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<h4>Background</h4>Liver synthesizes hepcidin in response to iron overload, leading to down-regulation of duodenal iron absorption. The pathophysiology of type 2 diabetes is associated with increased iron stores. We aimed to study circulating prohepcidin in association with insulin sensitivity and parameters of glucose and iron metabolism.<h4>Methods</h4>Serum prohepcidin was evaluated in three cohorts: 1) a cross-sectional study (cohort 1, men from the general population; n = 135); 2) after decreasing iron stores in men with "high-ferritin" type 2 diabetes (cohort 2; n = 13); and 3) after decreasing iron stores in men carrying HFE gene mutations (cohort 3; n = 16). Insulin sensitivity was measured using either the minimal model or the clamp technique.<h4>Results</h4>Circulating prohepcidin correlated significantly with glycated hemoglobin (P < 0.0001), fasting glucose (P = 0.002), triglycerides (P = 0.007), high-density lipoprotein-cholesterol (P = 0.01), ferritin (P = 0.01), and soluble transferrin receptor concentration (P = 0.001) in subjects from cohort 1. Prohepcidin decreased significantly after iron depletion in patients with type 2 diabetes (P = 0.04) (cohort 2) and in carriers of HFE gene mutations (P = 0.03) (cohort 3). In the latter subjects, the change in serum prohepcidin after iron depletion was associated with the change in both fasting glucose transferrin (r = 0.58; P = 0.02) and saturation (r = 0.68; P = 0.005). The changes in insulin sensitivity were associated with those of liver iron content (r = -0.64; P = 0.007) and with those of serum prohepcidin (r = -0.50; P = 0.04) (cohort 3).<h4>Conclusions</h4>These associations suggest that circulating prohepcidin concentration is pathophysiologically associated with parameters of glucose and iron metabolism. A failure to increase prohepcidin synthesis is hypothesized to contribute to iron-induced disorders of glucose metabolism.

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

It is becoming increasingly evident that discrete genetic alterations in neoplastic cells alone cannot explain multistep carcinogenesis whereby tumor cells are able to express diverse phenotypes during the complex phases of tumor development and progression. The epigenetic model posits that the host microenvironment exerts an initial, inhibitory constraint on tumor growth that is followed by acceleration of tumor progression through complex cell-matrix interactions. This review emphasizes the epigenetic aspects of breast cancer development in light of such interactions between epithelial cells ("seed") and the tumor microenvironment ("soil"). Our recent research findings suggest that epigenetic perturbations induced by the tumor microenvironment may play a causal role in promoting breast cancer development. It is believed that abrogation of these initiators could offer a promising therapeutic strategy.

Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8.Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as ATP-independent molecular chaperones that can bind unfolded proteins and facilitate their processing via the help of ATP-dependent chaperones such as the Hsp70 machine, in which Bag3 may act as a molecular link between HspB, Hsp70, and the ubiquitin ligases. However, here we show that HspB8 and Bag3 act in a non-canonical manner unrelated to the classical chaperone model. Rather, HspB8 and Bag3 induce the phosphorylation of the alpha-subunit of the translation initiator factor eIF2, which in turn causes a translational shut-down and stimulates autophagy. This function of HspB8.Bag3 does not require Hsp70 and also targets fully folded substrates. HspB8.Bag3 activity was independent of the endoplasmic reticulum (ER) stress kinase PERK, demonstrating that its action is unrelated to ER stress and suggesting that it activates stress-mediated translational arrest and autophagy through a novel pathway.

Recent studies that alleles in the hemochromatosis gene may accelerate the onset of Alzheimer's disease by five years have validated interest in the model in which metals (particularly iron) accelerate disease course. Biochemical and biophysical measurements demonstrated the presence of elevated levels of neurotoxic copper zinc and iron in the brains of AD patients. Intracellular levels of APP holoprotein were shown to be modulated by iron by a mechanism that is similar to the translation control of the ferritin L- and H mRNAs by iron-responsive element (IRE) RNA stem loops in their 5' untranslated regions (5'UTRs). More recently a putative IRE-like sequence was hypothesized present in the Parkinsons's alpha synuclein (ASYN) transcript (see [A.L. Friedlich, R.E. Tanzi, J.T. Rogers, The 5'-untranslated region of Parkinson's disease alpha-synuclein messenger RNA contains a predicted iron responsive element, Mol. Psychiatry 12 (2007) 222-223. [6]]). Together with the demonstration of metal dependent translation of APP mRNA, the involvement of metals in the plaque of AD patients and of increased iron in striatal neurons in the substantia nigra (SN) of Parkinson's disease patients have stimulated the development of metal attenuating agents and iron chelators as a major new therapeutic strategy for the treatment of these neurodegenerative diseases. In the case of AD, metal based therapeutics may ultimately prove more cost effective than the use of an amyloid vaccine as the preferred anti-amyloid therapeutic strategy to ameliorate the cognitive decline of AD patients.

The beta-1,4-galactosyltransferase (beta-1,4-GalT) V whose human and mouse genes were cloned by us has been suggested to be involved in the biosynthesis of N-glycans and O-glycans, and lactosylceramide. To determine its biological function, beta-1,4-GalT V (B4galt5) mutant mice obtained by a gene trap method were analyzed. Analysis of pre- and post-implantation embryos revealed that the B4galt5(-/-) mice die by E10.5 while B4galt5(+/-) mice were born and grown normally. Histological study showed that most tissues are formed in B4galt5(-/-) embryos but their appearance at E10.5 is close to that of B4galt5(+/-) embryos at E9.0-9.5. The results indicate that the growth is delayed by one to one and half day in B4galt5(-/-) embryos when compared to B4galt5(+/-) embryos, which results in early death of the embryos by E10.5, probably due to hematopoietic and/or placental defects.

All cells possess surveillance and homeostatic mechanisms to adjust protein biogenesis to the demands of growth, differentiation, ageing and environmental stress. However, under certain circumstances, these mechanisms fail to adequately respond to proteotoxic imbalances and result in the accumulation of misfolded proteins. In humans, this can lead to neurodegeneration and other protein conformational diseases. To protect itself, the cell employs highly conserved stress responses and chaperone networks to maintain protein-folding homeostasis (proteostasis). Although the regulation of stress responses, such as the heat-shock response, and of proteostasis have been widely considered to be cell autonomous, recent studies using Caenorhabditis elegans have shown that these processes are regulated by neuronal signaling and endocrine pathways and integrated into other functions of the organism. The hierarchical control of the cellular proteostasis machinery affords insight into the organization of stress regulatory networks in multicellular organisms and offers novel targets for the treatment of human protein conformational diseases.

Suicidal behavior is a multifactorial phenomenon, with a significant genetic predisposition. To assess the contribution of genes in the 4p region to suicide risk, we genotyped 36 single nucleotide polymorphisms from a 49Mb region on the chromosome arm 4p11-16 in a total of 288 male suicide victims and 327 healthy male volunteers. The nonsynonymous variants rs1383180 in EVC gene, rs6811863 in TBC1D1 gene, rs362272 in HTT gene, and rs734312 in WFS1 gene were associated to the male completed suicide. However, only EVC polymorphism remained significant after correcting for multiple comparisons (P < .05 after 10 K permutations). The function of these genes is not clear yet. WFS1 and HTT are related to the unfolded protein response and endoplasmic reticulum stress, and TBC1D1 is a GTPase activator. EVC is a protein with transmembrane and leucine zipper domains, its function has not been elucidated yet. Further studies are required in order to reveal the role of these four polymorphisms in the pathoetiology of suicide.

At spinal levels, sensory information pertaining to body positioning (proprioception) is relayed to the cerebellum by the spinocerebellar tracts (SCTs). In the past we revealed the basic helix-loop-helix transcription factor Atoh1 (Math1) to be important for establishing Dorsal Progenitor 1 (DP1) commissural interneurons, which comprise a subset of proprioceptive interneurons. Given there exists multiple subdivisions of the SCT we asked whether Atoh1 may also play a role in specifying other cell types in the spinal cord. Here, we reveal the generation of at least three DP1 derived interneuron populations that reside at spatially restricted positions along the rostral-caudal axis. Each of these cell populations expresses distinct markers and anatomically coincides with the cell bodies of the various subdivisions of the SCT. In addition, we found that as development proceeds (e.g. by E13.5) Atoh1 expression becomes apparent in the dorsal midline in the region of the roof plate (RP). Interestingly, we find that cells derived from Atoh1 expressing RP progenitors express SSEA-1, and in the absence of Atoh1 these progenitors become SOX9 positive. Altogether we reveal the existence of multiple Atoh1 dependent cell types in the spinal cord, and uncover a novel progenitor domain that arises late in development.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by systemic iron overload with consequent tissue damage. The vast majority of HH patients are homozygous for the C282Y mutation in HFE, a non-classical MHC class-I gene located in chromosome 6, whose role in the regulation of systemic iron metabolism is still not completely understood. Iron enhances the formation of reactive oxygen species, with increasing risk of DNA damage induced by oxidative stress, and consequently an increased susceptibility to chromosome instability. In the present work we examined spontaneous and diepoxybutane (DEB)-induced chromosome instability in PHA-stimulated lymphocyte cultures from 23 HH patients, all homozygous for the C282Y HFE mutation, in comparison to 29 normal controls. In addition, three patients with secondary forms of iron overload, not related to HFE, were studied as controls to test the role of iron overload on DEB-induced chromosome instability. Our results show a significantly higher frequency of spontaneous chromosome breaks in lymphocytes from the HH patients, when compared with lymphocytes from normal controls (p<0.0001). In addition, there is a significant correlation between the percentage of cells with spontaneous chromosomal breaks and the transferrin saturation (r=0.53, p=0.0041) or serum-ferritin levels (r=0.66, p=0.0001) in patients. Surprisingly, the frequency of DEB-induced chromosome breaks was significantly lower in lymphocytes from HH patients than in lymphocytes from both controls and patients with secondary forms of hemochromatosis (p=0.0029). No correlation was observed between the percentage of cells with DEB-induced chromosome breaks and the transferrin saturation or serum-ferritin values. These results suggest that lymphocytes from HH patients may have an increased capacity to respond to DEB-induced chromosome breakage, and that this capacity is somehow related to the presence of the C282Y HFE mutation.

Endothelial nitric oxide synthase-derived NO and its derivative, peroxynitrite (ONOO(-)), suppresses oxygen consumption by nitration of mitochondrial proteins after reperfusion. However, very few nitrated proteins are identified to date. In this paper, ischemia/reperfusion (I/R) injury was induced in mouse heart by ligation and release of the left anterior descending coronary artery. Western blotting showed that tyrosine nitration was higher in I/R hearts. Nitrated proteins were identified by capillary-liquid chromatography-nanospray tandem mass spectrometry. A total of 23 proteins were identified as being nitrated after I/R and 10 of them were from mitochondria. The nitrated mitochondrial proteins included 4 subunits from the oxidative phosphorylation system (the 24 and the 30 kDa subunits of complex I, the Rieske ISP of complex III, and the alpha subunit of ATP synthase), five enzymes in the matrix, and voltage-dependent anion channel. In purified complex I treated with ONOO(-), 3-NT was identified locating at the residue of Y247 of the 30 kDa subunit and the residues of Y47, Y53 of the 49 kDa subunit. In conclusion, I/R induced protein nitration and mitochondrial proteins were the major targets. Selective nitration of proteins from the oxidative phosphorylation system at the beginning of reperfusion may contribute to the suppression of oxygen consumption.

Members of the GDNF family of ligands, including neurturin (NTN), have been implicated as potential therapeutic agents for Huntington's disease (HD). The present study examined the ability of CERE-120 (AAV2-NTN) to provide structural and functional protection in the N171-82Q transgenic HD mouse model. AAV2-NTN therapy attenuated rotorod deficits in this mutant relative to control treated transgenics (p<0.01). AAV2-NTN treatment significantly reduced the number of transgenic mice that exhibited clasping behavior and partially restored their stride lengths (both p<0.05). Stereological counts of NeuN-ir neurons revealed a significant neuroprotection in the striatum of AAV2-NTN treated relative to control treated transgenics (p<0.001). Most fascinating, stereological counts of NeuN-labeled cells in layers V-VI of prefrontal cortex revealed that intrastriatal AAV2-NTN administration prevented the loss of frontal cortical NeuN-ir neurons seen in transgenic mice (p<0.01). These data indicate that gene delivery of NTN may be a viable strategy for the treatment of this incurable disease.

<h4>Background</h4>Atopic eczema (AE) is a common chronic inflammatory skin disorder. In order to dissect the genetic background several linkage and genetic association studies have been performed. Yet very little is known about specific genes involved in this complex skin disease, and the underlying molecular mechanisms are not fully understood.<h4>Methodology/findings</h4>We used human DNA microarrays to identify a molecular picture of the programmed responses of the human genome to AE. The transcriptional program was analyzed in skin biopsy samples from lesional and patch-tested skin from AE patients sensitized to Malassezia sympodialis (M. sympodialis), and corresponding biopsies from healthy individuals. The most notable feature of the global gene-expression pattern observed in AE skin was a reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. The overall transcriptional response in M. sympodialis patch-tested AE skin was similar to the gene-expression signature identified in lesional AE skin. In the constellation of genes differentially expressed in AE skin compared to healthy control skin, we have identified several potential susceptibility genes that may play a critical role in the pathological condition of AE. Many of these genes, including genes with a role in immune responses, lipid homeostasis, and epidermal differentiation, are localized on chromosomal regions previously linked to AE.<h4>Conclusions/significance</h4>Through genome-wide expression profiling, we were able to discover a distinct reciprocal expression pattern of induced inflammatory genes and repressed lipid metabolism genes in skin from AE patients. We found a significant enrichment of differentially expressed genes in AE with cytobands associated to the disease, and furthermore new chromosomal regions were found that could potentially guide future region-specific linkage mapping in AE. The full data set is available at http://microarray-pubs.stanford.edu/eczema.

<h4>Background</h4>Hepcidin, a key regulator of iron homeostasis, is increased in response to inflammation and some infections, but the in vivo role of hepcidin, particularly in children with iron deficiency anemia (IDA) is unclear. We investigated the relationships between hepcidin, cytokines and iron status in a pediatric population with a high prevalence of both anemia and co-morbid infections.<h4>Methodology/principal findings</h4>African refugee children <16 years were consecutively recruited at the initial post-resettlement health check with 181 children meeting inclusion criteria. Data on hematological parameters, cytokine levels and co-morbid infections (Helicobacter pylori, helminth and malaria) were obtained and urinary hepcidin assays performed. The primary outcome measure was urinary hepcidin levels in children with and without iron deficiency (ID) and/or ID anaemia (IDA). The secondary outcome measures included were the relationship between co-morbid infections and (i) ID and IDA, (ii) urinary hepcidin levels and (iii) cytokine levels. IDA was present in 25/181 (13.8%). Children with IDA had significantly lower hepcidin levels (IDA median hepcidin 0.14 nmol/mmol Cr (interquartile range 0.05-0.061) versus non-IDA 2.96 nmol/mmol Cr, (IQR 0.95-6.72), p<0.001). Hemoglobin, log-ferritin, iron, mean cell volume (MCV) and transferrin saturation were positively associated with log-hepcidin levels (log-ferritin beta coefficient (beta): 1.30, 95% CI 1.02 to 1.57) and transferrin was inversely associated (beta: -0.12, 95% CI -0.15 to -0.08). Cytokine levels (including IL-6) and co-morbid infections were not associated with IDA or hepcidin levels.<h4>Conclusions/significance</h4>This is the largest pediatric study of the in vivo associations between hepcidin, iron status and cytokines. Gastro-intestinal infections (H. pylori and helminths) did not elevate urinary hepcidin or IL-6 levels in refugee children, nor were they associated with IDA. Longitudinal and mechanistic studies of IDA will further elucidate the role of hepcidin in paediatric iron regulation.

<h4>Background</h4>Histone H1x is a ubiquitously expressed member of the H1 histone family. H1 histones, also called linker histones, stabilize compact, higher order structures of chromatin. In addition to their role as structural proteins, they actively regulate gene expression and participate in chromatin-based processes like DNA replication and repair. The epigenetic contribution of H1 histones to these mechanisms makes it conceivable that they also take part in malignant transformation.<h4>Methods</h4>Based on results of a Blast data base search which revealed an accumulation of expressed sequence tags (ESTs) of H1x in libraries from neuroendocrine tumours (NETs), we evaluated the expression of H1x in NETs from lung and the gastrointestinal tract using immunohistochemisty. Relative protein and mRNA levels of H1x were analysed by Western blot analysis and quantitative real-time RT-PCR, respectively. Since several reports describe a change of the expression level of the replacement subtype H1.0 during tumourigenesis, the analysis of this subtype was included in this study.<h4>Results</h4>We found an increased expression of H1x but not of H1.0 in NET tissues in comparison to corresponding normal tissues. Even though the analysed NETs were heterogenous regarding their grade of malignancy, all except one showed a considerably higher protein amount of H1x compared with corresponding non-neoplastic tissue. Furthermore, double-labelling of H1x and chromogranin A in sections of pancreas and small intestine revealed that H1x is highly expressed in neuroendocrine cells of these tissues.<h4>Conclusion</h4>We conclude that the high expression of histone H1x in NETs is probably due to the abundance of this protein in the cells from which these tumours originate.

A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC(50) values of 9.0 and 18.8 microM against Plasmodia.

<h4>Background</h4>We sought to describe the integrity of human genomic DNA extracted from saliva saturated cotton spit wads stored at -20 degrees C for approximately 11 years. 783 spit wad samples were collected from an ADHD sample population (Vermont Family Study) during 1996-2000. Human genomic DNA was extracted from the spit wads using a commercially available kit; QIAamp DNA Blood Midi Kit (Qiagen, Inc., Valencia, CA.) with a few modifications.<h4>Results</h4>The resulting DNA yield was more than adequate for genetic analysis and ranged from approximately 1 mug to a total of 80 mug (mean 17.3 mugs +/- 11.9 mugs). A260/A280 ratios for the human genomic DNA extracted from the spit wads was consistently within the generally acceptable values of 1.7-2.0, with the lowest purity being 1.70, and a mean value of 1.937 +/- 0.226 for the 783 samples. The DNA also was suitable for PCR reactions as evidenced by the amplification of the serotonin-transporter-linked polymorphic region, 5HTTLPR. 5HTTLPR is a functional polymorphism in the promoter region of the serotonin transporter gene (HTT, SLC6A4, or SERT), consisting of two intensively studied alleles. 770 of the 783 samples (98.3%) produced fragments after PCR of the expected size with primers specific for 5HTTLPR.<h4>Conclusion</h4>High quality and abundant genomic DNA can be successfully retrieved from saliva saturated cotton spit wads using the commercially available kit, QIAamp DNA Blood Midi Kit from Qiagen, Inc. Furthermore, the DNA can be extracted in less than 3 hours and multiple samples can be processed simultaneously thus reducing processing time.

The pineal gland plays an essential role in vertebrate chronobiology by converting time into a hormonal signal, melatonin, which is always elevated at night. Here we have analyzed the rodent pineal transcriptome using Affymetrix GeneChip(R) technology to obtain a more complete description of pineal cell biology. The effort revealed that 604 genes (1,268 probe sets) with Entrez Gene identifiers are differentially expressed greater than 2-fold between midnight and mid-day (false discovery rate <0.20). Expression is greater at night in approximately 70%. These findings were supported by the results of radiochemical in situ hybridization histology and quantitative real time-PCR studies. We also found that the regulatory mechanism controlling the night/day changes in the expression of most genes involves norepinephrine-cyclic AMP signaling. Comparison of the pineal gene expression profile with that in other tissues identified 334 genes (496 probe sets) that are expressed greater than 8-fold higher in the pineal gland relative to other tissues. Of these genes, 17% are expressed at similar levels in the retina, consistent with a common evolutionary origin of these tissues. Functional categorization of the highly expressed and/or night/day differentially expressed genes identified clusters that are markers of specialized functions, including the immune/inflammation response, melatonin synthesis, photodetection, thyroid hormone signaling, and diverse aspects of cellular signaling and cell biology. These studies produce a paradigm shift in our understanding of the 24-h dynamics of the pineal gland from one focused on melatonin synthesis to one including many cellular processes.

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

Golgi fragmentation is a process that is necessary to allow its redistribution into daughter cells during mitosis, a process controlled by serine-threonine kinases. This Golgi fragmentation is activated by MEK1 and Plk3. Plk3 is a kinase that is a downstream target in the Golgi fragmentation pathway induced by MEK1 or by nocodazole. In this work, we have identified that Plk3 and VRK1 are two consecutive steps in this signaling pathway. Plk3 interacts with VRK1, forming a stable complex detected by reciprocal immunoprecipitations and pull-down assays; VRK1 colocalizes with giantin in the Golgi apparatus, as Plk3 also does, forming clearly detectable granules. VRK1 does not phosphorylate Plk3, but Plk3 phosphorylates the C-terminal region of VRK1 in Ser342. VRK1 with substitutions in S342 is catalytically active but blocks Golgi fragmentation, indicating that its specific phosphorylation is necessary for this process. The induction of Golgi fragmentation by MEK1 and Plk3 can be inhibited by kinase-dead VRK1, the knockdown of VRK1 by siVRK1, kinase-dead Plk3, or PD98059, a MEK1 inhibitor. The Plk3-VRK1 kinase module might represent two consecutive steps of a signaling cascade that participates in the regulation of Golgi fragmentation.

Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. We characterized female-sterile alleles of the condensin complex component dcap-g and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, we demonstrate a role for dcap-g in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I-arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. We used the QA26 allele of passenger component incenp to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenp(QA26) mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient toward only one pole in prometaphase and metaphase I. We show that incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions.

Despite their profound importance in the development of cancer, the extracellular cues that target cell invasion through basement membrane barriers remain poorly understood. A central obstacle has been the difficulty of studying the interactions between invading cells and basement membranes in vivo. Using the genetically and visually tractable model of Caenorhabditis elegans anchor cell (AC) invasion, we show that UNC-6 (netrin) signalling, a pathway not previously implicated in controlling cell invasion in vivo, is a key regulator of this process. Site of action studies reveal that before invasion, localized UNC-6 secretion directs its receptor, UNC-40, to the plasma membrane of the AC, in contact with the basement membrane. There, UNC-40 polarizes a specialized invasive membrane domain through the enrichment of actin regulators, F-actin and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Cell ablation experiments indicate that UNC-6 promotes the formation of invasive protrusions from the AC that break down the basement membrane in response to a subsequent vulval cue. Together, these results characterize an invasive membrane domain in vivo, and reveal a role for UNC-6 (netrin) in polarizing this domain towards its basement membrane target.

<h4>Aims</h4>To screen for differentially tyrosine-phosphorylated proteins between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal epithelial tissues (NNET) to provide a basis for elucidate the molecular mechanisms of NPC carcinogenesis.<h4>Methods</h4>Two-dimensional (2-D) electrophoresis was applied to separate proteins from NPC and NNET, respectively, and 2-D Western blotting was performed to detect tyrosine-phosphorylated proteins using antiphosphotyrosine antibody. Differentially tyrosine-phosphorylated proteins were identified by electrospray ionization-quadrupole time-of-flight MS (ESI-Q-TOF MS). NetPhos software was used to predict the tyrosine-phosphorylation sites of the identified proteins, and Western blotting was used to detect the tyrosine-phosphorylated levels of RKIP in NPC and NNET.<h4>Results</h4>Twenty-five differentially tyrosine-phosphorylated proteins in the two types of tissues were found, 13 of which were identified by ESI-Q-TOF MS. Among the 13 identified proteins, tyrosine-phosphorylated levels of 7 proteins were increased, and those of 6 proteins were decreased in NPC compared with NNET. NetPhos software prediction showed that all the 13 identified proteins contained tyrosine phosphorylation sites, and the differentially tyrosine-phosphorylated level of RKIP in NPC and NNET was confirmed.<h4>Conclusion</h4>The 13 differentially tyrosine-phosphorylated proteins may be involved in the development and progression of NPC.

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 microm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.

We used a systems biology approach to identify and score protein interaction subnetworks whose activity patterns are discriminative of late stage human colorectal cancer (CRC) versus control in colonic tissue. We conducted two gel-based proteomics experiments to identify significantly changing proteins between normal and late stage tumor tissues obtained from an adequately sized cohort of human patients. A total of 67 proteins identified by these experiments was used to seed a search for protein-protein interaction subnetworks. A scoring scheme based on mutual information, calculated using gene expression data as a proxy for subnetwork activity, was developed to score the targets in the subnetworks. Based on this scoring, the subnetwork was pruned to identify the specific protein combinations that were significantly discriminative of late stage cancer versus control. These combinations could not be discovered using only proteomics data or by merely clustering the gene expression data. We then analyzed the resultant pruned subnetwork for biological relevance to human CRC. A number of the proteins in these smaller subnetworks have been associated with the progression (CSNK2A2, PLK1, and IGFBP3) or metastatic potential (PDGFRB) of CRC. Others have been recently identified as potential markers of CRC (IFITM1), and the role of others is largely unknown in this disease (CCT3, CCT5, CCT7, and GNA12). The functional interactions represented by these signatures provide new experimental hypotheses that merit follow-on validation for biological significance in this disease. Overall the method outlines a quantitative approach for integrating proteomics data, gene expression data, and the wealth of accumulated legacy experimental data to discover significant protein subnetworks specific to disease.

<h4>Background</h4>Revealing the molecular changes in chronic ethanol-impaired neuronal differentiation may be of great importance for understanding ethanol-related pathology in embryonic development but also in the adult brain. In this study, both acute and long-term effects of ethanol on neuronal differentiation of human neuroblastoma cells were investigated. We focused on several aspects of brain-derived neurotrophic factor (BDNF) signaling because BDNF activates the extracellular signal-regulated kinase (ERK) cascade, promoting neuronal differentiation including neurite outgrowth.<h4>Methods</h4>The effects of ethanol exposure on morphological differentiation, cellular density, neuronal marker proteins, basal ERK activity, and ERK responsiveness to BDNF were measured over 2 to 4 weeks. qRT-PCR and Western blotting were performed to investigate the expression of neurotrophin receptor tyrosin kinase B (TrkB), members of the ERK-cascade, protein kinase C (PKC) isoforms and Raf-Kinase-Inhibitor-Protein (RKIP).<h4>Results</h4>Chronic ethanol interfered with the development of a neuronal network consisting of cell clusters and neuritic bundles. Furthermore, neuronal and synaptic markers were reduced, indicating impaired neuronal differentiation. BDNF-mediated activation of the ERK cascade was found to be continuously impaired by ethanol. This could not be explained by expressional changes monitored for TrkB, Raf-1, MEK, and ERK. However, BDNF also activates PKC signaling which involves RKIP, which finally leads to ERK activation as well. Therefore, we hypothesized that ethanol impairs this branch of BDNF signaling. Indeed, both PKC and RKIP were significantly down-regulated.<h4>Conclusions</h4>Chronic ethanol exposure impaired neuronal differentiation of neuroblastoma cells and BDNF signaling, particularly the PKC-dependent branch. RKIP, acting as a signaling switch at the merge of the PKC cascade and the Raf/MEK/ERK cascade, was associated with neuronal differentiation and significantly reduced in ethanol treatment. Moreover, PKC expression itself was even more strongly reduced. In contrast, members of the Raf-1/MEK/ERK cascade were less affected and the observed changes were not associated with impaired differentiation. Thus, reduced RKIP and PKC levels and subsequently reduced positive feedback on ERK activation provide an explanation for the striking effects of long-term ethanol exposure on BDNF signal transduction and neuronal differentiation, respectively.

<h4>Background</h4>Using an experimental model of airway fibrosis following lung transplantation, we recently showed that chronic alcohol ingestion by donor rats amplifies airway fibrosis in the recipient. Associated with alcohol-mediated amplification of airway fibrosis is increased transforming growth factor beta-1(TGFbeta(1)) and alpha-smooth muscle actin expression. Other studies have shown that interleukin-13 (IL-13) modulates TGFbeta(1) signaling during experimentally-induced airway fibrosis. Therefore, we hypothesized that IL-13 is a component of alcohol-mediated amplification of pro-fibrotic mediators in the alcoholic lung.<h4>Methods</h4>To test this hypothesis, we analyzed tracheal epithelial cells and type II alveolar cells from control- or alcohol-fed rats, alcohol-treated mouse lung fibroblasts, and human bronchial epithelial cells in vitro for expression of various components of the IL-13 signaling pathway. Signaling via the IL-13 pathway was assessed by measuring levels of phosphorylated signal transducers and activators of transcription-6 (STAT6). In addition, we performed heterotopic tracheal transplantation using control-fed and alcohol-fed donor rats and analyzed tracheal allografts for expression of components of the IL-13 signaling pathway by RT-PCR and immunocytochemical analyses.<h4>Results</h4>Interleukin-13 expression was detected in type II alveolar epithelial cells and human bronchial epithelial cells, but not in lung fibroblasts. IL-13 expression was decreased in whole lung and type II cells in response to alcohol exposure. In all cell types analyzed, expression of IL-13 signaling receptor (IL-13R alpha(1)) mRNA was markedly increased. In contrast, mRNA and protein expression of the IL-13 decoy receptor (IL-13R alpha(2)) were decreased in all cells analyzed. Exposure to alcohol also increased STAT6 phosphorylation in response to IL-13 and lipopolysaccharide.<h4>Conclusions</h4>Data from multiple cell types in the pulmonary system suggest that IL-13 and its receptors play a role in alcohol-mediated activation of pro-fibrotic pathways. Taken together, these data suggest that alcohol primes the airway for increased IL-13 signaling and subsequent tissue remodeling upon injury such as transplantation.

The tumor necrosis factor ligand superfamily member 4 gene (TNFSF4) encodes the OX40 ligand (OX40L), a costimulatory molecule involved in T-cell activation. A recent study demonstrated the association of TNFSF4 haplotypes located in the upstream region with risk for or protection from systemic lupus erythematosus (SLE). To replicate this association, five single nucleotide polymorphisms (SNPs) tagging the previously associated haplotypes and passing the proper quality-control filters were tested in 1312 cases and 1801 controls from Germany, Italy, Spain and Argentina. The association of TNFSF4 with SLE was replicated in all the sets except Spain. There was a unique risk haplotype tagged by the minor alleles of the SNPs rs1234317 (pooled odds ratio (OR)=1.39, P=0.0009) and rs12039904 (pooled OR=1.38, P=0.0012). We did not observe association to a single protective marker (rs844644) or haplotype as the first study reported; instead, we observed different protective haplotypes, all carrying the major alleles of both SNPs rs1234317 and rs12039904. Association analysis conditioning on the haplotypic background confirmed that these two SNPs explain the entire haplotype effect. This first replication study confirms the association of genetic variation in the upstream region of TNFSF4 with susceptibility to SLE.

<h4>Background</h4>Dependences on addictive substances are substantially-heritable complex disorders whose molecular genetic bases have been partially elucidated by studies that have largely focused on research volunteers, including those recruited in Baltimore. Maryland. Subjects recruited from the Baltimore site of the Epidemiological Catchment Area (ECA) study provide a potentially-useful comparison group for possible confounding features that might arise from selecting research volunteer samples of substance dependent and control individuals. We now report novel SNP (single nucleotide polymorphism) genome wide association (GWA) results for vulnerability to substance dependence in ECA participants, who were initially ascertained as members of a probability sample from Baltimore, and compare the results to those from ethnically-matched Baltimore research volunteers.<h4>Results</h4>We identify substantial overlap between the home address zip codes reported by members of these two samples. We find overlapping clusters of SNPs whose allele frequencies differ with nominal significance between substance dependent vs control individuals in both samples. These overlapping clusters of nominally-positive SNPs identify 172 genes in ways that are never found by chance in Monte Carlo simulation studies. Comparison with data from human expressed sequence tags suggests that these genes are expressed in brain, especially in hippocampus and amygdala, to extents that are greater than chance.<h4>Conclusion</h4>The convergent results from these probability sample and research volunteer sample datasets support prior genome wide association results. They fail to support the idea that large portions of the molecular genetic results for vulnerability to substance dependence derive from factors that are limited to research volunteers.

The objective of this work was to identify peptide sequences with high affinity to bone-like mineral (BLM) to provide alternative design methods for functional bone regeneration peptides. Adsorption of preferential binding peptide sequences on four apatite-based substrates [BLM and three sintered apatite disks pressed from powders containing 0% CO(3)(2-) (HA), 5.6% CO(3)(2-) (CA5), 10.5% CO(3)(2-) (CA10)] with varied compositions and morphologies was investigated. A combination of phage display, ELISA, and computational modeling was used to elucidate three 12-mer peptide sequences APWHLSSQYSRT (A), STLPIPHEFSRE (S), and VTKHLNQISQSY (V), from 243 candidates with preferential adsorption on BLM and HA. Overall, peptides S and V have a significantly higher adsorption to the apatite-based materials in comparison to peptide A (for S vs. A, BLM p=0.001, CA5 p<0.001, CA10 p<0.001, HA p=0.038; for V vs. A, BLM p=0.006, CA5 p=0.033, CA10 p=0.029). FT-IR analysis displayed carbonate levels in CA5 and CA10 dropped to approximately 1.1-2.2% after sintering, whereas SEM imaging displayed CA5 and CA10 possess distinct morphologies. Adsorption results normalized to surface area indicate that small changes in carbonate percentage at a similar morphological scale did not provide enough carbonate incorporation to show statistical differences in peptide adsorption. Because the identified peptides (S and V) have preferential binding to apatite, their use can now be investigated in bone and dentin tissue engineering, tendon and ligament repair, and enamel formation.

UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which we show here are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with our finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. We discuss the remarkable balance between SMD and the two types of NMD in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis.

The use of lignin peroxidase (LIP) as an alternative method for the removal of four catechols (1,2-dihydroxybenzene): catechol (CAT), 4-chlorocatechol (4-CC), 4,5-dichlorocatechol (4,5-DCC) and 4-methylcatechol (4-MC) typical pollutants in wastewater derived from oil and paper industries, was evaluated. The removal of 2mM catecholic substrates by 1 microM LIP after 1h was in the following order: 4,5-DCC (95%)>4-CC(90%)>CAT(55%)>4-MC(43%). Except for 4-MC, all reactions were accompanied by the formation of insoluble products, leading to LIP precipitation. LIP was exposed to soluble or insoluble product-dependent inactivation, depending on the substrates tested, immediately at the start of the reactions. Despite immediate enzyme inactivation, removal of catecholic substrates continued, resulting in oligomeric product formation. Major oxidation products analyzed were compatible with dimeric, trimeric and tetrameric structures. Ether linkages and a benzoquinone structure were detected in two purified oligochlorocatechols. Catechol derivatives removal initiated by LIP, seems to be different for each catecholic substrate in terms of substrate consumption and transformation, and of enzyme activity.

Human mesothelial cells (LP9/TERT-1) were exposed to low and high (15 and 75 microm(2)/cm(2) dish) equal surface area concentrations of crocidolite asbestos, nonfibrous talc, fine titanium dioxide (TiO2), or glass beads for 8 or 24 hours. RNA was then isolated for Affymetrix microarrays, GeneSifter analysis and QRT-PCR. Gene changes by asbestos were concentration- and time-dependent. At low nontoxic concentrations, asbestos caused significant changes in mRNA expression of 29 genes at 8 hours and of 205 genes at 24 hours, whereas changes in mRNA levels of 236 genes occurred in cells exposed to high concentrations of asbestos for 8 hours. Human primary pleural mesothelial cells also showed the same patterns of increased gene expression by asbestos. Nonfibrous talc at low concentrations in LP9/TERT-1 mesothelial cells caused increased expression of 1 gene Activating Transcription Factor 3 (ATF3) at 8 hours and no changes at 24 hours, whereas expression levels of 30 genes were elevated at 8 hours at high talc concentrations. Fine TiO2 or glass beads caused no changes in gene expression. In human ovarian epithelial (IOSE) cells, asbestos at high concentrations elevated expression of two genes (NR4A2, MIP2) at 8 hours and 16 genes at 24 hours that were distinct from those elevated in mesothelial cells. Since ATF3 was the most highly expressed gene by asbestos, its functional importance in cytokine production by LP9/TERT-1 cells was assessed using siRNA approaches. Results reveal that ATF3 modulates production of inflammatory cytokines (IL-1 beta, IL-13, G-CSF) and growth factors (VEGF and PDGF-BB) in human mesothelial cells.

<h4>Background</h4>Starvation triggers a complex array of adaptative metabolic responses including energy-metabolic responses, a process which must imply tissue specific alterations in gene expression and in which the liver plays a central role. The present study aimed to describe the evolution of global gene expression profiles in liver of 4-week-old male chickens during a 48 h fasting period using a chicken 20 K oligoarray.<h4>Results</h4>A large number of genes were modulated by fasting (3532 genes with a pvalue corrected by Benjamini-Hochberg < 0.01); 2062 showed an amplitude of variation higher than +/- 40% among those, 1162 presented an human ortholog, allowing to collect functional information. Notably more genes were down-regulated than up-regulated, whatever the duration of fasting (16 h or 48 h). The number of genes differentially expressed after 48 h of fasting was 3.5-fold higher than after 16 h of fasting. Four clusters of co-expressed genes were identified by a hierarchical cluster analysis. Gene Ontology, KEGG and Ingenuity databases were then used to identify the metabolic processes associated to each cluster. After 16 h of fasting, genes involved in ketogenesis, gluconeogenesis and mitochondrial or peroxisomal fatty acid beta-oxidation, were up-regulated (cluster-1) whereas genes involved in fatty acid and cholesterol synthesis were down-regulated (cluster-2). For all genes tested, the microarray data was confirmed by quantitative RT-PCR. Most genes were altered by fasting as already reported in mammals. A notable exception was the HMG-CoA synthase 1 gene, which was up-regulated following 16 and 48 h of fasting while the other genes involved in cholesterol metabolism were down-regulated as reported in mammalian studies. We further focused on genes not represented on the microarray and candidates for the regulation of the target genes belonging to cluster-1 and -2 and involved in lipid metabolism. Data are provided concerning PPARa, SREBP1, SREBP2, NR1H3 transcription factors and two desaturases (FADS1, FADS2).<h4>Conclusion</h4>This study evidences numerous genes altered by starvation in chickens and suggests a global repression of cellular activity in response to this stressor. The central role of lipid and acetyl-CoA metabolisms and its regulation at transcriptional level are confirmed in chicken liver in response to short-term fasting. Interesting expression modulations were observed for NR1H3, FADS1 and FADS2 genes. Further studies are needed to precise their role in the complex regulatory network controlling lipid metabolism.

<h4>Unlabelled</h4>Disorders of neuronal migration are a heterogeneous group of disorders of nervous system development. One of the most frequent disorders is lissencephaly, characterized by a paucity of normal gyri and sulci resulting in a 'smooth brain'. There are two pathologic subtypes: classical and cobblestone. Six different genes could be responsible for this entity (LIS1, DCX, TUBA1A, VLDLR, ARX, RELN), although co-delection of YWHAE gene with LIS1 could result in Miller-Dieker Syndrome. Heterotopia is defined as a cluster of normal neurons in abnormal locations, and divided into three main groups: periventricular nodular heterotopia, subcortical heterotopia and marginal glioneural heterotopia. Genetically, heterotopia is related to Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2) genes mutations. Polymicrogyria is described as an augmentation of small circonvolutions separated by shallow enlarged sulci; bilateral frontoparietal form is characterized by bilateral, symmetric polymicrogyria in the frontoparietal regions. Bilateral perisylvian polymicrogyria results in a clinical syndrome manifested by mild mental retardation, epilepsy and pseudobulbar palsy. Gene mutations linked to this disorder are SRPX2, PAX6, TBR2, KIAA1279, RAB3GAP1 and COL18A1. Schizencephaly, consisting in a cleft of cerebral hemisphere connecting extra-axial subaracnoid spaces and ventricles, is another important disorder of neuronal migration whose clinical characteristics are extremely variable. EMX2 gene could be implicated in its genesis. Focal cortical dysplasia is characterized by three different types of altered cortical laminations, and represents one of most severe cause of epilepsy in children. TSC1 gene could play a role in its etiology.<h4>Conclusion</h4>This review reports the main clinical, genetical and neuroradiological aspects of these disorders. It is hoped that accumulating data of the development mechanisms underlying the expanded network formation in the brain will lead to the development of therapeutic options for neuronal migration disorders.

Sporadic colon cancer is caused predominantly by dietary factors. We have selected bile acids as a focus of this review since high levels of hydrophobic bile acids accompany a Western-style diet, and play a key role in colon carcinogenesis. We describe how bile acid-induced stresses cause cell death in susceptible cells, contribute to genomic instability in surviving cells, impose Darwinian selection on survivors and enhance initiation and progression to colon cancer. The most likely major mechanisms by which hydrophobic bile acids induce stresses on cells (DNA damage, endoplasmic reticulum stress, mitochondrial damage) are described. Persistent exposure of colon epithelial cells to hydrophobic bile acids can result in the activation of pro-survival stress-response pathways, and the modulation of numerous genes/proteins associated with chromosome maintenance and mitosis. The multiple mechanisms by which hydrophobic bile acids contribute to genomic instability are discussed, and include oxidative DNA damage, p53 and other mutations, micronuclei formation and aneuploidy. Since bile acids and oxidative stress decrease DNA repair proteins, an increase in DNA damage and increased genomic instability through this mechanism is also described. This review provides a mechanistic explanation for the important link between a Western-style diet and associated increased levels of colon cancer.

The intracellular accumulation of unfolded or misfolded proteins is believed to contribute to aging and age-related neurodegenerative diseases. However, the links between age-dependent proteotoxicity and cellular protein degradation systems remain poorly understood. Here, we show that 26S proteasome activity and abundance attenuate with age, which is associated with the impaired assembly of the 26S proteasome with the 19S regulatory particle (RP) and the 20S proteasome. In a genetic gain-of-function screen, we characterized Rpn11, which encodes a subunit of the 19S RP, as a suppressor of expanded polyglutamine-induced progressive neurodegeneration. Rpn11 overexpression suppressed the age-related reduction of the 26S proteasome activity, resulting in the extension of flies' life spans with suppression of the age-dependent accumulation of ubiquitinated proteins. On the other hand, the loss of function of Rpn11 caused an early onset of reduced 26S proteasome activity and a premature age-dependent accumulation of ubiquitinated proteins. It also caused a shorter life span and an enhanced neurodegenerative phenotype. Our results suggest that maintaining the 26S proteasome with age could extend the life span and suppress the age-related progression of neurodegenerative diseases.

The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

Individuals with the birth defect synpolydactyly (SPD) have 1 or more digit duplicated and 2 or more digits fused together. One form of SPD is caused by polyalanine expansions in homeobox d13 (Hoxd13). Here we have used the naturally occurring mouse mutant that has the same mutation, the SPD homolog (Spdh) allele, and a similar phenotype, to investigate the molecular pathogenesis of SPD. A transgenic approach and crossing experiments showed that the Spdh allele is a combination of loss and gain of function. Here we identify retinaldehyde dehydrogenase 2 (Raldh2), the rate-limiting enzyme for retinoic acid (RA) synthesis in the limb, as a direct Hoxd13 target and show decreased RA production in limbs from Spdh/Spdh mice. Intrauterine treatment with RA restored pentadactyly in Spdh/Spdh mice. We further show that RA and WT Hoxd13 suppress chondrogenesis in mesenchymal progenitor cells, whereas Hoxd13 encoded by Spdh promotes cartilage formation in primary cells isolated from Spdh/Spdh limbs, and that this was associated with increased expression of Sox6/9. Increased Sox9 expression and ectopic cartilage formation in the interdigital mesenchyme of limbs from Spdh/Spdh mice suggest uncontrolled differentiation of these cells into the chondrocytic lineage. Thus, we propose that mutated Hoxd13 causes polydactyly in SPD by inducing extraneous interdigital chondrogenesis, both directly and indirectly, via a reduction in RA levels.

The role of chronic hepatitis C virus (HCV) in the pathogenesis of HCV-associated hepatocellular carcinoma (HCC) remains controversial. To understand the transition from benign to malignant, we studied the gene expression patterns in liver tissues at different stages, including normal, cirrhosis, and different HCC stages. We studied 108 liver tissue samples obtained from 88 distinct patients (41 HCV-cirrhotic tissues, 17 HCV-cirrhotic tissues from patients with HCC, and 47 HCV-HCC tissues). Differentially expressed genes (DEG) were studied by use of high-density oligonucleotide arrays. Among probe sets identified as differentially expressed via the F test, all pairwise comparisons were performed. Cirrhotic tissues with and without concomitant HCC were further evaluated, and a classifier was used to predict whether the tissue type was associated with HCC. Differential expression profiles were analyzed using Interaction Networks and Functional Analysis. Characteristic gene signatures were identified when normal tissue was compared with cirrhosis, cirrhosis with early HCC, and normal with HCC. Pathway analysis classified the cellular and biological functions of the DEG as related to cellular growth and proliferation, cell death and inflammatory disease in cirrhosis; cell death, cell cycle, DNA replication, and immune response in early HCCs; and cell death, cell growth and proliferation, cell cycle, and DNA repair in advanced HCCs. Characteristic gene signatures were identified at different stages of HCV-HCC progression. A set of genes were identified to predict whether the cirrhotic tissue was associated with HCC.

Living donor liver transplantation (LDLT) may have better immunological outcomes compared to deceased donor liver transplantation (DDLT). The aim of this study was to analyze the incidence of acute cellular rejection (ACR) after LDLT and DDLT. Data from the adult-to-adult living donor liver transplantation (A2ALL) retrospective cohort study on 593 liver transplants done between May 1998 and March 2004 were studied (380 LDLT; 213 DDLT). Median LDLT and DDLT follow-up was 778 and 713 days, respectively. Rates of clinically treated and biopsy-proven ACR were compared. There were 174 (46%) LDLT and 80 (38%) DDLT recipients with >/=1 clinically treated episodes of ACR, whereas 103 (27%) LDLT and 58 (27%) DDLT recipients had >/=1 biopsy-proven ACR episode. A higher proportion of LDLT recipients had clinically treated ACR (p = 0.052), but this difference was largely attributable to one center. There were similar proportions of biopsy-proven rejection (p = 0.97) and graft loss due to rejection (p = 0.16). Longer cold ischemia time was associated with a higher rate of ACR in both groups despite much shorter median cold ischemia time in LDLT. These data do not show an immunological advantage for LDLT, and therefore do not support the application of unique posttransplant immunosuppression protocols for LDLT recipients.

Bisamides are key fragments for the introduction of gem-diaminoalkyl residues into retroinverso pseudopeptide derivatives and in the synthesis of peptidomimetic compounds. The literature methods for these types of compounds have certain drawbacks. In particular, when amides react with electrophile-activated DMSO, the yields are rather low. We have found new electrophiles, 2,4,6-trichloro[1,3,5]triazine (CC) and 2,4-dichloro-6-methoxy[1,3,5]triazine (DCMT), which activate DMSO in the presence of amides to yield methylenebisamides in good to fair yields. The amides can be aromatic or aliphatic. The operation is simple and the reagents are inexpensive.

<h4>Background</h4>This study examines the prevalence and nature of bereavement help-seeking among the population who experienced an "expected" death in the five years before their survey response. Such whole population data are not limited by identification through previous access to specific services nor practitioners.<h4>Methods</h4>In a randomised, cross-sectional, state-wide population-based survey, 6034 people over two years completed face-to-face interviews in South Australia by trained interviewers using piloted questions (74.2% participation rate). Respondent demographics, type of grief help sought, and circumstantial characteristics were collected. Uni- and multi-variate logistic regression models were created.<h4>Results</h4>One in three people (1965/6034) had experienced an 'expected' death of someone close to them in the last five years. Thirteen per cent sought help for their grief from one or more: friend/family members (10.7%); grief counselors (2.2%); spiritual advisers (1.9%); nurses/doctors (1.5%). Twenty five respondents (1.3%) had not sought, but would have valued help with their grief.In multi-variate regression modeling, those who sought professional help (3.4% of the bereaved) had provided more intense care (OR 5.39; CI 1.94 to14.98; p < 0.001), identified that they were less able to 'move on' with their lives (OR 7.08; CI 2.49 to 20.13; p = 0.001) and were more likely not to be in full- or part-time work (OR 3.75; CI 2.31 - 11.82; p = 0.024; Nagelkerke's R2 = 0.33).<h4>Conclusion</h4>These data provide a whole-of-population baseline of bereavement help-seeking. The uniquely identified group who wished they had sought help is one where potentially significant health gains could be made as we seek to understand better any improved health outcomes as a result of involving bereavement services.

Common variants at only two loci, FTO and MC4R, have been reproducibly associated with body mass index (BMI) in humans. To identify additional loci, we conducted meta-analysis of 15 genome-wide association studies for BMI (n > 32,000) and followed up top signals in 14 additional cohorts (n > 59,000). We strongly confirm FTO and MC4R and identify six additional loci (P < 5 x 10(-8)): TMEM18, KCTD15, GNPDA2, SH2B1, MTCH2 and NEGR1 (where a 45-kb deletion polymorphism is a candidate causal variant). Several of the likely causal genes are highly expressed or known to act in the central nervous system (CNS), emphasizing, as in rare monogenic forms of obesity, the role of the CNS in predisposition to obesity.

Aggression is a heterogeneous heritable psychological trait, also influenced by environmental factors. Previous studies, mostly conducted on male population, have found some associations of the aggression with the polymorphisms of genes, regulating the activity of serotonin (5-HT) in the brain. However, psychological as well as biochemical manifestations of the aggression are different in males and females. Our study aimed to investigate the association of 5-HTT gene polymorphism with different facets of aggression (BDHI) in females. Two groups: the synchronized swimming and non-athlete control, - were examined to study the possible modulation effect of sport on the association between 5-HTT gene polymorphism and aggression. It was found that in both groups the low-active 5-HTT polymorphism (SS) was associated with increased scores on Indirect Hostility scale and decreased scores on Negativism scale, compared to LL genotype. No interaction effect between sport and 5-HTT polymorphism was found. The higher percentage of LL-carriers and lower of LS-carriers in the synchronized swimming group compared to the control one was observed. This may be the sign of the importance of LL polymorphism of 5-HTT gene, previously associated with higher resistance to stress factors, for being an athlete, although this result has to be taken cautiously keeping in mind the stratification problem. Synchronized swimmers had lower scores on Assault, Negativism, Irritability and Verbal Hostility compared to age-matched control girls (in general and for each 5-HTT genotype separately), suggesting that they may have more matured emotional system (older control group has also lower scores on these scales).

Potentiation of serotonergic transmission increases cognitive flexibility, but can in other circumstances increase sensitivity to stressful environmental cues. The personality trait Openness to Experience reflects and is also associated with an increased risk for mood disorders. We hypothesized that the personality trait has an association with a biomarker of serotonergic transmission, the plasma membrane serotonin transporter (5-HTT). In 50 healthy volunteers, we tested for correlations between scores on the NEO-PI-R scale Openness to Experience and its subscales, and cerebral binding of the 5-HTT selective PET radioligand [11C]DASB. Subjects were genotyped for the 5-HTT long/short polymorphism, and for a single nucleotide polymorphism in the long allele, designated LA/LG. Midbrain [11C]DASB binding correlated negatively with scores for Openness to Experience and its two subscales, Openness to Actions and Openness to Values. The latter subscore was negatively correlated with [11C]DASB binding in all brain regions in which [11C]DASB binding was quantified. Genetic analysis showed that homozygote LA carriers had significantly higher [11C]DASB binding in the caudate nucleus, but no significant differences in openness scores. Thus, high scores in personality facets indicative of cognitive flexibility and openness to change are associated with lower [11C]DASB binding. Lower abundance of 5-HTT sites may result in potentiation of serotonergic signaling, which occurs during treatment with SSRIs. We speculate that the set-point of serotonergic signaling in an individual represents a trade-off between flexibility and vulnerability when exposed to environmental stress.

BRMS1 and SUDS3 are related members of SIN3-HDAC chromatin remodeling complexes. We hypothesized that they might have overlapping functions and that SUDS3 over-expression could compensate for BRMS1 deficiency. SUDS3 expression was ubiquitous in seven breast cell lines, regardless of metastatic potential. SUDS3 over-expression in BRMS1-non-expressing metastatic cells did not suppress metastasis, motility, osteopontin secretion, or EGF receptor expression, phenotypes associated with BRMS1-mediated metastasis suppression. This study demonstrates functional differences for BRMS1 family members and highlights how the composition of SIN3-HDAC (BRMS1/SUDS3) complexes uniquely affects protein expression and biological behaviors.

We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear. To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes (1) highly sensitive to zinc; (2) associated with zinc homeostasis, i.e., metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs); (3) relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR. Results showed that zinc effect on genome-wide expression patterns was cell-type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1953 in HPR-1; 3534 in PC-3) with a threshold of +/-2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes' expression provided evidence for the cell type-dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes MT-1J and MT-1M, denoted previously as "nonfunctional" MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g., Fos, Akt1, Jak3 and PI3K, were highly regulated by zinc with cell-type specificity. This work provided an extensive database on zinc-related prostate cancer research. The strategy of data analysis was devoted to finding genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.

An increasing number of studies about the molecular basis of angiogenesis are rapidly disclosing novel signal pathways involved in the blood vessel formation process. This review will focus on bone morphogenic proteins, Hedgehog, Notch, ephrins, neuropilins, neurotrophins and netrins. These recently discovered angiogenesis mediators are involved in vascular development during embryogenesis and, interestingly, they are shared between the nervous and vascular systems. They represent new potential targets in the vasculature and suggest novel therapeutic opportunities.

Dosage compensation in male Drosophila relies on the X chromosome-specific recruitment of a chromatin-modifying machinery, the dosage compensation complex (DCC). The principles that assure selective targeting of the DCC are unknown. According to a prevalent model, X chromosome targeting is initiated by recruitment of the DCC core components, MSL1 and MSL2, to a limited number of so-called "high-affinity sites" (HAS). Only very few such sites are known at the DNA sequence level, which has precluded the definition of DCC targeting principles. Combining RNA interference against DCC subunits, limited crosslinking, and chromatin immunoprecipitation coupled to probing high-resolution DNA microarrays, we identified a set of 131 HAS for MSL1 and MSL2 and confirmed their properties by various means. The HAS sites are distributed all over the X chromosome and are functionally important, since the extent of dosage compensation of a given gene and its proximity to a HAS are positively correlated. The sites are mainly located on non-coding parts of genes and predominantly map to regions that are devoid of nucleosomes. In contrast, the bulk of DCC binding is in coding regions and is marked by histone H3K36 methylation. Within the HAS, repetitive DNA sequences mainly based on GA and CA dinucleotides are enriched. Interestingly, DCC subcomplexes bind a small number of autosomal locations with similar features.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, with about 170 million people infected worldwide. Up to 70% of patients will have persistent infection after inoculation, making this disease a significant cause of morbidity and mortality. The severity of disease varies widely, from asymptomatic chronic infection to cirrhosis and hepatocellular carcinoma. Since the discovery of HCV, the treatment of hepatitis C has considerably improved. Recently, combination of pegylated interferons with ribavirin gives a response rate of about 55%. Treatment is indicated in patients with moderate or severe fibrosis. The tolerability of combination treatment is relatively poor, with a frequent flu-like syndrome and an impaired quality of life. In addition to viral and environmental behavioural factors, host genetic diversity is believed to contribute to the spectrum of clinical outcomes in HCV infection. The sequencing of the human genome, together with the development of high-throughput technologies that measure the function of the genome, have afforded unique opportunities to develop profiles that can distinguish, identify and classify discrete subsets of disease, predict the disease outcome or predict the response to treatment. This paper reviews the published literature on gene expression associated with HCV infection (HCV infection, fibrosis progression), and also according to response to treatment.

Only a small proportion of genetic variation in complex traits has been explained by SNPs from genome-wide association studies (GWASs). We report the results from two GWASs for serum markers of iron status (serum iron, serum transferrin, transferrin saturation with iron, and serum ferritin), which are important in iron overload (e.g., hemochromatosis) and deficiency (e.g., anemia) conditions. We performed two GWASs on samples of Australians of European descent. In the first GWAS, 411 adolescent twins and their siblings were genotyped with 100K SNPs. rs1830084, 10.8 kb 3' of TF, was significantly associated with serum transferrin (p total association test = 1.0 x 10(-9); p within-family test = 2.2 x 10(-5)). In the second GWAS on an independent sample of 459 female monozygotic (MZ) twin pairs genotyped with 300K SNPs, we found rs3811647 (within intron 11 of TF, HapMap CEU r(2) with rs1830084 = 0.86) was significantly associated with serum transferrin (p = 3.0 x 10(-15)). In the second GWAS, we found two additional and independent SNPs on TF (rs1799852 and rs2280673) and confirmed the known C282Y mutation in HFE to be independently associated with serum transferrin. The three variants in TF (rs3811647, rs1799852 and rs2280673) plus the HFE C282Y mutation explained approximately 40% of genetic variation in serum transferrin (p = 7.8 x 10(-25)). These findings are potentially important for our understanding of iron metabolism and of regulation of hepatic protein secretion, and also strongly support the hypothesis that the genetic architecture of some endophenotypes may be simpler than that of disease.

<h4>Background</h4>X-linked sideroblastic anemia (XLSA) is associated with iron overload and mutations in ALAS2, which encodes 5-aminolevulinate synthase. There are few reports of XLSA in persons of sub-Saharan African descent.<h4>Methods</h4>A 47-year-old African-American man had microcytic anemia, elevated iron measures, cardiomyopathy, hepatic cirrhosis, diabetes mellitus, a history of cocaine use and hepatitis C. We amplified and directly sequenced his genomic DNA to detect mutations of SLC40A1, HFE, TFR2, HAMP, HJV and ALAS2.<h4>Results</h4>The subject's transferrin saturation was 100% and his serum ferritin was 2,960 ng/ml. An MRI scan revealed diffusely decreased T(2) signals of the heart, liver and pancreas. Transjugular right endomyocardial and liver biopsy specimens revealed marked iron deposition in cardiac myocytes and hepatocytes, and cirrhosis. He died of progressive cardiomyopathy. He was hemizygous for ALAS2 R452S (exon 9; c.1354C-->A) and heterozygous for SLC40A1 R561G (exon 8; c.1681A-->G). He did not have coding region mutations in HFE, TFR2, HAMP or HJV.<h4>Conclusions</h4>ALAS2 R452S largely explains this patient's microcytic anemia and multi-organ iron overload and dysfunction. SLC40A1 R561G may have increased his iron absorption and overload further. Acquired factors, especially cocaine use and hepatitis C, may have contributed to his clinical phenotype.

<h4>Background</h4>Periventricular heterotopia (PH) is an etiologically heterogeneous disorder characterized by nodules of neurons ectopically placed along the lateral ventricles. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. At present, two genes have been identified to cause PH when mutated. Mutations in FLNA (Xq28) and ARFGEF2 (20q13) are responsible for X-linked bilateral PH and a rare autosomal recessive form of PH with microcephaly. Chromosomal rearrangements involving the 1p36, 5p15, and 7q11 regions have also been reported in association with PH but the genes implicated remain unknown. Fourteen additional distinct anatomoclinical PH syndromes have been described, but no genetic insights into their causes have been gleaned.<h4>Methods</h4>We report the clinical and imaging features of three unrelated patients with epilepsy, mental retardation, and bilateral PH in the walls of the temporal horns of the lateral ventricles, associated with a de novo deletion of the 5q14.3-15 region. We used microarray-based comparative genomic hybridization to define the boundaries of the deletions.<h4>Results</h4>The three patients shared a common deleted region spanning 5.8 Mb and containing 14 candidate genes.<h4>Conclusion</h4>We identified a new syndrome featuring bilateral periventricular heterotopia (PH), mental retardation, and epilepsy, mapping to chromosome 5q14.3-q15. This observation reinforces the extreme clinical and genetic heterogeneity of PH. Array comparative genomic hybridization is a powerful diagnostic tool for characterizing causative chromosomal rearrangements of limited size, identifying potential candidate genes for, and improving genetic counseling in, malformations of cortical development.

The accumulation of mutant protein in intracellular aggregates is a common feature of neurodegenerative disease. In Huntington disease, mutant huntingtin leads to inclusion body (IB) formation and neuronal toxicity. Impairment of the ubiquitin-proteasome system (UPS) has been implicated in IB formation and Huntington disease pathogenesis. However, IBs form asynchronously in only a subset of cells with mutant huntingtin, and the relationship between IB formation and UPS function has been difficult to elucidate. Here, we applied single-cell longitudinal acquisition and analysis to monitor mutant huntingtin IB formation, UPS function, and neuronal toxicity. We found that proteasome inhibition is toxic to striatal neurons in a dose-dependent fashion. Before IB formation, the UPS is more impaired in neurons that go on to form IBs than in those that do not. After forming IBs, impairment is lower in neurons with IBs than in those without. These findings suggest IBs are a protective cellular response to mutant protein mediated in part by improving intracellular protein degradation.

Astrocytes play an important role in neuronal development through the release of soluble factors that affect neuronal maturation. Shotgun proteomics followed by gene ontology analysis was used in this study to identify proteins present in the conditioned medium of primary rat astrocytes. One hundred and thirty three secreted proteins were identified, the majority of which were never before reported to be produced by astrocytes. Extracellular proteins were classified based on their biological and molecular functions; most of the identified proteins were involved in neuronal development. Semi-quantitative proteomic analysis was carried out to identify changes in the levels of proteins released by astrocytes after stimulation with the cholinergic agonist carbachol, as we have previously reported that carbachol-treated astrocytes elicit neuritogenesis in hippocampal neurons through the release of soluble factors. Carbachol up-regulated secretion of 15 proteins and down-regulated the release of 17 proteins. Changes in the levels of four proteins involved in neuronal differentiation (thrombospondin-1, fibronectin, plasminogen activator inhibitor-1, and plasminogen activator urokinase) were verified by western blot or ELISA. In conclusion, this study identified a large number of proteins involved in neuronal development in the astrocyte secretome and implicated extracellular matrix proteins and protease systems in neuronal development induced by astrocyte cholinergic stimulation.

<h4>Background</h4>Viral epidemics or pandemics such as of influenza or severe acute respiratory syndrome (SARS) pose a significant threat. Antiviral drugs and vaccination may not be adequate to prevent catastrophe in such an event.<h4>Objectives</h4>To systematically review the evidence of effectiveness of interventions to interrupt or reduce the spread of respiratory viruses (excluding vaccines and antiviral drugs, which have been previously reviewed).<h4>Search strategy</h4>We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (<i>The Cochrane Library</i> 2006, issue 4); MEDLINE (1966 to November 2006); OLDMEDLINE (1950 to 1965); EMBASE (1990 to November 2006); and CINAHL (1982 to November 2006).<h4>Selection criteria</h4>We scanned 2300 titles, excluded 2162 and retrieved the full papers of 138 trials, including 49 papers of 51 studies. The quality of three randomised controlled trials (RCTs) was poor; as were most cluster RCTs. The observational studies were of mixed quality. We were only able to meta-analyse case-control data. We searched for any interventions to prevent viral transmission of respiratory viruses (isolation, quarantine, social distancing, barriers, personal protection and hygiene). Study design included RCTs, cohort studies, case-control studies, cross-over studies, before-after, and time series studies.<h4>Data collection and analysis</h4>We scanned the titles, abstracts and full text articles using a standardised form to assess eligibility. RCTs were assessed according to randomisation method, allocation generation, concealment, blinding, and follow up. Non-RCTs were assessed for the presence of potential confounders and classified as low, medium, and high risk of bias.<h4>Main results</h4>The highest quality cluster RCTs suggest respiratory virus spread can be prevented by hygienic measures around younger children. Additional benefit from reduced transmission from children to other household members is broadly supported in results of other study designs, where the potential for confounding is greater. The six case-control studies suggested that implementing barriers to transmission, isolation, and hygienic measures are effective at containing respiratory virus epidemics. We found limited evidence that the more uncomfortable and expensive N95 masks were superior to simple surgical masks. The incremental effect of adding virucidals or antiseptics to normal handwashing to decrease respiratory disease remains uncertain. The lack of proper evaluation of global measures such as screening at entry ports and social distancing prevent firm conclusions about these measures.<h4>Authors' conclusions</h4>Many simple and probably low-cost interventions would be useful for reducing the transmission of epidemic respiratory viruses. Routine long-term implementation of some of the measures assessed might be difficult without the threat of a looming epidemic.<h4>Plain language summary</h4><b>Interventions to interrupt or reduce the spread of respiratory viruses</b> Although respiratory viruses usually only cause minor disease, they can cause epidemics. Approximately 10% to 15% of people worldwide contract influenza annually, with attack rates as high as 50% during major epidemics. Global pandemic viral infections have been devastating because of their wide spread. In 2003 the severe acute respiratory syndrome (SARS) epidemic affected ˜8,000 people, killed 780, and caused an enormous social and economic crisis. A new avian influenza pandemic caused by the H5N1 strain might be more catastrophic. Single measures (particularly the use of vaccines or antiviral drugs) may be insufficient to interrupt the spread.We found 51 studies including randomised controlled trials (RCTs) and observational studies with a mixed risk of bias.Respiratory virus spread might be prevented by hygienic measures around younger children. These might also reduce transmission from children to other household members. Implementing barriers to transmission, isolation, and hygienic measures may be effective at containing respiratory virus epidemics. There was limited evidence that (more uncomfortable and expensive) N95 masks were superior to simple ones. Adding virucidals or antiseptics to normal handwashing is of uncertain benefit. There is insufficient evaluation of global measures such as screening at entry ports and social distancing.

<h4>Background</h4>Carriers of the Huntington disease (HD) mutation develop a progressive neurodegenerative disorder after a pre-clinical phase. We examined the value of (11)C-raclopride PET (RAC) as a biomarker for pre-clinical HD pathophysiology.<h4>Methods</h4>In a prospective cohort study with clinical and neuropsychological assessment we collected complete RAC data in 18 pre-clinical mutation carriers (HD-PMC) and 11 controls. Follow-up was 2 years. We calculated striatal RAC binding potential (BP) to measure dopamine D2 receptor availability.<h4>Results</h4>No HD-PMC had overt neuropsychological dysfunction. RAC-BP in putamen was abnormal in up to 44% of HD-PMC. The rate of RAC-BP decline (2.6% per year) was not significantly higher than in controls. Follow-up putaminal BP correlated weakly with predicted distance to onset of clinical HD (P = 0.034), but the rate of decline did not. Three HD-PMC developed motor abnormalities suspect for HD but did not show an increased rate of decline of putaminal BP.<h4>Conclusions</h4>Many HD-PMC have striatal abnormalities but we found no clearly increased rate of D2 receptor changes around the onset of clinical HD. A longer follow-up of the present study cohort is needed to establish the value of RAC-BP in assessing the risk of clinical conversion from striatal D2 binding data.

The presence of 3-O-sulfated glucosamine residues in heparin or heparan sulfate plays a role in binding to antithrombin III and HSV infection. In this study, tandem mass spectrometry was used to differentiate between two heparin disaccharide isomers containing variable sulfate at C6 in a common disaccharide and C3 in a more rare one. The dissociation patterns shown by MS(2) and MS(3) were clearly distinguishable between the isomers, allowing their differentiation and quantitation. Using this technique, we show that an octasaccharide with 11 sulfate groups with high affinity for inflammatory chemokine CCL2 does not contain 3-O-sulfated disaccharides.

<h4>Background</h4>The trajectory of corticospinal tract (CST) axons from cortex to spinal cord involves a succession of choice points, each of which is controlled by multiple guidance molecules. To assess the involvement of transmembrane semaphorins and their plexin receptors in the guidance of CST axons, we have examined this tract in mutants of Semaphorin-6A (Sema6A), Plexin-A2 (PlxnA2) and Plexin-A4 (PlxnA4).<h4>Results</h4>We describe defects in CST guidance in Sema6A mutants at choice points at the mid-hindbrain boundary (MHB) and in navigation through the pons that dramatically affect how many axons arrive to the hindbrain and spinal cord and result in hypoplasia of the CST. We also observe defects in guidance within the hindbrain where a proportion of axons aberrantly adopt a ventrolateral position and fail to decussate. This function in the hindbrain seems to be mediated by the known Sema6A receptor PlxnA4, which is expressed by CST axons. Guidance at the MHB, however, appears independent of this and of the other known receptor, PlxnA2, and may depend instead on Sema6A expression on CST axons themselves at embryonic stages.<h4>Conclusion</h4>These data identify Sema6A as a major contributor to the guidance of CST axons at multiple choice points. They highlight the active control of guidance at the MHB and also implicate the inferior olive as an important structure in the guidance of CST axons within the hindbrain. They also suggest that Sema6A, which is strongly expressed by oligodendrocytes, may affect CST regeneration in adults.

<h4>Background</h4>Understanding the molecular control of cell lineages and fate determination in complex tissues is key to not only understanding the developmental biology and cellular homeostasis of such tissues but also for our understanding and interpretation of the molecular pathology of diseases such as cancer. The prerequisite for such an understanding is detailed knowledge of the cell types that make up such tissues, including their comprehensive molecular characterisation. In the mammary epithelium, the bulk of the tissue is composed of three cell lineages, namely the basal/myoepithelial, luminal epithelial estrogen receptor positive and luminal epithelial estrogen receptor negative cells. However, a detailed molecular characterisation of the transcriptomic differences between these three populations has not been carried out.<h4>Results</h4>A whole transcriptome analysis of basal/myoepithelial cells, luminal estrogen receptor negative cells and luminal estrogen receptor positive cells isolated from the virgin mouse mammary epithelium identified 861, 326 and 488 genes as highly differentially expressed in the three cell types, respectively. Network analysis of the transcriptomic data identified a subpopulation of luminal estrogen receptor negative cells with a novel potential role as non-professional immune cells. Analysis of the data for potential paracrine interacting factors showed that the basal/myoepithelial cells, remarkably, expressed over twice as many ligands and cell surface receptors as the other two populations combined. A number of transcriptional regulators were also identified that were differentially expressed between the cell lineages. One of these, Sox6, was specifically expressed in luminal estrogen receptor negative cells and functional assays confirmed that it maintained mammary epithelial cells in a differentiated luminal cell lineage.<h4>Conclusion</h4>The mouse mammary epithelium is composed of three main cell types with distinct gene expression patterns. These suggest the existence of a novel functional cell type within the gland, that the basal/myoepithelial cells are key regulators of paracrine signalling and that there is a complex network of differentially expressed transcription factors controlling mammary epithelial cell fate. These data will form the basis for understanding not only cell fate determination and cellular homeostasis in the normal mammary epithelium but also the contribution of different mammary epithelial cell types to the etiology and molecular pathology of breast disease.

We report a cluster of patients from a Karaite Jew community with a movement disorder suggestive of Huntington disease (HD), in some cases associated with repeat lengths below the edge of 36 CAG repeats. The study describes the clinical and genetic features of four patients who were followed over several years. Patients belonged to an inbred family in whom progressive chorea, manifesting predominantly with dystonia and cerebellar features, developed during middle age. Although severe psychiatric symptoms ultimately developed in two of the four patients, cognitive function remained reasonably well preserved in all of them even after several disease years. Moderate cognitive deficits were limited to the visuomotor organization and abstract thinking subtests in three of the four patients. Qualitative brain imaging showed atrophy of brain predominantly involving cortex and cerebellum. Genetic testing revealed a variable mutation penetrance among family members, some affected members showing an upper allele size ranging from 34 to 49, whereas others remained unaffected despite the presence of the full mutation beyond 40 CAG repeats. Co-morbidity with recessive hereditary inclusion body myopathy was found in two subjects from one family. Although the main diagnosis of HD remains to be confirmed by further neuropathological studies, these cases may suggest that HD could manifest with as few as 34 CAG repeats, in some geographic areas, the disease phenotype most probably being influenced by additional, as yet unidentified, genes.

Normal homeostatic adjustment of elevated intraocular pressure (IOP) involves remodeling the extracellular matrix (ECM) of the trabecular meshwork (TM). This entails sensing elevated IOP, releasing numerous activated proteinases to degrade existing ECM and concurrent biosynthesis of replacement ECM components. To increase or decrease IOP, the quantity, physical properties and/or organization of new components should be somewhat different from those replaced in order to modify outflow resistance. ECM degradation and replacement biosynthesis in the outflow pathway must be tightly controlled and focused to retain the complex structural organization of the tissue. Recently identified podosome- or invadopodia-like structures (PILS) may aid in the focal degradation of ECM and organization of replacement components.

CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in the development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPbeta target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPbeta binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPbeta to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPalpha, beta and delta. Analysis of the hippocampal transcriptome of C/EBPbeta knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain.

Human serum transferrin (hTF) is a bilobal glycoprotein that transports iron to cells. At neutral pH, diferric hTF binds with nM affinity to the transferrin receptor (TFR) on the cell surface. The complex is taken into the cell where, at the acidic pH of the endosome ( approximately pH 5.6), iron is released. Since iron coordination strongly quenches the intrinsic tryptophan fluorescence of hTF, the increase in the fluorescent signal reports the rate constant(s) of iron release. At pH 5.6, the TFR considerably enhances iron release from the C-lobe (with little effect on iron release from the N-lobe). The recombinant soluble TFR is a dimer with 11 tryptophan residues per monomer. In the hTF/TFR complex these residues could contribute to and compromise the readout ascribed to iron release from hTF. We report that compared to Fe(C) hTF alone, the increase in the fluorescent signal from the preformed complex of Fe(C) hTF and the TFR at pH 5.6 is significantly quenched (75%). To dissect the contributions of hTF and the TFR to the change in fluorescence, 5-hydroxytryptophan was incorporated into each using our mammalian expression system. Selective excitation of the samples at 280 or 315 nm shows that the TFR contributes little or nothing to the increase in fluorescence when ferric iron is released from Fe(C) hTF. Quantum yield determinations of TFR, Fe(C) hTF and the Fe(C) hTF/TFR complex strongly support our interpretation of the kinetic data.

Glial-derived neurotrophic factor (GDNF) promotes both sensory and motor neuron survival. The delivery of GDNF to the peripheral nervous system has been shown to enhance regeneration following injury. In this study, we evaluated the effect of affinity-based delivery of GDNF from a fibrin matrix in a nerve guidance conduit on nerve regeneration in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated which received GDNF or nerve growth factor (NGF) with the delivery system within the conduit, control groups excluding one or more components of the delivery system, and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry and electron microscopy. The use of the delivery system (DS) with either GDNF or NGF resulted in a higher frequency of nerve regeneration vs. control groups, as evidenced by a neural structure spanning the 13 mm gap. The GDNF DS and NGF DS groups were also similar to the nerve isograft group in measures of nerve fiber density, percent neural tissue and myelinated area measurements, but not in terms of total fiber counts. In addition, both groups contained a significantly greater percentage of larger diameter fibers, with GDNF DS having the largest in comparison to all groups, suggesting more mature neural content. The delivery of GDNF via the affinity-based delivery system can enhance peripheral nerve regeneration through a silicone conduit across a critical nerve gap and offers insight into potential future alternatives to the treatment of peripheral nerve injuries.

No abstract available.

Eukaryotic chromosomes terminate in telomeres, complex nucleoprotein structures that are required for chromosome integrity that are implicated in cellular senescence and cancer. The chromatin at the telomere is unique with characteristics of both heterochromatin and euchromatin. The end of the chromosome is capped by a structure that protects the end and is required for maintaining proper chromosome length. Immediately proximal to the cap are the telomere associated satellite-like (TAS) sequences. Genes inserted into the TAS sequences are silenced indicating the chromatin environment is incompatible with transcription. This silencing phenomenon is called telomeric position effect (TPE). Two other silencing mechanisms have been identified in eukaryotes, suppressors position effect variegation [Su(var)s, greater than 30 members] and Polycomb group proteins (PcG, approximately 15 members). We tested a large number of each group for their ability to suppress TPE [Su(TPE)]. Our results showed that only three Su(var)s and only one PcG member are involved in TPE, suggesting silencing in the TAS sequences occurs via a novel silencing mechanism. Since, prior to this study, only five genes have been identified that are Su(TPE)s, we conducted a candidate screen for Su(TPE) in Drosophila by testing point mutations in, and deficiencies for, proteins involved in chromatin metabolism. Screening with point mutations identified seven new Su(TPE)s and the deficiencies identified 19 regions of the Drosophila genome that harbor suppressor mutations. Chromatin immunoprecipitation experiments on a subset of the new Su(TPE)s confirm they act directly on the gene inserted into the telomere. Since the Su(TPE)s do not overlap significantly with either PcGs or Su(var)s, and the candidates were selected because they are involved generally in chromatin metabolism and act at a wide variety of sites within the genome, we propose that the Su(TPE) represent a third, widely used, silencing mechanism in the eukaryotic genome.

To determine if changes in endometrial expression of the enzymes and receptors involved in prostaglandin (PG) synthesis and action might provide insights into the PGs involved in the initiation of decidualization, ovariectomized steroid-treated rats at the equivalent of day 5 of pseudopregnancy were given a deciduogenic stimulus and killed at various times up to 32 h thereafter. The expression of PG-endoperoxide synthases (PTGS1 and PTGS2), microsomal PGE synthases (PTGES and PTGES2), cytosolic PGE synthase (PTGES3), prostacyclin synthase (PTGIS), prostacyclin receptor, peroxisome proliferator-activated receptor delta (PPARD) and retinoid x receptor alpha (RXRA) in endometrium was assessed by semiquantitative RT-PCR, western blot analyses and immunohistochemistry. In addition, to determine which PG is involved in mediating decidualization, we compared the ability of PGE(2), stable analogues of PGI(2), L165041 (an agonist of PPARD), and docasahexanoic acid (an agonist of RXRA) to increase endometrial vascular permeability (EVP, an early event in decidualization), and decidualization when infused into the uterine horns of rats sensitized for the decidual cell reaction (DCR). EVP was assessed by uterine concentrations of Evans blue 10 h after initiation of infusions. DCR was assessed by the uterine mass 5 days after the initiation of the infusions. Because enzymes associated with the synthesis of PGE(2), including PTGS2, are up-regulated in response to a deciduogenic stimulus and because PGE(2) was more effective than the PGI(2) analogues and PPARD and RXRA agonists in increasing EVP and inducing decidualization, we suggest that PGE(2) is most likely the PG involved in the initiation of decidualization in the rat.

In addition to the differences between populations in transcriptional and translational regulation of genes, alternative pre-mRNA splicing (AS) is also likely to play an important role in regulating gene expression and generating variation in mRNA and protein isoforms. Recently, the genetic contribution to transcript isoform variation has been reported in individuals of recent European descent. We report here results of an investigation of the differences in AS patterns between human populations. AS patterns in 176 HapMap lymphoblastoid cell lines derived from individuals of European and African ancestry were evaluated using the Affymetrix GeneChip Human Exon 1.0 ST Array. A variety of biological processes such as response to stimulus and transcription were found to be enriched among the differentially spliced genes. The differentially spliced genes also include some involved in human diseases that have different prevalence or susceptibility between populations. The genetic contribution to the population differences in transcript isoform variation was then evaluated by a genome-wide association using the HapMap genotypic data on single nucleotide polymorphisms (SNPs). The results suggest that local and distant genetic variants account for a substantial fraction of the observed transcript isoform variation between human populations. Our findings provide new insights into the complexity of the human genome as well as the health disparities between the two populations.

Hereditary Haemochromatosis is an iron overload disorder associated with mutations in the HFE gene, and to a lesser degree, the gene encoding its chaperone protein beta-2 microglobulin (beta2M). Here, we report that knockdown of beta2M by RNAi restricts HFE distribution to the endoplasmic reticulum (ER). Additionally, we demonstrate that hepcidin, an iron homeostasis-associated protein, localises predominantly to LBPA-positive late endosomes. Interestingly, we show that knockdown of beta2M by RNAi perturbs hepcidin localisation to late endosomes. In summary, our data suggest that beta2M is essential for the correct subcellular distribution of both HFE and hepcidin, two proteins, which are critical for iron homeostasis.

By applying a method that combines end-sequence profiling and massively parallel sequencing, we obtained a sequence-level map of chromosomal aberrations in the genome of the MCF-7 breast cancer cell line. A total of 157 distinct somatic breakpoints of two distinct types, dispersed and clustered, were identified. A total of 89 breakpoints are evenly dispersed across the genome. A majority of dispersed breakpoints are in regions of low copy repeats (LCRs), indicating a possible role for LCRs in chromosome breakage. The remaining 68 breakpoints form four distinct clusters of closely spaced breakpoints that coincide with the four highly amplified regions in MCF-7 detected by array CGH located in the 1p13.1-p21.1, 3p14.1-p14.2, 17q22-q24.3, and 20q12-q13.33 chromosomal cytobands. The clustered breakpoints are not significantly associated with LCRs. Sequences flanking most (95%) breakpoint junctions are consistent with double-stranded DNA break repair by nonhomologous end-joining or template switching. A total of 79 known or predicted genes are involved in rearrangement events, including 10 fusions of coding exons from different genes and 77 other rearrangements. Four fusions result in novel expressed chimeric mRNA transcripts. One of the four expressed fusion products (RAD51C-ATXN7) and one gene truncation (BRIP1 or BACH1) involve genes coding for members of protein complexes responsible for homology-driven repair of double-stranded DNA breaks. Another one of the four expressed fusion products (ARFGEF2-SULF2) involves SULF2, a regulator of cell growth and angiogenesis. We show that knock-down of SULF2 in cell lines causes tumorigenic phenotypes, including increased proliferation, enhanced survival, and increased anchorage-independent growth.

Many studies have highlighted the pathological involvement of iron accumulation and iron-related oxidative stress (OS) in Alzheimer's disease (AD). Iron was further demonstrated to modulate expression of the Alzheimer's amyloid precursor holo-protein (APP) by a mechanism similar to that of regulation of ferritin-L and -H mRNA translation through an iron-responsive element (IRE) in their 5' untranslated regions (UTRs). Here, we discuss two aspects of the link between iron and AD, in relation to the recently discovered IRE in the 5'UTR of APP mRNA. The first is the physiological aspect: a compensatory neuroprotective response of amyloid-beta protein (Abeta) in reducing iron-induced neurotoxicity. Thus, given that Abeta possesses iron chelation sites, it is hypothesized that OS-induced intracellular iron may stimulate APP holo-protein translation (via the APP 5'UTR) and subsequently the generation of its cleavage product, Abeta, as a compensatory response that eventually reduces OS. The second is the pathological aspect: iron chelating compounds target the APP 5'UTR and possess the capacity to reduce APP translation, and subsequently Abeta levels, and thus represent molecules with high potential in the development of drugs for the treatment of AD.

<h4>Background</h4>Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.<h4>Methodology/principal findings</h4>Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1(-/y) neurons versus L1(+/y) neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1(-/y) neurons. Moreover, treatment of L1(+/y) neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cgamma (PLCgamma) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.<h4>Conclusions</h4>Neuronal surface sialylation and fucosylation are regulated via PLCgamma by L1, modulating neurite outgrowth, cell survival and migration.

Lipid peroxidation yields a variety of electrophiles, which are thought to contribute to the molecular pathogenesis of diseases involving oxidative stress, yet little is known of the scope of protein damage caused by lipid electrophiles. We identified protein targets of the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE) in RKO cells treated with 50 or 100 mum HNE. HNE Michael adducts were biotinylated by reaction with biotinamidohexanoic acid hydrazide, captured with streptavidin, and the captured proteins were resolved by one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, digested with trypsin, and identified by liquid chromatography-tandem mass spectrometry. Of the 1500+ proteins identified, 417 displayed a statistically significant increase in adduction with increasing HNE exposure concentration. We further identified 18 biotin hydrazide-modified, HNE-adducted peptides by specific capture using anti-biotin antibody and analysis by high resolution liquid chromatography-tandem mass spectrometry. A subset of the identified HNE targets were validated with a streptavidin capture and immunoblotting approach, which enabled detection of adducts at HNE exposures as low as 1 mum. Protein interaction network analysis indicated several subsystems impacted by endogenous electrophiles in oxidative stress, including the 26 S proteasomal and chaperonin containing TCP-1 (CCT) systems involved in protein-folding and degradation, as well as the COP9 signalosome, translation initiation complex, and a large network of ribonucleoproteins. Global analyses of protein lipid electrophile adducts provide a systems-level perspective on the mechanisms of diseases involving oxidative stress.

<h4>Aim</h4>The purpose of this study was to investigate the role of serotonergic genes in the development of alcohol dependence. The manifestation of alcohol withdrawal symptoms related to serotonergic polymorphisms in alcoholics was also examined.<h4>Methods</h4>The role of polymorphisms in the serotonin receptor 1A (5-HT1A), serotonin receptor 2A (5-HT2A), and the serotonin transporter gene (5-HTT) promotor region (5-HTTLPR) in the manifestation of individual alcohol withdrawal symptoms was investigated in 97 Korean male inpatients with alcohol dependence and 76 Korean healthy male subjects. The patient's alcohol withdrawal symptoms were assessed with the Clinical Institute Withdrawal Assessment for Alcohol (CIWA-Ar) scale.<h4>Results</h4>In the 5-HT1A receptor, the frequency of G- genotype (CC) was significantly higher in patients with alcohol dependence than in normal controls (chi(2) = 5.03, p = 0.025). The CIWA-Ar subscale scores of nausea, anxiety, and headache, and total CIWA-Ar scale scores were significantly higher in G+ genotypes (CG+ GG) than in G- genotype (p = 0.005, p = 0.004, p = 0.008, and p = 0.008, respectively).<h4>Conclusion</h4>The results suggest that the genetic polymorphism of the 5-HT1A receptor may play a role in alcohol dependence and polymorphisms of serotonergic genes may be important in withdrawal symptoms of patients with alcohol dependence.

<h4>Background</h4>Exposures of pregnant women to natural and manmade chemicals can lead to negative health effects in the baby, ranging from low birth weight to developmental defects. In some cases, diseases were postulated to have their basis in toxic exposure in utero or in early childhood. Therefore, an understanding of fetal responses to environmental exposures is essential. To that end, cord blood is a readily accessible biofluid whose proteomic makeup remains mostly unexplored when compared with that of adults.<h4>Objectives</h4>Our goal was an initial global assessment of the fetal serum proteome and for the identification of protein biomarkers indicative of toxic in utero exposures related to maternal cigarette smoking.<h4>Methods</h4>Drawing from a repository of 300 samples, we selected umbilical cord blood sera from 12 babies born to six smokers and six nonsmokers and analyzed both sample pools by tandem mass spectrometry in conjunction with isobaric tags (iTRAQ) for protein quantification.<h4>Results</h4>We identified 203 proteins, 17 of which were differentially expressed between the cigarette smoke-exposed and control populations. Most of the identified candidate biomarkers were biologically plausible, thereby underscoring the feasibility of screening neonates with global proteomic techniques for biomarkers of exposure and early biological effects triggered by in utero chemical exposures.<h4>Conclusions</h4>This validation study provides an initial view of the proteome of human cord blood sera; it demonstrates the feasibility of identifying therein by use of proteomics, biomarkers of environmental, toxic exposures.

<h4>Purpose</h4>Adiponectin possesses anti-inflammatory and insulin-sensitizing properties. Little is known about the role of adiponectin in hepatitis B-related liver disease.<h4>Methods</h4>Serum adiponectin and hepatitis B viral factors were cross-sectionally assayed in 280 patients with chronic hepatitis B virus (HBV) infection including 120 patients with chronic HBV infection, 40 patients with cirrhosis, and 120 patients with hepatocellular carcinoma (HCC); 116 healthy adults were used as controls. The dynamics of serum adiponectin level was also studied longitudinally in 25 patients with hepatitis B e antigen (HBeAg) seroconversion (SC).<h4>Results</h4>We found that serum adiponectin level in patients with chronic HBV infection was similar to that in healthy controls and was significantly lower than patients with cirrhosis and HCC. In univariate analysis, high serum adiponectin level significantly correlated with the presence of HBV-related cirrhosis or HCC, abnormal serum ALT level, and HBV genotype C. Multivariate analysis revealed that high serum adiponectin level significantly correlated with the development of HCC. Serum adiponectin levels remained stationary in patients experiencing HBeAg SC.<h4>Conclusions</h4>Our findings suggest that HBV infection itself does not affect adiponectin levels. Serum adiponectin level correlates with the progression of HBV-related liver diseases but not with the development of HBeAg SC.

Repeated measures data for rotavirus infection in children within 14 day care centres (DCCs) in the Oxfordshire area, UK, are used to explore aspects of rotavirus transmission and immunity. A biologically realistic model for the transmission of infection is presented as a set of probability models suitable for application to the data. Two transition events are modelled separately: incidence and recovery. The complexity of the underlying mechanistic model is reflected in the choice of the fixed variables in the probability models. Parameter estimation was carried out using a Bayesian Markov chain Monte Carlo method. We use the parameter estimates obtained to build a profile of the natural history of rotavirus reinfection in an individual child. We infer that rotavirus transmission in children in DCCs is dependent on the DCC prevalence, with symptomatic infection of longer duration, but no more infectious per day of infectious period, than asymptomatic infection. There was evidence that a recent previous infection reduces the risk of disease and, to a lesser extent, reinfection, but not duration of infection. The results provide evidence that partial immunity to rotavirus infection develops over several time scales.

Two of the most common signalling pathways in breast cancer are the ER (oestrogen receptor) ligand activation pathway and the E-cadherin snai1 slug EMT (epithelial-mesenchymal transition) pathway. Although these pathways have been thought to interact indirectly, the present study is the first to observe direct interactions between these pathways that involves the regulation of slug expression. Specifically we report that ligand-activated ERalpha suppressed slug expression directly by repression of transcription and that knockdown of ERalpha with RNA interference increased slug expression. More specifically, slug expression was down-regulated in ERalpha-negative MDA-MB-468 cells transfected with ERalpha after treatment with E2 (17beta-oestradiol). The down-regulation of slug in the ERalpha-positive MCF-7 cell line was mediated by direct repression of slug transcription by the formation of a co-repressor complex involving ligand-activated ERalpha protein, HDAC1 (histone deacetylase 1) and N-CoR (nuclear receptor co-repressor). This finding was confirmed by sequential ChIP (chromatin immunoprecipitation) studies. In the MCF-7 cell line, slug expression normally was low. In addition, knockdown of ERalpha with RNA interference in this cell line increased slug expression. This effect could be partially reversed by treatment of the cells with E2. The efficacy of the effect of ERalpha on slug repression was dependent on the overall level of ERalpha. These observations confirmed that slug was an E2-responsive gene.

The promise of nutrigenomics is of personalized nutrition that will lead to optimization or maintenance of good health and/or prevention of the development of chronic diseases. Type 2 diabetes mellitus (T2DM) is a leading health problem throughout the world. Adherence to a Mediterranean-style diet, regulation of carbohydrate intake, and regular exercise may be desirable. Four key genes were originally identified: KCNJ11, potassium inwardly rectifying channel, subfamily J, member 11 gene; PPAR-gamma, peroxisome proliferator activated receptor-gamma; TCF2, transcription factor 2, hepatic; WFS1, Wolfram syndrome 1. However, genome-wide association studies are accelerating our knowledge of the genetics of complex diseases, and have identified seven other key genes in T2DM: CDKAL1, CDK5 regulatory subunit associated protein-like 1; CDKN2, cyclin-dependent kinase inhibitor 2A; FTO, fat mass and obesity associated; HHEX, haematopoietically expressed homeobox; IDE, insulin-degrading enzyme; IGF2BP2, insulin-like growth factor 2 mRNA-binding protein 2; SLC30A8, solute carrier family 30 (zinc transporter), member 8; TCF7L2, transcription factor 7-like 2 (T-cell specific, HMG-box). Gene-nutrient or gene-environment interactions may be important. For example, the PPAR-gamma variant genotype is responsive to different types and levels of lipids, while the effect of the FTO variant can be partly overcome by exercise. Several of these genes act through their effect on the gastrointestinal tract. There are analytical challenges in analyzing the high-dimensional datasets relating genes, nutrients, and other variables to their influence on health and disease processes. An even greater challenge may be in implementing population level changes in diet and behavior to fully exploit the potential of this field.

The two main histological types of infiltrating breast cancer, lobular (ILC) and the more common ductal (IDC) carcinoma are morphologically and clinically distinct. To assess the molecular alterations associated with these breast cancer subtypes, we conducted a whole-genome study of 166 archival estrogen receptor (ER)-positive tumors (89 IDC and 77 ILC) using the Affymetrix GeneChip(R) Mapping 10K Array to identify sites of loss of heterozygosity (LOH) that either distinguished, or were shared by, the two phenotypes. We found single nucleotide polymorphisms (SNPs) of high-frequency LOH (>50%) common to both ILC and IDC tumors predominately in 11q, 16q, and 17p. Overall, IDC had a slightly higher frequency of LOH events across the genome than ILC (fractional allelic loss = 0.186 and 0.156). By comparing the average frequency of LOH by chromosomal arm, we found IDC tumors with significantly (P < 0.05) higher frequency of LOH on 3p, 5q, 8p, 9p, 20p, and 20q than ILC tumors. We identified additional chromosomal arms differentiating the subtypes when tumors were stratified by tumor size, mitotic rate, or DNA content. Of 5,754 informative SNPs (>25% informativity), we identified 78 and 466 individual SNPs with a higher frequency of LOH (P < 0.05) in ILC and IDC tumors, respectively. Hierarchical clustering of these 544 SNPs grouped tumors into four major groups based on their patterns of LOH and retention of heterozygosity. LOH in chromosomal arms 8p and 5q was common in higher grade IDC tumors, whereas ILC and low-grade IDC grouped together by virtue of LOH in 16q.

Hypoxia-inducible factor (HIF) is a transcriptional activator that promotes death or survival in neurons. The regulators and targets of HIF-1alpha-mediated death remain unclear. We found that prodeath effects of HIF-1 are not attributable to an imbalance in HIF-1alpha and HIF-1beta expression. Rather, the synergistic death caused by oxidative stress and by overexpression of an oxygen-resistant HIF-VP16 in neuroblasts was attributable to transcriptional upregulation of BH3-only prodeath proteins, PUMA or BNIP3. By contrast, overexpression of BNIP3 was not sufficient to potentiate oxidative death. As acidosis is known to activate BNIP3-mediated death, we examined other secondary stresses, such as oxidants or prolyl hydroxylase activity are necessary for exposing the prodeath functions of HIF in neurons. Antioxidants or prolyl hydroxylase inhibition prevented potentiation of death by HIF-1alpha. Together, these studies suggest that antioxidants and PHD inhibitors abrogate the ability of HIF-mediated transactivation of BH3-only proteins to potentiate oxidative death in normoxia. The findings offer strategies for minimizing the prodeath effects of HIF-1 in neurologic conditions associated with hypoxia and oxidative stress, such as stroke and spinal cord injury.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.

<h4>Aim</h4>The purpose of this study was to assess the level of satisfaction and understanding of test results, by a sample of non-C282Y homozygous participants in the hemochromatosis and iron overload screening (HEIRS) study, who received serum ferritin (SF), transferrin saturation (TS), and HFE gene test results by mail.<h4>Methods</h4>Approximately 1 month after receiving test results by mail, participants were surveyed about understanding of and satisfaction with results notification.<h4>Results</h4>Overall, participants were satisfied with receiving test results by mail. Participants receiving results with one or two HFE mutations or TS and/or SF levels outside the normal range (an "alert value") were less likely to be satisfied with this method of notification. Participants with normal HFE test results understood their results and recommendations better than those with one or two mutations. Although all participants received results letters in their native language, English-speaking participants had higher mean understanding scores than Mandarin, Vietnamese, or Spanish-speaking participants.<h4>Conclusion</h4>Participants were satisfied with receiving test results by mail. However, the level of understanding of the results was not sufficient for this mode of results notification to stand alone, especially for non-English speaking participants, and all participants with one or more test results outside the normal range.

Smoking behavior has been associated in two independent European cohorts with the most common Caucasian human leukocyte antigen (HLA) haplotype (A1-B8-DR3). We aimed to test whether polymorphic members of the two odorant receptor (OR) clusters within the extended HLA complex might be responsible for the observed association, by genotyping a cohort of Hungarian women in which the mentioned association had been found. One hundred and eighty HLA haplotypes from Centre d'Etude du Polymorphisme Humain families were analyzed in silico to identify single-nucleotide polymorphisms (SNPs) within OR genes that are in linkage disequilibrium with the A1-B8-DR3 haplotype, as well as with two other haplotypes indirectly linked to smoking behavior. A nonsynonymous SNP within the OR12D3 gene (rs3749971(T)) was found to be linked to the A1-B8-DR3 haplotype. This polymorphism leads to a (97)Thr --> Ile exchange that affects a putative ligand binding region of the OR12D3 protein. Smoking was found to be associated in the Hungarian cohort with the rs3749971(T) allele (p = 1.05 x 10(-2)), with higher significance than with A1-B8-DR3 (p = 2.38 x 10(-2)). Our results link smoking to a distinct OR allele, and demonstrate that the rs3749971(T) polymorphism is associated with the HLA haplotype-dependent differential recognition of cigarette smoke components, at least among Caucasian women.

<h4>Background and aims</h4>Malignant complications of celiac disease (CD) include carcinomas and lymphomas. The genetic basis behind cancer development in CD is not known, but acquisition of genetic abnormalities and genomic instability has been involved. The aim of this study was to explore molecular characteristics of genomic instability in CD patients by analyzing microsatellite instability (MSI) and loss of heterozygosis (LOH) with carefully selected microsatellites.<h4>Methods</h4>We genotyped small bowel biopsies and peripheral blood samples from 20 untreated CD patients using five microsatellites related to MMR genes (panel A), and five repeats associated with tumor suppressor genes, chromosome instability, inflammation, and cancer (panel B).<h4>Results</h4>Genomic instability was found in seven out of 20 (35%) cases at: D5S107, D18S58, GSTP, TP53 or DCC, being TP53 the most frequently affected (five out of seven cases; 71%). Microsatellite alterations were significantly found using panel B markers (P=0.04). No cases with high frequency of MSI and replication error phenotype were detected. Only one case displayed MSI-L alone. Three patients exhibited LOH and three other cases showed LOH with low level of MSI, being classified as having chromosome instability phenotype.<h4>Conclusion</h4>Two novel observations were found in this study: first, the finding that non-neoplastic cells from a group of untreated CD patients present genomic instability at nucleotide level; and second, the advantage to use carefully selected microsatellites to identify celiac patients with molecular instability. Our data support the existence of chromosome instability phenotype in CD, suggesting that stable and unstable patients are genomically distinct subtypes that may follow a different evolution.

We describe a patient with an unusual combination of hypersideremia (700 microg/dL), hypertransferrinemia (570 mg/dL), hyperferritinemia (800 microg/L), and monoclonal gammopathy of undetermined significance (MGUS), in which the monoclonal immunoglobulin showed specific transferrin-binding activity. Liver histology revealed hepatic iron overload, prominent in periportal hepatocytes, suggesting intestinal iron hyperabsorption. We demonstrate that low urinary hepcidin, likely due to impaired iron delivery to erythroid cells via the transferrin cycle pathway over time, may be the mechanism for iron loading. We suggest that MGUS associated with monoclonal antibodies with antitransferrin activity should be added to the list of acquired causes of hemochromatosis.

Transcription factors initiate programs of gene expression and are catalysts in downstream molecular cascades that modulate a variety of cellular processes. Pax3 is a transcription factor that is important in the melanocyte and influences melanocytic proliferation, resistance to apoptosis, migration, lineage specificity and differentiation. In this review, we focus on Pax3 and the molecular pathways that Pax3 is a part of during melanogenesis and in the melanocyte stem cell. These roles of Pax3 are emphasized during the development of diseases and syndromes resulting from either too much or too little Pax3 function. Due to its key task in melanocyte stem cells and tumors, the Pax3 pathway may provide an ideal target for either stem cell or cancer therapies.

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is an RNA-mediated disease. Dramatically expanded (CUG) repeats accumulate in nuclei and sequester RNA-binding proteins such as the splicing regulator MBNL1. We have employed resin-bound dynamic combinatorial chemistry (RBDCC) to identify the first examples of compounds able to inhibit MBNL1 binding to (CUG) repeat RNA. Screening an RBDCL with a theoretical diversity of 11 325 members yielded several molecules with significant selectivity for binding to (CUG) repeat RNA over other sequences. These compounds were also able to inhibit the interaction of GGG-(CUG)(109)-GGG RNA with MBNL1 in vitro, with K(i) values in the low micromolar range.

<h4>Aim</h4>To compare maternal and neonatal outcomes of Tibetan and Han Chinese women delivering vaginally at high altitude (3650 meters) in Lhasa, Tibet Autonomous Region, People's Republic of China.<h4>Method</h4>Comparative analysis of data from a prospective observational study of Tibetan (n = 938) and Han Chinese (n = 146) women delivering at three hospitals between January 2004 and May 2005.<h4>Results</h4>Han Chinese women had higher rates of pre-eclampsia/gestational hypertension than Tibetan women, (10.3% vs 5.9%, P = 0.04). There was no difference in rates of postpartum hemorrhage between Tibetan and Han women (12.8% vs 17.1%, P = 0.15). Han newborns weighed significantly less than Tibetan newborns (P < 0.01), and were twice as likely to be small for gestational age, (24.5% vs 11.6%, P < 0.01). Tibetan newborns were less likely to have poor neonatal outcomes than Han newborns (P < 0.01).<h4>Conclusion</h4>In high altitude deliveries in Tibet, adverse outcomes were significantly more common among Han Chinese.

Transactivation-responsive DNA-binding protein 43 (TDP-43) is a component of pathological inclusions in amyotrophic lateral sclerosis and several forms of sporadic and familial frontotemporal lobar degeneration. Transactivation-responsive DNA-binding protein 43-immunostained inclusions have also been found in other neurodegenerative disorders including Alzheimer disease, dementia with Lewy bodies, and parkinsonism dementia complex of Guam. Here, we analyzed the occurrence of TDP-43 immunostaining in Huntington disease, which is characterized by inclusions containing mutated huntingtin. In all Huntington disease cases studied, TDP-43 was frequently colocalized with huntingtin in dystrophic neurites and various intracellular inclusions, but not in intranuclear inclusions; the latter were only stained with huntingtin and anti-ubiquitin antibodies. Two phosphorylation-dependent TDP-43 antibodies proved to be superior for detecting pathological inclusions because they did not stain nonphosphorylated TDP-43 in normal nuclei; staining of normal nuclei with phosphorylation-independent antibodies obscured the inclusions. Our results further add to the hypothesis that TDP-43 may be involved in the pathology of a variety of neurodegenerative disorders.

The essential metals iron, zinc and copper deposit near the Abeta (amyloid beta-peptide) plaques in the brain cortex of AD (Alzheimer's disease) patients. Plaque-associated iron and zinc are in neurotoxic excess at 1 mM concentrations. APP (amyloid precursor protein) is a single transmembrane metalloprotein cleaved to generate the 40-42-amino-acid Abetas, which exhibit metal-catalysed neurotoxicity. In health, ubiquitous APP is cleaved in a non-amyloidogenic pathway within its Abeta domain to release the neuroprotective APP ectodomain, APP(s). To adapt and counteract metal-catalysed oxidative stress, as during reperfusion from stroke, iron and cytokines induce the translation of both APP and ferritin (an iron storage protein) by similar mechanisms. We reported that APP was regulated at the translational level by active IL (interleukin)-1 (IL-1-responsive acute box) and IRE (iron-responsive element) RNA stem-loops in the 5' untranslated region of APP mRNA. The APP IRE is homologous with the canonical IRE RNA stem-loop that binds the iron regulatory proteins (IRP1 and IRP2) to control intracellular iron homoeostasis by modulating ferritin mRNA translation and transferrin receptor mRNA stability. The APP IRE interacts with IRP1 (cytoplasmic cis-aconitase), whereas the canonical H-ferritin IRE RNA stem-loop binds to IRP2 in neural cell lines, and in human brain cortex tissue and in human blood lysates. The same constellation of RNA-binding proteins [IRP1/IRP2/poly(C) binding protein] control ferritin and APP translation with implications for the biology of metals in AD.

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45beta and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-kappaB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

The positive association between body iron stores and risk of coronary heart disease (CHD) initially observed among a Finnish male population has not been corroborated by studies conducted in other populations. The soluble transferrin receptor (sTfR):ferritin ratio has been suggested to be a better index than ferritin to measure body iron stores. Because sTfR is sensitive to iron deficiency, this ratio can distinguish individuals with similar ferritin levels with respect to their iron status. To evaluate this novel index in relation to CHD risk, we prospectively identified and confirmed 242 incident CHD cases and randomly selected 483 controls matched for age, smoking, and fasting status among women that provided blood samples in the Nurses' Health Study during 9 y of follow-up. In both crude and multivariate analyses, neither the sTfR:ferritin ratio nor ferritin was significantly associated with an elevated risk of CHD. After multivariate adjustment for established and potential CHD risk factors, compared with women in the lowest quartile of the sTfR:ferritin ratio, women in the 2nd to 4th quartiles had relative risks (RR) (95% CI) of 1.39 (0.82, 2.36), 1.12 (0.66, 1.91), and 1.13 (0.65, 1.97; P-trend = 0.61), respectively. The multivariate RR (95% CI) for ferritin were 1.05 (0.62, 1.77), 1.19 (0.69, 2.03), and 1.05 (0.60, 1.85; P-trend = 0.90) across quartiles. Our data do not support the hypothesis that excessive body iron stores are associated with risk of CHD.

<h4>Introduction</h4>The safety of antidepressants following overdose is critical because of the high risk of suicide attempts in depressed patients. This study was conducted to decrease the fatality rate of antidepressant overdoses by providing data to shift prescribing toward safer antidepressants.<h4>Methods</h4>US poison control data for 2000-2004 were analyzed by 25 antidepressant types. Medical outcome differences were quantified using a hazard index (number of major or fatal outcomes per 1000 reported antidepressant ingestions).<h4>Results</h4>Of 82,802 suicidal single-agent ingestions of identifiable antidepressants approved for use in the US, cases occurred predominantly in females and peaked in teens. Fatal cases peaked at 40 to 49 years of age. Suicidal ingestions of the SSRIs, SNRIs, and other antidepressants peaked in teens, lithium in the twenties, tricyclics and tetracyclics in the thirties, and MAO inhibitors in the forties. There were 40 major or fatal outcomes per 1000 cases. Weighted by antidepressant type, the mean hazard index for the 25 antidepressants was 79 (range: 0 to 292). Amoxapine (292), maprotiline (211), and desipramine (187) had the highest hazard indices. The tricyclic antidepressants, MAO inhibitors, maprotiline, and bupropion were in the more severe half of antidepressants, ranked by hazard index. All SSRIs had low hazard indices. Hazard index and exposure frequency were inversely correlated (R = -0.423, p = 0.035), while hazard index and use of critical care were positively correlated for the 25 antidepressant types (R = 0.797, p < 0.001). Clinical effect profiles for each antidepressant type are presented.<h4>Conclusion</h4>Suicidal overdose severity varied considerably by antidepressant type. Prescribing decisions should be informed by regularly updated comparative overdose severity data.

Hereditary hemochromatosis (HH) refers to several inherited disorders of iron metabolism leading to tissue iron overload. Classical HH is associated with mutations in HFE (C282Y homozygotes or C282Y/H63D compound heterozygotes) and is almost exclusively found in populations of northern European descent. Non-HFE associated HH is caused by mutations in other recently identified genes involved in iron metabolism. Hepcidin is an iron regulatory hormone that inhibits ferroportin-mediated iron export from enterocytes and macrophages. Defective hepcidin gene expression or function may underlie most forms of HH. Target organs and tissues affected by HH include the liver, heart, pancreas, joints, and skin, with cirrhosis and diabetes mellitus representing late signs of disease in patients with markedly elevated liver iron concentration. Compound heterozygotes have milder disease than C282Y homozygotes and clinical signs of HH in these patients are usually associated with other factors such as alcoholism and the dysmetabolic syndrome. The most frequent causes of death in HH are liver cancer, cirrhosis, cardiomyopathy, and diabetes, but patients who undergo successful iron depletion before the development of cirrhosis or diabetes can have normal survival. Classical HH is characterized by incomplete penetrance and variable expressivity, and women are less affected than men by iron overload and iron overload-related disease. The diagnosis of HH is established by genetic testing in patients with elevated transferrin saturation values. Patients with an established diagnosis of HH and iron overload should be treated with phlebotomy to achieve body iron depletion followed by maintenance phlebotomy. Population screening for HH is controversial principally because of incomplete penetrance, but screening of selected, high risk populations and first-degree relatives of affected probands may be cost effective.

<h4>Objective</h4>Recent reports have confirmed association of single-nucleotide polymorphisms (SNPs) mapping to the interleukin-23 receptor (IL-23R) and IL-12beta genes with psoriasis susceptibility. The aim of this study was to determine whether these variants are also associated with susceptibility to psoriatic arthritis (PsA).<h4>Methods</h4>Two IL23R SNPs (rs7530511 and rs11209026) and 2 IL12B SNPs (rs3212227 and rs6887695) were genotyped in DNA samples from 520 white patients with PsA and 2,260 control subjects, all of whom resided in the UK. For SNP rs3212227, data on a larger group of controls (n = 4,681) were publicly available; this information was used in the analysis. Genotype counts were compared between patients with PsA and population controls, using the trend test.<h4>Results</h4>A haplotype comprising carriage of the common variants of both IL23R SNPs was associated with PsA susceptibility (adjusted P = 0.013 [1,000 permutations]). Both IL12B SNPs were independently associated with PsA susceptibility, and this association was strongest under a dominant model, with homozygosity for the common allele being more frequent in patients with PsA than in control subjects: for rs3212227, the odds ratio (OR) for carriage of AA versus other genotypes was 1.43 (95% confidence interval [95% CI] 1.17-1.76); for rs6887695, the OR for carriage of GG versus other genotypes was 1.43 (95% CI 1.18-1.74).<h4>Conclusion</h4>Variation within IL23R and IL12B is associated with susceptibility to both psoriasis and PsA. The effect sizes observed in patients with PsA appear to be smaller than those previously reported in patients with psoriasis, suggesting that both loci are primarily associated with psoriasis susceptibility. However, this does support the idea that the genetic etiology of the psoriasis present in patients with PsA has susceptibility loci in common with those observed in patients with uncomplicated psoriasis.

No abstract available.

<h4>Background</h4>The etiologies of variable antidepressant response remain elusive. Aging and age-related illness add to the complexity and heterogeneity of late-life depression. The serotonin transporter (5-HTT) is the principal site of initial action for several antidepressants, including serotonin re-uptake inhibitors (SSRIs). The serotonin transporter-linked polymorphic region (5-HTTLPR) is the most widely studied polymorphism of the 5-HTT gene, SLC6A4, and is suspected of conferring vulnerability to elderly depression and resistance to treatment.<h4>Objective</h4>To present an up-to-date account of the influence of 5-HTT polymorphisms on elderly depression, antidepressant response and susceptibility to medication side effects.<h4>Method</h4>A Medline search (1993 - 2008) of 5-HTT gene variation studies and analyses that included elderly depressed subjects was performed using the terms: 'serotonin transporter'; '5-HTT'; 'SERT'; '5-HTTLPR'; 'late-life depression'; 'elderly depression'; 'geriatric depression'; 'antidepressants' and 'SSRIs'. Reference sections were gleaned for relevant articles that may have been overlooked by the search strategy.<h4>Conclusion</h4>5-HTTLPR may influence treatment response variability in late-life depression in a number of ways. Indirectly, 5-HTTLPR seems to influence the likelihood of adverse effects and non-adherence. Directly, the promoter region may contribute to response variability during the initial stages of treatment, which is explained, in part, by a gene-concentration interaction for paroxetine. Subjects with the S allele may be at an increased risk of adverse drug reactions and may require higher initial SSRI plasma concentrations to maximize response. Conversely, patients with the L/L genotype may respond even at lower concentrations.

Growth differentiation factor 15 (GDF15) is a divergent member of the transforming growth factor-beta superfamily and has been identified in different contexts as a hypoxia-inducible gene product and as a molecule involved in hepcidin regulation. The biology of iron and oxygen is closely related, and known regulatory pathways involving hypoxia-inducible factor (HIF) and iron-regulatory proteins (IRPs) are responsive to both these stimuli. We therefore sought to characterize the regulation of GDF15 by iron and oxygen and to define the involvement or otherwise of HIF and IRP pathways. Here we show that GDF15 is strongly up-regulated by stimuli that deplete cells of iron and that this response is specifically antagonized by the reprovision of iron. GDF15 exhibits greater sensitivity to iron depletion than hypoxia, and responses to hypoxia and iron depletion are independent of HIF and IRP activation, suggesting a novel mechanism of regulation. We also report significant induction of serum GDF15 in iron-deficient subjects and after administration of an iron chelator to normal subjects. These findings indicate that GDF15 can be induced by pathophysiologic changes in iron availability, raising important questions about the mechanism of regulation and its role in iron homeostasis.

The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a-dependent pathway in the zebrafish embryo.

Current ovarian cancer biomarkers are inadequate because of their relatively low diagnostic sensitivity and specificity. There is a need to discover and validate novel ovarian cancer biomarkers that are suitable for early diagnosis, monitoring, and prediction of therapeutic response. We performed an in-depth proteomics analysis of ovarian cancer ascites fluid. Size exclusion chromatography and ultrafiltration were used to remove high abundance proteins with molecular mass >/=30 kDa. After trypsin digestion, the subproteome (</=30 kDa) of ascites fluid was determined by two-dimensional liquid chromatography-tandem mass spectrometry. Filtering criteria were used to select potential ovarian cancer biomarker candidates. By combining data from different size exclusion and ultrafiltration fractionation protocols, we identified 445 proteins from the soluble ascites fraction using a two-dimensional linear ion trap mass spectrometer. Among these were 25 proteins previously identified as ovarian cancer biomarkers. After applying a set of filtering criteria to reduce the number of potential biomarker candidates, we identified 52 proteins for which further clinical validation is warranted. Our proteomics approach for discovering novel ovarian cancer biomarkers appears to be highly efficient because it was able to identify 25 known biomarkers and 52 new candidate biomarkers that warrant further validation.

The PAX2 gene encodes a transcription factor expressed during development. In humans, PAX2 mutations cause the renal-coloboma syndrome, whereas homozygous mutations are lethal, causing severe organ malformation, notably in the brain and kidney. Wilms tumor (WT) of the kidney results from a failure in the mesenchymal-epithelial transition, a crucial step partly controlled by PAX2. Downstream target genes regulated by PAX2 are still undefined. We therefore hypothesized that identification and characterization of the genes regulated by PAX2 may improve our understanding of developmentally related malignancies including WT. We used nickel agarose chromatin enrichment, chromatin immunoprecipitation, and the human embryonic kidney-derived cell line HEK293 to identify regulatory elements responding to PAX2. Among others, we identified WNT5A as a gene potentially regulated by PAX2. Here, we demonstrate that WNT5A is a direct target of PAX2 in HEK293 cells, using both transactivation and electrophoretic mobility shift assays. We were unable to find any WNT5A disease-associated mutations after screening a panel of 99 WT samples. However, quantitative reverse transcription-polymerase chain reaction in human favorable-histology WT revealed that approximately 66% of the cases expressed significantly less WNT5A than human fetal kidney. Moreover, the WiT9 WT cell line revealed a weak expression of the WNT5A gene. A correlation of decreased WNT5A expression with predominant blastemal histology tumors suggests a possible inhibitory role in WT pathogenesis. This study underlines the importance of PAX2 in the regulation of WNT5A. Further in vivo study is necessary to determine whether the PAX2 and WNT5A are truly involved in WT pathogenesis.

Ovarian carcinoma arises from the ovarian surface epithelium, which undergoes phenotypic changes characteristic of müllerian epithelium during the first stages of tumorigenesis. The variant isoform of the hepatocyte nuclear factor 1 (vHNF1) is a transcription factor involved in the development of tissues derived from the müllerian duct. Here, we show that vHNF1 knockdown in two ovarian carcinoma cell lines, SKOV3 and IGROV1, leads to reduced E-cadherin (E-cadh) expression and decreased proliferation rate. Accordingly, SKOV3 cells ectopically expressing a dominant-negative (DN) vHNF1 mutant undergo an epithelial-mesenchymal-like transition, acquiring a spindle-like morphology, loss of E-cadh, and disrupted cell-cell contacts. Gene expression profiling of DNvHNF1 cells on the basis of a newly compiled list of epithelial-mesenchymal transition-related genes revealed a correlation between vHNF1 loss-of-function and acquisition of the mesenchymal phenotype. Indeed, phenotypic changes were associated with increased Slug transcription and functionality. Accordingly, vHNF1-transfected immortalized ovarian surface epithelial cells showed down-regulation of Snail and Slug transcripts. In DNvHNF1-transfected SKOV3 cells, growth rate decreased, and in vHNF1-transfected immortalized ovarian surface epithelial cells, growth rate increased. By immunohistochemistry, we found a strong association of vHNF1 with E-cadh in clear cell and in a subset of serous carcinomas, data that could potentially contribute in distinguishing different types of ovarian tumors. Our results may help in understanding the biology of ovarian carcinoma, identifying early detection markers, and opening potential avenues for therapeutic intervention.

Forming and removing epigenetic histone marks at gene loci are central processes in differentiation. Here, we explored mechanisms establishing long-range H4 acetylation marks at the Ifng locus during Th1 lineage commitment. In Th0 cells, histone deacetylase (HDAC)-Sin3A complexes recruited to the Ifng locus actively prevented accumulation of H4 acetylation marks. Th1 differentiation caused loss of HDAC-Sin3A complexes by T-bet-dependent mechanisms and accumulation of H4 acetylation marks. HDAC-Sin3A complexes were absent from the locus in NOD Th0 cells, obviating the need for Th1 differentiation signals to establish histone marks and Th1 differentiation. Thus, Ifng transcription is actively prevented in Th0 cells via epigenetic mechanisms and epigenetic defects allow unregulated Ifng transcription that may contribute to autoimmunity.

Respiratory exposure to allergens can lead to airway tolerance. Factors that antagonize tolerance mechanisms in the lung might result in susceptibility to diseases such as asthma. We show that inhalation of endotoxin/LPS with Ag prevented airway tolerance and abolished protection from T cell-driven asthmatic lung inflammation. Under conditions leading to tolerance, adaptive Ag-specific CD4(+)Foxp3(+) T regulatory cells (Treg) were generated following exposure to intranasal Ag and outnumbered IL-4- and IFN-gamma-producing CD4 T cells by 100:1 or greater. Inhaled LPS altered the ratio of Treg to IL-4(+) or IFN-gamma(+) T cells by concomitantly suppressing Treg generation and promoting effector T cell generation. LPS induced OX40L expression on dendritic cells and B cells that resulted in a synergistic activity between TLR4 and OX40 signals, leading to production of IL-4, IFN-gamma, and IL-6, which blocked Treg development. Furthermore, inhibiting OX40/OX40L interactions prevented LPS from suppressing tolerance, and resulted in the generation of greater numbers of adaptive Treg. Thus, cooperation between TLR4 and OX40 controls susceptibility to developing airway disease via modulating the balance between adaptive Treg and IL-4(+) or IFN-gamma(+) T cells. Targeting OX40L then has the potential to improve the efficacy of Ag immunotherapy to promote tolerance.

We have previously reported that mGluR5 signaling via PLC-beta1 regulates the development of whisker patterns within S1 (barrel) cortex of mice (Hannan et al., 2001). However, whether these defects arise from the loss of postsynaptic mGluR5 signaling, and whether the level of mGluR5 is important for barrel formation, was not examined. Furthermore, whether mGluR5 regulates other developmental processes that occur before or after barrel development is not known. We now show that mGluR5 is present postsynaptically at thalamocortical synapses during barrel formation. In addition, Mglur5(+/-) mice exhibit normal TCA patch formation but reduced cellular segregation in layer 4, indicating a dose-dependent role for mGluR5 in the regulation of pattern formation. Furthermore Mglur5(-/-) and Mglur5(+/-) mice display normal cortical arealization, layer formation, and size of PMBSF indicating the defects within S1 do not result from general abnormalities of cortical mapping during earlier stages of development. At P21 layer 4 neurons from Mglur5(-/-) and Mglur5(+/-) mice show a significant reduction in spine density but normal dendritic complexity compared with Mglur5(+/+) mice indicating a role in synaptogenesis during cortical development. Finally, mGluR5 regulates pattern formation throughout the trigeminal system of mice as the representation of the AS whiskers in the PrV, VpM, and S1 cortex was disrupted in Mglur5(-/-) mice. Together these data indicate a key role for mGluR5 at both early and late stages of neuronal development in the trigeminal system of mice.

A variety of neurological disorders and polyglutamine (polyQ) diseases are caused by misfolded proteins. The common feature of these diseases is late-onset cellular degeneration that selectively affects neurons in distinct brain regions. polyQ diseases, including Huntington's disease (HD), present a clear case of selective neurodegeneration caused by polyQ expansion-induced protein misfolding, which also leads to predominant inclusions in neuronal nuclei. It remains unclear how these ubiquitously expressed disease proteins selectively kill neurons. In HD, mutant huntingtin accumulates in both neurons and glia, but more neuronal cells display huntingtin aggregates. These aggregates colocalize with components of the ubiquitin-proteasome system (UPS), which plays a critical role in clearing misfolded proteins. Using fluorescent reporters that reflect cellular UPS activity, we found that UPS activity in cultured neurons and glia decreases in a time-dependent manner. Importantly, UPS activity is lower in neurons than in glia and also lower in the nucleus than the cytoplasm. By expressing the UPS reporters in glia and neurons in the mouse brain, we also observed an age-dependent decrease in UPS activity, which is more pronounced in neurons than glial cells. Although brain UPS activities were similar between wild-type and HD 150Q knock-in mice, inhibiting the UPS markedly increases the accumulation of mutant htt in cultured glial cells. These findings suggest that the lower neuronal UPS activity may account for the preferential accumulation of misfolded proteins in neurons, as well as their selective vulnerability.

N-Acetyl-L-cysteine (NAC) is an antioxidant and anti-inflammatory agent with significant potential in clinical applications including stroke and neuroinflammation. The drug shows high plasma binding upon IV administration, requiring high doses and associated side effects. Through the use of an appropriate delivery vehicle, the stability and efficacy of NAC can be significantly improved. Dendrimers are an emerging class of nanoscale drug delivery vehicles, which enable high drug payloads and intracellular delivery. Poly(amidoamine) (PAMAM) dendrimer-NAC conjugates having cleavable disulfide linkages are designed for intracellular delivery based on glutathione levels. We have successfully synthesized two conjugates with a cationic G4-NH(2) and an anionic G3.5-COOH PAMAM dendrimer with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by (1)H NMR and MALDI-TOF analysis. NAC release from the conjugates at intracellular and extracellular glutathione (GSH) concentrations were evaluated by reverse phase HPLC (RP-HPLC) analysis, and approximately 70% of NAC payload was released within one hour at intracellular GSH concentrations (approximately 10 mM), whereas negligible NAC release was observed at extracellular GSH levels (2 microM). FITC-labeled conjugates showed that they enter cells rapidly and localize in the cytoplasm of lipopolysaccharide (LPS)-activated microglial cells (the target cells in vivo). The significantly improved efficacies of dendrimer-NAC conjugates in activated microglial cells was confirmed by measuring the nitrite inhibition in the cell culture medium, which is an indication of the antioxidative property of the drug. Both G4-NH(2) and G3.5-COOH conjugates showed significantly better nitrite inhibition both at 24 and 72 h compared to free NAC, by as much as a factor of 16. The results indicate that PAMAM dendrimer conjugates produce higher local NAC concentration inside the cells, with GSH-sensitive disulfide linker enabling efficient and rapid cellular release of the drug.

Previous studies showed that the mixed monoamine transporter inhibitor (6, RTI-112) reduced cocaine self-administration at a high level of serotonin transporter (5-HTT) occupancy with no detectable dopamine transporter (DAT) occupancy. In this study, a series of 3beta-(substituted phenyl)tropane-2beta-carboxylic acid methyl esters (7a-g), 3beta-(4-methoxyphenyl)tropane-2beta-carboxylic acid esters (8a-j), and 3beta-(4-methoxyphenyl)-2beta-[3-(4'-methylphenyl)isoxazol-5-yl]tropane (9) were synthesized and evaluated for their monoamine transporter binding affinities to identify potent and selective compounds for both the DAT and 5-HTT relative to the norepinephrine transporter (NET). A number of compounds showed high binding affinities for both the DAT and 5-HTT and low affinity for the NET. 3Beta-(4-methoxyphenyl)tropane-2beta-carboxylic acid 2-(3-iodo-4-aminophenyl)ethyl ester (8i) with an IC(50) value of 2.5 nM for the DAT and K(i) values of 3.5 and 2040 nM for the 5-HTT and NET, respectively, is the most potent and selective compound for the DAT and 5-HTT relative to the NET in this study.

The design and synthesis of a series of urea-based nonpolycyclic aromatic ligands with alkylaminoanilino side chains as telomeric and genomic G-quadruplex DNA interacting agents are described. Their interactions with quadruplexes have been examined by means of fluorescent resonance energy transfer melting, circular dichroism, and surface plasmon resonance-based assays. These validate the design concept for such urea-based ligands and also show that they have significant selectivity over duplex DNA, as well as for particular G-quadruplexes. The ligand-quadruplex complexes were investigated by computational molecular modeling, providing further information on structure-activity relationships. Preliminary biological studies using short-term cell growth inhibition assays show that some of the ligands have cancer cell selectivity, although they appear to have low potency for intracellular telomeric G-quadruplex structures, suggesting that their cellular targets may be other, possibly oncogene-related quadruplexes.

An imbalance between peptidases and their inhibitors leads to pulmonary disease. Imbalances occur in the adult and the neonate at risk for a specific set of lung pathologies. Serpins (serine peptidase inhibitors) make up the major source of antipeptidase activity in the lung. The purpose of this review is to describe the serpin mechanism of inhibition, their roles in the normal and pathological lung and their potential as therapeutic agents.

Hereditary hemochromatosis (HH) is caused by chronic hyperabsorption of dietary iron. Progressive accumulation of excess iron within tissue parenchymal cells may lead to severe organ damage. The most prevalent type of HH is linked to mutations in the HFE gene, encoding an atypical major histocompatibility complex class I molecule. Shortly after its discovery in 1996, the hemochromatosis protein HFE was shown to physically interact with transferrin receptor 1 (TfR1) and impair the uptake of transferrin-bound iron in cells. However, these findings provided no clue why HFE mutations associate with systemic iron overload. It was later established that all forms of HH result from misregulation of hepcidin expression. This liver-derived circulating peptide hormone controls iron efflux from duodenal enterocytes and reticuloendothelial macrophages by promoting the degradation of the iron exporter ferroportin. Recent studies with animal models of HH uncover a crucial role of HFE as a hepatocyte iron sensor and upstream regulator of hepcidin. Thus, hepatocyte HFE is indispensable for signaling to hepcidin, presumably as a constituent of a larger iron-sensing complex. A working model postulates that the signaling activity of HFE is silenced when the protein is bound to TfR1. An increase in the iron saturation of plasma transferrin leads to displacement of TfR1 from HFE and assembly of the putative iron-sensing complex. In this way, iron uptake by the hepatocyte is translated into upregulation of hepcidin, reinforcing the concept that the liver is the major regulatory site for systemic iron homeostasis, and not merely an iron storage depot.

Primary liver cancer remains one of the most lethal malignancies worldwide. Due to differences in prevalence of etiological factors the incidence of primary liver cancer varies among the world, with a peak in East-Asia. As this disease is still lethal in most of the cases, research has to be done to improve our understanding of the disease, offering insights for possible treatment options. For this purpose, animal models are widely used, especially mouse models. In this review, we describe the different types of mouse models used in liver cancer research, with emphasis on genetically engineered mice used in this field. We focus on hepatocellular carcinoma (HCC), as this is by far the most common type of primary liver cancer, accounting for 70%-85% of cases.

<h4>Background</h4>Long-term success in lung transplantation is limited by obliterative bronchiolitis (OB), yet the mechanism for this disease is not well understood. Chemokine SDF-1 and its receptor, CXCR4, have been reported to be involved in several fibrogenic processes by recruiting inflammatory and fibroblast progenitor cells into injured tissues. We hypothesized that the SDF-1/CXCR4 axis also plays a role in the pathogenesis of OB.<h4>Methods</h4>Using the mouse heterotopic allogeneic airway transplant model, we transplanted mouse tracheas from BALB/c donors into C57BL/6 recipients. At Day 10 after transplant, we found high expression of SDF-1 in cells in the sub-epithelial layers of the allograft. Approximately 26% of cells infiltrating the allograft were CD45(+)CXCR4(+), as determined by flow cytometry analysis.<h4>Results</h4>Treatment of the recipients with a CXCR4 antagonist, TN14003, decreased cell infiltration into the grafts at Day 10 post-implantation. At Day 42, a significant reduction in luminal occlusion was found in the TN14003-treated animals compared with controls (57.40% vs 98.21%, p < 0.01). To demonstrate the relevance of the SDF-1/CXCR4 axis in OB, sections of lung tissue obtained from lung transplant patients with OB were examined for SDF-1 and CXCR4 expression. We found a higher number of CXCR4- and SDF-1-positive cells in samples from patients with OB as compared with normal lungs.<h4>Conclusions</h4>These findings provide new insights into the mechanisms of lung chronic rejection and may lead to new intervention tools for the treatment of OB.

The neuronal gene repressor REST/NRSF recruits corepressors, including CoREST, to modify histones and repress transcription. REST also functions as a tumor suppressor, but the mechanism remains unclear. We identified chromodomain on Y-like (CDYL) as a REST corepressor that physically bridges REST and the histone methylase G9a to repress transcription. Importantly, RNAi knockdown of REST, CDYL, and G9a, but not CoREST, induced oncogenic transformation of immortalized primary human cells and derepression of the proto-oncogene TrkC. Significantly, transgenic expression of TrkC also induced transformation. This implicates CDYL-G9a, but not CoREST, in REST suppression of transformation, possibly by oncogene repression. CDYL knockdown also augments transformation in a cell culture model of cervical cancer, where loss of heterozygosity of the CDYL locus occurs. These findings demonstrate molecular strategies by which REST carries out distinct biological functions via different corepressors and provide critical insights into the role of histone-modifying complexes in regulating cellular transformation.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease linked to a polyQ (polyglutamine) expansion in the huntingtin protein. Although general brain atrophy is found in HD patients, the striatum is the most severely affected region. Loss or mutant forms of huntingtin were reported to disrupt fast axonal transport in Drosophila, squid, and mice. However, previous work did not resolve whether mutant huntingtin affects global axonal transport or only a subset of cargoes, nor did it resolve whether striatal neurons are preferentially sensitive to huntingtin-mediated defects. We used amyloid precursor protein (APP)-yellow fluorescent protein and brain-derived neurotrophic factor (BDNF)-mCherry fusion proteins as markers for fast axonal transport when huntingtin is altered. We found that movement of APP and BDNF is impaired in striatal and hippocampal, but not cortical, neurons from presymptomatic homozygous mutant mice carrying 150Q huntingtin knock-in mutations. In addition, loss of huntingtin disrupts APP axonal transport, whereas overexpression of wild-type, but not mutant, huntingtin enhances APP transport in all three types of neurons tested. These data suggest that a loss of wild-type huntingtin function in fast axonal transport plays important roles in the development of cell-type-specific defects in HD.

Although numerous studies have underlined the role of histone deacetylases (HDAC) in breast physiology and tumorigenesis, little is known on the particular contribution of the various classes of HDACs in these processes. Using estrogen receptor-alpha (ERalpha)-positive MCF-7 breast cancer cells, the effects of MC1575 and MC1568, two novel class II-specific HDAC inhibitors, were analyzed on cell proliferation, apoptosis, and estrogen signaling. The specificity of these HDAC inhibitors was validated by measuring histone and alpha-tubulin acetylation and by the specific in vitro inhibition of recombinant HDAC4 using histone and nonhistone substrates, contrasting with the lack of inhibition of class I HDACs. In addition, MC1575 did not inhibit class I HDAC gene expression, thus confirming the specific targeting of class II enzymes. Similar to trichostatin A (TSA), MC1575 displayed a dose-dependent antiproliferative effect and induced cell cycle arrest although this blockade occurred at a different level than TSA. Moreover, and in contrast to TSA, MC1575 had no effect on MCF-7 cells apoptosis. Interestingly, MC1575 was able to increase p21(waf1/CIP1) mRNA levels but did not regulate the expression of other genes such as cyclin D1, p27, p14(ARF), Bcl2, Baxalpha, Trail-R1, and Trail-R2. Finally, MC1575 strongly induced ERbeta gene expression but did not decrease ERalpha expression, nor did it switch hydroxytamoxifen to an agonist activity. Altogether, these data suggest that the class II HDAC subfamily may exert specific roles in breast cancer progression and estrogen dependence.

The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells cocultured with cancer-associated fibroblasts (CAF). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulting from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contact between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Because this two-way interaction is anticipated, the described coculture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.

Epigenetic changes are strongly associated with cancer development. DNA hypermethylation is associated with gene silencing and is often observed in CpG islands. Recently, it was suggested that aberrant CpG island methylation in tumors is directed by Polycomb (PcG) proteins. However, specific mechanisms responsible for methylation of PcG target genes in cancer are not known. Chronic infection and inflammation contribute to up to 25% of all cancers worldwide. Using glutathione peroxidase, Gpx1 and Gpx2, double knockout (Gpx1/2-KO) mice as a model of inflammatory bowel disease predisposing to intestinal cancer, we analyzed genome-wide DNA methylation in the mouse ileum during chronic inflammation, aging, and cancer. We found that inflammation leads to aberrant DNA methylation in PcG target genes, with 70% of the approximately 250 genes methylated in the inflamed tissue being PcG targets in embryonic stem cells and 59% of the methylated genes being marked by H3K27 trimethylation in the ileum of adult wild-type mice. Acquisition of DNA methylation at CpG islands in the ileum of Gpx1/2-KO mice frequently correlates with loss of H3K27 trimethylation at the same loci. Inflammation-associated DNA methylation occurs preferentially in tissue-specific silent genes and, importantly, is much more frequently represented in tumors than is age-dependent DNA methylation. Sixty percent of aberrant methylation found in tumors is also present in the inflamed tissue. In summary, inflammation creates a signature of aberrant DNA methylation, which is observed later in the malignant tissue and is directed by the PcG complex.

A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2-oxoglutarate-dependent dioxygenases, known as the HIF prolyl 4-hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF-1 in some of the salutary effects of iron chelation-induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development of HIF activators that do not bind iron a high priority. Here we report the development of a high throughput screen to develop novel HIF activators and/or PHD inhibitors for therapeutic use in the central nervous system (CNS). We show that tilorone, a low-molecular weight, antiviral, immunomodulatory agent is the most effective activator of the HIF pathway in a neuronal line. We also show that tilorone enhances HIF protein levels and increases the expression of downstream target genes independent of iron chelation and HIF PHD inhibition in vitro. We further demonstrate that tilorone can activate an HIF-regulated reporter gene in the CNS. These studies confirm that tilorone can penetrate the blood-brain barrier to activate HIF in the CNS. As expected from these findings, we show that tilorone provides effective prophylaxis against permanent ischemic stroke and traumatic spinal cord injury in male rodents. Altogether these findings identify tilorone as a novel and potent modulator of HIF-mediated gene expression in neurons with neuroprotective properties.

A large body of evidence from postmortem brain tissue and genetic analysis in humans and biochemical and pathological studies in animal models (transgenic and toxin) of neurodegeneration suggest that mitochondrial dysfunction is a common pathological mechanism. Mitochondrial dysfunction from oxidative stress, mitochondrial DNA deletions, pathological mutations, altered mitochondrial morphology, and interaction of pathogenic proteins with mitochondria leads to neuronal demise. Therefore, therapeutic approaches targeting mitochondrial dysfunction and oxidative damage hold great promise in neurodegenerative diseases. This review discusses the potential therapeutic efficacy of creatine, coenzyme Q10, idebenone, synthetic triterpenoids, and mitochondrial targeted antioxidants (MitoQ) and peptides (SS-31) in in vitro studies and in animal models of Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. We have also reviewed the current status of clinical trials of creatine, coenzyme Q10, idebenone, and MitoQ in neurodegenerative disorders. Further, we discuss newly identified therapeutic targets, including peroxisome proliferator-activated receptor-gamma-coactivator and sirtuins, which provide promise for future therapeutic developments in neurodegenerative disorders.

<h4>Aim</h4>To rapidly detect molecular alterations in different malignancies and investigate the possible role of Tp53, C-myc, and CCND1 genes in development of tumors in human organs and their adjacent normal tissues, as well as the possible relation between well- and poorly-differentiated tumors.<h4>Methods</h4>A tissue array consisting of seven different tumors was generated. The tissue array included 120 points of esophagus, 120 points of stomach, 80 points of rectum, 60 points of thyroid gland, 100 points of mammary gland, 80 points of liver, and 80 points of colon. Expressions of Tp53, C-myc, and CCND1 were determined by RNA in situ hybridization. 3' terminal digoxin-labeled anti-sense single stranded oligonucleotide and locked nucleic acid modifying probe were used.<h4>Results</h4>The expression level of Tp53 gene was higher in six different carcinoma tissue samples than in paracancerous tissue samples with the exception in colon carcinoma tissue samples (P < 0.05). The expression level of CCND1 gene was significantly different in different carcinoma tissue samples with the exception in esophagus and colon carcinoma tissue samples. The expression level of C-myc gene was different in esophagus carcinoma tissue samples (chi2 = 18.495, P = 0.000), stomach carcinoma tissue samples (chi2 = 23.750, P = 0.000), and thyroid gland tissue samples (chi2 = 10.999, P = 0.004). The intensity of signals was also different in different carcinoma tissue samples and paracancerous tissue samples.<h4>Conclusion</h4>Over-expression of the Tp53, CCND1, and C-myc genes appears to play a role in development of human cancer by regulating the expression of mRNA. Tp53, CCND1 and C-myc genes are significantly correlated with the development of different carcinomas.

<h4>Aim</h4>To evaluate the safety and efficacy of Vitamin E in children with chronic hepatitis B.<h4>Methods</h4>We randomly assigned patients with chronic hepatitis B, positive for hepatitis B e antigen (HBeAg), to receive either Vitamin E or placebo once daily for 6 mo in a 3:1 ratio and double-blind manner. The primary end point was HBeAg seroconversion, defined as the loss of HBeAg, undetectable levels of serum hepatitis B virus DNA, and the appearance of antibodies against HBeAg 12 mo after therapy.<h4>Results</h4>At baseline visit, 49 patients had normal and 43 had increased serum aminotransferase levels. Twenty-nine patients did not respond to previous treatment with interferon-alpha or lamivudine. Seventy-six children completed the study; 16 were non-compliant (n = 7), lost to follow-up (n = 7), or started another antiviral treatment (n = 3). Intention-to-treat analysis showed HBeAg seroconversion in 16 children (23.2%) treated with Vitamin E and two (8.7%) in the placebo group (P = 0.13). Vitamin E was well tolerated.<h4>Conclusion</h4>There is only a tendency that Vitamin E may promote HBeAg seroconversion. Therefore larger studies are needed to clarify the role of antioxidants in the therapy of chronic hepatitis B.

No abstract available.

Sustained progress in defining the molecular pathophysiology of hepatic fibrosis has led to a comprehensive framework for developing antifibrotic therapies. Indeed, the single greatest limitation in bringing new drugs to the clinical setting is a lack of clarity regarding clinical trial and treatment end points, not a lack of promising agents. A range of treatments, including those developed for other indications, as well as those specifically developed for hepatic fibrosis, are nearing or in clinical trials. Most are focused on attacking features of either hepatic injury and/or activated stellate cells and myofibroblasts, which are the primary sources of extracellular matrix (scar) proteins. Thus, features of injury and stellate cell activation provide a useful template for classifying these emerging agents and point to a new class of therapies for patients with fibrosing liver disease.

Prolyl hydroxylase domain (PHD) proteins play a major role in regulating the hypoxia-inducible factor (HIF) that induces expression of genes involved in angiogenesis, erythropoiesis, and cell metabolism, proliferation, and survival. Germ-line mutations in the prolyl hydroxylase domain 2 gene (PHD2) have been reported in patients with familial erythrocytosis but not in association with tumors. We describe a patient with erythrocytosis and recurrent paraganglioma who carries a newly discovered PHD2 mutation. This mutation affects PHD2 function and stabilizes HIF-alpha proteins. In addition, we demonstrate loss of heterozygosity of PHD2 in the tumor, suggesting that PHD2 could be a tumor-suppressor gene.

Insights into the host factors that contribute to an effective antiviral immune response may be obtained by examining global gene expression in simian human immunodeficiency virus (SHIV)-infected nonhuman primates that exhibit different virological outcomes. Immune responses and gene expression profiles in peripheral blood mononuclear cells (PBMCs) were compared between animals that controlled or did not control viremia after infection. Rectal inoculation of eight rhesus macaques with R5-tropic SHIV(SF162P3) resulted in a high level of plasma viremia during the acute phase of infection. The viremia was controlled to below levels of detection in six of these animals at the set point (controllers), whereas two animals had persistent viremia throughout the 140 wk that the animals were monitored (non-controllers). CD4(+) T-cell counts declined slightly in both controllers and non-controllers in the acute phase of infection, but CD4(+) T-cell counts continued to decline only in the non-controllers. Neutralizing antibodies to the challenge virus were variable and could not account for the control of viremia. However, analysis of the cellular gene expression profiles in the PBMCs from both groups of animals revealed distinctive gene expression patterns between controllers and non-controllers. Using the paired LPE test, 59 genes with p values <0.01 were identified and specific differences in the gene expression profiles in PBMCs from controllers versus non-controllers were detected.

The transcription factor REST silences neuronal gene expression in non-neuronal cells. In neurons, the protein is sequestered in the cytoplasm in part through binding to huntingtin. Polyglutamine expansions in huntingtin, which causes Huntington's disease (HD), abrogates REST-huntingtin binding. Consequently, REST translocates to the nucleus, occupies RE1 repressor sequences and decreases neuronal gene expression. In this work, we found that levels of several microRNAs (miRNAs) with upstream RE1 sites are decreased in HD patient cortices relative to healthy controls. Interestingly, one of these, the bifunctional brain enriched miR-9/miR-9*, targets two components of the REST complex: miR-9 targets REST and miR-9* targets CoREST. These data provide evidence for a double negative feedback loop between the REST silencing complex and the miRNAs it regulates.

Proteins containing extended polyglutamine repeats cause at least nine neurodegenerative disorders, but the mechanisms of disease-related neuronal death remain uncertain. We show that sympathetic neurons containing cytoplasmic inclusions formed by 97 glutamines expressed within human huntingtin exon1-enhanced green fluorescent protein (Q97) undergo a protracted form of nonapoptotic death that is insensitive to Bax deletion or caspase inhibition but is characterized by mitochondrial dysfunction. By treating the neurons with combined cytosine arabinoside and NGF withdrawal, we demonstrate that Q97 confers a powerful resistance to apoptosis at multiple levels: despite normal proapoptotic signaling (elevation of P-ser15-p53 and BimEL), there is no increase of Puma mRNA or Bax activation, both necessary for apoptosis. Even restoration of Bax translocation with overexpressed Puma does not activate apoptosis. We demonstrate that this robust inhibition of apoptosis is caused by Q97-mediated accumulation of Hsp70, which occurs through inhibition of proteasomal activity. Thus, apoptosis is reinstated by short hairpin RNA-mediated knockdown of Hsp70. These findings explain the rarity of apoptotic death in Q97-expressing neurons. Given the proteasomal blockade, we test whether enhancing lysosomal-mediated degradation with rapamycin reduces Q97 accumulation. Rapamycin reduces the amount of nonpathological Q25 by 70% over 3 d, but Q97 accumulation is unaffected. Interestingly, Q47 inclusions form more slowly as a result of constitutive lysosomal degradation, but faster-forming Q97 inclusions escape lysosomal control. Thus, cytoplasmic Q97 inclusions are refractory to clearance by proteasomal and lysosomal systems, leading to a toxicity that dominates over neuroprotective Hsp70. Our findings may explain the rarity of apoptosis but the inevitable cell death associated with polyQ inclusion diseases.

Genes play a major role in behavioral adaptation to challenging environmental stimuli, but the complexity of their contribution remains unclear. There is growing evidence linking disease phenotypes with genes on the one hand, and the genesis of stress-related disorders like major depression, as a result of exposure to stressful environmental pathogens on the other. Here we illustrate the convergent role of monoaminergic genes in regulating the underlying biological mechanisms of stress and the emotions. By reviewing data that support a role of monoaminergic and other related genes in environmental adaptation, we conclude by advocating the use of convergent approaches in examining the genetic modulation of disease phenotypes.

The etiology of type 1 diabetes (T1D) remains unknown, but a growing body of evidence points to infectious agents and/or components of early childhood diet. The National Institutes of Health has established the TEDDY Study consortium of six clinical centers in the United States and Europe and a data coordinating center to identify environmental factors predisposing to, or protective against, islet autoimmunity and T1D. From 2004-2009, TEDDY will screen more than 360,000 newborns from both the general population and families already affected by T1D to identify an estimated 17,804 children with high-risk HLA-DR,DQ genotypes. Of those, 7,801 (788 first-degree relatives and 7,013 newborns with no family history of T1D) will be enrolled in prospective follow-up beginning before the age of 4.5 months. As of May 2008, TEDDY has screened more than 250,000 newborns and enrolled nearly 5,000 infants--approximately 70% of the final cohort. Participants are seen every 3 months up to 4 years of age, with subsequent visits every 6 months until the subject is 15 years of age. Blood samples are collected at each visit for detection of candidate infectious agents and nutritional biomarkers; monthly stool samples are collected for infectious agents. These samples are saved in a central repository. Primary endpoints include (1) appearance of one or more islet autoantibodies (to insulin, GAD65 or IA-2) confirmed at two consecutive visits; (2) development of T1D. By age 15, an estimated 800 children will develop islet autoimmunity and 400 will progress to T1D; 67 and 27 children have already reached these endpoints.

<h4>Background</h4>Scholars acknowledge that both biologically-founded child temperament and environmental factors are influential in determining the quality of infant-mother attachment. We present evidence for gene by environment (G x E) interaction in the organization of attachment.<h4>Methods</h4>Participants were 88 typically developing infants and their mothers. Molecular genetic measures of the infants focused on the polymorphism in the serotonin transporter gene (5-HTTLPR, ss/sl vs. ll genotype). Mothers' responsiveness to their infants at 7 months was observed in lengthy naturalistic interactions, and was conceptualized as the environmental influence.<h4>Results</h4>For infants with a short allele (ss/sl ), variation in mothers' responsiveness was significantly associated with attachment security, assessed at 15 months in the Strange Situation. For those infants, low responsiveness predicted particularly high risk for insecure attachment, and high responsiveness offset that risk. For infants homozygous for the long allele (ll ), there was no association between responsiveness and attachment organization.<h4>Conclusions</h4>The findings show that the quality of early care serves to amplify or offset the risk conferred by genotype.

<h4>Aim</h4>To study the frequency of HFE gene mutations (C282Y, H63D, S65C) in a group of 54 sporadic PCT patients and in a group of healthy controls (blood donors) from Guipúzcoa, Spain. We studied the association of PCT with HCV, HBV, alcohol abuse, and other established risk factors.<h4>Methods</h4>The analysis of mutations was made by PCR. Allelic and genotypic frequencies were compared. Probability was determined and a Chi-squared test was performed.<h4>Results</h4>No association was observed between C282Y mutation and PCT (5.76 vs. 5% in controls). A high H63D mutation frequency was observed in PCT (34.25%) but was not statistically significant (controls 29.31%) because of the high prevalence of this mutation in the Basque general population. The S65C mutation was lower in PCT than in controls. There is a similar presence for H63D heterozygosis in PCT (38.8 vs. 38.8%). HCV association was observed in 35.18% of patients with PCT. HBV infected 7.4% of patients. Heavy alcohol intake (> 60 g/day) was present in 55.55% of patients. No HIV-infected patients were detected. The study of other risk factors revealed only one of the five women with PCT taking estrogens.<h4>Conclusion</h4>Our results found no relevant role for C282Y and H63D mutations. External factors such as HCV and alcohol could be determinant in the development of PCT in the Basque population.

Anemia is a very common clinical problem in patients with chronic kidney disease (CKD) and is associated with increased morbidity and mortality in these patients. Erythropoietin is a hormone synthesized that is deficient in the majority of patients with advanced kidney disease, thereby predisposing these patients to anemia. The other cause of anemia is deficiency of iron. Iron deficiency anemia is common in people with CKD and its importance in supporting erythropoiesis is unquestioned, especially in those patients treated with erythropoietin. Intravenous iron is frequently used to treat anemia in CKD patients and is very efficacious in increasing hemoglobin but at the same time there are some safety issues associated with it. The objective of this review is to assess the frequency of adverse drug events associated with four different iron formulations: two iron dextran products known as high and low molecular weight iron dextran, iron sucrose, and sodium ferric gluconate complex. Several electronic databases were searched. In general, with the exception of high molecular weight iron dextran, serious or life-threatening adverse events appeared rare. Iron sucrose has the least reported adverse events and high molecular weight iron dextran has the highest number of reported adverse events. Low molecular weight iron dextran and ferric gluconate fall in between these two for number of adverse drug events.

To identify novel proteins associated with the pathogenesis of nasopharyngeal carcinoma (NPC), a proteomic approach was used to screen for differential proteins between NPC and adjacent noncancerous nasopharyngeal epithelial tissue (ANNET). As a result, 21 differential proteins were identified by two-dimensional electrophoresis and mass spectrometer. Raf kinase inhibitor protein (RKIP), one of the downregulated proteins in NPC compared to ANNET, was investigated for its role in the metastasis of NPC. Western blot analysis and immunohistochemistry were used to detect RKIP expression in 5-8F and 6-10B NPC cell lines with the different metastatic potentials, and in NNET, primary NPC and NPC metastasis. Furthermore, high metastatic 5-8F with low RKIP expression and nonmetastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA, respectively, or with empty vector. The effects of RKIP expression on in vitro cell invasion, and the activity of Raf-1/MEK/ERK signaling pathway were analyzed in the transfected cells. The results showed that RKIP was significantly downregulated in 5-8F compared with 6-10B, in NPC compared with NNET, and not detectable in NPC metastasis. Overexpressed RKIP in 5-8F could decrease its in vitro cell invasion, whereas downregulated RKIP in 6-10B could increase its in vitro cell invasion. RKIP negatively regulated Raf-1/MEK/ERK signaling pathway in NPC cells, and activation of this signaling pathway by RKIP downregulation increased in vitro invasion of NPC cells. Taken together, our results suggest that RKIP may be a NPC metastasis suppressor, and decreased RKIP expression is associated with the increased invasive capability of NPC cells possibly through the activation of Raf-1/MEK/ERK pathway.

Peroxiredoxin 6 (PRX6) belongs to the 1-Cys class of peroxiredoxins and is recognized as an important antioxidant protein in tissues such as cardiac muscle, skin, and lung. Preliminary in vivo proteomic data have revealed that PRX6 is adducted by 4-hydroxynonenal (4HNE) in the livers of rats chronically fed an ethanol-containing diet. The goals of this study were to evaluate the in vitro effect of aldehyde adduction on PRX6 peroxidase activity, identify specific sites of aldehyde modification using mass spectrometry, and predict conformational changes due to adduction using molecular modeling. PRX6 was found to be resistant to inactivation via aldehyde modification; however, Western blots of adducted protein revealed that both 4HNE and 4-oxononenal (4ONE) caused extensive cross-linking, resulting in high molecular mass species. Tandem mass spectrometry (ESI-LC-MS/MS) analysis demonstrated multiple sites of modification, but adduction of the active site Cys47 was not observed. Molecular modeling simulations indicated that adduction at Cys91 results in a change in protein active site conformation, which potentially restricts access of 4-HNE to Cys47. The Cys91-Lys209 cross-linked adducts could provide the conformational changes required to inactivate the protein by either restricting access to electrophiles or preventing important amino acid interactions within the catalytic triad.

Quantitative mass spectrometry is often performed using isotopically labeled samples. Although the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e., a heavy form containing nine deuteriums and a light form without deuterium). In the present report, two additional forms containing three and six deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents coelute from reversed-phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these four tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis, should allow these labels to be more widely used for proteomics and peptidomics analyses.

Metalloporphyrin-based phosphorescent nanoprobes are being developed for two-photon microscopy of oxygen. In these molecular constructs generation of porphyrin triplet states upon two-photon excitation is induced upon the intramolecular Förster-type resonance energy transfer from a covalently attached 2P antenna. In the earlier developed prototypes, electron transfer between the antenna and the metalloporphyrin strongly interfered with the phosphorescence, reducing the sensitivity and the dynamic range of the sensors. By tuning the distances between the antenna and the core and adjusting their redox potentials the unwanted electron transfer could be prevented. An array of phosphorescent Pt porphyrins (energy transfer acceptors) and 2P dyes (energy transfer donors) was screened using dynamic quenching of phosphorescence, and the FRET-pair with the minimal ET rate was identified. This pair, consisting of Coumarin-343 and Pt meso-tetra-(4-alkoxyphenyl)porphyrin, was used to construct a probe in which the antenna fragments were linked to the termini of G3 poly(arylglycine) (AG) dendrimer with PtP core. The folded dendrimer formed an insulating layer between the porphyrin and the antenna, simultaneously controlling the rate of oxygen quenching (Stern-Volmer oxygen quenching constant). Modification of the dendrimer periphery with oligoethyleneglycol residues made the probe's signal insensitive to the presence of proteins and other macromolecular solutes.

Anomalous choline metabolic patterns have been consistently observed in vivo using Magnetic Resonance Spectroscopy (MRS) analysis of patients with neurodegenerative diseases and tissues from cancer patient. It remains unclear; however, what signaling events may have triggered these choline metabolic aberrancies. This study investigates how changes in choline and phospholipid metabolism are regulated by distinct changes in the mitochondrial electron transport system (ETS). We used specific inhibitors to down regulate the function of individual protein complexes in the ETS of SH-SY5Y neuroblastoma cells. Interestingly, we found that dramatic elevation in the levels of phosphatidylcholine metabolites could be induced by the inhibition of individual ETS complexes, similar to in vivo observations. Such interferences produced divergent metabolic patterns, which were distinguishable via principal component analysis of the cellular metabolomes. Functional impairments in ETS components have been reported in several central nervous system (CNS) diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases.

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19(ARF) gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

An enantioselective synthesis of the ABD-ring of (-)-phomactin A is described here. The sequence features Rawal's asymmetric Diels-Alder cycloaddition. The overall length is significantly reduced from our previous attempt.

A facile synthetic approach for the preparation of well-defined (co)polymers bearing pendent alkene functionalities was established by selective reversible addition-fragmentation chain transfer (RAFT) (co)polymerization. A divinyl monomer 4-(3'-buten-1'-oxy)-2,3,5,6-tetrafluorostyrene (1) with a styrenyl group and a pendent alkene group was synthesized. Due to a very high reactivity of the styrenyl group relative to the alkene group in 1, functional fluoro(co)polymers with both well-defined structures and pendent alkene groups were prepared by RAFT polymerizations of 1 and copolymerization of 1 with pentafluorostyrene (PFS). Alkene-functionalized diblock copolymers were also prepared by RAFT copolymerization of 1 with PFS or styrene, extending from a poly(styrene-alt-maleic anhydride) macro-chain transfer agent. Hydrolysis and ammonolysis of these copolymers resulted in amphiphilic diblock fluorocopolymers with alkene-functionalized hydrophobic segments, which were shown to form internally-functionalized micelles in THF-water.

INTRODUCTIONCore histones can be purified from a variety of cell sources, including Drosophila embryos, HeLa tissue culture cells, calf thymus, or chicken erythrocytes. Chick erythrocytes are an excellent source of cellular histones: Large quantities of source material are readily obtainable, the purified histones have low levels of post-translational modifications, and linker histones can also be purified from the same cell sample. Also, avian histones have an amino acid sequence identical to that of human histones. Histone stocks can be stored successfully for more than a year at 4°C and for several years at -20°C. With this protocol, 200 mL of blood usually yields in excess of 50 mg of purified histone octamers. Additional optional procedures are also presented for the purification of H1 and H5 linker histones, as well as for the preparation of H3/H4 tetramers and H2A/H2B dimers.

An enantioselective synthesis of the ABD-ring of (-)-phomactin A is described here. The sequence features Rawal's asymmetric Diels-Alder cycloaddition. The overall length is significantly reduced from our previous attempt.

No abstract available.

No abstract available.