPD susceptibility gene and an animal model to determine the potential role of SOX5 in lung development and COPD.<h4>Methods</h4>On chromosome 12p, we genotyped 1,387 SNPs in 386 COPD cases from the National Emphysema Treatment Trial and 424 control smokers from the Normative Aging Study. SNPs with significant associations were then tested in the BEOCOPD study and the International COPD Genetics Network. Based on the human results, we assessed histology and gene expression in the lungs of Sox5(-/-) mice.<h4>Measurements and main results</h4>In the case-control analysis, 27 SNPs were significant at P ≤ 0.01. The most significant SNP in the BEOCOPD replication was rs11046966 (National Emphysema Treatment Trial-Normative Aging Study P = 6.0 × 10(-4), BEOCOPD P = 1.5 × 10(-5), combined P = 1.7 × 10(-7)), located 3' to the gene SOX5. Association with rs11046966 was not replicated in the International COPD Genetics Network. Sox5(-/-) mice showed abnormal lung development, with a delay in maturation before the saccular stage, as early as E16.5. Lung pathology in Sox5(-/-) lungs was associated with a decrease in fibronectin expression, an extracellular matrix component critical for branching morphogenesis.<h4>Conclusions</h4>Genetic variation in the transcription factor SOX5 is associated with COPD susceptibility. A mouse model suggests that the effect may be due, in part, to its effects on lung development and/or repair processes.

This study presents a novel network methodology to identify prognostic gene signatures. Implication networks based on prediction logic are used to construct genome-wide coexpression networks for different disease states. From the differential components associated with specific disease states, candidate genes that are co-expressed with major disease signal hallmarks are selected. From these candidate genes, top genes that are the most predictive of clinical outcome are identified using univariate Cox model and Relief algorithm. Using this approach, a 13-gene lung cancer prognosis signature was identified, which generated significant prognostic stratifications (log-rank P < 0.05) in Director's Challenge Study (n = 442).

<h4>Background</h4>Cell numbers in above-ground meristems of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex. CLV3 produced in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 and WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. Overexpression of CLV3 eliminates the stem cells, causing meristem termination, and loss of CLV3 function allows meristem overproliferation. There are many questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype.<h4>Results</h4>Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model.<h4>Conclusions</h4>This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants.

Induction of autoantibodies towards immune regulatory proteins, such as cytokines or their receptors, is a powerful strategy for functional studies on the role of these factors in vivo. Here we describe a new procedure to elicit autoantibodies by taking advantage of tumor cells as a vaccine against peptides presented at their surface in fusion with the human CD134L transmembrane protein. P1.HTR, an immunogenic variant of the P815 mastocytoma cell line, was used to generate stably transfected cell clones with expression vectors encoding the human CD134L transmembrane protein fused with either mouse IL-22BP or IL-9. Following repeated injections of living tumor cells expressing the mIL-22BP construct, mice developed autoantibodies that bind to mIL-22BP and inhibit its interaction with IL-22 in vitro. Mice similarly immunized against mIL-9 produced high titers of autoantibodies that block the activity of this cytokine in the TS1 bioassay. This procedure also inhibits IL-9 activity in vivo as no increase of serum MMCP-1 mast cell protease concentration was observed following IL-9 administration to immunized mice. As an alternative to the injection of living tumor cells expressing the CD134L-antigen fusion protein, intramuscular electrotransfer of the corresponding DNA construct also induced autoantibodies. These results validate this method as a simple and convenient approach to knock down the in vivo activity of soluble regulatory proteins, including cytokines and their receptors.

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. Recent studies have greatly expanded the number of established SLE risk alleles, but the distribution of multiple risk alleles in cases versus controls and their relationship to subphenotypes have not been studied. We studied 22 SLE susceptibility polymorphisms with previous genome-wide evidence of association (p < 5 x 10⁻¹²⁸) in 1919 SLE cases from 9 independent Caucasian SLE case series and 4813 independent controls. The mean number of risk alleles in cases was 15.1 (SD 3.1) while the mean in controls was 13.1 (SD 2.8), with trend p = 4 x 10⁻⁸. We defined a genetic risk score (GRS) for SLE as the number of risk alleles with each weighted by the SLE risk odds ratio (OR). The OR for high-low GRS tertiles, adjusted for intra-European ancestry, sex, and parent study, was 4.4 (95% CI 3.8-5.1). We studied associations of individual SNPs and the GRS with clinical manifestations for the cases: age at diagnosis, the 11 American College of Rheumatology classification criteria, and double-stranded DNA antibody (anti-dsDNA) production. Six subphenotypes were significantly associated with the GRS, most notably anti-dsDNA (OR(high-low) = 2.36, p = 9e-9), the immunologic criterion (OR(high-low) = 2.23, p = 3e-7), and age at diagnosis (OR(high-low) = 1.45, p = 0.0060). Finally, we developed a subphenotype-specific GRS (sub-GRS) for each phenotype with more power to detect cumulative genetic associations. The sub-GRS was more strongly associated than any single SNP effect for 5 subphenotypes (the above plus hematologic disorder and oral ulcers), while single loci are more significantly associated with renal disease (HLA-DRB1, OR = 1.37, 95% CI 1.14-1.64) and arthritis (ITGAM, OR = 0.72, 95% CI 0.59-0.88). We did not observe significant associations for other subphenotypes, for individual loci or the sub-GRS. Thus our analysis categorizes SLE subphenotypes into three groups: those having cumulative, single, and no known genetic association with respect to the currently established SLE risk loci.

A new class of proteasome inhibitors was synthesized using lithocholic acid as a scaffold. Modification at the C-3 position of lithocholic acid with a series of acid acyl groups yielded compounds with a range of potency on proteasome inhibition. Among them, the phenylene diacetic acid hemiester derivative (13) displayed the most potent proteasome inhibition with IC(50) = 1.9 μM. Enzyme kinetic analysis indicates that these lithocholic acid derivatives are noncompetitive inhibitors of the proteasome.

We develop a statistical framework to study the relationship between chromatin features and gene expression. This can be used to predict gene expression of protein coding genes, as well as microRNAs. We demonstrate the prediction in a variety of contexts, focusing particularly on the modENCODE worm datasets. Moreover, our framework reveals the positional contribution around genes (upstream or downstream) of distinct chromatin features to the overall prediction of expression levels.

<h4>Background</h4>The processing ability of poultry meat is highly related to its ultimate pH, the latter being mainly determined by the amount of glycogen in the muscle at death. The genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. In this study, Chicken Genome Arrays (20 K) were used to compare muscle gene expression profiles of chickens from Fat (F) and Lean (L) lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high (G+) or low (G-) muscle glycogen content originating from the F2 cross between the Fat and Lean lines. Real-time RT-PCR was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known.<h4>Results</h4>Among the genes found to be expressed in chicken P. major muscle, 197 and 254 transcripts appeared to be differentially expressed on microarrays for the F vs. L and the G+ vs. G- comparisons, respectively. Some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time RT-PCR. We confirmed that, as in mammals, the down-regulation of CEBPB and RGS2 coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the cAMP-dependent signalling pathway. Several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. PDK4 may act as a glycogen sensor in muscle, UGDH may compete for glycogen synthesis by using UDP-glucose for glucoronidation, and PRKAB1, PRKAG2, and PHKD may impact on glycogen turnover in muscle, through AMP-activated signalling pathways.<h4>Conclusions</h4>This study is the first stage in the understanding of molecular mechanisms underlying variations in poultry meat quality. Large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices.

<h4>Introduction</h4>suPAR is the soluble form of the urokinase plasminogen activator receptor (uPAR), which is expressed in various immunologically active cells. High suPAR serum concentrations are suggested to reflect the activation of the immune system in circumstances of inflammation and infection, and have been associated with increased mortality in different populations of non-intensive care patients. In this study we sequentially analyzed suPAR serum concentrations within the first week of intensive care in a large cohort of well characterized intensive care unit (ICU) patients, in order to investigate potential regulatory mechanisms and evaluate the prognostic significance in critically ill patients.<h4>Methods</h4>A total of 273 patients (197 with sepsis, 76 without sepsis) were studied prospectively upon admission to the medical intensive care unit (ICU), on Day 3 and Day 7, and compared to 43 healthy controls. Clinical data, various laboratory parameters as well as investigational inflammatory cytokine profiles were assessed. Patients were followed for approximately one year.<h4>Results</h4>Upon admission to the ICU suPAR serum concentrations were elevated in critically ill patients as compared with healthy controls. In sepsis patients suPAR levels were higher than in non-sepsis patients (with or without systemic inflammatory response syndrome (SIRS)). During the first week after admission to the ICU serum suPAR concentrations remained stably elevated. suPAR serum concentrations measured upon admission were closely and independently correlated to various laboratory parameters, specifically biomarkers of inflammation (tumor necrosis factor (TNF), C-reactive protein (CRP)), hepatic and renal dysfunction. High suPAR levels at admission and at Day 3 were a strong independent predictor for both ICU and long-term mortality in critically ill patients.<h4>Conclusions</h4>In sepsis and non-sepsis patients suPAR serum concentrations are increased upon admission to the ICU, likely reflecting the activation state of the immune system, and remain stably elevated in the initial course of treatment. Low suPAR levels are a positive predictor of ICU- and overall survival in critically ill patients, including sepsis and non-sepsis patients. Aside from its value as a promising new prognostic biomarker, both experimental and clinical studies are required in order to understand the specific effects and regulatory mechanisms of suPAR in SIRS and sepsis, and may reveal new therapeutic options.

<h4>Background and aims</h4>Elevated iron biomarkers are associated with diabetes and other cardiometabolic abnormalities in the general population. It is unclear whether they are associated with an increased risk of all-cause or cause-specific mortality. The purpose of the current analysis was to evaluate the association of ferritin and transferrin saturation levels with all-cause, cardiovascular, and cancer mortality in the general US adult population.<h4>Methods and results</h4>A prospective cohort study was conducted with 12,258 adults participating in the Third National Health and Nutrition Examination Survey (NHANES III), a nationally representative sample of the US population. Study participants were recruited in 1988-1994 and followed through December 31, 2006 for all-cause, cardiovascular disease, and cancer mortality. The multivariable-adjusted hazard ratios (95% confidence interval) for all-cause mortality comparing the fourth versus the second quartiles of ferritin and transferrin saturation were 1.09 (0.82-1.44; p-trend across quartiles = 0.92) and 1.08 (0.82-1.43; p-trend across quartiles = 0.62), respectively, for men, 1.43 (0.63-3.23; p-trend across quartiles = 0.31) and 1.48 (0.70-3.11; p-trend across quartiles = 0.60), respectively, for premenopausal women, and 1.03 (0.79-1.34; p-trend across quartiles = 0.95) and 1.17 (0.92-1.49; p-trend across quartiles = 0.63), respectively, for postmenopausal women. Quartile of ferritin and transferrin saturation also showed no association between biomarkers of iron status and mortality.<h4>Conclusions</h4>In a large nationally representative sample of US adults, within the spectrum of normal iron metabolism, ferritin and transferrin saturation were not associated with risk of mortality among people who were not taking iron supplements and did not have a baseline history of cardiovascular disease or cancer.

It is well recognised that there are pitfalls when defining the subcellular localisation of a protein with immunocytochemistry. Accurate protein localisation to particular cellular micro-architecture is crucial in defining its role within the cell. Huntingtin (HTT), the protein mutated in the neurodegenerative disorder Huntington's disease (HD) is a large protein of ill-defined function. Bearing little resemblance to other proteins, its function has been difficult to assign, therefore localising this protein with precision within the cell may provide further clues as to its normal and pathological function. Lack of consistency between methods employed in different studies has resulted in varying conclusions as to its subcellular localisation. This technical review investigates the effects that different immunocytological methods can have upon the apparent subcellular localisation of the huntingtin protein, and discusses the implications this may have.

<h4>Background</h4>Diet and exercise promote cardiovascular health but their relative contributions to atherosclerosis are not fully known. The transition from a sedentary to active lifestyle requires increased caloric intake to achieve energy balance. Using atherosclerosis-prone ApoE-null mice we sought to determine whether the benefits of exercise for arterial disease are dependent on the food source of the additional calories.<h4>Methods and results</h4>Mice were fed a high-fat diet (HF) for 4.5 months to initiate atherosclerosis after which time half were continued on HF while the other half were switched to a high protein/fish oil diet (HP). Half of each group underwent voluntary running. Food intake, running distance, body weight, lipids, inflammation markers, and atherosclerotic plaque were quantified. Two-way ANOVA tests were used to assess differences and interactions between groups. Exercised mice ran approximately 6-km per day with no difference between groups. Both groups increased food intake during exercise and there was a significant main effect of exercise F((1,44) = 9.86, p<0.01) without interaction. Diet or exercise produced significant independent effects on body weight (diet: F(1,52) = 6.85, p = 0.012; exercise: F(1,52) = 9.52, p<0.01) with no significant interaction. The combination of HP diet and exercise produced a greater decrease in total cholesterol (F(1, 46) = 7.9, p<0.01) and LDL (F(1, 46) = 7.33, p<0.01) with a large effect on the size of the interaction. HP diet and exercise independently reduced TGL and VLDL (p<0.05 and 0.001 respectively). Interleukin 6 and C-reactive protein were highest in the HF-sedentary group and were significantly reduced by exercise only in this group. Plaque accumulation in the aortic arch, a marker of cardiovascular events was reduced by the HP diet and the effect was significantly potentiated by exercise only in this group resulting in significant plaque regression (F1, 49 = 4.77, p<0.05).<h4>Conclusion</h4>In this model exercise is beneficial to combat dyslipidemia and protect from atherosclerosis only when combined with diet.

Up to 60% of depressed patients do not respond completely to antidepressants (ADs) and up to 30% do not respond at all. Genetic factors contribute for about 50% of the AD response. During the recent years the possible influence of a set of candidate genes as genetic predictors of AD response efficacy was investigated by us and others. They include the cytochrome P450 superfamily, the P-glycoprotein (ABCB1), the tryptophan hydroxylase, the catechol-O-methyltransferase, the monoamine oxidase A, the serotonin transporter (5-HTTLPR), the norepinephrine transporter, the dopamine transporter, variants in the 5-hydroxytryptamine receptors (5-HT1A, 5-HT2A, 5-HT3A, 5-HT3B, and 5-HT6), adrenoreceptor beta-1 and alpha-2, the dopamine receptors (D2), the G protein beta 3 subunit, the corticotropin releasing hormone receptors (CRHR1 and CRHR2), the glucocorticoid receptors, the c-AMP response-element binding, and the brain-derived neurotrophic factor. Marginal associations were reported for angiotensin I converting enzyme, circadian locomotor output cycles kaput protein, glutamatergic system, nitric oxide synthase, and interleukin 1-beta gene. In conclusion, gene variants seem to influence human behavior, liability to disorders and treatment response. Nonetheless, gene × environment interactions have been hypothesized to modulate several of these effects.

We have recently proposed a new model for understanding tumor metabolism, termed: "The Autophagic Tumor Stroma Model of Cancer Metabolism". In this new paradigm, catabolism (autophagy) in the tumor stroma fuels the anabolic growth of aggressive cancer cells. Mechanistically, tumor cells induce autophagy in adjacent cancer-associated fibroblasts via the loss of caveolin-1 (Cav-1), which is sufficient to promote oxidative stress in stromal fibroblasts. To further test this hypothesis, here we created human Cav-1 deficient immortalized fibroblasts using a targeted sh-RNA knock-down approach. Relative to control fibroblasts, Cav-1 deficient fibroblasts dramatically promoted tumor growth in xenograft assays employing an aggressive human breast cancer cell line, namely MDA-MB-231 cells. Co-injection of Cav-1 deficient fibroblasts, with MDA-MB-231 cells, increased both tumor mass and tumor volume by ~4-fold. Immuno-staining with CD31 indicated that this paracrine tumor promoting effect was clearly independent of angiogenesis. Mechanistically, proteomic analysis of these human Cav-1 deficient fibroblasts identified > 40 protein biomarkers that were upregulated, most of which were associated with i) myofibroblast differentiation, or ii) oxidative stress/hypoxia. In direct support of these findings, the tumor promoting effects of Cav-1 deficient fibroblasts could be functionally suppressed (nearly 2-fold) by the recombinant over-expression of SOD2 (superoxide dismutase 2), a known mitochondrial enzyme that de-activates superoxide, thereby reducing mitochondrial oxidative stress. In contrast, cytoplasmic soluble SOD1 had no effect, further highlighting a specific role for mitochondrial oxidative stress in this process. In summary, here we provide new evidence directly supporting a key role for a loss of stromal Cav-1 expression and oxidative stress in cancer-associated fibroblasts, in promoting tumor growth, which is consistent with "The Autophagic Tumor Stroma Model of Cancer". The human Cav-1 deficient fibroblasts that we have generated are a new genetically tractable model system for identifying other suppressors of the cancer-associated fibroblast phenotype, via a genetic "complementation" approach. This has important implications for understanding the pathogenesis of triple negative and basal breasts cancers, as well as tamoxifen-resistance in ER+ breast cancers, which are all associated with a Cav-1 deficient "lethal" tumor micro-environment, driving poor clinical outcome.

<h4>Introduction</h4>Aplastic anemia is a rare disease caused by destruction of pluripotent stem cells in bone marrow. During pregnancy it could be life-threatening for both mother and child. The only causal therapy for aplastic anemia is bone marrow transplantation, which is contraindicated during pregnancy because of potential embryo toxicity. Treatment options are erythrocytes and platelet transfusions and immunosuppressive therapy. There is, however, no agreement about the optimal supportive care and treatment regime for this disorder during pregnancy.<h4>Case presentation</h4>A 26-year-old nulliparous Asian woman with an uneventful medical history was admitted to the hospital at 14 weeks' gestation because of excessive vomiting. Routine laboratory tests showed pancytopenia (Hb 3.5 mmol/L, leukocytes 3.5 *109/L, platelets 45 *109L). A bone marrow biopsy confirmed aplastic anemia. Methylprednisolon, cyclosporine A, packed cells and platelet transfusions were initiated. At 33 weeks she developed neutropenia (0.1 *109/L) for which oral colistin and tobramycin were given prophylactically. At 35 weeks labor was induced, during which she developed a fever of 38.2°C. She gave birth spontaneously to a healthy son weighing 2415 grams, who had no signs of pancytopenia. After delivery the blood count of the patient did not recover and did not respond to medication. Eighteen weeks after delivery she died of sepsis complicated by cerebral bleeding and infarction due to severe thrombocytopenia and neutropenia, despite optimal supportive treatment.<h4>Conclusion</h4>This potential life-threatening disease has a relatively good prognosis for both mother and child after optimal treatment. Transfusion during pregnancy is the first choice treatment with recommended hemoglobin levels of >5.5 mmol/L and platelet counts of >20 *109/L. Cyclosporine A seems a reasonable alternative therapy with a reported success rate in non-pregnant patients of 70% when combined with antithymocyte globuline. Our patient died 18 weeks postpartum from cerebral bleeding and infarction due to severe thrombocytopenia despite intensive supportive treatment, methylprednisolon and cyclosporine A.

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

<h4>Background</h4>Understanding variation in the normal electric activity of the heart, assessed by the ECG, may provide a starting point for studies of susceptibility to serious arrhythmias such as sudden cardiac death during myocardial infarction or drug therapy. Recent genetic association studies of one ECG trait, the QT interval, have identified common variation in European-descent populations, but little is known about the genetic determinants of ECG traits in populations of African descent.<h4>Methods and results</h4>To identify genetic risk factors, we have undertaken a candidate gene study of ECG traits in collaboration with the Jackson Heart Study, a longitudinal study of 5301 blacks ascertained from the Jackson, Mississippi, area. Nine quantitative ECG traits were evaluated: P, PR, QRS, QT, and QTc durations, heart rate, and P, QRS, and T axes. We genotyped 72 variations in the predominant sodium channel gene expressed in heart, SCN5A, encoding the Na(v)1.5 voltage-gated sodium channel in 4558 subjects. Both rare and common variants in this gene have previously been associated with inherited arrhythmia syndromes and variable conduction. Adjusting for age, sex, and European ancestry, we performed tests of association in 3054 unrelated participants and identified 14 significant associations (P<1.0×10(-4)), of which 13 are independent, based on linkage disequilibrium. These variants explain up to 2% of the variation in ECG traits in the Jackson Heart Study.<h4>Conclusions</h4>These results suggest that SCN5A variation contributes to ECG trait distributions in blacks, and these same variations may be risk or protective factors associated with susceptibility to arrhythmias.

<h4>Background</h4>MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV).<h4>Methods</h4>Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature.<h4>Results</h4>One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%.<h4>Conclusion</h4>MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

<h4>Background</h4>In adult women with retrospective data, childhood adiposity, pubertal growth and development were associated with benign breast disease (BBD) and/or breast cancer. The authors prospectively evaluated these childhood/adolescent characteristics and BBD risk.<h4>Methods</h4>The Growing Up Today Study (GUTS) included females, aged 9-15 years in 1996, who completed annual questionnaires through 2001, then 2003, 2005, and 2007. Participants annually/biennially provided information on menarche, height, and weight, from which the authors derived body mass index (BMI in kg/m(2) ). Peak height growth velocity (PHV in cm/year) was estimated from longitudinal data. On 2005-2007 surveys, 6899 females (18-27 years of age) reported whether a healthcare provider ever diagnosed BBD (n = 147), and whether it was confirmed by biopsy (n = 67). Logistic models investigated risk factors adjusted for age, alcohol, pregnancy, and maternal history.<h4>Results</h4>More childhood adiposity (odds ratio [OR], 0.91/[kg/m(2) ]; P = .04) and shorter adult height (OR, 0.93/inch shorter; P = .07) were associated with lower risk of biopsy-confirmed BBD. Girls with most rapid height growth were at increased risk (OR, 2.12; P = .09) relative to those with the slowest growth. Age at menarche was not associated (OR, 1.11/year; P = .32) nor was adult BMI (adjusted for childhood BMI: OR, 1.01/[kg/m(2) ]; P = .98); larger BMI increases (childhood to adulthood) were not protective (OR + 1.04/[kg/m(2) ]; P = .37). Among girls with maternal breast cancer, those with more rapid growth had higher risk (OR, 1.47/[cm/year]; P = .02). All estimates were age-adjusted.<h4>Conclusions</h4>Increased BBD risk (likely evolving to elevated breast cancer risk) was observed in thinner girls, girls with the most rapid growth, and taller women. Contrary to expectations, later menarche age was not protective against BBD, consistent with studies that found BBD patients are not protected against breast cancer by later menarche.

Ovarian cancer remains a leading cause of cancer death. A comparative proteomic study was performed on normal ovarian tissue (n=5) and grade 3 ovarian tumours (n=5) to search for differentially expressed proteins. In contrast to other studies, here we extracted proteins in soluble and insoluble protein fractions using commercial kits and also utilised three medium-range IPG strips that encompassed the broad pH range of 3-10 (pH 3-6, 5-8 and 7-10). Protein fractions were compared by 2D-PAGE and MALDI-TOF/TOF-MS. Nineteen differentially expressed proteins were identified: HSP60, Grp78, CK19, EF-Tu, MRLC2, prohibitin, Stress-70 protein, TPI and tubulin α6 were up-regulated in grade 3 tumours whereas annexin A2 and A5, antithrombin-III precursor, CBR1, GSTM2, GSTM3, RALDH1, serum albumin precursor, transthyretin precursor and vimentin were found to be down-regulated in grade 3 ovarian tumours. These proteins are associated with cytoskeleton rearrangement, cell metabolism, tumour suppression function, apoptosis and induction of host response.

Imaging studies of serotonin transporter binding or tryptophan retention in autistic patients suggest that the brain serotonin system is decreased. However, treatment with drugs which increase serotonin (5-HT) levels, specific serotonin reuptake inhibitors (SSRIs), commonly produce a worsening of the symptoms. In this study we examined 5-HT axons that were immunoreactive to a serotonin transporter (5-HTT) antibody in a number of postmortem brains from autistic patients and controls with no known diagnosis who ranged in age from 2 to 29 years. Fine, highly branched, and thick straight fibers were found in forebrain pathways (e.g. medial forebrain bundle, stria terminalis and ansa lenticularis). Many immunoreactive varicose fine fibers were seen in target areas (e.g. globus pallidus, amygdala and temporal cortex). Morphometric analysis of the stained axons at all ages studied indicated that the number of serotonin axons was increased in both pathways and terminal regions in cortex from autism donors. Our findings provide morphological evidence to warrant caution when using serotonin enhancing drugs (e.g. SSRIs and receptor agonist) to treat autistic children. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

<h4>Background & aims</h4>Iron may influence severity and progression of non-hemochromatotic liver diseases. Our aim was to assess the relationship of iron and HFE genetic variations to progression and outcomes in the HALT-C Trial and whether PegIFN therapy influenced iron variables.<h4>Methods</h4>Participants were randomized to receive long-term PegIFN [n = 400] or no therapy [n = 413] for 3.5 y, with follow-up for up to 8.7 y [median 6.0 y]. Associations of patient characteristics with iron variables at baseline and over time were carried out using Kaplan-Meier analyses, Cox regression models, and repeated measures analysis of covariance.<h4>Results</h4>Participants who developed clinical outcomes [CTP > 7, ascites, encephalopathy, variceal bleeding, SBP, HCC, death] had significantly higher baseline scores for stainable iron in hepatocytes and in portal tract cells than those without. There were significant direct correlations between stainable iron in portal triads and lobular and total Ishak inflammatory and fibrosis scores [P < 0.0001]. Iron in triads at baseline increased risk of outcomes (HR = 1.35, P = 0.02). Stainable iron in hepatocytes decreased, whereas that in portal stromal cells increased significantly [P < 0.0001] over time. Serum iron and TIBC fell significantly over time [P < 0.0001], as did serum ferritin [P = 0.0003]. Chronic PegIFN treatment did not affect stainable iron. HFE genetic variations did not correlate with outcomes, including development of hepatocellular carcinoma.<h4>Conclusions</h4>Stainable iron in hepatocytes and portal tract cells is a predictor of progression and clinical and histological outcomes in advanced chronic hepatitis C. Chronic low-dose PegIFN therapy did not improve outcomes, nor iron variables.

Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs), RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. It is significantly elevated in the majority of human tumors and functionally linked to cellular proliferation, survival and migration. In this study, we used two experimental-based strategies to search for novel miR-21 targets. On the one hand, we performed a proteomic approach using two-dimensional differential gel electrophoresis (2D-DIGE) to identify proteins suppressed upon enhanced miR-21 expression in LNCaP human prostate carcinoma cells. The tumor suppressor acidic nuclear phosphoprotein 32 family, member A (ANP32A) (alias pp32 or LANP) emerged as the most strongly downregulated protein. On the other hand, we applied a mathematical approach to select correlated gene sets that are negatively correlated with primary-miR-21 (pri-miR-21) expression in published transcriptome data from 114 B-cell lymphoma cases. Among these candidates, we found tumor suppressor SMARCA4 (alias BRG1) together with the already validated miR-21 target, PDCD4. ANP32A and SMARCA4, which are both involved in chromatin remodeling processes, were confirmed as direct miR-21 targets by immunoblot analysis and reporter gene assays. Furthermore, knock down of ANP32A mimicked the effect of enforced miR-21 expression by enhancing LNCaP cell viability, whereas overexpression of ANP32A in the presence of high miR-21 levels abrogated the miR-21-mediated effect. In A172 glioblastoma cells, enhanced ANP32A expression compensated for the effects of anti-miR-21 treatment on cell viability and apoptosis. In addition, miR-21 expression clearly increased the invasiveness of LNCaP cells, an effect also seen in part upon downregulation of ANP32A. In conclusion, these results suggest that downregulation of ANP32A contributes to the oncogenic function of miR-21.

No abstract available.

Low folate status increases colorectal cancer risk. Paradoxically, overly abundant folate supplementation, which is not uncommon in the United States, may increase risk. The mechanisms of these effects are unknown. We conducted two translational studies to define molecular pathways in the human colon altered either by folate supplementation or by dietary folate depletion (followed by repletion). In the first study, 10 healthy, at-risk volunteers (with documented stable/normal folate intake) received supplemental folic acid (1 mg/d) for 8 weeks. In the second study, 10 similar subjects were admitted to a hospital as inpatients for 12 weeks to study folate depletion induced by a low folate diet. A repletion regimen of folic acid (1 mg/d) was provided for the last 4 of these weeks. Both studies included an 8-week run-in period to ensure stabilized folate levels prior to intervention. We obtained 12 rectosigmoid biopsies (from 4 quadrants of normal-appearing mucosa 10-15 cm from the anal verge) at baseline and at measured intervals in both studies for assessing the primary endpoints: genome-wide gene expression, genomic DNA methylation, promoter methylation (depletion/repletion study only), and p53 DNA strand breaks. Serum and rectosigmoid folate concentrations accurately tracked all changes in folate delivery (P < 0.05). In the first study, gene array analysis revealed that supplementation upregulated multiple inflammation- and immune-related pathways in addition to altering several 1-carbon-related enzymes (P < 0.001). In the second study, folate depletion downregulated genes involved in immune response, inflammation, the cell cycle, and mitochondrial/energy pathways; repletion reversed most of these changes. However, changes in gene expression after repletion in the second study (involving immune response and inflammation) did not reach the levels seen after supplementation in the first study. Neither genomic nor promoter-specific DNA methylation changed during the course of the depletion/repletion protocol, and genomic methylation did not change with supplementation in the first study. p53 DNA strand breaks increased with depletion after 12 weeks. In sum, depletion downregulates, whereas repletion or supplementation upregulates pathways related to inflammation and immune response. These findings provide novel support to the concept that excessive folate supplementation might promote colorectal carcinogenesis by enhancing proinflammatory and immune response pathways. These results indicate that modest changes in folate delivery create substantial changes in the molecular milieu of the human colon.

The interplay between pathogens and their hosts has been studied for decades using targeted approaches, such as the analysis of mutants and host immunological responses. Although much has been learned from such studies, they focus on individual pathways and fail to reveal the global effects of infection on the host. To alleviate this issue, high-throughput methods, such as transcriptomics and proteomics, have been used to study host-pathogen interactions. Recently, metabolomics was established as a new method to study changes in the biochemical composition of host tissues. We report a metabolomic study of Salmonella enterica serovar Typhimurium infection. Our results revealed that dozens of host metabolic pathways are affected by Salmonella in a murine infection model. In particular, multiple host hormone pathways are disrupted. Our results identify unappreciated effects of infection on host metabolism and shed light on mechanisms used by Salmonella to cause disease and by the host to counter infection.

Asian patients with chronic hepatitis C (CHC) are known to have better virological responses to pegylated (Peg) IFN-based therapy than Western patients. Although IL28B gene polymorphisms may contribute to this difference, whether favorable hepatitis C virus (HCV) kinetics during treatment plays a role remains unclear. We enrolled 145 consecutive Taiwanese patients with CHC receiving Peg-IFN α-2a plus ribavirin for the study. Blood samples were taken more frequently at defined intervals in the first 3 d. Peg-IFN was administered at week 1. It was then administered weekly in combination with daily ribavirin for 24 or 48 wk. A mathematical model fitted to the observed HCV kinetics was constructed, which could interpret the transient HCV titer elevation after Peg-IFN treatment. The results demonstrated a comparable viral clearance rate (c = 3.45 ± 3.73) (day(-1), mean ± SD) but lower daily viral production rate (P = 10(6)-10(12)) in our patients than those reported previously in Western patients. Of 110 patients with a sustained virological response (SVR), 47 (43%) had a transient elevation of viral titer within 12 h (proportion of 12 h/3 d: 44% in non-SVR vs. 70% in SVR; P = 0.029). Among 91 patients with available rs8099917 data, patients with the TT genotype had an early surge of viral titer after therapy and a higher SVR and viral clearance rate than those with the GT genotype. In conclusion, Taiwanese patients with CHC receiving Peg-IFN plus ribavirin therapy have a lower daily viral production rate than Western patients, and the rs8099917 TT genotype may contribute to the increased viral clearance rate and better virological responses in these patients.

Myosin X (MyoX), encoded by Myo10, is a representative member of the MyTH4-FERM domain-containing myosins, and this family of unconventional myosins shares common functions in promoting formation of filopodia/stereocilia structures in many cell types with unknown mechanisms. Here, we present the structure of the MyoX MyTH4-FERM tandem in complex with the cytoplasmic tail P3 domain of the netrin receptor DCC. The structure, together with biochemical studies, reveals that the MyoX MyTH4 and FERM domains interact with each other, forming a structural and functional supramodule. Instead of forming an extended β-strand structure in other FERM binding targets, DCC_P3 forms a single α-helix and binds to the αβ-groove formed by β5 and α1 of the MyoX FERM F3 lobe. Structure-based amino acid sequence analysis reveals that the key polar residues forming the inter-MyTH4/FERM interface are absolutely conserved in all MyTH4-FERM tandem-containing proteins, suggesting that the supramodular nature of the MyTH4-FERM tandem is likely a general property for all MyTH4-FERM proteins.

In humans, embryonic, fetal, and adult hemoglobins are sequentially expressed in developing erythroblasts during ontogeny. For the past 40 years, this process has been the subject of intensive study because of its value to enlighten the biology of developmental gene regulation and because fetal hemoglobin can significantly ameliorate the clinical manifestations of both sickle cell disease and β-thalassemia. Understanding the normal process of loss of fetal globin expression and activation of adult globin expression could potentially lead to new therapeutic approaches for these hemoglobin disorders. Herein, we briefly review the history of the study of hemoglobin switching and then focus on recent discoveries in the field that now make new therapeutic approaches seem feasible in the future. Erythroid-specific knockdown of fetal gene repressors or enforced expression of fetal gene activators may provide clinically applicable approaches for genetic treatment of hemoglobin disorders that would benefit from increased fetal hemoglobin levels.

<h4>Background</h4>Daily inhaled corticosteroids are an effective treatment for mild persistent asthma, but some children have exacerbations even with good day-to-day control, and many discontinue treatment after becoming asymptomatic. We assessed the effectiveness of an inhaled corticosteroid (beclomethasone dipropionate) used as rescue treatment.<h4>Methods</h4>In this 44-week, randomised, double-blind, placebo-controlled trial we enrolled children and adolescents with mild persistent asthma aged 5-18 years from five clinical centres in the USA. A computer-generated randomisation sequence, stratified by clinical centre and age group, was used to randomly assign participants to one of four treatment groups: twice daily beclomethasone with beclomethasone plus albuterol as rescue (combined group); twice daily beclomethasone with placebo plus albuterol as rescue (daily beclomethasone group); twice daily placebo with beclomethasone plus albuterol as rescue (rescue beclomethasone group); and twice daily placebo with placebo plus albuterol as rescue (placebo group). Twice daily beclomethasone treatment was one puff of beclomethasone (40 μg per puff) or placebo given in the morning and evening. Rescue beclomethasone treatment was two puffs of beclomethasone or placebo for each two puffs of albuterol (180 μg) needed for symptom relief. The primary outcome was time to first exacerbation that required oral corticosteroids. A secondary outcome measured linear growth. Analysis was by intention to treat. This study is registered with clinicaltrials.gov, number NCT00394329.<h4>Results</h4>843 children and adolescents were enrolled into this trial, of whom 288 were assigned to one of four treatment groups; combined (n=71), daily beclomethasone (n=72), rescue beclomethasone (n=71), and placebo (n=74)-555 individuals were excluded during the run-in, according to predefined criteria. Compared with the placebo group (49%, 95% CI 37-61), the frequency of exacerbations was lower in the daily (28%, 18-40, p=0·03), combined (31%, 21-43, p=0·07), and rescue (35%, 24-47, p=0·07) groups. Frequency of treatment failure was 23% (95% CI 14-43) in the placebo group, compared with 5·6% (1·6-14) in the combined (p=0·012), 2·8% (0-10) in the daily (p=0·009), and 8·5% (2-15) in the rescue (p=0·024) groups. Compared with the placebo group, linear growth was 1·1 cm (SD 0·3) less in the combined and daily arms (p<0·0001), but not the rescue group (p=0·26). Only two individuals had severe adverse events; one in the daily beclomethasone group had viral meningitis and one in the combined group had bronchitis.<h4>Interpretation</h4>Children with mild persistent asthma should not be treated with rescue albuterol alone and the most effective treatment to prevent exacerbations is daily inhaled corticosteroids. Inhaled corticosteroids as rescue medication with albuterol might be an effective step-down strategy for children with well controlled, mild asthma because it is more effective at reducing exacerbations than is use of rescue albuterol alone. Use of daily inhaled corticosteroid treatment and related side-effects such as growth impairment can therefore be avoided.<h4>Funding</h4>National Heart, Lung and Blood Institute.

Therapy-related acute leukemia (t-AML), is a severe complication of cytotoxic therapy used for primary cancer treatment. The outcome of these patients is poor, compared to people who develop de novo acute leukemia (p-AML). Cytogenetic abnormalities in t-AML are similar to those found in p-AML but present more frequent unfavorable karyotypes depending on the inducting agent. Losses of chromosome 5 or 7 are observed after alkylating agents while balanced translocations are found after topoisomerase II inhibitors. This study compared t-AML to p-AML using high resolution array CGH in order to find copy number abnormalities (CNA) at a higher resolution than conventional cytogenetics. More CNAs were observed in 30 t-AML than in 36 p-AML: 104 CNAs were observed with 63 losses and 41 gains (mean number 3.46 per case) in t-AML, while in p-AML, 69 CNAs were observed with 32 losses and 37 gains (mean number of 1.9 per case). In primary leukemia with a previously "normal" karyotype, 18% exhibited a previously undetected CNA, whereas in the (few) t-AML with a normal karyotype, the rate was 50%. Several minimal critical regions (MCRs) were found in t-AML and p-AML. No common MCRs were found in the two groups. In t-AML a 40 kb deleted MCR pointed to RUNX1 on 21q22, a gene coding for a transcription factor implicated in frequent rearrangements in leukemia and in familial thrombocytopenia. In de novo AML, a 1 Mb MCR harboring ERG and ETS2 was observed from patients with complex aCGH profiles. High resolution cytogenomics obtained by aCGH and similar techniques already published allowed us to characterize numerous non random chromosome abnormalities. This work supports the hypothesis that they can be classified into several categories: abnormalities common to all AML; those more frequently found in t-AML and those specifically found in p-AML.

Most of the colorectal cancer (CRC) cases are sporadic, only 25% of the patients have a family history of the disease, and major genes causing syndromes predisposing to CRC only account for 5-6% of the total cases. The following subtypes can be recognized: MIN (microsatellite instability), CIN (chromosomal instability), and CIMP (CpG island methylator phenotype). CIN occurs in 80-85% of CRC. Chromosomal instability proceeds through two major mechanisms, missegregation that results in aneuploidy through the gain or loss of whole chromosomes, and unbalanced structural rearrangements that lead to the loss and/or gain of chromosomal regions. The loss of heterozygosity that occur in the first phases of the CRC cancerogenesis (in particular for the genes on 18q) as well as the alteration of methylation pattern of multiple key genes can drive the development of colorectal cancer by facilitating the acquisition of multiple tumor-associated mutations and the instability phenotype.

Because polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously, we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that over a short range of repeat lengths, from Q(23) to Q(26), the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex antiparallel β-sheet framework for the nucleus, and it further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in the size of aggregation nuclei may have a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.

Huntington's disease (HD) is a neurodegenerative disorder involving progressive motor disturbances, cognitive decline, and desynchronized sleep-wake rhythms. Recent studies revealed that restoring normal sleep-wake cycles can improve cognitive function in HD mice, suggesting that some sleep/wake systems remain operational and thus represent potential therapeutic targets for HD. Hypothalamic neurons expressing orexins/hypocretins (orexin neurons) are fundamental orchestrators of arousal in mammals, but it is unclear whether orexin circuits operate normally in HD. Here we analyzed the electrophysiology, histology, and gene expression of orexin circuits in brain slices from R6/2 mice, a transgenic model of HD with a progressive neurological phenotype. We report that in R6/2 mice, the size of an electrically distinct subpopulation of orexin neurons is reduced, as is the number of orexin-immunopositive cells in some hypothalamic regions. R6/2 orexin cells display altered glutamatergic inputs, and have an abnormal circadian profile of activity, despite normal circadian rhythmicity of the suprachiasmatic nucleus (SCN), the "master clock" of the brain. Nevertheless, even at advanced stages of HD, intrinsic firing properties of orexin cells remain normal and suppressible by serotonin, noradrenaline, and glucose. Furthermore, histaminergic neurons (key cells required for the propagation of orexin-induced arousal) also display normal responses to orexin. Together, these data suggest that the orexin system remains functional and modifiable in HD mice, although its circadian activity profile is disrupted and no longer follows that of the SCN.

<h4>Background & aims</h4>Pazopanib has demonstrated clinical benefit in patients with advanced renal cell carcinoma (RCC) and is generally well tolerated. However, transaminase elevations have commonly been observed. This 2-stage study sought to identify genetic determinants of alanine transaminase (ALT) elevations in pazopanib-treated white patients with RCC.<h4>Methods</h4>Data from two separate clinical studies were used to examine the association of genetic polymorphisms with maximum on-treatment ALT levels.<h4>Results</h4>Of 6852 polymorphisms in 282 candidate genes examined in an exploratory dataset of 115 patients, 92 polymorphisms in 40 genes were significantly associated with ALT elevation (p<0.01). Two markers (rs2858996 and rs707889) in the HFE gene, which are not yet known to be associated with hemochromatosis, showed evidence for replication. Because of multiple comparisons, there was a 12% likelihood the replication occurred by chance. These two markers demonstrated strong linkage disequilibrium (r(2)=0.99). In the combined dataset, median (25-75th percentile) maximum ALT values were 1.2 (0.7-1.9), 1.1 (0.8-2.5), and 5.4 (1.9-7.6)×ULN for rs2858996 GG (n=148), GT (n=82), and TT (n=1 2) genotypes, respectively. All 12 TT patients had a maximum ALT>ULN, and 8 (67%) had ALT≥3×ULN. The odds ratio (95% CI) for ALT≥3×ULN for TT genotype was 39.7 (2.2-703.7) compared with other genotypes. As a predictor of ALT≥3×ULN, the TT genotype had a negative predictive value of 0.83 and positive predictive value of 0.67. No TT patients developed liver failure.<h4>Conclusions</h4>The rs2858996/rs707889 polymorphisms in the HFE gene may be associated with reversible ALT elevation in pazo-panib-treated patients with RCC.

<h4>Background</h4>The onset of major depressive disorder is likely precipitated by a combination of heredity and life stress. The present study tested the hypothesis that rats selectively bred on a trait related to emotional reactivity would show differential susceptibility or resilience to the development of depression-like signs in response to chronic mild variable intermittent stress (CMS).<h4>Methods</h4>Male Sprague-Dawley rats that were bred based on the trait of either high or low locomotor activity in response to a novel environment were exposed to 4 weeks of CMS or control conditions. Changes in hedonic behavior were assessed using weekly sucrose preference tests and anxiety-like behavior was evaluated using the novelty-suppressed feeding test.<h4>Results</h4>During 4 weeks of CMS, bred low responder (bLR) rats became anhedonic at a faster rate and to a larger degree than bred high responder (bHR) rats, based on weekly sucrose preference tests. Measures of anxiety-like behavior in the novelty-suppressed feeding test were also significantly increased in the CMS-exposed bLR rats, though no differences were observed between CMS-exposed bHR rats and their unstressed controls.<h4>Conclusions</h4>These findings present further evidence that increased emotional reactivity is an important factor in stress susceptibility and the etiology of mood disorders, and that bHR and bLR rats provide a model of resistance or vulnerability to stress-induced depression. Furthermore, exposing bHR and bLR rats to CMS provides an excellent way to study the interaction of genetic and environmental factors in the development of depression-like behavior.

The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Recent advances in RNA nanotechnology have led to the emergence of a new field and brought vitality to the area of therapeutics [P. Guo, The emerging field of RNA nanotechnology, Nat. Nanotechnol., 2010]. Due to the complementary nature of the four nucleotides and its special catalytic activity, RNA can be manipulated with simplicity characteristic of DNA, while possessing versatile structure and diverse function similar to proteins. Loops and tertiary architecture serve as mounting dovetails or wedges to eliminate external linking dowels. Unique features in transcription, termination, self-assembly, self-processing, and acid-resistance enable in vivo production of nanoparticles harboring aptamer, siRNA, ribozyme, riboswitch, or other regulators for therapy, detection, regulation, and intracellular computation. The unique property of noncanonical base-pairing and stacking enables RNA to fold into well-defined structures for constructing nanoparticles with special functionalities. Bacteriophage phi29 DNA packaging motor is geared by a ring consisting of six packaging RNA (pRNA) molecules. pRNA is able to form a multimeric complex via the interaction of two reengineered interlocking loops. This unique feature makes it an ideal polyvalent vehicle for nanomachine fabrication, pathogen detection, and delivery of siRNA or other therapeutics. This review describes methods in using pRNA as a building block for the construction of RNA dimers, trimers, and hexamers as nanoparticles in medical applications. Methods for industrial-scale production of large and stable RNA nanoparticles will be introduced. The unique favorable PK (pharmacokinetics) profile with a half life (T(1/2)) of 5-10h comparing to 0.25 of conventional 2'-F siRNA, and advantageous in vivo features such as non-toxicity, non-induction of interferons or non-stimulating of cytokine response in animals will also be reviewed.

Spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, is a late-onset motor neuron disease (MND) caused by an abnormal expansion of the CAG repeat in the androgen receptor (AR) gene on the X-chromosome, encoding a polyglutamine (poly-Q) sequence in the protein product. Mutant poly-Q-expanded AR protein is widely expressed but leads to selective lower motoneuron death. Although the mechanisms that underlie SBMA remain unclear, defective axonal transport has been implicated in MND and other forms of poly-Q disease. Transcriptional dysregulation may also be involved in poly-Q repeat pathology. We therefore examined axonal transport in a mouse model of SBMA recapitulating many aspects of the human disease. We found no difference in the expression levels of motor and the microtubule-associated protein tau, in the spinal cord and sciatic nerve of wild-type (WT) and SBMA mice at various stages of disease progression. Furthermore, we found no alteration in binding properties of motor proteins and tau to microtubules. Moreover, analysis of axonal transport rates both in cultured primary motoneurons in vitro and in vivo in the sciatic nerve of adult WT and mutant SBMA mice demonstrated no overt axonal transport deficits in these systems. Our results therefore indicate that unlike other motoneuron and poly-Q diseases, axonal transport deficits do not play a significant role in the pathogenesis of SBMA.

We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.

<h4>Background</h4>In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.<h4>Methodology/principal findings</h4>We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions. At least some of these features are hairpin loops, as judged by cross-sectional analysis and sensitivity to cleavage by mung bean nuclease. Single-molecule force measurements showed that the convoluted DNA was very resistant to untangling. In vitro replication by PCR was markedly reduced, and TseI restriction enzyme digestion was also hindered by the abnormal DNA structures. However, significantly, the DNA gained sensitivity to cleavage by the Type III restriction-modification enzyme, EcoP15I.<h4>Conclusions/significance</h4>"Super-long" CAG repeats are found in a number of neurological diseases and may also appear through CAG repeat instability. We suggest that unusual DNA structures associated with super-long CAG repeats decrease transcriptional efficiency in vitro. We also raise the possibility that if these structures occur in vivo, they may play a role in the aetiology of CAG repeat diseases such as HD.

<h4>Background</h4>The tripartite motif (TRIM) proteins are a family of more than 70 members in human. However, only a few of them have been well studied. The TRIM proteins contain the conserved RING, B-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination. TRIM38 is a member of the TRIM protein family, which we studied in more detail here as its functions are largely unknown.<h4>Results</h4>Our study shows that, similar to other TRIM family members, TRIM38 is localized in the cytoplasm. TRIM38 increases ubiquitination of other cellular proteins and catalyzes self-ubiquitination. TRIM38 also promotes K63- and K48-linked ubiquitination of cellular proteins. An intact RING domain is important for the functions of TRIM38. In addition, enterovirus 71 infection induces TRIM38 degradation.<h4>Conclusions</h4>Our observations demonstrate that TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection. These findings may provide insight into innate immune signaling pathways.

<h4>Background</h4>Airway management remains a fundamental component of optimal care of the severely injured patient, with endotracheal intubation representing the definitive strategy for airway control. However, multiple studies document an association between out-of-hospital intubation and increased mortality for severe traumatic brain injury.<h4>Objectives</h4>To explore the relationship between out-of-hospital intubation attempts and outcome among trauma patients with Glasgow Coma Scale (GCS) scores ≤ 8 across sites participating in the Resuscitation Outcomes Consortium (ROC).<h4>Methods</h4>The ROC Epistry-Trauma, an epidemiologic database of prehospital encounters with critically injured trauma victims, was used to identify emergency medical services (EMS)-treated patients with GCS scores ≤ 8. Multiple logistic regression was used to explore the association between intubation attempts and vital status at discharge, adjusting for the following covariates: age, gender, GCS score, hypotension, mechanism of injury, and ROC site. Sites were then stratified by frequency of intubation attempts and chi-square test for trend was used to associate the frequency of intubation attempts with outcome.<h4>Results</h4>A total of 1,555 patients were included in this analysis; intubation was attempted in 758 of these. Patients in whom intubation was attempted had higher mortality (adjusted odds ratio [OR] 2.91, 95% confidence interval [CI] 2.13-3.98, p < 0.01). However, sites with higher rates of attempted intubation had lower mortality across all trauma victims with GCS scores ≤ 8 (OR 1.40, 95% CI 1.15-1.72, p < 0.01).<h4>Conclusions</h4>Patients in whom intubation is attempted have higher adjusted mortality. However, sites with a higher rate of attempted intubation have lower adjusted mortality across the entire cohort of trauma patients with GCS scores ≤ 8. Coma Scale score.

Our aim is to present a working model that may serve as a valuable heuristic to predict enduring effects of drugs when administered during development. Our primary tenet is that a greater understanding of neurodevelopment can lead to improved treatment that intervenes early in the progression of a given disorder and prevents symptoms from manifesting. The immature brain undergoes significant changes during the transitions between childhood, adolescence, and adulthood. Such changes in innervation, neurotransmitter levels, and their respective signaling mechanisms have profound and observable changes on typical behavior, but also increase vulnerability to psychiatric disorders when the maturational process goes awry. Given the remarkable plasticity of the immature brain to adapt to its external milieu, preventive interventions may be possible. We intend for this review to initiate a discussion of how currently used psychotropic agents can influence brain development. Drug exposure during sensitive periods may have beneficial long-term effects, but harmful delayed consequences may be possible as well. Regardless of the outcome, this information needs to be used to improve or develop alternative approaches for the treatment of childhood disorders. With this framework in mind, we present what is known about the effects of stimulants, antidepressants, and antipsychotics on brain maturation (including animal studies that use more clinically-relevant dosing paradigms or relevant animal models). We endeavor to provocatively set the stage for altering treatment approaches for improving mental health in non-adult populations.

Childhood-onset psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), mood disorders, obsessive compulsive spectrum disorders (OCSD), and schizophrenia (SZ), affect many school-age children, leading to a lower quality of life, including difficulties in school and personal relationships that persist into adulthood. Currently, the causes of these psychiatric disorders are poorly understood, resulting in difficulty diagnosing affected children, and insufficient treatment options. Family and twin studies implicate a genetic contribution for ADHD, ASD, mood disorders, OCSD, and SZ. Identification of candidate genes and chromosomal regions associated with a particular disorder provide targets for directed research, and understanding how these genes influence the disease state will provide valuable insights for improving the diagnosis and treatment of children with psychiatric disorders. Transgenic mouse models are one important approach in the study of human diseases, allowing for the use of a variety of experimental approaches to dissect the contribution of a specific chromosomal or genetic abnormality in human disorders. While it is impossible to model an entire psychiatric disorder in a single mouse model, these models can be extremely valuable in dissecting out the specific role of a gene, pathway, neuron subtype, or brain region in a particular abnormal behavior. In this review we discuss existing transgenic mouse models for childhood-onset psychiatric disorders. We compare the strength and weakness of various transgenic mouse models proposed for each of the common childhood-onset psychiatric disorders, and discuss future directions for the study of these disorders using cutting-edge genetic tools.

<h4>Background</h4>Laser surgery lacks haptic feedback, which is accompanied by the risk of iatrogenic nerve damage. It was the aim of this study to investigate diffuse reflectance spectroscopy for tissue differentiation as the base of a feedback control system to enhance nerve preservation in oral and maxillofacial laser surgery.<h4>Methods</h4>Diffuse reflectance spectra of nerve tissue, salivary gland and bone (8640 spectra) of the mid-facial region of ex vivo domestic pigs were acquired in the wavelength range of 350-650 nm. Tissue differentiation was performed using principal component (PC) analysis followed by linear discriminant analysis (LDA). Specificity and sensitivity were calculated using receiver operating characteristic (ROC) analysis and the area under curve (AUC).<h4>Results</h4>Five PCs were found to be adequate for tissue differentiation with diffuse reflectance spectra using LDA. Nerve tissue could be differed from bone as well as from salivary gland with AUC results of greater than 88%, sensitivity of greater than 83% and specificity in excess of 78%.<h4>Conclusions</h4>Diffuse reflectance spectroscopy is an adequate technique for nerve identification in the vicinity of bone and salivary gland. The results set the basis for a feedback system to prevent iatrogenic nerve damage when performing oral and maxillofacial laser surgery.

N-terminal proteolysis of huntingtin is thought to be an important mediator of HD pathogenesis. The formation of short N-terminal fragments of huntingtin (cp-1/cp-2, cp-A/cp-B) has been demonstrated in cells and in vivo. We previously mapped the cp-2 cleavage site by mass spectrometry to position Arg167 of huntingtin. The proteolytic enzymes generating short N-terminal fragments of huntingtin remain unknown. To search for such proteases, we conducted a genome-wide screen using an RNA-silencing approach and an assay for huntingtin proteolysis based on the detection of cp-1 and cp-2 fragments by Western blotting. The primary screen was carried out in HEK293 cells, and the secondary screen was carried out in neuronal HT22 cells, transfected in both cases with a construct encoding the N-terminal 511 amino acids of mutant huntingtin. For additional validation of the hits, we employed a complementary assay for proteolysis of huntingtin involving overexpression of individual proteases with huntingtin in two cell lines. The screen identified 11 enzymes, with two major candidates to carry out the cp-2 cleavage, bleomycin hydrolase (BLMH) and cathepsin Z, which are both cysteine proteases of a papain-like structure. Knockdown of either protease reduced cp-2 cleavage, and ameliorated mutant huntingtin induced toxicity, whereas their overexpression increased the cp-2 cleavage. Both proteases partially co-localized with Htt in the cytoplasm and within or in association with early and late endosomes, with some nuclear co-localization observed for cathepsin Z. BLMH and cathepsin Z are expressed in the brain and have been associated previously with neurodegeneration. Our findings further validate the cysteine protease family, and BLMH and cathepsin Z in particular, as potential novel targets for HD therapeutics.

Huntington disease (HD) is a neurodegenerative disorder caused by an elongated polyglutamine tract in huntingtin (htt). htt normally undergoes different posttranslational modifications (PTMs), including phosphorylation, SUMOylation, ubiquitination, acetylation, proteolytic cleavage, and palmitoylation. In the presence of the HD mutation, some PTMs are significantly altered and can result in changes in the clinical phenotype. A rate-limiting PTM is defined as one that can result in significant effects on the phenotype in animal models. For example, the prevention of proteolysis at D586 as well as constitutive phosphorylation at S13 and S16 can obviate the expression of phenotypic features of HD. The enzymes involved in these modifications such as caspase-6, the IκB kinase (IKK) complex, and still to be characterized phosphatases therefore represent promising therapeutic targets for HD. Identifying and testing specific modulators of PTMs now constitute the next big challenges in order to further validate these targets and proceed towards the goal of a mechanism-based treatment for HD.

<h4>Background</h4>It is well known that many malignancies, including pancreatic cancer (PC), possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility.<h4>Methods and findings</h4>PBMC samples from 26 PC patients and 33 matched healthy controls were analyzed by whole genome cDNA microarray. Three hundred eighty-three genes were found to be significantly different between PC and healthy controls, with 65 having at least a 1.5 fold change in expression. Pathway analysis revealed that many of these genes fell into pathways responsible for hematopoietic differentiation, cytokine signaling, and natural killer (NK) cell and CD8+ T-cell cytotoxic response. Unsupervised hierarchical clustering analysis identified an eight-gene predictor set, consisting of SSBP2, Ube2b-rs1, CA5B, F5, TBC1D8, ANXA3, ARG1, and ADAMTS20, that could distinguish PC patients from healthy controls with an accuracy of 79% in a blinded subset of samples from treatment naïve patients, giving a sensitivity of 83% and a specificity of 75%.<h4>Conclusions</h4>In summary, we report the first in-depth comparison of global gene expression profiles of PBMCs between PC patients and healthy controls. We have also identified a gene predictor set that can potentially be developed further for use in diagnostic algorithms in PC. Future directions of this research should include analysis of PBMC expression profiles in patients with chronic pancreatitis as well as increasing the number of early-stage patients to assess the utility of PBMCs in the early diagnosis of PC.

Cytokine signaling is critical for proliferation, survival and differentiation of hematopoietic cell, and interleukin-3 (IL-3) is required for maintenance of many hematopoietic cell lines, such as BaF3. We have isolated apoptosis-resistant clones of BaF3 using retroviral insertional mutagenesis and the Xbp1 locus was identified as a retroviral integration site. Expression and splicing of the Xbp1 transcript was conserved in the resistant clone but was promptly disappeared on IL-3 withdrawal in parental BaF3. IL-3 stimulation of BaF3 cells enhanced Xbp1 promoter activity and induced phosphorylation of the endoplasmic reticulum stress sensor protein IRE1, resulting in the increase in Xbp1S that activates unfolded protein response. When downstream signaling from IL-3 was blocked by LY294002 and/or dn-Stat5, Xbp1 expression was downregulated and IRE1 phosphorylation was suppressed. Inhibition of IL-3 signaling as well as knockdown of Xbp1-induced apoptosis in BaF3 cells. In contrast, constitutive expression of Xbp1S protected BaF3 from apoptosis during IL-3 depletion. However, cell cycle arrest at the G1 stage was observed in BaF3 and myeloid differentiation was induced in IL-3-dependent 32Dcl3 cells. Expression of apoptosis-, cell cycle- and differentiation-related genes was modulated by Xbp1S expression. These results indicate that the proper transcriptional and splicing regulation of Xbp1 by IL-3 signaling is important in homeostasis of hematopoietic cells.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized pathologically by aggregates composed of N-terminal fragments of the mutant form of the protein huntingtin (htt). The role of these N-terminal fragments in disease pathogenesis has been questioned based in part on studies in transgenic mice. In one important example, mice that express an N-terminal fragment of mutant htt terminating at the C-terminus of exon 2 (termed the Shortstop mouse) were reported to develop robust inclusion pathology without developing phenotypic abnormalities seen in the R6/2 or N171-82Q models of HD, which are also based on expression of mutant N-terminal htt fragments. To further explore the capacity of mutant exon-2 htt fragments to produce neurologic abnormalities (N-terminal 118 amino acids; N118), we generated transgenic mice expressing cDNA that encodes htt N118-82Q with the mouse prion promoter vector. In mice generated in this manner, we demonstrate robust inclusion pathology accompanied by early death and failure to gain weight. These phenotypes are the most robust abnormalities identified in the R6/2 and N171-82Q models. We conclude that the lack of an overt phenotype in the initial Shortstop mice cannot be completely explained by the properties of mutant htt N118 fragments.

Helper-dependent adenovirus (hdAd) vectors have shown tremendous potential in animal models of human disease in numerous preclinical studies. Expression of a therapeutic transgene can be maintained for several years after a single administration of the hdAd vector. However, despite the long-term persistence of hdAd DNA in the transduced cell, little is known of the fate and structure of hdAd DNA within the host nucleus. In this study, we have characterized the assembly of hdAd DNA into chromatin in tissue culture. Eviction of the Ad DNA-packaging protein VII, histone deposition, and vector-associated gene expression all began within 2 to 6 h of host cell transduction. Inhibition of transcription elongation through the vector DNA template had no effect on the loss of VII, suggesting that transcription was not necessary for removal of the majority of protein VII. Vector DNA assembled into physiologically spaced nucleosomes within 6 h. hdAd vectors incorporated the histone H3 variant H3.3, which was dependent on the histone chaperone HIRA. Knockdown of HIRA reduced hdAd association with histones and reduced expression of the vector-carried transgene by 2- to 3-fold. Our study elucidates an essential role for hdAd DNA chromatinization for optimal vector gene expression.

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.

Estrogen (E) plays a pivotal role in regulating the female reproductive system, particularly the ovary. However, the number and type of ovarian genes influenced by estrogen remain to be fully elucidated. In this study, we have utilized wild-type (WT) and aromatase knockout (ArKO; estrogen free) mouse ovaries as an in vivo model to profile estrogen dependent genes. RNA from each individual ovary (n = 3) was analyzed by a microarray-based screen using Illumina Sentrix Mouse WG-6 BeadChip (45,281 transcripts). Comparative analysis (GeneSpring) showed differential expression profiles of 450 genes influenced by E, with 291 genes up-regulated and 159 down-regulated by 2-fold or greater in the ArKO ovary compared to WT. Genes previously reported to be E regulated in ArKO ovaries were confirmed, in addition to novel genes not previously reported to be expressed or regulated by E in the ovary. Of genes involved in 5 diverse functional processes (hormonal processes, reproduction, sex differentiation and determination, apoptosis and cellular processes) 78 had estrogen-responsive elements (ERE). These analyses define the transcriptome regulated by E in the mouse ovary. Further analysis and investigation will increase our knowledge pertaining to how E influences follicular development and other ovarian functions.

Pancreatic cancer is difficult to cure, so its prevention is very important. For this purpose, animal model studies are necessary to develop effective methods. Injection of N-nitrosobis(2-oxopropyl)amine (BOP) into Syrian golden hamsters is known to induce pancreatic ductal adenocarcinomas, the histology of which is similar to human tumors. Moreover, K-ras activation by point mutations and p16 inactivation by aberrant methylation of 5' CpG islands or by homozygous deletions have been frequently observed in common in both the hamster and humans. Thus, this chemical carcinogenesis model has an advantage of histopathological and genetic similarity to human pancreatic cancer, and it is useful to study promotive and suppressive factors. Syrian golden hamsters are in a hyperlipidemic state even under normal dietary conditions, and a ligand of peroxizome proliferator-activated receptor gamma was found to improve the hyperlipidemia and suppress pancreatic carcinogenesis. Chronic inflammation is a known important risk factor, and selective inhibitors of inducible nitric oxide synthase and cyclooxygenase-2 also have protective effects against pancreatic cancer development. Anti-inflammatory and anti-hyperlipidemic agents can thus be considered candidate chemopreventive agents deserving more attention.

<h4>Context</h4>The axon guidance cues netrin-1 is a secreted protein overexpressed in many different cancer tissues.<h4>Objectives</h4>To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer.<h4>Materials and methods</h4>A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry.<h4>Results</h4>Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples.<h4>Discussion and conclusion</h4>Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.

<h4>Background</h4>Plasma levels of high-density lipoprotein cholesterol (HDL-C) are known to be heritable, but only a fraction of the heritability is explained. We used a high-density genotyping array containing single-nucleotide polymorphisms (SNPs) from HDL-C candidate genes selected on known biology of HDL-C metabolism, mouse genetic studies, and human genetic association studies. SNP selection was based on tagging SNPs and included low-frequency nonsynonymous SNPs.<h4>Methods and results</h4>Association analysis in a cohort containing extremes of HDL-C (case-control, n=1733) provided a discovery phase, with replication in 3 additional populations for a total meta-analysis in 7857 individuals. We replicated the majority of loci identified through genome-wide association studies and present on the array (including ABCA1, APOA1/C3/A4/A5, APOB, APOE/C1/C2, CETP, CTCF-PRMT8, FADS1/2/3, GALNT2, LCAT, LILRA3, LIPC, LIPG, LPL, LRP4, SCARB1, TRIB1, ZNF664) and provide evidence that suggests an association in several previously unreported candidate gene loci (including ABCG1, GPR109A/B/81, NFKB1, PON1/2/3/4). There was evidence for multiple, independent association signals in 5 loci, including association with low-frequency nonsynonymous variants.<h4>Conclusions</h4>Genetic loci associated with HDL-C are likely to harbor multiple, independent causative variants, frequently with opposite effects on the HDL-C phenotype. Cohorts comprising subjects at the extremes of the HDL-C distribution may be efficiently used in a case-control discovery of quantitative traits.

Raf kinase inhibitory protein (RKIP) is a physiologic inhibitor of c-RAF kinase and nuclear factor κB signaling that represses tumor invasion and metastasis. Glycogen synthase kinase-3β (GSK3β) suppresses tumor progression by downregulating multiple oncogenic pathways including Wnt signaling and cyclin D1 activation. Here, we show that RKIP binds GSK3 proteins and maintains GSK3β protein levels and its active form. Depletion of RKIP augments oxidative stress-mediated activation of the p38 mitogen activated protein kinase, which, in turn, inactivates GSK3β by phosphorylating it at the inhibitory T390 residue. This pathway de-represses GSK3β inhibition of oncogenic substrates causing stabilization of cyclin D, which induces cell-cycle progression and β-catenin, SNAIL, and SLUG, which promote epithelial to mesenchymal transition. RKIP levels in human colorectal cancer positively correlate with GSK3β expression. These findings reveal the RKIP/GSK3 axis as both a potential therapeutic target and a prognosis-based predictor of cancer progression.

The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (β(s) mice). Enhanced microvascular thrombosis in β(s) mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from β(s) mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in β(s) mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in β(s) mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Amyloid proteins constitute a chemically heterogeneous group of proteins, which share some biophysical and biological characteristics, the principal of which are the high propensity to acquire an incorrect folding and the tendency to aggregate. A number of diseases are associated with misfolding and aggregation of proteins, although only in some of them-most notably Alzheimer's disease (AD) and transmissible spongiform encephalopathies (TSEs)-a pathogenetic link with misfolded proteins is now widely recognized. Lipid rafts (LRs) have been involved in the pathophysiology of diseases associated with protein misfolding at several levels, including aggregation of misfolded proteins, amyloidogenic processing, and neurotoxicity. Among the pathogenic misfolded proteins, the AD-related protein amyloid β (Aβ) is by far the most studied protein, and a large body of evidence has been gathered on the role played by LRs in Aβ pathogenicity. However, significant amount of data has also been collected for several other amyloid proteins, so that their ability to interact with LRs can be considered an additional, shared feature characterizing the amyloid protein family. In this paper, we will review the evidence on the role of LRs in the neurotoxicity of huntingtin, α-synuclein, prion protein, and calcitonin.

Hereditary hemochromatosis is a common-recessive-autosomal disease characterized by progressive iron overload, and its prevalence correlates with c.845G>A (p. C282Y) mutation of the HFE gene. Two other variants c.187C>G and c.193A>T are associated with a mild iron overload phenotype. The correlation studies have revealed incompletely penetrance of the HFE mutations, as well as the lack of mutation on some chromosomes from patients. We screened for SNPs before examining allele and haplotype association with elevated iron parameters. We confirmed that the c.845G>A mutation is in complete linkage disequilibrium with a unique haplotype, whereas two haplotypes proved to account for 79.8 and 20.2% of the c.187G chromosomes whose only difference was the g.4694C>G variation. A greater prevalence of the g.4694G allele among patients' chromosomes, compared to controls, was observed. In addition, among non-mutant chromosomes the analyses revealed a risk haplotype and a protective haplotype, and the g.4694G and the c.1007-47A alleles were associated with a higher risk of elevated iron parameters. We determined that the g.4694C allele was located within a putative hypoxia-response element, protein binding was evidenced and was reduced with the g.4694C>G change. In addition, IVS4 was not spliced as well in the c.1007-47A allele compared to the c.1007-47G allele.

Spinocerebellar ataxia-3 (also known as Machado-Joseph disease) is an incurable neurodegenerative disorder caused by expression of a mutant variant of ataxin-3 (ATX3) protein. Inhibiting expression of ATX3 would provide a therapeutic strategy, but indiscriminant inhibition of both wild-type and mutant ATX3 might lead to undesirable side effects. An ideal silencing agent would block expression of mutant ATX3 while leaving expression of wild-type ATX3 intact. We have previously observed that peptide nucleic acid (PNA) conjugates targeting the expanded CAG repeat within ATX3 mRNA block expression of both alleles. We have now identified additional PNAs capable of inhibiting ATX3 expression that vary in length and in the nature of the conjugated cation chain. We can also achieve potent and selective inhibition using duplex RNAs containing one or more mismatches relative to the CAG repeat. Anti-CAG antisense bridged nucleic acid oligonucleotides that lack a cationic domain are potent inhibitors but are not allele-selective. Allele-selective inhibitors of ATX3 expression provide insights into the mechanism of selectivity and promising lead compounds for further development and in vivo investigation.

DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling and inhibits prostate tumour growth. However, the antitumour activity depends on the efficient delivery of si-Stat3. The effects on the growth of mouse prostate cancer cells of si-Stat3 delivered by hydroxyapatite were determined in this study. RM-1 tumour blocks were transplanted into C57BL/6 mice. CaCl₂-modified hydroxyapatite carrying si-Stat3 plasmids were injected into tumours, and tumour growth and histology were determined. The expression levels of Stat3, pTyr-Stat3, Bcl-2, Bax, Caspase3, VEGF and cyclin D1 were measured by western blot analysis. Amounts of apoptosis in cancer cells were analysed with immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay. The results showed that hydroxyapatite-delivered si-Stat3 significantly suppressed tumour growth up to 74% (P < 0.01). Stat3 expression was dramatically downregulated in the tumours. The immunohistochemistry and TUNEL results showed that si-Stat3-induced apoptosis (up to 42%, P < 0.01). The Stat3 downstream genes Bcl-2, VEGF and cyclin D1 were also strongly downregulated in the tumour tissues that also displayed significant increases in Bax expression and Caspase3 activity. These results suggest that hydroxyapatite can be used for the in vivo delivery of plasmid-based siRNAs into tumours.

<h4>Introduction</h4>Current hormonal adjuvant therapies for breast cancer including tamoxifen treatment and estrogen depletion are overall tumoristatic and are severely limited by the frequent recurrence of the tumors. Regardless of the resistance mechanism, development and progression of the resistant tumors requires the persistence of a basal level of cycling cells during the treatment for which the underlying causes are unclear.<h4>Methods</h4>In estrogen-sensitive breast cancer cells the effects of hormone depletion and treatment with estrogen, tamoxifen, all-trans retinoic acid (ATRA), fulvestrant, estrogen receptor α (ER) siRNA or retinoic acid receptor α (RARα) siRNA were studied by examining cell growth and cycling, apoptosis, various mRNA and protein expression levels, mRNA profiles and known chromatin associations of RAR. RARα subtype expression was also examined in breast cancer cell lines and tumors by competitive PCR.<h4>Results</h4>Basal proliferation persisted in estrogen-sensitive breast cancer cells grown in hormone depleted conditioned media without or with 4-hydroxytamoxifen (OH-Tam). Downregulating ER using either siRNA or fulvestrant inhibited basal proliferation by promoting cell cycle arrest, without enrichment for ErbB2/3+ overexpressing cells. The basal expression of RARα1, the only RARα isoform that was expressed in breast cancer cell lines and in most breast tumors, was supported by apo-ER but was unaffected by OH-Tam; RAR-β and -γ were not regulated by apo-ER. Depleting basal RARα1 reproduced the antiproliferative effect of depleting ER whereas its restoration in the ER depleted cells partially rescued the basal cycling. The overlapping tamoxifen-insensitive gene regulation by apo-ER and apo-RARα1 comprised activation of mainly genes promoting cell cycle and mitosis and suppression of genes involved in growth inhibition; these target genes were generally insensitive to ATRA but were enriched in RAR binding sites in associated chromatin regions.<h4>Conclusions</h4>In hormone-sensitive breast cancer, ER can support a basal fraction of S-phase cells (i) without obvious association with ErbB2/3 expression, (ii) by mechanisms unaffected by hormone depletion or OH-Tam and (iii) through maintenance of the basal expression of apo-RARα1 to regulate a set of ATRA-insensitive genes. Since isoform 1 of RARα is genetically redundant, its targeted inactivation or downregulation should be further investigated as a potential means of enhancing hormonal adjuvant therapy.

<h4>Background</h4>Traumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.<h4>Methods</h4>Through two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.<h4>Results</h4>Our results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin+ immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.<h4>Conclusions</h4>Our findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.

A key barrier to the realization of personalized medicine for cancer is the identification of biomarkers. Here we describe a two-stage strategy for the discovery of serum biomarker signatures corresponding to specific cancer-causing mutations and its application to prostate cancer (PCa) in the context of the commonly occurring phosphatase and tensin homolog (PTEN) tumor-suppressor gene inactivation. In the first stage of our approach, we identified 775 N-linked glycoproteins from sera and prostate tissue of wild-type and Pten-null mice. Using label-free quantitative proteomics, we showed that Pten inactivation leads to measurable perturbations in the murine prostate and serum glycoproteome. Following bioinformatic prioritization, in a second stage we applied targeted proteomics to detect and quantify 39 human ortholog candidate biomarkers in the sera of PCa patients and control individuals. The resulting proteomic profiles were analyzed by machine learning to build predictive regression models for tissue PTEN status and diagnosis and grading of PCa. Our approach suggests a general path to rational cancer biomarker discovery and initial validation guided by cancer genetics and based on the integration of experimental mouse models, proteomics-based technologies, and computational modeling.

During an analysis of T-cell responses against human renal cell carcinoma (RCC), we identified a CD4(+) T-cell line that showed TCR-mediated recognition and lysis of nearly all RCC lines regardless of MHC type. We have now elucidated the nature of the ligand for this α/β TCR, and it contains no MHC-related moiety and does not involve classic peptide processing. First, matrix metalloproteinase 14 (MMP14) expressed on RCC cells releases membrane-bound TRAIL expressed by the T cell; then, soluble TRAIL binds to its receptor DR4 (TRAIL-R1), which is expressed on tumor cells, and this TRAIL-DR4 complex is recognized by the TCR through a complementarity-determining region 3α (CDR3α)-mediated interaction. Direct and specific antigen-TCR interaction was demonstrated when the immobilized recombinant TRAIL/DR4 complex stimulated the TCR. In addition, amino acid substitutions in the CDR3α of the TCR either obliterated or enhanced target-specific recognition. This description of the molecular nature of a non-MHC target structure recognized by a naturally occurring α/β TCR not only broadens our concept of what the TCR can recognize, but also raises the question of whether such a T cell could be of clinical utility against RCC.

<h4>Background</h4>Moderate prenatal alcohol exposure can contribute to neurodevelopmental impairments and disrupt several neurotransmitter systems. We examined the timing of moderate level alcohol exposure, serotonin transporter gene polymorphic region variation (rh5-HTTLPR), and levels of primary serotonin and dopamine (DA) metabolites in cerebrospinal fluid (CSF) in rhesus monkeys.<h4>Methods</h4>Thirty-two 30-month old rhesus monkeys (Macaca mulatta) from 4 groups of females were assessed: (i) early alcohol-exposed group (n = 9), in which mothers voluntarily consumed 0.6 g/kg/d alcohol solution on gestational days 0 to 50; (ii) middle-to-late gestation alcohol-exposed group (n = 6), mothers consumed 0.6 g/kg/d alcohol solution on gestational days 50 to 135; (iii) a continuous-exposure group (n = 8), mothers consumed 0.6 g/kg/d alcohol solution on gestational days 0 to 135; and (iv) controls (n = 9), mothers consumed an isocaloric control solution on gestational days 0 to 50, 50 to 135, or 0 to 135. Serotonin transporter promoter region allelic variants (homozygous s/s or heterozygous s/l vs. homozygous l/l) were determined. We examined CSF concentrations of the 5-HT and DA metabolites, 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA), respectively, at baseline and 50 hours after separation from cage-mates, when the monkeys were 30 months old.<h4>Results</h4>Early- and middle-to-late gestation-alcohol exposed monkeys carrying the short allele had lower concentrations of 5-HIAA in CSF relative to other groups. Concentrations of 5-HIAA in CSF were lower for s allele carriers and increased from baseline relative to pre-separation values, whereas 5-HIAA levels in l/l allele carriers were not affected by separation. Monkeys carrying the short allele had lower basal concentrations of HVA in CSF compared with monkeys homozygous for the long allele.<h4>Conclusion</h4>Carrying the s allele of the 5-HT transporter increased the probability of reduced 5-HIAA in early- and middle-to-late gestation alcohol-exposed monkeys and reduced HVA at baseline. These findings that prenatal alcohol exposure altered central 5-HT activity in genetically sensitive monkeys raise questions about whether abnormal serotonin biological pathways could underlie some of the psychiatric disorders reported in fetal alcohol spectrum disorder.

Inflammation of the gastrointestinal tract increases the risk of developing colon cancer especially in younger adults. Dietary compounds are not only associated with the etiology of inflammation and colon cancer but also in their prevention. Sphingolipid metabolites have been shown to play a role in the initiation and perpetuation of inflammatory responses. In the present study, we investigated the suppression of dextran sodium sulfate-induced colitis and azoxymethane-induced colon cancer by dietary sphingomyelin (SM) in mice that lack functional peroxisome proliferator-activated receptor γ (PPAR-γ) in intestinal epithelial and immune cells. Dietary SM decreased disease activity and colonic inflammatory lesions in mice of both genotypes but more efficiently in mice expressing PPAR-γ. The increased survival and suppression of tumor formation in the SM-fed mice appeared to be independent of PPAR-γ expression in immune and epithelial cells. Using a real-time polymerase chain reaction array, we detected an up-regulation in genes involved in Th1 (interferon γ) and Th17 (interleukin [IL]-17 and IL-23) responses despite the reduced inflammation scores. However, the genes involved in Th2 (IL-4, IL-13 and IL-13ra2) and Treg (IL-10rb) anti-inflammatory responses were up-regulated in a PPAR-γ-dependent manner. In line with the PPAR-γ dependency of our in vivo findings, treatment of RAW macrophages with sphingosine increased the PPAR-γ reporter activity. In conclusion, dietary SM modulated inflammatory responses at the early stages of the disease by activating PPAR-γ, but its anticarcinogenic effects followed a PPAR-γ-independent pattern.

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.

Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li(2)O-CaO-B(2)O(3) glass (106-150 μm) in K(2)HPO(4) solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K(2)HPO(4) solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m(2)/g), and a mesoporous shell wall (pore size ≈ 13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24-48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1-24 h at 600-900°C). Sustained release of BSA was achieved over 7-14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs.

The IGF pathway stimulates malignant behavior of breast cancer cells. Herein we identify the mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) axis as a critical component of IGF and estrogen receptor (ER)α cross talk. The insulin receptor substrate (IRS) adaptor molecules function downstream of IGF-I receptor and dictate a specific biological response, in which IRS-1 drives proliferation and IRS-2 is linked to motility. Although rapamycin-induced mTOR inhibition has been shown to block IGF-induced IRS degradation, we reveal differential effects on motility (up-regulation) and proliferation (down-regulation). Because a positive correlation between IRS-1 and ERα expression is thought to play a central role in the IGF growth response, we investigated the potential role of ERα as a downstream mTOR target. Small molecule inhibition and targeted knockdown of S6K1 blocked the IGF-induced ERα(S167) phosphorylation and did not influence ligand-dependent ERα(S118) phosphorylation. Inhibition of S6K1 kinase activity consequently ablated IGF-stimulated S6K1/ERα association, estrogen response element promoter binding and ERα target gene transcription. Moreover, site-specific ERα(S167) mutation reduced ERα target gene transcription and blocked IGF-induced colony formation. These findings support a novel link between the IGF pathway and ERα, in which the translation factor S6K1 affects transcription of ERα-regulated genes.

Ataxia-Telangiectasia (A-T) is an autosomal recessive disorder resulting in a myriad of abnormalities, including progressive neurodegeneration and cancer predisposition. At the cellular level, A-T is a disease of chronic oxidative stress (OS) causing damage to proteins, lipids, and DNA. OS is contributed to by pro-oxidative transition metals such as iron that catalyze the conversion of weakly reactive oxygen species to highly reactive hydroxyl radicals. Iron-associated OS has been linked to neurodegeneration in Alzheimer's and Parkinson's diseases and development of lymphoid tumors (which afflict ∼30% of A-T patients). To investigate iron regulation in A-T, iron indexes, regulatory genes, and OS markers were studied in livers of wild-type and Ataxia telangiectasia mutated (Atm) null mice on control or high-iron diets. Atm(-/-) mice had increased serum iron, hepatic iron, and ferritin and significantly higher Hepcidin compared with wild-type mice. When challenged with the high-iron diet, Bmp6 and Hfe expression was significantly increased. Atm(-/-) mice had increased protein tyrosine nitration and significantly higher Heme Oxygenase (decycling) 1 levels that were substantially increased by a high-iron diet. Ferroportin gene expression was significantly increased; however, protein levels were unchanged. We demonstrate that Atm(-/-) mice have a propensity to accumulate iron that is associated with a significant increase in hepatic OS. The iron-induced increase in hepcidin peptide in turn suppresses ferroportin protein levels, thus nullifying the upregulation of mRNA expression in response to increased OS. Our results suggest that increased iron status may contribute to the chronic OS seen in A-T patients and development of disease pathology.

The genetic mechanisms underlying tissue maintenance of the gastrointestinal tract are critical for the proper function of the digestive system under normal physiological stress. The identification of transcription factors and related signal transduction pathways that regulate stem cell maintenance and lineage allocation is attractive from a clinical standpoint in that it may provide targets for novel cell- or drug-based therapies. Sox [sex-determining region Y (Sry) box-containing] factors are a family of transcription factors that are emerging as potent regulators of stem cell maintenance and cell fate decisions in multiple organ systems and might provide valuable insight toward the understanding of these processes in endodermally derived tissues of the gastrointestinal tract. In this review, we focus on the known genetic functions of Sox factors and their roles in epithelial tissues of the esophagus, stomach, intestine, colon, pancreas, and liver. Additionally, we discuss pathological conditions in the gastrointestinal tract that are associated with a dysregulation of Sox factors. Further study of Sox factors and their role in gastrointestinal physiology and pathophysiology may lead to advances that facilitate control of tissue maintenance and development of advanced clinical therapies.

We have leveraged a Drosophila model relevant to Alzheimer disease (AD) for functional screening of findings from a genome-wide scan for loci associated with a quantitative measure of AD pathology in humans. In six of the 15 genomic regions evaluated, we successfully identified a causal gene for the association, on the basis of in vivo interactions with the neurotoxicity of Tau, which forms neurofibrillary tangles in AD. Among the top results, rs10845990 within SLC2A14, encoding a glucose transporter, showed evidence of replication for association with AD pathology, and gain and loss of function in glut1, the Drosophila ortholog, was associated with suppression and enhancement of Tau toxicity, respectively. Our strategy of coupling genome-wide association in humans with functional screening in a model organism is likely to be a powerful approach for gene discovery in AD and other complex genetic disorders.

We conducted a comprehensive study of copy number variants (CNVs) well-tagged by SNPs (r(2)≥ 0.8) by analyzing their effect on gene expression and their association with disease susceptibility and other complex human traits. We tested whether these CNVs were more likely to be functional than frequency-matched SNPs as trait-associated loci or as expression quantitative trait loci (eQTLs) influencing phenotype by altering gene regulation. Our study found that CNV-tagging SNPs are significantly enriched for cis eQTLs; furthermore, we observed that trait associations from the NHGRI catalog show an overrepresentation of SNPs tagging CNVs relative to frequency-matched SNPs. We found that these SNPs tagging CNVs are more likely to affect multiple expression traits than frequency-matched variants. Given these findings on the functional relevance of CNVs, we created an online resource of expression-associated CNVs (eCNVs) using the most comprehensive population-based map of CNVs to inform future studies of complex traits. Although previous studies of common CNVs that can be typed on existing platforms and/or interrogated by SNPs in genome-wide association studies concluded that such CNVs appear unlikely to have a major role in the genetic basis of several complex diseases examined, our findings indicate that it would be premature to dismiss the possibility that even common CNVs may contribute to complex phenotypes and at least some common diseases.

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins.

Silicon membranes with highly uniform nanopore sizes fabricated using microelectromechanical systems (MEMS) technology allow for the development of miniaturized implants such as those needed for renal replacement therapies. However, the blood compatibility of silicon has thus far been an unresolved issue in the use of these substrates in implantable biomedical devices. We report the results of hemocompatibility studies using bare silicon, polysilicon, and modified silicon substrates. The surface modifications tested have been shown to reduce protein and/or platelet adhesion, thus potentially improving biocompatibility of silicon. Hemocompatibility was evaluated under four categories-coagulation (thrombin-antithrombin complex, TAT generation), complement activation (complement protein, C3a production), platelet activation (P-selectin, CD62P expression), and platelet adhesion. Our tests revealed that all silicon substrates display low coagulation and complement activation, comparable to that of Teflon and stainless steel, two materials commonly used in medical implants, and significantly lower than that of diethylaminoethyl (DEAE) cellulose, a polymer used in dialysis membranes. Unmodified silicon and polysilicon showed significant platelet attachment; however, the surface modifications on silicon reduced platelet adhesion and activation to levels comparable to that on Teflon. These results suggest that surface-modified silicon substrates are viable for the development of miniaturized renal replacement systems.

Traits related to fatness, important as economic factors in pork production, are associated with serious diseases in humans. Genetical genomics is a useful approach for studying the effects of genetic variation at the molecular level in biological systems. Here we applied a whole-genome association analysis to hepatic gene expression traits, focusing on transcripts with expression levels that correlated with fatness traits in a porcine model. A total of 150 crossbred pigs [Pietrain × (German Large White × German Landrace)] were studied for transcript levels in the liver. The 24K Affymetrix expression microarrays and 60K Illumina single nucleotide polymorphism (SNP) chips were used for genotyping. A total of 663 genes, whose expression significantly correlated with the trait "fat area," were analyzed for enrichment of functional annotation groups as defined in the Ingenuity Pathways Knowledge Base (IPKB). Genes involved in metabolism of various macromolecules and nutrients as well as functions related to dynamic cellular processes correlated with fatness traits. Regions affecting the transcription levels of these genes were mapped and revealed 4,727 expression quantitative trait loci (eQTL) at P < 10⁻⁵, including 448 cis-eQTL. In this study, genome-wide association analysis of trait-correlated expression was successfully used in a porcine model to display molecular networks and list genes relevant to fatness traits.

Hepcidin negatively regulates systemic iron homeostasis in response to inflammation and elevated serum iron. Conversely, hepcidin expression is diminished in response to hypoxia, oxidative stress, and increased erythropoietic demand, though the molecular intermediates involved are incompletely understood. To address this, we have investigated hypoxic hepcidin regulation in HuH7 hepatoma cells either cultured alone or cocultured with activated THP-1 macrophages. HuH7 hepcidin mRNA expression was determined using quantitative polymerase chain reaction (Q-PCR). Hepcidin promoter activity was measured using luciferase reporter constructs containing a 0.9 kb fragment of the wild-type human hepcidin promoter, and constructs containing mutations in bone morphogenetic protein (BMP)/SMAD4, signal transducer and activator of transcription 3 (STAT3), CCAAT/enhancer-binding protein (C/EBP), and E-box-responsive elements. Hepatic expression of bone morphogenetic proteins BMP2 and BMP6 and the BMP inhibitor noggin was determined using Q-PCR, and the protein expression of hemojuvelin (HJV), pSMAD 1/5/8, and SMAD4 was determined by western blotting. Following exposure to hypoxia or H(2)O(2), hepcidin mRNA expression and promoter activity increased in HuH7 cells monocultures but were decreased in HuH7 cells cocultured with THP-1 macrophages. This repression was attenuated by mutation of the BMP/SMAD4-response element, suggesting that modulation of SMAD signaling mediated the response to hypoxia. No changes in hepatocyte BMP2, BMP6 or noggin mRNA, or protein expression of HJV or pSMAD 1/5/8 were detected. However, treatment with hypoxia caused a marked decrease in nuclear and cytosolic SMAD4 protein and SMAD4 mRNA expression in cocultured HuH7 cells. Together these data indicate that hypoxia represses hepcidin expression through inhibition of BMP/SMAD signaling.

T lymphocyte activation requires signal 1 from the TCR and signal 2 from costimulatory receptors. For long-lasting immunity, growth and survival signals imparted through the Akt/protein kinase B (PKB) pathway in activated or effector T cells are important, and these can be strongly influenced by signaling from OX40 (CD134), a member of the TNFR superfamily. In the absence of OX40, T cells do not expand efficiently to Ag, and memory formation is impaired. How most costimulatory receptors integrate their signals with those from Ag through the TCR is not clear, including whether OX40 directly recruits PKB or molecules that regulate PKB. We show that OX40 after ligation by OX40L assembled a signaling complex that contained the adapter TNFR-associated factor 2 as well as PKB and its upstream activator phosphoinositide 3-kinase (PI3K). Recruitment of PKB and PI3K were dependent on TNFR-associated factor 2 and on translocation of OX40 into detergent-insoluble membrane lipid microdomains but independent of TCR engagement. However, OX40 only resulted in strong phosphorylation and functional activation of the PI3K-PKB pathway when Ag was recognized. Therefore, OX40 primarily functions to augment PKB signaling in T cells by enhancing the amount of PI3K and PKB available to the TCR. This highlights a quantitative role of this TNFR family second signal to supplement signal 1.

This study primarily focused on the anti-metastatic activity of doxorubicin (DOX) loaded in a pH-sensitive mixed polymeric micelle formed from two block polymers: poly(L-lactide) (PLLA) (Mn 3000)-b-poly(ethylene glycol) (PEG) (Mn 2000)-folate and poly(L-histidine) (PHis) (Mn 4700)-b-PEG (Mn 2000). Tumor formation and metastasis in mice were examined using a murine mammary carcinoma cell of 4T1 which is one of the most aggressive metastatic cancer cell lines. The efficacy was evaluated by tumor size, body weight change, survival rate, dorsal skin fold window chamber model, and histological observation of the lung, heart, liver and spleen after treatment with various DOX formulations. When the tumor reached 50-100 mm³ in size, the mice were treated 4 times at a 3-day interval at a dose of 10 mg DOX/kg. The mixed micelle formulation resulted in retarded tumor growth, no weight loss, and no death for 4-5 weeks. In another set of the in vivo test for histological evaluation of the organs, the mice were similarly treated but the formulations were injected one day after 4T1 cell inoculation. The treatment by DOX loaded mixed micelle showed no apparent metastasis till 28 days. However, significant metastasis to the lung and heart was observed on Day 28 when the mice were treated with DOX carried by PBS, PLLA-b-PEG micelle and PHis-b-PEG micelle.

Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value  = 0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value  = 0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.

We examined the genetic associations of previously identified sequence variants with type 2 diabetes mellitus (T2DM) and its potentially genetic heterogeneity by gender in a large-scale cohort. A total of 613 T2DM patients and 8221 control subjects from the Korea Association REsource (KARE) cohort were included in the analysis of genetic association of T2DM with 33 nucleotide polymorphic markers identified by previous studies. The association analysis was further conducted with data partitioned by gender. The association analysis resulted in five nucleotide sequence variants associated with the susceptibility of T2DM after Bonferonni correction (P < 0.0015). One was located near the gene of hematopoietically expressed homeobox (HHEX), and the others were all in the gene of cyclin-dependent kinase 5 regulatory subunit-associated protein 1-like 1 (CDKAL1). Further analysis revealed that the sequence variant (rs5015480) near HHEX and two SNPs (rs7756992 and rs9465871) in CDKAL1 were associated with the susceptibility of T2DM in females (P<0.005), but not in males (P>0.005). We suggested heterogeneous genetic associations of the T2DM susceptibility with the CDKAL1 and HHEX genes by gender.

The asymmetric synthesis of gymnastatin H has been achieved by using the photoisomerisation of a conjugated ester to its β,γ-unsaturated isomer through the protonation of a in situ generated dienol as key step. Thanks to diacetone D-glucose used as a chiral alkoxy group, the protonation occurred well onto one of the two diastereotopic faces with very high yields and selectivities. Moreover, by this way the configuration of the C-6 centre of the target molecule was controlled.

Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16-months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome-c-oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox-positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox-activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB- and mtTFA-antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non-homogenous defect pattern.

<h4>Objective</h4>Melanocortin 1 receptor (MC1R) is involved in various functions, such as pigmentation, antipyretic and anti-inflammatory actions, development of melanoma, susceptibility to ultraviolet-induced sun damage, modification of oculocutaneous albinism, development of freckles, and mediation of female-specific mechanisms of analgesia. MC1R's natural agonists include α-melanocyte-stimulating hormone and corticotrophin (ACTH1-39), which are important components of hypothalamic pituitary adrenal axis and increase in response to stress. Given the multiple relevant roles of MC1R, we studied whether the MC1R gene would be associated with susceptibility to major depressive disorder or with response to antidepressant treatment.<h4>Methods</h4>The human MC1R gene is highly polymorphic; therefore, we sequenced the entire MC1R coding region of 1122 bp in 181 depressed Mexican-American patients and 185 Mexican-American controls.<h4>Results</h4>A total of 23 single nucleotide polymorphisms (SNPs, 15 known and eight new) were found within the sequenced region. Among the common SNPs, the nonsynonymous SNP, rs885479 (R163Q) was associated with the diagnosis of depression (P=0.04). The nonsynonymous SNP, rs2228479 (V92M) and the synonymous SNP, rs2228478 were found to be associated with the remission with desipramine treatment. No associations were found for remission with fluoxetine treatment or for the combined sample treated with fluoxetine or desipramine. The frequency of one (H2) of the five haplotypes identified was higher in depressed patients when compared with controls (P=0.05). In-silico functional analysis indicates that SNPs rs885479 and rs2228479 have significant impact on the protein function.<h4>Conclusion</h4>The MC1R gene might be associated with major depressive disorder and with treatment response to desipramine.

<h4>Objective</h4>There are theoretical reasons why comparing marker allele frequencies between cases of different diseases, rather than with controls, may offer benefits. The samples may be better matched, especially for background risk factors common to both diseases. Genetic loci may also be detected which influence which of the two diseases occurs if common risk factors are present.<h4>Method</h4>We used samples of UK bipolar and schizophrenic cases that had earlier been subject to genome-wide association studies and compared marker allele frequencies between the two samples. When these differed for a marker, we compared the case sample allele frequencies with those of a control sample.<h4>Results</h4>Eight markers were significant at P value of less than 10(-5). Of these, the most interesting finding was for rs17645023, which was significant at P value of less than 10(-6) and which lies 36 kb from CACNG5. Control allele frequencies for this marker were intermediate between those for bipolar and schizophrenic cases.<h4>Conclusion</h4>The application of this approach suggests that it does have some merits. The finding for CACNG5, taken together with the earlier implication of CACNA1C and CACNA1B, strongly suggests a key role for voltage-dependent calcium channel genes in the susceptibility to bipolar disorder and/or schizophrenia.

<h4>Purpose of review</h4>To discuss inherited iron disorders, their pathophysiology and clinical implications in the light of the recent advances in our knowledge of iron metabolism and its regulation.<h4>Recent findings</h4>In previous years the molecular mechanisms of cellular iron uptake and release and the cellular and systemic iron homeostasis have been substantially clarified. New proteins (hepcidin, hemojuvelin, HFE, TFR2 and ferroportin), mutated in hereditary hemochromatosis, have been identified with a crucial role in iron regulation. These advances have modified our understanding of the pathophysiology of hemochromatosis, now considered a disorder either due to hepcidin deficiency or (rarely) due to hepcidin resistance. Novel genetic forms of iron-related microcytic anemia have been identified, due to defects of iron transport/utilization or to TMPRSS6 deficiency and hepcidin hyperproduction, as occurs in iron-refractory iron deficiency anemia (IRIDA). A role for hepcidin has been identified also in acquired conditions, as in iron-loading anemias and in anemia of chronic diseases and inflammation.<h4>Summary</h4>Advances in basic research have improved the classification and diagnosis of genetic anemias and iron overload and are paving the way towards the development of drugs that target the molecular lesions.

<h4>Purpose of review</h4>The developmental switch from fetal to adult hemoglobin has long fascinated biologists and attracted hematologists given its importance for patients with hemoglobin disorders. New discoveries have reinvigorated the field of globin gene regulation. These results hold promise for improved treatment of the major hemoglobinopathies.<h4>Recent findings</h4>Both genome-wide association studies and traditional linkage studies have identified several genetic loci involved in silencing fetal hemoglobin. BCL11A is a potent silencer of fetal hemoglobin in both mouse and humans. It controls the beta-globin gene cluster in concert with other factors. KLF1, a vital erythroid transcription factor, activates BCL11A and assists in coordinating the switch from fetal to adult hemoglobin. A regulatory network of cell-intrinsic and cell-extrinsic factors maintains the epigenetic homeostasis of the beta-globin cluster and accounts for the precise lineage-specific and developmental stage-specific regulation of the globin genes.<h4>Summary</h4>With an improved understanding of pathways involved in the switch from fetal to adult hemoglobin, new targets have emerged for the treatment of the common hemoglobin disorders, sickle cell anemia and beta-thalassemia.

A variety of neurological diseases including Huntington's disease (HD), Alzheimer's disease and Parkinson's disease share common neuropathology, primarily featuring the presence of abnormal protein inclusions containing specific misfolded proteins. Mutations leading to expansion of a poly-glutamine track in Huntingtin cause HD, and trigger its misfolding and aggregation. Recent evidence indicates that alterations in the secretory pathway, in particular the endoplasmic reticulum (ER), are emerging features of HD. Although it is not clear how cytoplasmic/nuclear located mutant Huntingtin alters the function of the ER, several reports indicate that mutant Huntingtin affects many essential processes related to the secretory pathway, including inhibition of ER-associated degradation, altered ER/Golgi vesicular trafficking and axonal transport, disrupted autophagy and abnormal ER calcium homeostasis. All these alterations are predicted to have a common pathological outcome associated to disturbance of protein folding and maturation pathways at the ER, generating chronic ER stress and neuronal dysfunction. Here, we review recent evidence involving ER stress in HD pathogenesis and discuss possible therapeutic strategies to target organelle function in the context of disease.

Late-onset/low-vulnerability alcoholics (LOAs) appear to drink less when treated with a selective serotonin reuptake inhibitor than placebo, whereas early-onset/high-vulnerability alcoholics (EOAs) show the opposite effect. We conducted a 12-week, parallel-group, placebo-controlled trial of the efficacy of sertraline in alcohol dependence (AD). We compared the effects in LOAs versus EOAs and examined the moderating effects of a functional polymorphism in the serotonin transporter gene. Patients (N = 134, 80.6% male, 34.3% EOAs) with Diagnostic and Statistical Manual of Mental Disorders-IV AD received up to 200 mg of sertraline (n = 63) or placebo (n = 71) daily. We used urn randomization, and patients were genotyped for the tri-allelic 5-hydroxytryptamine transporter protein linked promoter region polymorphism. Planned analyses included main and interaction effects of medication group, age of onset (≤ 25 years vs >25 years), and genotype (L'/L' vs S' carriers) on drinking outcomes. Results showed that the moderating effect of age of onset on the response to sertraline was conditional on genotype. There were no main or interaction effects among S' allele carriers. However, in L' homozygotes, the effects of medication group varied by age of onset (P = 0.002). At the end of treatment, LOAs reported fewer drinking and heavy drinking days when treated with sertraline (P = 0.011), whereas EOAs had fewer drinking and heavy drinking days when treated with placebo (P < 0.001). The small cell sizes and high rate of attrition, particularly for L' homozygotes, render these findings preliminary and their replication in larger samples necessary. Because AD is common, particularly in medical settings, and selective serotonin reuptake inhibitors are widely prescribed by practitioners, these findings have potential public health significance and warrant further evaluation.

<h4>Background aims</h4>Bone marrow (BM)-derived progenitor cells are under investigation for cardiovascular repair but may be altered by disease. Our aim was to identify differences in gene expression in CD133(+) cells of patients with coronary artery disease (CAD) and healthy controls, and determine whether exercise modifies gene expression.<h4>Methods</h4>CD133(+) cells were flow-sorted from 10 CAD patients and four controls, and total RNA was isolated for microarray-based gene expression profiling. Genes that were found to be differentially regulated in patients were analyzed further to investigate whether exercise had any normalizing effect on CD133(+) cells in CAD patients following 3 months of an exercise program.<h4>Results</h4>Improvement in effort tolerance and increases in the number of CD133(+) cells were observed in CAD patients after 3 months of exercise. Gene expression analysis of the CD133(+) cells identified 82 differentially expressed genes (2-fold cut-off, 25% false-discovery rate and % present calls) in patients compared with controls, of which 59 were found to be up-regulated and 23 down-regulated. These genes were found to be involved in carbohydrate metabolism, cell cycle, cellular development and signaling, and molecular transport. Following completion of the exercise program, gene expression patterns resembled those of controls in seven of 10 patients.<h4>Conclusions</h4>Alterations in gene expression of BM-derived CD133(+) progenitor cells were found in CAD patients, which in part may be normalized by exercise.

Sickle cell anemia (SCA, HBB glu6val) is characterized by multiple complications and a high degree of phenotypic variability: some subjects have only sporadic pain crises and few acute hospitalizations, while others experience multiple serious complications, high levels of morbidity, and accelerated mortality [1]. The tumor necrosis factor-α (TNF-α) signaling pathway plays important roles in inflammation and the immune response; variation in this pathway might be expected to modify the overall severity of SCA through the pathway's effects on the vascular endothelium [2,3]. We examined plasma biomarkers of TNF-α activity and endothelial cell activation for associations with SCA severity in 24 adults (12 mild, 12 severe). Two biomarkers, tumor necrosis factor-α receptor-1 (TNF-R1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly higher in subjects with severe SCA. Along with these biomarker differences, we also examined data from a genome-wide association study (GWAS) using SCA severity as a disease phenotype, and found evidence of genetic association between disease severity and a single nucleotide polymorphism (SNP) in VCAM1, which codes for VCAM-1, and several SNPs in ARFGEF2, a gene involved in TNF-R1 release [4].

MicroRNAs (miRNAs) have emerged as critical regulators of numerous biological processes by modulating gene expression at the post-transcriptional level. It has become increasingly clear that almost all aspects of skeletal muscle development involve regulation by miRNAs. Many of these miRNAs have distinct expression profiles in skeletal muscles, under the regulation by the myogenic program. In the last few years the field has seen a rapid expansion of our knowledge of myogenic miRNAs that target a wide range of muscle genes to coordinately control the myogenic process. In this review we provide an up-to-date list of reported myogenic miRNAs and survey their expression patterns, regulation of biogenesis, and gene targets in skeletal muscles. Emerging themes of miRNA regulation in the context of skeletal myogenesis will also be discussed.

<h4>Introduction</h4>Olfactomedin 4 (OLFM4) is expressed in gastrointestinal cancers and related to progression and differentiation of these malignancies. However, whether OLFM4 contributes to tumorigenesis of other tissues has not been thoroughly investigated. The purpose of the study was to investigate OLFM4 expression in cervical epithelium and its association with progression of cervical neoplasia and differentiation of cervical carcinomas.<h4>Methods</h4>Immunohistochemistry and real-time reverse transcription-polymerase chain reaction were used to evaluate the expression and distribution of OLFM4 in cervical intraepithelial neoplasia (CIN) and invasive squamous cell carcinomas (ISCCs).<h4>Results</h4>The overall positive OLFM4 staining levels in normal cervical epithelia, CIN I, CIN II, CIN III, and ISCCs are 22.0%, 94.2%, 93.7%, 94.6%, and 96.7%, respectively. The intensity of OLFM4 staining was enhanced according to increased pathologic grade of cervical squamous intraepithelial lesion. The immunoreactivity to OLFM4 was stronger in well-differentiated ISCCs than in poorly differentiated ISCCs.<h4>Conclusions</h4>Olfactomedin 4 expression has been associated with progression of CIN and differentiation of cervical cancer. The results provide new evidence that OLFM4 plays an important role in tumorigenesis in the female reproductive tract.

The nervous system is a complex network with many types of neurons and numerous synaptic connections. The present knowledge on how neurons recognize specific targets and form such an intricate network is still limited. The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily and contributes to defects in the central nervous system in Down syndrome patients. DSCAM plays important roles in neural development, including dendritic patterning and self-avoidance, axon guidance and branching, axon target recognition and synaptic formation. However, the functional mechanisms and the underlying signaling pathways are still largely unknown. Here the functions of DSCAM in neural development were reviewed. Future research for better understanding DSCAM function and the relevance of DSCAM to human diseases was also discussed.

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. Under physiological conditions, half of the inhibitor transitions to a latent state within 1-2 h. The interaction between PAI-1 and the plasma protein vitronectin prolongs this active lifespan by ∼50%. Previously, our group demonstrated that PAI-1 binds to resins using immobilized metal affinity chromatography (Day, U.S. Pat. 7,015,021 B2, March 21, 2006). In this study, the effect of these metals on function and stability was investigated by measuring the rate of the transition from the active to latent conformation. All metals tested showed effects on stability, with the majority falling into one of two types depending on their effects. The first type of metal, which includes magnesium, calcium and manganese, invoked a slight stabilization of the active conformation of PAI-1. A second category of metals, including cobalt, nickel and copper, showed the opposite effects and a unique vitronectin-dependent modulation of PAI-1 stability. This second group of metals significantly destabilized PAI-1, although the addition of vitronectin in conjunction with these metals resulted in a marked stabilization and slower conversion to the latent conformation. In the presence of copper and vitronectin, the half-life of active PAI-1 was extended to 3 h, compared to a half-life of only ∼30 min with copper alone. Nickel had the largest effect, reducing the half-life to ∼5 min. Together, these data demonstrate a heretofore-unknown role for metals in modulating PAI-1 stability.

We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions and leads to localized complex formation with FAK to regulate the dynamics of nascent adhesions.

β-Thalassemia and pseudoxanthoma elasticum (PXE) are distinct genetic disorders. Yet, a dystrophic mineralization phenotype similar to PXE has frequently been associated with β-thalassemia or sickle cell anemia patients of Mediterranean descent. These calcifications are clinically and structurally identical to inherited PXE. As we previously excluded the presence of PXE-causing mutations in the ABCC6 gene of β-thalassemia patients with PXE manifestations, we hypothesized that a molecular mechanism independent of gene mutations either altered the ABCC6 gene expression or disrupted the biologic properties of its product in the liver or kidneys, which are the tissues with the highest levels of expression. To test this possibility, we investigated Abcc6 synthesis in the liver and kidneys of a β-thalassemia mouse model (Hbb(th3/+)). We found a progressive liver-specific down-regulation of the Abcc6 gene expression and protein levels by quantitative PCR, Western blotting, and immunofluorescence. The levels of Abcc6 protein decreased significantly at 6 months of age and stabilized at 10 months and older ages at ∼25% of the wild-type protein levels. We studied the transcriptional regulation of the Abcc6 gene in wild-type and Hbb(th3/+) mice, and we identified the erythroid transcription factor NF-E2 as the main cause of the transcriptional down-regulation using transcription factor arrays and chromatin immunoprecipitation. The Hbb(th3/+) mice did not develop spontaneous calcification as seen in the Abcc6(-/-) mice probably because the Abcc6 protein decrease occurred late in life and was probably insufficient to promote mineralization in the Hbb(th3/+) mouse C57BL/6J genetic background. Nevertheless, our result suggested that a similar decrease of ABCC6 expression occurs in the liver of β-thalassemia patients and may be responsible for their frequent PXE-like manifestations.

<h4>Background</h4>Recent meta-analyses have raised concerns about the replicability of gene × environment interactions involving the serotonin transporter gene (5-HTTLPR) in moderating the associations between adverse life events and mental disorders.<h4>Aims</h4>To use data gathered over the course of a 30-year longitudinal study of a New Zealand birth cohort to test the hypothesis that the presence of short ('s') alleles of 5-HTTLPR are associated with an increased response to life stress.<h4>Method</h4>Participants were 893 individuals from the Christchurch Health and Development Study who had complete data on: the 5-HTTLPR genotype; psychiatric disorders up to the age of 30; and exposure to childhood and adult adverse life events.<h4>Results</h4>A series of 104 regression models were fitted to four mental health outcomes (depressive symptoms, major depression, anxiety disorder and suicidal ideation) observed at ages 18, 21, 25 and 30 using 13 measures of life-course stress that spanned childhood and adult stressors. Both multiplicative and additive models were fitted to the data. No evidence was found that would support the hypothesis that 's' alleles of 5-HTTLPR are associated with increased responsivity to life stressors.<h4>Conclusions</h4>The present findings add to the evidence suggesting that it is unlikely that there is a stable gene × environment interaction involving 5-HTTLPR, life stress and mental disorders.

Osteoporosis, a syndrome characterized by thin bones and fractures, has become more prevalent in both women and men. Established therapies for treating this disorder consist primarily of drugs that prevent bone loss, such as the bisphosphonates and selective oestrogen receptor modulators. Although these drugs have been shown to reduce fractures in randomized trials, there is an urgent need for treatments that could lower fracture risk further without additional adverse effects. The introduction of parathyroid hormone (teriparatide), which significantly increases bone mineral density, albeit for a relatively short duration, raised expectations that drugs that stimulate bone formation might cure osteoporosis. After outlining current approaches for treating osteoporosis, this Review focuses on emerging therapeutic opportunities for osteoporosis that are based on recent insights into skeletal physiology. Such novel strategies offer promise not only for reducing age-related bone loss and the associated risk of fractures but also for restoring bone mineral density to healthy levels.

<h4>Background</h4>Protein deformation has been extensively analysed through global methods based on RMSD, torsion angles and Principal Components Analysis calculations. Here we use a local approach, able to distinguish among the different backbone conformations within loops, α-helices and β-strands, to address the question of secondary structures' shape variation within proteins and deformation at interface upon complexation.<h4>Results</h4>Using a structural alphabet, we translated the 3 D structures of large sets of protein-protein complexes into sequences of structural letters. The shape of the secondary structures can be assessed by the structural letters that modeled them in the structural sequences. The distribution analysis of the structural letters in the three protein compartments (surface, core and interface) reveals that secondary structures tend to adopt preferential conformations that differ among the compartments. The local description of secondary structures highlights that curved conformations are preferred on the surface while straight ones are preferred in the core. Interfaces display a mixture of local conformations either preferred in core or surface. The analysis of the structural letters transition occurring between protein-bound and unbound conformations shows that the deformation of secondary structure is tightly linked to the compartment preference of the local conformations.<h4>Conclusion</h4>The conformation of secondary structures can be further analysed and detailed thanks to a structural alphabet which allows a better description of protein surface, core and interface in terms of secondary structures' shape and deformation. Induced-fit modification tendencies described here should be valuable information to identify and characterize regions under strong structural constraints for functional reasons.

Huntington disease (HD) is a dominantly inherited neurodegenerative disorder that results from expansion of the polyglutamine repeat in the huntingtin (HTT) gene. There are currently no effective treatments for this devastating disease. Given its monogenic nature, disease modification therapies for HD should be theoretically feasible. Currently, pharmacological therapies aimed at disease modification by altering levels of HTT protein are in late-stage preclinical development. Here, we review current efforts to develop new treatments for HD based on our current understanding of HTT function and the main pathological mechanisms. We emphasize the need to enhance translational efforts and highlight the importance of aligning the clinical and basic research communities to validate existing hypotheses in clinical studies. Human and animal therapeutic trials are presented with an emphasis on cellular and molecular mechanisms relevant to disease progression.

Recent studies have focused on understanding the neural mechanisms underlying the emergence of clinical signs and symptoms in early stage Huntington disease (HD). Although cell-based assays have focused on cell autonomous effects of mutant huntingtin, animal HD models have revealed alterations in the function of neuronal networks, particularly those linking the cerebral cortex and striatum. These findings are complemented by metabolic imaging studies of disease progression in premanifest subjects. Quantifying metabolic progression at the systems level may identify network biomarkers to aid in the objective assessment of new disease-modifying therapies and identify new regions that merit mechanistic study in HD models.

Huntington disease (HD) is an autosomal dominant neurodegenerative disease with complete penetrance. Although the understanding of the cellular mechanisms that drive neurodegeneration in HD and account for the characteristic pattern of neuronal vulnerability is incomplete, defects in energy metabolism, particularly mitochondrial function, represent a common thread in studies of HD pathogenesis in humans and animal models. Here we review the clinical, biochemical, and molecular evidence of an energy deficit in HD and discuss the mechanisms underlying mitochondrial and related alterations.

Huntington disease is an autosomal dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene. Oligonucleotide approaches based on RNAi and antisense oligonucleotides provide promising new therapeutic strategies for direct intervention through reduced production of the causative mutant protein. Allele-specific and simultaneous mutant and wild-type allele-lowering strategies are being pursued with local delivery to the brain, each with relative merits. Delivery remains a key challenge for translational success, especially with chronic therapy. The potential of disease-modifying oligonucleotide approaches for Huntington disease will be revealed as they progress into clinical trials.

MicroRNAs (miRNAs) were shown to be important for pancreas development, yet their roles in differentiated β-cells remain unclear. Here, we show that miRNA inactivation in β-cells of adult mice results in a striking diabetic phenotype. While islet architecture is intact and differentiation markers are maintained, Dicer1-deficient β-cells show a dramatic decrease in insulin content and insulin mRNA. As a consequence of the change in insulin content, the animals become diabetic. We provide evidence for involvement of a set of miRNAs in regulating insulin synthesis. The specific knockdown of miR-24, miR-26, miR-182 or miR-148 in cultured β-cells or in isolated primary islets downregulates insulin promoter activity and insulin mRNA levels. Further, miRNA-dependent regulation of insulin expression is associated with upregulation of transcriptional repressors, including Bhlhe22 and Sox6. Thus, miRNAs in the adult pancreas act in a new network that reinforces insulin expression by reducing the expression of insulin transcriptional repressors.

By virtue of the presence of multiple protein-protein interaction and signaling domains, PDZ proteins play important roles in assembling protein complexes that participate in diverse cell biological processes. GIPC is a versatile PDZ protein that binds a variety of target proteins in different cell types. In previous studies we showed that, in epidermal melanocytes, GIPC interacts with newly synthesized melanosomal protein TRP1 in the Golgi region and proposed that this interaction may facilitate intracellular trafficking of TRP1. However, since GIPC contains a single PDZ domain and no other known protein interaction motifs, it is not known how GIPC-TRP1 interaction affects melanosome biogenesis and/or melanin pigmentation. Here, we show that in human primary melanocytes GIPC interacts with AKT-binding protein APPL (adaptor protein containing pleckstrin homology, leucine zipper and phosphotyrosine binding domains), which readily co-precipitates with newly synthesized TRP1. Knockdown of either GIPC or APPL inhibits melanogenesis by decreasing tyrosinase protein levels and enzyme activity. In melanocytes, APPL exists in a complex with GIPC and phospho-AKT. Inhibition of AKT phosphorylation using a PI3-kinase inhibitor abolishes this interaction and results in retardation TRP1 in the Golgi. These data suggest that interactions between TRP1-GIPC and GIPC-APPL-AKT provide a potential link between melanogenesis and PI3 kinase signaling.

The somatosensory barrel cortex in the rodent forms during the first postnatal week setting up a periphery related map with each whisker represented as a bundle of thalamocortical axons (TCAs) in layer IV. The centers of each barrel (hollows) contain the densely packed TCAs, while the areas between each barrel (septa) form a boundary between each barrel. NG2 chondroitin sulfate proteoglycan (CSPG) expressing cells (NG2 cells, polydendrocytes) make up a unique population of glial cells that receive synaptic like input and form close contacts with growing axons. In the present study we investigated the developmental distribution of NG2 cells in the barrel cortex to determine if they display preferential septa distribution similar to other extracellular and cell surface CSPGs. Immunohistochemistry for NG2 and platelet-derived growth factor receptor alpha (PDGFRα) in NG2DsRedBAC transgenic mice showed uniform distribution of NG2 cells and processes in barrel hollows and septa at postnatal (P) days 5, 6, 7, 8, 14, and 30. Changes in the barrel pattern formation caused by cauterization of one row of whiskers at P1 resulted in corresponding changes in extracellular and cell surface CSPG distribution at P7 but no detectable changes in NG2 cell bodies and processes. Furthermore, no abnormalities in barrel formation or reorganization were detected in NG2 knockout mice. These observations suggest that NG2 cells are unlikely to play an inhibitory boundary role on TCA growth and that NG2 expression is not necessary for normal barrel formation.

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells.

Although imatinib revolutionized the management of chronic myeloid leukemia (CML), recent data indicate a transformation in the treatment approach likely in the near future. The superiority of second-generation tyrosine kinase inhibitors (TKIs) over imatinib in newly diagnosed disease has been recognized. Several investigational agents specific for those patients with the T315I mutation remain under evaluation. In Philadelphia-positive (Ph-positive) acute lymphoblastic leukemia (ALL), the addition of imatinib improved response rates. However, short remission durations with single agent therapy limit the benefit on survival. Early molecular remissions achieved with dasatinib will enable more patients to proceed to stem cell transplant (SCT), with increased likelihood of positive outcomes post-SCT.

The asymmetric polarization of cells allows specialized functions to be performed at discrete subcellular locales. Spatiotemporal coordination of polarization between groups of cells allowed the evolution of metazoa. For instance, coordinated apical-basal polarization of epithelial and endothelial cells allows transport of nutrients and metabolites across cell barriers and tissue microenvironments. The defining feature of such tissues is the presence of a central, interconnected luminal network. Although tubular networks are present in seemingly different organ systems, such as the kidney, lung, and blood vessels, common underlying principles govern their formation. Recent studies using in vivo and in vitro models of lumen formation have shed new light on the molecular networks regulating this fundamental process. We here discuss progress in understanding common design principles underpinning de novo lumen formation and expansion.

Imaging is essential in accurately detecting, staging, and treating primary liver cancer (hepatocellular carcinoma [HCC]), one of the most prevalent and lethal malignancies. We developed a novel multifunctional nanoparticle (NP) specifically targeting glypican-3 (GPC3), a proteoglycan implicated in promotion of cell growth that is overexpressed in most HCCs. Quantitative real-time polymerase chain reaction was performed to confirm the differential GPC3 expression in two human HCC cells, Hep G2 (high) and HLF (negligible). These cells were treated with biotin-conjugated GPC3 monoclonal antibody (αGPC3) and subsequently targeted using superparamagnetic iron oxide NPs conjugated to streptavidin and Alexa Fluor 647. Flow cytometry demonstrated that only GPC3-expressing Hep G2 cells were specifically targeted using this αGPC3-NP conjugate (fourfold mean fluorescence over nontargeted NP), and magnetic resonance imaging (MRI) experiments showed similar findings (threefold R2 relaxivity). Confocal fluorescence microscopy localized the αGPC3 NPs only to the cell surface of GPC3-expressing Hep G2 cells. Further characterization of this construct demonstrated a negatively charged, monodisperse, 50 nm NP, ideally suited for tumor targeting. This GPC3-specific NP system, with dual-modality imaging capability, may enhance pretreatment MRI, enable refined intraoperative HCC visualization by near-infrared fluorescence, and be potentially used as a carrier for delivery of tumor-targeted therapies, improving patient outcomes.

The cerebellum receives its input from multiple precerebellar nuclei located in the brainstem and sends processed information to other brain structures via the deep cerebellar neurons. Guidance molecules that regulate the complex migrations of precerebellar neurons and the initial guidance of their leading processes have been identified. However, the molecules necessary for dorsal guidance of precerebellar axons to the developing cerebellum or for guidance of decussating axons of the deep nuclei are not known. To determine whether Unc5c plays a role in the dorsal guidance of precerebellar and deep cerebellar axons, we studied axonal trajectories of these neurons in Unc5c(-/-) mice. Our results show that Unc5c is expressed broadly in the precerebellar and deep cerebellar neurons. Unc5c deletion disrupted long-range dorsal guidance of inferior olivary and pontine axons after crossing the midline. In addition, dorsal guidance of the axons from the medial deep cerebellar and external cuneate neurons was affected in Unc5c(-/-) mice, as were anterior migrations of pontine neurons. Coincident with the guidance defects of their axons, degeneration of neurons in the external cuneate nucleus and subdivisions of the inferior olivary nucleus was observed in Unc5c(-/-) mice. Lastly, transgenic expression of Unc5c in deep neurons and pontine neurons by the Atoh1 promoter rescued defects of the medial deep cerebellar and pontine axons observed in Unc5c(-/-) embryos, demonstrating that Unc5c acts cell autonomously in the guidance of these axons. Our results suggest that Unc5c plays a broad role in dorsal guidance of axons in the developing hindbrain.

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).

<h4>Objective</h4>To observe the change in number of circulating endothelial progenitor cells (cEPCs) and analyze its significance in septic rat.<h4>Methods</h4>Septic model of male Sprague-Dawley (SD) rats was reproduced by cecum ligation and puncture (n=80), and the normal control group (n=16) and sham operation group (n=80) were established. Nine rats in each group were used, and the cEPCs numbers in peripheral blood mononuclear cells (PBMCs, by flow cytometry), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), D-dimer (by enzyme linked immunosorbent assay, ELISA), antithrombase-III ( AT-III , by immunonephelometry), wet/dry (W/D) ratio of liver, kidney and lung were determined at 0, 6, 12, 18 hours and 1, 2, 3, 7 days after reproduction of model. Eight rats in each group were used, and the pathologic changes in liver, kidney and lung at 1 day were observed, and the injury scores were evaluated.<h4>Results</h4>The cEPCs number was markedly increased, reaching the peak [(7 161.9±689.8)/10(6) PBMCs]at 18 hours. Circulating TNF-α, IL-10, D-dimer, AT-III were found to be increased, and the levels reached the peak at 12 hours, 12 hours, 3 days, 18 hours, respectively[(51.3±6.8) ng/L, (77.9±8.6) ng/L, (93.5±11.5) mg/L, (193.8±43.0) mg/L]. W/D ratio was elevated and signs of injury to the liver,kidney, lung became more obvious (18-hour W/D of liver: 3.79±0.09, kidney: 4.25±0.08, lung: 4.91±0.09; 1-day tissue evaluation of liver:1.86±0.26, kidney: 5.14±0.34, lung: 6.57±0.37). The levels of all parameters in model group were significantly higher than those in sham operation group[18-hour cEPCs numbers: (2 235.5±472.7)/10(6) PBMCs, 12-hour TNF-α: (14.3±5.8) ng/L, 12-hour IL-10: (35.0±5.8) ng/L, 3-day D-dimer: (14.2±4.4) mg/L, 18-hour AT-III: (100.1±12.8) mg/L; 18-hour liver W/D ratio: 3.50±0.07, kidney: 3.96±0.04, lung: 4.54±0.14; 1-day tissue evaluation of liver:0.29±0.18,kidney: 0.57±0.20, lung: 1.14±0.51, P<0.05 or P<0.01]. There was positive correlation between cEPCs numbers and TNF-α (r=0.587), IL-10 (r=0.497), D-dimer (r=0.294), AT-III (r=0.690), and W/D ratio of liver, kidney, lung (r(1)=0.532, r(2)=0.532, r(3)=0.679, all P<0.01).<h4>Conclusion</h4>The cEPCs number markedly increases in septic rats, and it shows positive correlation with the degree of inflammatory reaction, blood clotting activation, capillary leakage and tissue damage. The increase of number of cEPCs is the result of reaction to sepsis, and its change in number might be valuable in evaluating the pathogenesis of sepsis.

Mouse models for Huntington's Disease (HD) and HD patients demonstrate motor and behavioral dysfunctions, such as progressive loss of coordination and memory, and share similar transcriptional profiles and striatal neuron atrophy. Clear differences between the mouse and human diseases include almost complete striatal degeneration and rarity of intranuclear inclusions in HD, and the fact that mice expressing full-length mutant huntingtin do not demonstrate a shortened life span characteristic of HD. While no clinical interventions tested in mouse models to date have delayed disease progression, the mouse models provide an invaluable tool for both investigating the underlying pathogenic processes and developing new effective therapies. Inherent differences between humans and mice must be considered in the search for efficacious treatments for HD, but the striking similarities between human HD and mouse models support the view that these models are a biologically relevant system to support the identification and testing of potential clinical therapies.

Although a growing body of evidence supports that Wnt-Frizzled signaling controls axon guidance from vertebrates to worms, whether and how this is mediated by planar cell polarity (PCP) signaling remain elusive. We show here that the core PCP components are required for Wnt5a-stimulated outgrowth and anterior-posterior guidance of commissural axons. Dishevelled1 can inhibit PCP signaling by increasing hyperphosphorylation of Frizzled3 and preventing its internalization. Vangl2 antagonizes that by reducing Frizzled3 phosphorylation and promotes its internalization. In commissural axon growth cones, Vangl2 is predominantly localized on the plasma membrane and is highly enriched on the tips of the filopodia as well as in patches of membrane where new filopodia emerge. Taken together, we propose that the antagonistic functions of Vangl2 and Dvl1 (over Frizzled3 hyperphosphorylation and endocytosis) allow sharpening of PCP signaling locally on the tips of the filopodia to sense directional cues, Wnts, eventually causing turning of growth cones.

Voltage sensors (VS) domains couple the activation of ion channels/enzymes to changes in membrane voltage. We used molecular dynamics simulations to examine interactions with lipids of several VS homologs. VSs in intact channels in the activated state are exposed to phospholipids, leading to a characteristic local distortion of the lipid bilayer which decreases its thickness by ∼10 Å. This effect is mediated by a conserved hydrophilic stretch in the S4-S5 segment linking the VS and the pore domains, and may favor gating charges crossing the membrane. In cationic lipid bilayers lacking phosphate groups, VSs form fewer contacts with lipid headgroups. The S3-S4 paddle motifs show persistent interactions of individual lipid molecules, influenced by the hairpin loop. In conclusion, our results suggest common interactions with phospholipids for various VS homologs, providing insights into the molecular basis of their stabilization in the membrane and how they are altered by lipid modification.

Linker histones are multifunctional proteins that are involved in a myriad of processes ranging from stabilizing the folding and condensation of chromatin to playing a direct role in regulating gene expression. However, how this class of enigmatic proteins binds in chromatin and accomplishes these functions remains unclear. Here we review data regarding the H1 structure and function in chromatin, with special emphasis on the C-terminal domain (CTD), which typically encompasses approximately half of the mass of the linker histone and includes a large excess of positively charged residues. Owing to its amino acid composition, the CTD was previously proposed to function in chromatin as an unstructured polycation. However, structural studies have shown that the CTD adopts detectable secondary structure when interacting with DNA and macromolecular crowding agents. We describe classic and recent experiments defining the function of this domain in chromatin folding and emerging data indicating that the function of this protein may be linked to intrinsic disorder.

On September 18, 2009, the Chicago Department of Public Health (CDPH) was notified by a local hospital of a suspected case of fatal laboratory-acquired infection with Yersinia pestis, the causative agent of plague. The patient, a researcher in a university laboratory, had been working along with other members of the laboratory group with a pigmentation-negative (pgm-) attenuated Y. pestis strain (KIM D27). The strain had not been known to have caused laboratory-acquired infections or human fatalities. Other researchers in a separate university laboratory facility in the same building had contact with a virulent Y. pestis strain (CO92) that is considered a select biologic agent; however, the pgm- attenuated KIM D27 is excluded from the National Select Agent Registry. The university, CDPH, the Illinois Department of Public Health (IDPH), and CDC conducted an investigation to ascertain the cause of death. This report summarizes the results of that investigation, which determined that the cause of death likely was an unrecognized occupational exposure (route unknown) to Y. pestis, leading to septic shock. Y. pestis was isolated from premortem blood cultures. Polymerase chain reaction (PCR) identified the clinical isolate as a pgm- strain of Y. pestis. Postmortem examination revealed no evidence of pneumonic plague. A postmortem diagnosis of hereditary hemochromatosis was made on the basis of histopathologic, laboratory, and genetic testing. One possible explanation for the unexpected fatal outcome in this patient is that hemochromatosis-induced iron overload might have provided the infecting KIM D27 strain, which is attenuated as a result of defects in its ability to acquire iron, with sufficient iron to overcome its iron-acquisition defects and become virulent. Researchers should adhere to recommended biosafety practices when handling any live bacterial cultures, even attenuated strains, and institutional biosafety committees should implement and maintain effective surveillance systems to detect and monitor unexpected acute illness in laboratory workers.

A three-stage approach was undertaken using genome-wide, case-control, and case-only association studies to identify genetic variants associated with heart failure mortality. In an Amish founder population (n = 851), cardiac hypertrophy, a trait integral to the adaptive response to failure, was found to be heritable (h² = 0.28, p = 0.0002) and GWAS revealed 21 candidate hypertrophy SNPs. In a case (n = 1,610)-control (n = 463) study in unrelated Caucasians, one of the SNPs associated with hypertrophy (rs2207418, p = 8 × 10⁻⁶), was associated with heart failure, RR = 1.85(1.25-2.73, p = 0.0019). In heart failure cases rs2207418 was associated with increased mortality, HR = 1.51(1.20-1.97, p = 0.0004). There was consistency between studies, with the GG allele being associated with increased ventricular mass (~13 g/m²) in the Amish, heart failure risk, and heart failure mortality. This SNP is in a gene desert of chromosome 20p12. Five genes are within 2.0 mbp of rs2207418 but with low LD between their SNPs and rs2207418. A region near this SNP is highly conserved in multiple vertebrates (lod score = 1,208). This conservation and the internal consistency across studies suggests that this region has biologic importance in heart failure, potentially acting as an enhancer or repressor element. rs2207418 may be useful for predicting a more progressive form of heart failure that may require aggressive therapy.

We present a case of bilateral severe retinal edema with subretinal fluid in an infant diagnosed with neonatal hemochromatosis and liver failure. A macular cherry-red spot in each eye mimicked the clinical appearance of many metabolic storage diseases. Both the clinical retinal appearance and the anatomic abnormalities observed on spectral domain optical coherence tomography resolved after successful liver transplant.

Heterogeneity of central serotonin (5-HT) raphe neurons is suggested by numerous lines of evidence, but its genetic basis remains elusive. The transcription factor Pet1 is required for the acquisition of serotonergic identity in a majority of neurons in the raphe nuclei. Nevertheless, a subset of 5-HT neurons differentiates in Pet1 knock-out mice. We show here that these residual 5-HT neurons outline a unique subpopulation of raphe neurons with highly selective anatomical targets and characteristic synaptic differentiations. In Pet1 knock-out mice, 5-HT innervation strikingly outlines the brain areas involved in stress responses with dense innervation to the basolateral amygdala, the paraventricular nucleus of the hypothalamus, and the intralaminar thalamic nuclei. In these regions, 5-HT terminals establish asymmetric synaptic junctions. This target selectivity could not be related to altered growth of the remaining 5-HT neurons, as indicated by axon tracing and cell culture analyses. The residual 5-HT axon terminals are functional with maintained release properties in vitro and in vivo. The functional consequence of this uneven distribution of 5-HT innervation on behavior was characterized. Pet1 knock-out mice showed decreased anxiety behavior in novelty exploration and increased fear responses to conditioned aversive cues. Overall, our findings lead us to propose the existence of Pet1-dependent and Pet1-resistant 5-HT neurons targeting different brain centers that might delineate the anatomical basis for a dual serotonergic control on stress responses.

Basement membranes are widely distributed extracellular matrices that coat the basal aspect of epithelial and endothelial cells and surround muscle, fat, and Schwann cells. These extracellular matrices, first expressed in early embryogenesis, are self-assembled on competent cell surfaces through binding interactions among laminins, type IV collagens, nidogens, and proteoglycans. They form stabilizing extensions of the plasma membrane that provide cell adhesion and that act as solid-phase agonists. Basement membranes play a role in tissue and organ morphogenesis and help maintain function in the adult. Mutations adversely affecting expression of the different structural components are associated with developmental arrest at different stages as well as postnatal diseases of muscle, nerve, brain, eye, skin, vasculature, and kidney.

<h4>Purpose</h4>To investigate the benefits and limitations of patient-phantom matching for determining organ dose during fluoroscopy guided interventions.<h4>Methods</h4>In this study, 27 CT datasets representing patients of different sizes and genders were contoured and converted into patient-specific computational models. Each model was matched, based on height and weight, to computational phantoms selected from the UF hybrid patient-dependent series. In order to investigate the influence of phantom type on patient organ dose, Monte Carlo methods were used to simulate two cardiac projections (PA/left lateral) and two abdominal projections (RAO/LPO). Organ dose conversion coefficients were then calculated for each patient-specific and patient-dependent phantom and also for a reference stylized and reference hybrid phantom. The coefficients were subsequently analyzed for any correlation between patient-specificity and the accuracy of the dose estimate. Accuracy was quantified by calculating an absolute percent difference using the patient-specific dose conversion coefficients as the reference.<h4>Results</h4>Patient-phantom matching was shown most beneficial for estimating the dose to heavy patients. In these cases, the improvement over using a reference stylized phantom ranged from approximately 50% to 120% for abdominal projections and for a reference hybrid phantom from 20% to 60% for all projections. For lighter individuals, patient-phantom matching was clearly superior to using a reference stylized phantom, but not significantly better than using a reference hybrid phantom for certain fields and projections.<h4>Conclusions</h4>The results indicate two sources of error when patients are matched with phantoms: Anatomical error, which is inherent due to differences in organ size and location, and error attributed to differences in the total soft tissue attenuation. For small patients, differences in soft tissue attenuation are minimal and are exceeded by inherent anatomical differences. For large patients, difference in soft tissue attenuation can be large. In these cases, patient-phantom matching proves most effective as differences in soft tissue attenuation are mitigated. With increasing obesity rates, overweight patients will continue to make up a growing fraction of all patients undergoing medical imaging. Thus, having phantoms that better represent this population represents a considerable improvement over previous methods. In response to this study, additional phantoms representing heavier weight percentiles will be added to the UFHADM and UFHADF patient-dependent series.

<h4>Objectives</h4>Oxidative stress is suggested to play an important role in the pathogenesis of nonalcoholic steatohepatitis (NASH). The present study was aimed to compare plasma levels of antioxidants in patients suffering from NASH and healthy controls.<h4>Methods</h4>Plasma levels of the antioxidants α-tocopherol, γ-tocopherol, lutein, zeaxanthin, β-cryptoxanthin, lycopene, α-carotene β-carotene were determined in 57 patients with biopsy-proven NASH and 40 healthy controls.<h4>Results</h4>Levels of α-tocopherol (22.4 vs. 26.8 nmol/ ml; p<0.01), lutein (0.19 vs. 0.33 nmol/ml; p<0.0001), zeaxanthin (0.04 vs. 0.08 nmol/ml; p<0.0001), lyco?pene (0.15 vs. 0.42 nmol/ml; p<0.0001), α-carotene (0.03 vs. 0.06 nmol/ml; p<0.005) and β-carotene (0.25 vs. 0.39 nmol/ml; p<0.01) were significantly decreased in NASH patients compared to controls. Age, aminotransferase status (ALT, AST) and BMI were not correlated with the levels of tocopherols or caro?tenoids.<h4>Conclusions</h4>Given the decreased levels supplementation of lipophilic antioxidants might be a rational treatment option for patients with NASH.

<h4>Objective</h4>The tripartite motif (TRIM) proteins are a family of more than 70 human members, however only a few of them have been well studied. It has been shown that TRIM proteins are involved in various cellular processes such as cell proliferation, differentiation, apoptosis and antiviral defense. The functions of TRIM38 are largely unknown. In this study we explore the effect of TRIM38 on NF-kappaB signaling pathway.<h4>Methods</h4>293T cells were transfected with NF-kappaB-Luc and plasmids expressing TRIM38 and its mutants fused to Flag. 24 h after transfection, cells were harvested and luciferase activities were measured. Data are representative of three independent experiments with triplicate samples. The expression of proteins was analyzed by Western Blot.<h4>Results</h4>TRIM38 could activate NF-kappaB signaling pathway. The mutants of TRIM38 affected the function of TRIM38. Only the mutant of SPRY domain deletion had no obviously influence of the function of TRIM38.<h4>Conclusion</h4>Our study reveals that NF-kappaB is activated in response to TRIM38.