Gene Literature Dashboard

GnRH neurons follow a carefully orchestrated journey from their birth in the olfactory placode area. Initially, they migrate along with the vomeronasal nerve into the brain at the cribriform plate, then progress caudally to sites within the hypothalamus where they halt and send projections to the median eminence to activate pituitary gonadotropes. Many factors controlling this precise journey have been elucidated by the silencing or over-expression of candidate genes in mouse models. Importantly, a number of these factors may not only play a role in normal physiology of the hypothalamic-pituitary-gonadal axis but also be mis-expressed to cause human disorders of GnRH deficiency, presenting as a failure to undergo normal pubertal development. This review outlines the current cadre of candidates thought to modulate GnRH neuronal migration. The further elucidation and characterization of these factors that impact GnRH neuron development may shed new light on human reproductive disorders and provide potential targets to develop new pro-fertility or contraceptive agents.

A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis, and characterization of amino-reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 and >530nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 and 546nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells.

The transcription factor hypoxia-inducible factor-1α (HIF-1α) is a master regulator of the cellular response to low oxygen. HIF-1α protein accumulates in hypoxia due to inhibition of prolyl hydroxylase enzymes, which under normoxic conditions use molecular oxygen to hydroxylate HIF-1α on two conserved proline residues (Pro(402) and Pro(564)), thus targeting the protein for 26 S proteasome-dependent degradation. A functional mitochondrial electron transport chain is known to be necessary for HIF-1α stabilization in hypoxia. It has been reported that reactive oxygen species (ROS), produced under hypoxia by complex III of the mitochondrial electron transport chain, play a critical role in the stabilization of the HIF-1α protein, possibly by directly inhibiting prolyl hydroxylase enzymes. In contrast, we found that ROS production by complex III is not required for hypoxia-induced HIF-1α stabilization. Thus, reestablishing mitochondrial oxygen consumption in the presence of a complex III inhibitor by using an artificial electron donor to complex IV or by overexpressing Ciona intestinalis alternative oxidase results in HIF-1α protein stabilization in hypoxia. Furthermore, five inhibitors that target different sites of the mitochondrial electron transport chain have similar effects on the HIF-1α protein half-life in hypoxia but vary in their effects on mitochondrial ROS production. Finally, ROS do not regulate prolyl hydroxylase activity directly. We conclude that HIF-1α protein stabilization in hypoxia occurs independently of mitochondrial ROS production. However, mitochondria can modulate the cellular hypoxic response through altered respiratory activity, likely by regulating the cellular oxygen availability.

Sox9 belongs to the family of Sry-related high-mobility group box transcription factors controlling cell fate, cell proliferation and differentiation in various tissues, including cartilage, testis, the central nervous system, kidney, and gastrointestine. Mice conditionally lacking Sox9 have revealed essential roles for Sox9 in these tissues. To gain further insight into the role of Sox9 in different tissues and at different stages of development, we have generated a transgenic mouse line to express Sox9 in a Cre recombinase-dependent manner. The construct contained the human cytomegalovirus enhancer and chicken β-actin promoter, and a floxed monomeric red fluorescence protein 1 (mRFP1) cassette to direct ubiquitous expression of mRFP1. Cre expression removed the mRFP1 gene, allowing expression of Sox9 and enhanced green fluorescent protein. Expectedly, overexpression of Sox9 in chondrocytes using Col2a1-Cre mice suppressed chondrocyte hypertrophy, and delayed terminal differentiation and subsequent ossification. Misexpression of Sox9 in hypertrophic chondrocytes using Col10a1-Cre knock-in mice also delayed terminal differentiation. This Sox9 conditional transgenic mouse line will be a valuable tool to uncover tissue-specific and developmental stage-specific functions of Sox9.

Recently, a variant allele in the 3'UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as ovarian cancer, and was most enriched in ovarian cancer patients from hereditary breast and ovarian cancer families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, we have genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, we also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. Importantly, results from the current study suggest that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers.

RhoA/Rho-kinase pathway plays an important role in many pathological conditions. RhoA participates in the regulation of smooth muscle tone and activates many downstream kinases. The best characterized are the serine/threonine kinase isoforms (Rho-kinase or ROCK), ROCKα/ROCK2 and ROCKβ/ROCK1. ROCK is necessary for diverse functions such as local blood flow, arterial/pulmonary blood pressure, airway resistance and intestinal peristalsis. ROCK activation permits actin/myosin interactions and smooth muscle cells contraction by maintaining the activity of myosin light-chain kinase, independently of the free cytosolic calcium level. The sensitization of smooth muscle myofilaments to calcium has been implicated in many pathological states, such as hypertension, diabetes, heart attack, stroke, pulmonary hypertension, erectile dysfunction, and cancer. The focus of this review is on the involvement of RhoA/Rho-kinase in diseases. We will briefly describe the ROCK isoforms and the role of RhoA/Rho-kinase in the vasculature, before exploring the most recent findings regarding this pathway and various diseases.

<h4>Background</h4>It is believed that hereditary hemochromatosis (HH) might play a role in cardiac disease (heart failure (HF) and ischemia). Mutations within several genes are HH-associated, the most common being the HFE gene. In a large cohort of HF patients, we sought to determine the etiological role and the prognostic significance of HFE genotypes.<h4>Methods</h4>We studied 667 HF patients (72.7% men) with depressed systolic function, enrolled in a multicentre trial with a follow-up period of up to 5 years. All were genotyped for the known HFE variants C282Y, H63D and S65C.<h4>Results</h4>The genotype and allele frequencies in the HF group were similar to the frequencies determined in the general Danish population. In multivariable analysis mortality was not predicted by C282Y-carrier status (HR 1.2, 95% CI: 0.8-1.7); H63D-carrier status (HR 1.0, 95% CI: 0.7-1.3); nor S65C-carrier status (HR 1.2, 95% CI: 0.7-2.0). We identified 27 (4.1%) homozygous or compound heterozygous carriers of HFE variants. None of these carriers had a clinical presentation suggesting hemochromatosis, but hemoglobin and ferritin levels were higher than in the rest of the cohort. Furthermore, a trend towards reduced mortality was seen in this group in univariate analyses (HR 0.4, 95% CI: 0.2-0.9, p = 0.03), but not in multivariate (HR 0.5, 95% CI: 0.2-1.2).<h4>Conclusion</h4>HFE genotypes do not seem to be a significant contributor to the etiology of heart failure in Denmark. HFE variants do not affect mortality in HF.

Programmed genetic rearrangements in lymphocytes require transcription at antigen receptor genes to promote accessibility for initiating double-strand break (DSB) formation critical for DNA recombination and repair. Here, we showed that activated B cells deficient in the PTIP component of the MLL3 (mixed-lineage leukemia 3)-MLL4 complex display impaired trimethylation of histone 3 at lysine 4 (H3K4me3) and transcription initiation of downstream switch regions at the immunoglobulin heavy-chain (Igh) locus, leading to defective immunoglobulin class switching. We also showed that PTIP accumulation at DSBs contributes to class switch recombination (CSR) and genome stability independently of Igh switch transcription. These results demonstrate that PTIP promotes specific chromatin changes that control the accessibility of the Igh locus to CSR and suggest a nonredundant role for the MLL3-MLL4 complex in altering antibody effector function.

Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their function--female determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate.

<h4>Background</h4>Colorectal cancer (CRC) is one of the most frequently occurring cancers in Japan, and thus a wide range of methods have been deployed to study the molecular mechanisms of CRC. In this study, we performed a comprehensive analysis of CRC, incorporating copy number aberration (CRC) and gene expression data. For the last four years, we have been collecting data from CRC cases and organizing the information as an "omics" study by integrating many kinds of analysis into a single comprehensive investigation. In our previous studies, we had experienced difficulty in finding genes related to CRC, as we observed higher noise levels in the expression data than in the data for other cancers. Because chromosomal aberrations are often observed in CRC, here, we have performed a combination of CNA analysis and expression analysis in order to identify some new genes responsible for CRC. This study was performed as part of the Clinical Omics Database Project at Tokyo Medical and Dental University. The purpose of this study was to investigate the mechanism of genetic instability in CRC by this combination of expression analysis and CNA, and to establish a new method for the diagnosis and treatment of CRC.<h4>Materials and methods</h4>Comprehensive gene expression analysis was performed on 79 CRC cases using an Affymetrix Gene Chip, and comprehensive CNA analysis was performed using an Affymetrix DNA Sty array. To avoid the contamination of cancer tissue with normal cells, laser micro-dissection was performed before DNA/RNA extraction. Data analysis was performed using original software written in the R language.<h4>Result</h4>We observed a high percentage of CNA in colorectal cancer, including copy number gains at 7, 8q, 13 and 20q, and copy number losses at 8p, 17p and 18. Gene expression analysis provided many candidates for CRC-related genes, but their association with CRC did not reach the level of statistical significance. The combination of CNA and gene expression analysis, together with the clinical information, suggested UGT2B28, LOC440995, CXCL6, SULT1B1, RALBP1, TYMS, RAB12, RNMT, ARHGDIB, S1000A2, ABHD2, OIT3 and ABHD12 as genes that are possibly associated with CRC. Some of these genes have already been reported as being related to CRC. TYMS has been reported as being associated with resistance to the anti-cancer drug 5-fluorouracil, and we observed a copy number increase for this gene. RALBP1, ARHGDIB and S100A2 have been reported as oncogenes, and we observed copy number increases in each. ARHGDIB has been reported as a metastasis-related gene, and our data also showed copy number increases of this gene in cases with metastasis.<h4>Conclusion</h4>The combination of CNA analysis and gene expression analysis was a more effective method for finding genes associated with the clinicopathological classification of CRC than either analysis alone. Using this combination of methods, we were able to detect genes that have already been associated with CRC. We also identified additional candidate genes that may be new markers or targets for this form of cancer.

Intellectual disability (ID), also referred to as mental retardation (MR), is frequently the result of genetic mutation. Where ID is present together with additional clinical symptoms or physical anomalies, there is often sufficient information available for the diagnosing physician to identify a known syndrome, which may then educe the identification of the causative defect. However, where co-morbid features are absent, narrowing down a specific gene can only be done by 'brute force' using the latest molecular genetic techniques. Here we attempt to provide a systematic review of genetic causes of cases of ID where no other symptoms or co-morbid features are present, or non-syndromic ID. We attempt to summarize commonalities between the genes and the molecular pathways of their encoded proteins. Since ID is a common feature of autism, and conversely autistic features are frequently present in individuals with ID, we also look at possible overlaps in genetic etiology with non-syndromic ID.

A first case of clinical tenofovir (TDF) HBV resistance in an HIV/HBV coinfected patient who developed an acute flare of hepatitis B is reported. The clinical course was accompanied by signs of acute liver failure after being on successful HBV treatment with tenofovir and persistently undetectable HBV-DNA viral load for over five years.

The aim of this study was to characterize the affinity and permeability patterns of the amino acid ester prodrugs of acyclovir (ACV), L-alanine-ACV (AACV), L-serine-ACV (SACV), L-serine-succinate-ACV (SSACV) and L-cysteine-ACV (CACV) on rabbit primary corneal epithelial cell culture (rPCEC) and on rabbit cornea. Amino acid prodrugs of acyclovir, AACV, SACV, SSACV and CACV were synthesized in our laboratory. Chemical hydrolysis in aqueous buffer, enzymatic hydrolysis in corneal homogenates and transport across freshly excised rabbit cornea of these prodrugs were studied. SSACV inhibited the uptake of [(3)H] L-alanine on rPCEC and across the intact rabbit cornea. Lineweaver-Burk plot transformation revealed competitive inhibition between L-alanine and SSACV. In corneal tissue homogenate, the half lives of SSACV, SACV and CACV (t1/2) were observed to be 3.5 ± 0.4, 9.2 ± 0.6 and 1.8 ± 0.1 hr respectively, whereas AACV was readily converted to the active parent drug acyclovir exhibiting complete degradation before 5 min. Interestingly translocation of SACV across cornea was inhibited in the presence of 5 mM arginine (~51%), a specific substrate for cationic transport system and in presence of BCH (~38%), a substrate specific for large neutral amino acid transport system (LAT) or cationic and neutral amino acid transport system (B(0,+)). SACV exhibited higher permeability across cornea along with excellent antiviral activity against herpes simplex virus (HSV-1) and varicella-zoster virus (VZV) in comparison to ACV. Recognition by multiple transporters, stability in corneal homogenate and changes in physico-chemical properties contributed to the increased permeability of SACV across cornea.

<h4>Background</h4>Coronary heart disease (CHD) and stroke were key outcomes in the Women's Health Initiative (WHI) randomized trials of postmenopausal estrogen and estrogen plus progestin therapy. We recently reported a large number of changes in blood protein concentrations in the first year following randomization in these trials using an in-depth quantitative proteomics approach. However, even though many affected proteins are in pathways relevant to the observed clinical effects, the relationships of these proteins to CHD and stroke risk among postmenopausal women remains substantially unknown.<h4>Methods</h4>The same in-depth proteomics platform was applied to plasma samples, obtained at enrollment in the WHI Observational Study, from 800 women who developed CHD and 800 women who developed stroke during cohort follow-up, and from 1-1 matched controls. A plasma pooling strategy, followed by extensive fractionation prior to mass spectrometry, was used to identify proteins related to disease incidence, and the overlap of these proteins with those affected by hormone therapy was examined. Replication studies, using enzyme-linked-immunosorbent assay (ELISA), were carried out in the WHI hormone therapy trial cohorts.<h4>Results</h4>Case versus control concentration differences were suggested for 37 proteins (nominal P < 0.05) for CHD, with three proteins, beta-2 microglobulin (B2M), alpha-1-acid glycoprotein 1 (ORM1), and insulin-like growth factor binding protein acid labile subunit (IGFALS) having a false discovery rate < 0.05. Corresponding numbers for stroke were 47 proteins with nominal P < 0.05, three of which, apolipoprotein A-II precursor (APOA2), peptidyl-prolyl isomerase A (PPIA), and insulin-like growth factor binding protein 4 (IGFBP4), have a false discovery rate < 0.05. Other proteins involved in insulin-like growth factor signaling were also highly ranked. The associations of B2M with CHD (P < 0.001) and IGFBP4 with stroke (P = 0.005) were confirmed using ELISA in replication studies, and changes in these proteins following the initiation of hormone therapy use were shown to have potential to help explain hormone therapy effects on those diseases.<h4>Conclusions</h4>In-depth proteomic discovery analysis of prediagnostic plasma samples identified B2M and IGFBP4 as risk markers for CHD and stroke respectively, and provided a number of candidate markers of disease risk and candidate mediators of hormone therapy effects on CHD and stroke.<h4>Clinical trials registration</h4>ClinicalTrials.gov identifier: NCT00000611.

Monkeypox virus (MPV) is a zoonotic Orthopoxvirus and a potential biothreat agent that causes human disease with varying morbidity and mortality. Members of the Orthopoxvirus genus have been shown to suppress antiviral cell defenses, exploit host cell machinery, and delay infection-induced cell death. However, a comprehensive study of all host genes and virus-targeted host networks during infection is lacking. To better understand viral strategies adopted in manipulating routine host biology on global scale, we investigated the effect of MPV infection on Macaca mulatta kidney epithelial cells (MK2) using GeneChip rhesus macaque genome microarrays. Functional analysis of genes differentially expressed at 3 and 7 hours post infection showed distinctive regulation of canonical pathways and networks. While the majority of modulated histone-encoding genes exhibited sharp copy number increases, many of its transcription regulators were substantially suppressed; suggesting involvement of unknown viral factors in host histone expression. In agreement with known viral dependence on actin in motility, egress, and infection of adjacent cells, our results showed extensive regulation of genes usually involved in controlling actin expression dynamics. Similarly, a substantial ratio of genes contributing to cell cycle checkpoints exhibited concerted regulation that favors cell cycle progression in G1, S, G2 phases, but arrest cells in G2 phase and inhibits entry into mitosis. Moreover, the data showed that large number of infection-regulated genes is involved in molecular mechanisms characteristic of cancer canonical pathways. Interestingly, ten ion channels and transporters showed progressive suppression during the course of infection. Although the outcome of this unusual channel expression on cell osmotic homeostasis remains unknown, instability of cell osmotic balance and membrane potential has been implicated in intracellular pathogens egress. Our results highlight the role of histones, actin, cell cycle regulators, and ion channels in MPV infection, and propose these host functions as attractive research focal points in identifying novel drug intervention sites.

Liver disease is responsible for more than 42,000 deaths yearly. Elevated hepatic iron levels have been shown to play a role in chronic liver diseases including hereditary hemochromatosis, thalassemia, and chronic hepatitis C, whereas acetaminophen (APAP) is the leading cause of acute liver failure. The goal of this study was to determine whether increased hepatic iron affects APAP-induced cytotoxicity, reactive oxygen species (ROS) production, and/or mitochondrial dysfunction in primary mouse hepatocytes (PMHs) that are differentiated and have gap junctional intracellular integrity, properties associated with hepatocytes in vivo and important for conducting toxicant studies. Treatment of PMHs with the iron donor 3,5,5-trimethyl-hexanoyl ferrocene (TMHF) caused an elevation in ferritin, reduction in transferrin receptor 1, and accumulation of hemosiderin, but TMHF treatment alone did not induce ROS or cause mitochondrial dysfunction. The threshold APAP dose that induced PMH cell death after TMHF treatment of PMHs was lower than in the absence of TMHF. In addition, treatment with the iron chelator deferoxamine (DFO) protected from APAP and resulted in a higher threshold dose being needed to induce cell death. We also showed that after TMHF treatment, APAP induced ROS and mitochondrial dysfunction at earlier time points than treatment with APAP alone; treatment with DFO increased the length of time required for APAP to induce ROS and mitochondrial dysfunction; and treatment with DFO, subsequent to TMHF, partially protected against TMHF-potentiated APAP injury. We conclude that iron potentiates the effects of APAP on cytotoxicity, ROS production, and mitochondrial dysfunction in PMHs.

Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene. Here, we report that the transcript of the peroxisome proliferator-activated receptor-γ (PPARγ), a transcription factor that is critical for energy homeostasis, was markedly downregulated in multiple tissues of a mouse model (R6/2) of HD and in lymphocytes of HD patients. Therefore, downregulation of PPARγ seems to be a pathomechanism of HD. Chronic treatment of R6/2 mice with an agonist of PPARγ (thiazolidinedione, TZD) rescued progressive weight loss, motor deterioration, formation of mutant Htt aggregates, jeopardized global ubiquitination profiles, reduced expression of two neuroprotective proteins (brain-derived neurotrophic factor and Bcl-2) and shortened life span exhibited by these mice. By reducing HTT aggregates and, thus, ameliorating the recruitment of PPARγ into HTT aggregates, chronic TZD treatment also elevated the availability of the PPARγ protein and subsequently normalized the expression of two of its downstream genes (the glucose transporter type 4 and PPARγ coactivator-1 alpha genes). The protective effects described above appear to have been exerted, at least partially, via direct activation of PPARγ in the brain, as TZD was detected in the brains of mice treated with TZD and because a PPARγ agonist (rosiglitazone) protected striatal cells from mHTT-evoked energy deficiency and toxicity. We demonstrated that the systematic downregulation of PPARγ seems to play a critical role in the dysregulation of energy homeostasis observed in HD, and that PPARγ is a potential therapeutic target for this disease.

Autoimmune polyendocrinopathy syndrome type 1 (APS1) results from homozygous Aire mutations that cripple thymic deletion of organ-specific T cells. The clinical course in man and mouse is characterized by high variability both in the latent period before onset of autoimmune disease and in the specific organs affected, but the reasons for this are unknown. Here we test the hypothesis that the latent period reflects the failsafe action of discrete postthymic mechanisms for imposing self-tolerance in peripheral T cells. Aire-deficient mice were crossed with mice of a uniform major histocompatibility complex (MHC) haplotype and genetic background carrying specific genetic defects in one of four distinct peripheral tolerance mechanisms: activation-induced cell death (Fasl(gld/gld)), anergy and requirement for CD28 costimulation (Cblb(-/-)), inhibition of ICOS and T(FH) cells (Rc3h1(san/san)), or decreased numbers of Foxp3(+) T regulatory cells (Card11(unm/unm)). Cblb-deficiency was unique among these four in precipitating rapid clinical autoimmune disease when combined with Aire-deficiency, resulting in autoimmune exocrine pancreatitis with median age of survival of only 25 d. Massive lymphocytic infiltration selectively destroyed most of the exocrine acinar cells of the pancreas and submandibular salivary gland, and CD4(+) and CD8(+) subsets were necessary and sufficient to transfer the disease. Intrinsic regulation of peripheral T cells by CBL-B thus serves a uniquely critical role as a failsafe against clinical onset of autoimmune disease in AIRE deficiency, and multiple peripheral tolerance mechanisms may need to fail before onset of clinical autoimmunity to many organs.

SOX5 is a transcription factor with homology to the high mobility group box region of the testis-determining factor, SRY. Both the mouse and human SOX5 genes encode a 48-kDa SOX5 protein (S-SOX5) that is only present in tissues containing cells with motile cilia/flagella. The mammalian sperm-associated antigen 6 gene (SPAG6) encodes an axoneme central apparatus protein. Because human and mouse SPAG6 gene promoters contain multiple potential binding sites for SOX5, SPAG6 gene regulation by S-SOX5 was investigated in BEAS-2B cells, a line derived from human bronchial cells. Like FOXJ1, a transcription factor known to be essential for motile ciliogenesis, S-SOX5 stimulated mouse and human SPAG6 promoter function in BEAS-2B cells, but the effect was abrogated when the SOX5 binding sites were mutated or deleted. S-SOX5 and FOXJ1 functioned cooperatively in stimulating SPAG6 promoter activity. The SPAG6 message was up-regulated when S-SOX5 was overexpressed in BEAS-2B cells, and silencing of S-SOX5 by RNA interference down-regulated SPAG6 transcripts. Chromatin immunoprecipitation and EMSA experiments demonstrated that S-SOX5 associates with the SPAG6 promoter directly. The present study demonstrates that SPAG6 is a S-SOX5 target gene, indicating a key role for S-SOX5 in the formation and function of motile cilia.

A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.

<h4>Purpose</h4>Standard dose (13-15 mg/kg) ursodeoxycholic acid (UCDA) is ineffective in the treatment of nonalcoholic steatohepatitis (NASH), however, its immunomodulatory and hepatoprotective effects are dose related. Therefore, we examined the impact of high-dose (28-32 mg/kg) UCDA on aminotransaminase levels in a pilot study of patients with NASH.<h4>Methods</h4>Twelve patients with biopsy-proven NASH and elevated aminotransaminases were prescribed high-dose UCDA for 6 months. Liver function tests were monitored during and after treatment with the study endpoint defined as normalization of aminotransaminase levels.<h4>Results</h4>Normalization of aspartate aminotransaminase (AST) levels was observed in two (17%) patients, however, no patient normalized their alanine aminotransaminase (ALT) levels. A trend towards a minor reduction in median (range) ALT values from baseline to end of treatment was noted [124 (66-229) vs. 101 (53-188) IU/l, p = 0.07], whereas AST levels remained unchanged [85 (40-132) vs. 98 (28-147) IU/l, p = 0.83]. One patient discontinued treatment prematurely due to diarrhea. No significant change in fasting glucose, triglyceride or HDL cholesterol was observed with treatment. No significant change in ALT or AST levels was observed in the 6-month period after cessation of treatment.<h4>Conclusion</h4>High-dose UCDA does not normalize aminotransaminase levels in patients with NASH. Other inexpensive well-tolerated agents for the treatment of NASH need to be investigated.

Genetics, and more recently genomics, reveal striking conservation in the fundamental signaling pathways that underlie normal and aberrant cell processes. Consequently, various genetic model organisms are now attracting the interest of biomedical scientists who are focused on therapeutic approaches to human disease. There are now several examples of studies in which Drosophila seems likely to facilitate advances in potential therapies, and a recent report has demonstrated the utility of the fly model for understanding and treating human disease. Basic developmental genetic information first obtained in Drosophila was used to design a therapeutic block to oncogenic Notch signaling that was associated with leukemia in mice. The story of Notch signaling in Drosophila demonstrates the potential for standard Drosophila molecular genetics in developing therapeutic strategies that are relevant to human disease.

<h4>Background</h4>MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype.<h4>Methods</h4>We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1.<h4>Results</h4>We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17,295 correlated miRNA-mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction.<h4>Conclusions</h4>This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.

<h4>Background</h4>Cell-derived microvesicles (MVs) have been described as a new mechanism of cell-to-cell communication. MVs after internalization within target cells may deliver genetic information. Human bone marrow derived mesenchymal stem cells (MSCs) and liver resident stem cells (HLSCs) were shown to release MVs shuttling functional mRNAs. The aim of the present study was to evaluate whether MVs derived from MSCs and HLSCs contained selected micro-RNAs (miRNAs).<h4>Methodology/principal findings</h4>MVs were isolated from MSCs and HLSCs. The presence in MVs of selected ribonucleoproteins involved in the traffic and stabilization of RNA was evaluated. We observed that MVs contained TIA, TIAR and HuR multifunctional proteins expressed in nuclei and stress granules, Stau1 and 2 implicated in the transport and stability of mRNA and Ago2 involved in miRNA transport and processing. RNA extracted from MVs and cells of origin was profiled for 365 known human mature miRNAs by real time PCR. Hierarchical clustering and similarity analysis of miRNAs showed 41 co-expressed miRNAs in MVs and cells. Some miRNAs were accumulated within MVs and absent in the cells after MV release; others were retained within the cells and not secreted in MVs. Gene ontology analysis of predicted and validated targets showed that the high expressed miRNAs in cells and MVs could be involved in multi-organ development, cell survival and differentiation. Few selected miRNAs shuttled by MVs were also associated with the immune system regulation. The highly expressed miRNAs in MVs were transferred to target cells after MV incorporation.<h4>Conclusions</h4>This study demonstrated that MVs contained ribonucleoproteins involved in the intracellular traffic of RNA and selected pattern of miRNAs, suggesting a dynamic regulation of RNA compartmentalization in MVs. The observation that MV-highly expressed miRNAs were transferred to target cells, rises the possibility that the biological effect of stem cells may, at least in part, depend on MV-shuttled miRNAs. Data generated from this study, stimulate further functional investigations on the predicted target genes and pathways involved in the biological effect of human adult stem cells.

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

<h4>Background</h4>Pulmonary surfactant is required for lung function at birth and throughout life. Lung lipid and surfactant homeostasis requires regulation among multi-tiered processes, coordinating the synthesis of surfactant proteins and lipids, their assembly, trafficking, and storage in type II cells of the lung. The mechanisms regulating these interrelated processes are largely unknown.<h4>Results</h4>We integrated mRNA microarray data with array independent knowledge using Gene Ontology (GO) similarity analysis, promoter motif searching, protein interaction and literature mining to elucidate genetic networks regulating lipid related biological processes in lung. A Transcription factor (TF)-target gene (TG) similarity matrix was generated by integrating data from different analytic methods. A scoring function was built to rank the likely TF-TG pairs. Using this strategy, we identified and verified critical components of a transcriptional network directing lipogenesis, lipid trafficking and surfactant homeostasis in the mouse lung.<h4>Conclusions</h4>Within the transcriptional network, SREBP, CEBPA, FOXA2, ETSF, GATA6 and IRF1 were identified as regulatory hubs displaying high connectivity. SREBP, FOXA2 and CEBPA together form a common core regulatory module that controls surfactant lipid homeostasis. The core module cooperates with other factors to regulate lipid metabolism and transport, cell growth and development, cell death and cell mediated immune response. Coordinated interactions of the TFs influence surfactant homeostasis and regulate lung function at birth.

<h4>Background</h4>Neonatal growth is a complex process involving genetic and environmental factors. Polymorphisms in the hemochromatosis (HFE) iron regulatory genes have been shown to modify transport and toxicity of lead which is known to affect birth weight.<h4>Methods</h4>We investigated the role of HFE C282Y, HFE H63 D, and transferrin (TF) P570 S gene variants in modifying the association of lead and infant birthweight in a cohort of Mexican mother-infant pairs. Subjects were initially recruited between 1994-1995 from three maternity hospitals in Mexico City and 411 infants/565 mothers had archived blood available for genotyping. Multiple linear regression models, stratified by either maternal/infant HFE or TF genotype and then combined with interaction terms, were constructed examining the association of lead and birthweight after controlling for covariates.<h4>Results</h4>3.1%, 16.8% and 17.5% of infants (N=390) and 1.9%, 14.5% and 18.9% of mothers (N=533) carried the HFE C282Y, HFE H63D, and TF P570 S variants, respectively. The presence of infant HFE H63 D variants predicted 110.3 g (95% CI -216.1, -4.6) decreases in birthweight while maternal HFE H63 D variants predicted reductions of 52.0 g (95% CI -147.3 to 43.2). Interaction models suggest that both maternal and infant HFE H63 D genotype may modify tibia lead's effect on infant birthweight in opposing ways. In our interaction models, maternal HFE H63 D variant carriers had a negative association between tibia lead and birthweight.<h4>Conclusions</h4>These results suggest that the HFE H63 D genotype modifies lead's effects on infant birthweight in a complex fashion that may reflect maternal-fetal interactions with respect to the metabolism and transport of metals.

<h4>Background & aims</h4>Parenchymal liver siderosis is associated with increased fibrosis in patients with non-alcoholic fatty liver disease (NAFLD). The aim of this study was to assess whether a panel of genetic variants previously reported to influence iron metabolism, including the C282Y/H63D HFE, the PiZ/PiS alpha1-antitrypsin, the IVS1-24 ferroportin polymorphisms, and the beta-thalassemia trait, may be able to predict the presence of parenchymal siderosis and of progressive fibrosis in NAFLD.<h4>Methods</h4>We considered 274 Italian patients with biopsy-proven NAFLD. Genetic polymorphisms were searched for by sequence allele specific-polymerase chain reaction and restriction analysis, whereas beta-trait was determined according to blood count and HbA(2) determination.<h4>Results</h4>Parenchymal iron deposition was predominantly observed in 32 (11.7%) patients. Heterozygosity for the C282Y (OR 1.87, 95% CI 1.04-3.25), homozygosity for the H63D HFE (OR 2.31, 95% CI 1.04-4) mutations, and the beta-thalassemia trait (OR 2.57 95% CI 1.49-4.47) were all predominantly associated with parenchymal siderosis, independently of age, sex, body mass index, alcohol intake, ferritin, and transferrin saturation. Sixty-three percent of patients with hepatocellular siderosis were positive for at least one of the aforementioned genetic variants. The beta-thalassemia trait had the highest positive and the lowest negative likelihood ratios for predominantly parenchymal iron accumulation (5.05 and 0.74, respectively), and was independently associated with moderate/severe fibrosis (OR 2.50, 95% CI 1.26-5.19).<h4>Conclusions</h4>In patients with NAFLD, predominant hepatocellular iron deposition is often related to genetic factors, among which beta-globin mutations play a major role, predisposing to parenchymal iron accumulation and to progressive liver fibrosis.

No abstract available.

N-methyl-D-aspartate receptor (NMDAR) mediated excitotoxicity is a probable proximate mechanism of neurodegeneration in Huntington disease (HD). Striatal neurons express the NR2B-NMDAR subunit at high levels, and this subunit is thought to be instrumental in causing excitotoxic striatal neuron injury. We evaluated the efficacy of 3 NR2B-selective antagonists in the R6/2 transgenic fragment model of HD. We evaluated ifenprodil (10 mg/kg; 100 mg/kg), RO25,6981 (10 mg/kg), and CP101,606 (30 mg/kg). Doses were chosen on the basis of pilot acute maximally tolerated dose studies. Mice were treated with subcutaneous injections twice daily. Outcomes included survival; motor performance declines assessed with the rotarod, balance beam task, and activity measurements; and post-mortem striatal volumes. No outcome measure demonstrated any benefit of treatments. Lack of efficacy of NR2B antagonists in the R6/2 model has several possible explanations including blockade of beneficial NMDAR mediated effects, inadequacy of the R6/2 model, and the existence of multiple proximate mechanisms of neurodegeneration in HD.

Risky decision making is a complex process that involves weighing the probabilities of alternative options that can be desirable, undesirable, or neutral. Individuals vary greatly in how they make decisions either under ambiguity and/or under risk. Such individual differences may have genetic bases. Based on previous studies on the genetic basis of decision making, two decision making tasks [i.e., the Iowa Gambling Task (IGT) and Loss Aversion Task (LAT)] were used to test the effect of 5-HTTLPR polymorphism on decision making under ambiguity and under risk in a large Han Chinese sample (572 college students, 312 females). Basic intelligence and memory tests were also included to control for the influence of basic cognitive abilities on decision making. We found that 5-HTTLPR polymorphism significantly influenced performance in both IGT and LAT. After controlling for intelligence and memory abilities, subjects homozygous for s allele had lower IGT scores than l carriers in the first 40 trials of the IGT task. They also exhibited higher loss aversion than l carriers in the LAT task. Moreover, the effects of 5-HTTLPR were stronger for males than for females. These results extend the literature on the important role of emotion in decision making under ambiguity and risk, and shed additional lights on how decision making is influenced by culture as well as sex differences. Combining our results with existing literature, we propose that these effects might be mediated by a neural circuitry that comprises the amygdala, ventromedial prefrontal cortex, and insular cortex. Understanding the genetic factors affecting decision making in healthy subjects may allow us to better identify at-risk individuals, and better target the development of new potential treatments for specific disorders such as schizophrenia, addiction, and depression.

To characterize the humoral response to the unglycosylated central region of the respiratory syncytial virus (RSV) attachment (G) protein, we generated glutathione S-transferase (GST)-RSV G subdomains (central core (CC), residues 151-190; proximal central core (PCC), 151-172; and distal central core (DCC), 173-190) to screen paired sera from RSV subtype A- or B-infected adults in hospitalized or outpatient settings. Following RSV infection, a >or=4-fold increase in homo- and heterosubtypic IgG response was noted in most subjects against the RSV G CC and PCC regions; in contrast, such titer increases against the RSV G DCC was only noted in a homosubtypic manner. Our results have implications for RSV G-based serological diagnostics and vaccine development.

<h4>Purpose</h4>Temporal lobe epilepsy (TLE) is the most common epilepsy and about 30% of patients have poorly controlled seizures. Neurobiology underlying responsiveness to medical treatment in TLE patients is unclear and there are currently no biological tests to predict course of the disease. Animal and human studies repeatedly suggested serotonergic dysfunction in subjects with TLE. We investigated association of serotonin transporter (5-HTT) gene polymorphisms with medical treatment response in patients with TLE.<h4>Methods</h4>We analyzed 5-HTT gene linked polymorphic region (5-HTTLPR) in promoter and variable number of tandem repeats in the second intron of the 5-HTT gene (VNTR-2) in 101 consecutive subjects with TLE.<h4>Results</h4>TLE patients with the combination of transcriptionally more efficient genotypes, i.e. 5-HTTLPR L/L and VNTR-2 12/12, had increased seizure refractoriness to antiepileptic medication therapy and shorter periods of seizure freedom, than subjects with other combinations of the 5-HTT genotypes. There were no other clinical or demographic differences among patient groups based on the 5-HTT genotypes.<h4>Conclusion</h4>Combination of the 5-HTT genotypes linked with higher 5-HTT gene expression was found to be associated with worse response to optimal drug therapy. Further studies should determine potential role of this 5-HTT genotype polymorphism in epileptogenesis.

Peritoneal carcinomatosis has been considered a terminal disease with a median survival time of 5.2-12.6 months. Systemic chemotherapy and cytoreductive surgery (CRS) have long been used to treat macroscopic disease, with limited success. However, a comprehensive treatment approach involving cytroreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) has evolved into a novel approach for peritoneal carcinomatosis. Surgery removes the primary cancer and any dissemination within the peritoneal cavity and adjuvant HIPEC eradicates macroscopic or microscopic tumor residue, thus reducing the risk for recurrence. This approach offers a new potential treatment option for patients with metastatic disease confined to the peritoneum. The present review provides an update of the most recent data on the current therapy for pseudomyxoma peritonei (PMP) and mucinous colorectal adenocarcinoma (MCA) with metastatic disease confined to the peritoneum.

Although oxidative stress is deleterious to mammals, the mechanisms underlying oxidant susceptibility or tolerance remain to be elucidated. In this study, through a long-term laboratory selection over many generations, we generated a Drosophila melanogaster strain that can live and reproduce in very high O(2) environments (90% O(2)), a lethal condition to naïve flies. We demonstrated that tolerance to hyperoxia was heritable in these flies and that these hyperoxia-selected flies exhibited phenotypic differences from naïve flies, such as a larger body size and increased weight by 20%. Gene expression profiling revealed that 227 genes were significantly altered in expression and two third of these genes were down-regulated. Using a mutant screen strategy, we studied the role of some altered genes (up- or down-regulated in the microarrays) by testing the survival of available corresponding P-element or UAS construct lines under hyperoxic conditions. We report that down-regulation of several candidate genes including Tropomyosin 1, Glycerol 3 phosphate dehydrogenase, CG33129, and UGP as well as up-regulation of Diptericin and Attacin conferred tolerance to severe hyperoxia. In conclusion, we identified several genes that were not only altered in hyperoxia-selected flies but we also prove that these play an important role in hyperoxia survival. Thus our study provides a molecular basis for understanding the mechanisms of hyperoxia tolerance.

<h4>Purpose</h4>To improve understanding in the physical therapy (PT) community of hereditary hemochromatosis (HH), a common but little-known iron overload disorder, symptoms of which may mimic other orthopaedic conditions. Medical management typically involves phlebotomy to remove excess iron; however, there is little specific information in the literature on PT management of patients with HH after trauma.<h4>Case description</h4>The patient was a 65-year-old woman with multiple fall-related traumas, including right wrist, thumb, and patellar fractures and left thigh muscle strain with significant ecchymosis and effusion. Medical history included HH. Iron-related lab values had been analyzed 9 days prior to the fall and had demonstrated a steady increase over the previous 4 months since her last phlebotomy.<h4>Outcomes</h4>As the level of exercise and activity increased during the course of PT treatment, the patient developed shortness of breath and increased fatigue. The exercise level in therapy was reduced to accommodate the change in the patient's response. Blood values analyzed 7 weeks after the fall demonstrated a drop in haemoglobin and hematocrit values, while serum ferritin levels had risen.<h4>Implications</h4>Understanding early symptoms and management of a patient with manifestations of HH will better enable physical therapists to consider this disorder as a differential diagnosis or co-morbidity that affects treatment considerations.

<h4>Background</h4>The physiological function of the prion protein remains largely elusive while its key role in prion infection has been expansively documented. To potentially assess this conundrum, we performed a comparative transcriptomic analysis of the brain of wild-type mice with that of transgenic mice invalidated at this locus either at the zygotic or at the adult stages.<h4>Results</h4>Only subtle transcriptomic differences resulting from the Prnp knockout could be evidenced, beside Prnp itself, in the analyzed adult brains following microarray analysis of 24 109 mouse genes and QPCR assessment of some of the putatively marginally modulated loci. When performed at the adult stage, neuronal Prnp disruption appeared to sequentially induce a response to an oxidative stress and a remodeling of the nervous system. However, these events involved only a limited number of genes, expression levels of which were only slightly modified and not always confirmed by RT-qPCR. If not, the qPCR obtained data suggested even less pronounced differences.<h4>Conclusions</h4>These results suggest that the physiological function of PrP is redundant at the adult stage or important for only a small subset of the brain cell population under classical breeding conditions. Following its early reported embryonic developmental regulation, this lack of response could also imply that PrP has a more detrimental role during mouse embryogenesis and that potential transient compensatory mechanisms have to be searched for at the time this locus becomes transcriptionally activated.

The quality of the social environment can have profound influences on the development and activity of neural systems with implications for numerous behavioral and physiological responses, including the expression of emotionality. Though social experiences occurring early in development may be particularly influential on the developing brain, there is continued plasticity within these neural circuits amongst juveniles and into early adulthood. In this review, we explore the evidence derived from studies in rodents which illustrates the social modulation during development of neural systems, with a particular emphasis on those systems in which a long-term effect is observed. One possible explanation for the persistence of dynamic changes in these systems in response to the environment is the involvement of epigenetic mechanisms, and here we discuss recent studies which support the role of these mechanisms in mediating the link between social experiences, gene expression, neurobiological changes, and behavioral variation. This literature raises critical questions about the interaction between neural systems, the concordance between neural and behavioral changes, sexual dimorphism in effects, the importance of considering individual differences in response to the social environment, and the potential of an epigenetic perspective in advancing our understanding of the pathways leading to variations in mental health.

Insulin secretion and glucose homeostasis is implicated through serotonergic function. Pyridoxine is involved in decarboxylation step in synthesis of serotonin. The present study was carried out to find the role of insulin in combination with pyridoxine on the concentrations of 5-HT and 5-HIAA, 5-HT receptor binding, 5-HTT gene expression and immunohistochemistry studies in the cerebral cortex and brainstem of streptozotocin induced diabetic rats. 5-HT content showed a significant decrease with a significant increase in 5-HIAA in cerebral cortex (p<0.01) and brain stem (p<0.001) in diabetic rats. 5-HT receptor binding parameters, B(max) and K(d), showed a significant decrease (p<0.001) in diabetic rats in cerebral cortex whereas in brainstem it showed a significant increase (p<0.001) compared to control. Gene expression studies of 5-HTT in cerebral cortex showed a significant down regulation (p<0.001) and in brainstem an upregulation (p<0.001) in diabetic rats compared to control. Insulin and pyridoxine treatment to diabetic rats reversed the 5-HT content, B(max), K(d) and gene expression of 5-HTT confirmed by immunohistochemistry studies in cerebral cortex and brainstem to near control. Thus our results suggest that pyridoxine along with insulin has a role in the regulation of insulin synthesis and release through serotonergic function which has clinical significance in the management of diabetes.

The serotonin molecule plays a multifunctional role in mammalian homeostasis serving as a neurotransmitter in the central nervous system, a gut-derived mediator of peristalsis, and a circulating hormone that regulates appetite, cardiovascular function, and hemostasis. Recent evidence from the clinic and the bench highlight an unexpected target for serotonin action, the skeleton. Clinically, two classes of drugs, the second generation antipsychotic drugs (SGAs) and selective serotonin reuptake inhibitors (SSRIs), which modulate central and peripheral serotonin signaling, have been shown to alter bone remodeling although the mechanism is not clear. In contrast, genetically engineered mouse models have demonstrated a bimodal control system whereby gut-derived serotonin under the control of the Wnt/Lrp/beta-catenin system acts systemically to suppress bone formation, whereas CNS serotonin activated by leptin modulates sympathetic outflow to the skeleton. In this brief review, we will summarize recent findings linking serotonin to the skeleton and discuss future directions for this new but challenging aspect of this multidimensional molecule.

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

Neurodegeneration is a hallmark of many neurological diseases, including Alzheimer's, Parkinson's and the polyglutamine diseases, which are all caused by misfolded proteins that accumulate in neuronal cells of the brain. Although apoptosis is believed to contribute to neurodegeneration in these cases, genetic mouse models of these diseases often fail to replicate apoptosis and overt neurodegeneration in the brain. Using nuclear transfer, we generated transgenic Huntington's disease (HD) pigs that express N-terminal (208 amino acids) mutant huntingtin with an expanded polyglutamine tract (105Q). Postnatal death, dyskinesia and chorea-like movement were observed in some transgenic pigs that express mutant huntingtin. Importantly, the transgenic HD pigs, unlike mice expressing the same transgene, displayed typical apoptotic neurons with DNA fragmentation in their brains. Also, expression of mutant huntingtin resulted in more neurons with activated caspase-3 in transgenic pig brains than that in transgenic mouse brains. Our findings suggest that species differences determine neuropathology and underscore the importance of large mammalian animals for modeling neurological disorders.

Dipeptidyl (acyloxy)methyl ketones (AOMKs) have been identified as mechanism-based inhibitors of certain cysteine proteases. These compounds are also inhibitors of the integral membrane proteins Rce1p and Ste24p, which are proteases that independently mediate a cleavage step associated with the maturation of certain isoprenylated proteins. The enzymatic mechanism of Rce1p is ill-defined, whereas Ste24p is a zinc metalloprotease. Rce1p is required for the proper processing of the oncoprotein Ras and is viewed as a potential target for cancer therapy. In this study, we synthesized a small library of dipeptidyl AOMKs to investigate the structural elements that contribute to the inhibitor properties of this class of molecules toward Rce1p and Ste24p. The compounds were evaluated using a fluorescence-based in vitro proteolysis assay. The most potent dipeptidyl AOMKs contained an arginine residue and the identity of the benzoate group strongly influenced potency. A 'warhead' free AOMK inhibited Rce1p and Ste24p. The data suggest that the dipeptidyl AOMKs are not mechanism-based inhibitors of Rce1p and Ste24p and corroborate the hypothesis that Rce1p is not a cysteine protease.

The mechanism responsible for the development of hepatocellular carcinoma (HCC) in the setting of oxidative stress has yet to be clearly defined. We studied the role of oxidative stress in hepatocarcinogenesis in subjects without underlying chronic viral hepatitis. The subjects were 24 patients negative for serum hepatitis B surface antigen and hepatitis C antibody tests, who underwent hepatic resection for HCC (Group N). Subjects were excluded if diagnosed with liver disease predisposing to HCC. Immunohistochemical staining for oxidative stress-related markers was performed on non-cancerous liver regions. Resected liver tissues adjacent to HCC from 24 patients with chronic hepatitis B (Group B) and 21 patients with chronic hepatitis C (Group C) were also examined. The percentage of 8-hydroxydeoxyguanosine-positive hepatocytes in Group N was significantly lower than that in Group B and that in the combined population of Groups B and C. The percentage of the area positive for 4-hydroxynonenal in Group N was significantly higher than that in Groups B or C. Meanwhile, the percentage of the area positive for manganese superoxide dismutase in Group N was not different from that in Groups B and C. In conclusion, the mechanism of hepatocarcinogenesis through oxidative stress for patients without known liver disease predisposing to HCC may differ from that for patients with chronic viral hepatitis.

Glutamate excitotoxicity is thought to play an important role in Huntington's disease (HD), which is caused by a polyglutamine expansion in the HD protein huntingtin (htt). Overactivation of group I metabotropic glutamate receptors (mGluRs), which include mGluR1 as well as mGluR5 and are coupled via phospholipase C to the inositol phosphate pathway, is found to be involved in mutant htt-mediated neurotoxicity. However, activation of mGluR5 also leads to neuronal protection. Here, we report that mutant htt can activate both mGluR5-mediated ERK and JNK signaling pathways. While increased JNK signaling causes cell death, activation of ERK signaling pathway is protective against cell death. Expression of mutant htt in cultured cells causes greater activation of JNK than ERK. These findings suggest that selective inhibition of the JNK signaling pathway may offer an effective therapeutic approach for reducing htt-mediated excitotoxicity.

Stem cell research has been focused on niche signaling and epigenetic programming of stem cells. However, epigenetic programming of niche cells remains unexplored. We showed previously that Piwi plays a crucial role in Piwi-interacting RNA-mediated epigenetic regulation and functions in the niche cells to maintain germline stem cells (GSCs) in the Drosophila ovary. To investigate the epigenetic programming of niche cells by Piwi, we screened mutations in the Polycomb and trithorax group genes, and an enhancer of Polycomb and trithorax called corto, for their potential genetic interaction with piwi. corto encodes a chromatin protein. corto mutations restored GSC division in mutants of piwi and fs(1)Yb (Yb), a gene that regulates piwi expression in niche cells to maintain GSCs. Consistent with this, corto appears to be expressed in the niche cells and is not required in the germline. Furthermore, in corto-suppressed Yb mutants, the expression of hedgehog (hh) is restored in niche cells, which is likely responsible for corto suppression of the GSC and somatic stem cell defects of Yb mutants. These results reveal a novel epigenetic mechanism involving Corto and Piwi that defines the fate and signaling function of niche cells in maintaining GSCs.

Elevated serum phosphate is a risk factor for vascular calcification and cardiovascular events in kidney disease as well as in the general population. Elevated phosphate levels drive vascular calcification, in part, by regulating vascular smooth muscle cell (VSMC) gene expression, function, and fate. The type III sodium-dependent phosphate co-transporter, PiT-1, is necessary for phosphate-induced VSMC osteochondrogenic phenotype change and calcification, and has recently been shown to have unexpected functions in cell proliferation and embryonic development.

Mutations in PSEN genes, which encode presenilin proteins, cause familial early-onset Alzheimer's disease (AD). Transgenic mouse models based on coexpression of familial AD-associated presenilin and amyloid precursor protein variants successfully mimic characteristic pathological features of AD, including plaque formation, synaptic dysfunction, and loss of memory. Presenilins function as the catalytic subunit of gamma-secretase, the enzyme that catalyzes intramembraneous proteolysis of amyloid precursor protein to release beta-amyloid peptides. Familial AD-associated mutations in presenilins alter the site of gamma-secretase cleavage in a manner that increases the generation of longer and highly fibrillogenic beta-amyloid peptides. In addition to amyloid precursor protein, gamma-secretase catalyzes intramembrane proteolysis of many other substrates known to be important for synaptic function. This paper focuses on how various animal models have enabled us to elucidate the physiological importance of diverse gamma-secretase substrates, including amyloid precursor protein and discusses their roles in the context of cellular signaling and synaptic function.

Cephalexin is a well tolerated antimicrobial and hepatic injury is an infrequent occurrence with its use. We here describe a 21-year-old female who presented with jaundice and elevated liver enzymes after 4 weeks completion of 10 day course of cephalexin, prescribed prophylactically after mammoplasty. Extensive work up including all causes of hepatitis was within normal limits and she improved with conservative management. This case highlights the need to suspect drug induced liver injury in cases of jaundice and cephalexin use.

Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS--the 1000 Genome pilot alone includes nearly five terabases--make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.

<h4>Background</h4>Laminin alpha2 chain mutations cause congenital muscular dystrophy with dysmyelination neuropathy (MDC1A). Previously, we demonstrated that laminin alpha1 chain ameliorates the disease in mice. Dystroglycan and integrins are major laminin receptors. Unlike laminin alpha2 chain, alpha1 chain binds the receptors by separate domains; laminin globular (LG) domains 4 and LG1-3, respectively. Thus, the laminin alpha1 chain is an excellent tool to distinguish between the roles of dystroglycan and integrins in the neuromuscular system.<h4>Methodology/principal findings</h4>Here, we provide insights into the functions of laminin alpha1LG domains and the division of their roles in MDC1A pathogenesis and rescue. Overexpression of laminin alpha1 chain that lacks the dystroglycan binding LG4-5 domains in alpha2 chain deficient mice resulted in prolonged lifespan and improved health. Importantly, diaphragm and heart muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different requirements for LG4-5 domains. Furthermore, the regenerative capacity of the skeletal muscle did not depend on laminin alpha1LG4-5. However, this domain was crucial for preventing apoptosis in limb muscles, essential for myelination in peripheral nerve and important for basement membrane assembly.<h4>Conclusions/significance</h4>These results show that laminin alpha1LG domains and consequently their receptors have disparate functions in the neuromuscular system. Understanding these interactions could contribute to design and optimization of future medical treatment for MDC1A patients.

The molecular mechanism by which roquin controls the expression of inducible costimulator (ICOS) to prevent autoimmunity remains unsolved. Here we show that in helper T cells, roquin localized to processing (P) bodies and downregulated ICOS expression. The repression was dependent on the RNA helicase Rck, and roquin interacted with Rck and the enhancer of decapping Edc4, which act together in mRNA decapping. Sequences in roquin that confer P-body localization were essential for roquin-mediated ICOS repression. However, this process did not require microRNAs or the RNA-induced silencing complex (RISC). Instead, roquin bound ICOS mRNA directly, showing an intrinsic preference for a previously unrecognized sequence in the 3' untranslated region (3' UTR). Our results support a model in which roquin controls ICOS expression through binding to the 3' UTR of ICOS mRNA and by interacting with proteins that confer post-transcriptional repression.

The purpose of the present study is to examine HFE gene mutations in relation to newly diagnosed (incident) coronary heart disease (CHD). In a population-based follow-up study of 7,983 individuals aged 55 years and older, we compared the risk of incident CHD between HFE carriers and non-carriers, overall and stratified by sex and smoking status. HFE mutations were significantly associated with an increased risk of incident CHD in women but not in men (hazard ratio [HR] for women = 1.7, 95% confidence interval [CI] 1.2-2.4 versus HR for men = 0.9, 95% CI 0.7-1.2). This increased CHD risk associated with HFE mutations in women was statistically significant in never smokers (HR = 1.8, 95% CI 1.1-2.8) and current smokers (HR = 3.1, 95% CI 1.4-7.1), but not in former smokers (HR = 1.3, 95% CI 0.7-2.4). HFE mutations are associated with increased risk of incident CHD in women.

We identified and characterized enteroviruses by amplifying the partial VP1 gene from pediatric patients with aseptic meningitis and other enterovirus-related diseases from the Gyeong-Ju and Po-Hang provinces of Korea in 2003. We identified two strains each of coxsackievirus A (CA) 6, CA9, and CA10; three enterovirus 71 (EV71) strains; and six echovirus 30 (E30) strains. The three EV71 strains were characterized as genogroup C4. These results are the first documentation reporting the occurrence of CA10 and EV71 genogroup C4.

Peroxiredoxins (Prxs) are important peroxidases associated with both antioxidant protection and redox signaling. They use a conserved Cys residue to reduce peroxide substrates. The Prxs have a remarkably high catalytic efficiency that makes them a dominant player in cell-wide peroxide reduction, but the origins of their high activity have been mysterious. We present here a novel structure of human PrxV at 1.45 A resolution that has a dithiothreitol bound in the active site with its diol moiety mimicking the two oxygens of a peroxide substrate. This suggests diols and similar di-oxygen compounds as a novel class of competitive inhibitors for the Prxs. Common features of this and other structures containing peroxide, peroxide-mimicking ligands, or peroxide-mimicking water molecules reveal hydrogen bonding and steric factors that promote its high reactivity by creating an oxygen track along which the peroxide oxygens move as the reaction proceeds. Key insights include how the active-site microenvironment activates both the peroxidatic cysteine side chain and the peroxide substrate and how it is exquisitely well suited to stabilize the transition state of the in-line S(N)2 substitution reaction that is peroxidation.

Chaperone-mediated autophagy (CMA) contributes to selective degradation of individual soluble proteins in lysosomes. Unique to this type of autophagy is the fact that proteins reach the lysosomal lumen for degradation by directly crossing the lysosomal membrane, in contrast with the vesicle-mediated delivery characteristic of the other types of autophagy. These two characteristics--selective targeting and direct translocation of substrates--determine the contribution of CMA to different physiological functions and the type of pathological conditions associated with CMA dysfunction. In this review, we briefly revise recent findings on the molecular mechanisms behind CMA function, and describe the physiological relevance of the selective lysosomal degradation through this pathway. We also comment on the cellular consequences of CMA malfunction and on the connections already established between CMA dysfunction and different human disorders, with special emphasis on neurodegenerative diseases. This article is part of a Special Issue entitled "Autophagy and protein degradation in neurological diseases."

<h4>Background & aims</h4>Classical ferroportin disease is characterized by hyperferritinemia, normal transferrin saturation, and iron overload in macrophages. A non-classical form is characterized by additional hepatocellular iron deposits and a high transferrin saturation. Both forms demonstrate autosomal dominant transmission and are associated with ferroportin gene (SLC40A1) mutations. SLC40A1 encodes a cellular iron exporter expressed in macrophages, enterocytes, and hepatocytes. The aim of the analysis is to determine the penetrance of SLC40A1 mutations and to evaluate in silico tools to predict the functional impairment of ferroportin mutations as an alternative to in vitro studies.<h4>Methods</h4>We conducted a systematic review of the literature and meta-analysis of the biochemical presentation, genetics, and pathology of ferroportin disease.<h4>Results</h4>Of the 176 individuals reported with SLC40A1 mutations, 80 were classified as classical phenotype with hyperferritinemia and normal transferrin saturation. The non-classical phenotype with hyperferritinemia and elevated transferrin saturation was present in 53 patients. The remaining patients had normal serum ferritin or the data were reported incompletely. Despite an increased hepatic iron concentration in all biopsied patients, significant fibrosis or cirrhosis was present in only 11%. Hyperferritinemia was present in 86% of individuals with ferroportin mutations. Bio-informatic analysis of ferroportin mutations showed that the PolyPhen score has a sensitivity of 99% and a specificity of 67% for the discrimination between ferroportin mutations and polymorphisms.<h4>Conclusions</h4>In contrast to HFE hemochromatosis, ferroportin disease has a high penetrance, is genetically heterogeneous and is rarely associated with fibrosis. Non-classical ferroportin disease is associated with a higher risk of fibrosis and a more severe overload of hepatic iron.

Seizures are associated with high intracellular calcium levels. However, conditions characterized by high intracellular calcium levels, such as stroke or traumatic brain injury, do not always evoke epilepsy. We hypothesized that polymorphisms in calcium-related genes CACNA1E and Camk2d contribute to the individual variability in seizure susceptibility. The distribution of one single nucleotide polymorphism (SNP) in the CACNA1E and one in the Camk2d gene was determined in Sprague-Dawley rats that were subjected to amygdala kindling or hyperthermia-induced seizures. The pre-kindling afterdischarge threshold was significantly lower in rats with the CACNA1E GG genotype (45.2+/-6.7microA) than in the GT genotyped animals (79.3+/-53.7microA). Among hyperthermia treated rats, the Camk2d G allele was more frequent among rats that did not display behavioral seizures during hyperthermia (67%) than in animals that did show behavioral seizures during hyperthermia (52%, chi(2)(1)=3.847, p=0.05). SNPs in CACNA1E and Camk2d genes are associated with the individual variability in seizure susceptibility in two experimental seizure models.

K-RAS mutations are found in approximately 30% of lung cancers. The transcription factor Krüppel-like Factor 5 (KLF5) has been shown to mediate cellular transformation signaling events downstream of oncogenic RAS in other cancers, but a role for KLF5 in lung tumorigenesis has not been defined. We show here that knockdown of KLF5 expression significantly decreased anchorage-independent growth, but did not affect proliferation of human lung adenocarcinoma cells. Moreover, Klf5 is not required for lung tumor formation in an inducible oncogenic K-Ras(G12D) mouse model of lung tumorigenesis, and non-small cell lung cancer patients expressing high levels of KLF5 (21/258) have a significantly better disease-specific survival than those with intermediate to no KLF5 expression. Further, KLF5 knockdown in K-RAS-mutant human lung cancer cells resulted in a fivefold increase in ATP-binding cassette, subfamily G (WHITE), member 2 (ABCG2), an anthracycline drug transporter, which lead to significantly increased resistance to doxorubicin treatment, a chemotherapeutic agent clinically used to treat lung cancer. In summary, while KLF5 is not required for oncogenic mutant K-Ras-induced lung tumorigenesis, KLF5 regulation of ABCG2 expression may be important for chemotherapeutic resistance and patient survival.

Light-activated large ventral lateral clock neurons (large LNv) modulate behavioral arousal and sleep in Drosophila while their counterparts, the small LNv (s-LNv) are important for circadian behavior. Recently, it has been proposed that the pattern of day-night locomotor behavioral activity is mediated by two anatomically distinct oscillators composed of a morning oscillator in the small LNv and an evening oscillator in the lateral dorsal neurons and an undefined number of dorsal pacemaker neurons. This contrasts with a circuit described by network models which are not as anatomically constrained. By selectively ablating the small LNv while sparing the large LNv, we tested the relative importance of the small and large LNv for regulating morning behavior of animals living in standard light/dark cycles. Behavioral anticipation of the onset of morning and the high amplitude morning startle response which coincides with light onset are preserved in small LNv functionally-ablated animals. However, the amplitude of the morning behavioral peak is severely attenuated in these animals during the transition from regular light/dark cycles to constant darkness, providing further support that small LNv are necessary for circadian behavior. The large LNv, in combination with the network of other circadian neurons, in the absence of functional small LNv are sufficient for the morning anticipation and the high amplitude light-activated morning startle response.

OX40, a membrane-bound member of the tumor-necrosis-factor-receptor (TNFR) superfamily, plays an important role in proliferation, survival and infiltration of activated T cells via binding to OX40L. Recent studies indicate that OX40/OX40L system mediates the adhesion and infiltration of adult T cell leukemia (ATL). Previously, we detected OX40 expression in breast carcinoma cell lines and tissues. The correlation of expression of OX40 and OX40L and clinical features in breast carcinogenesis, however, has not been well characterized. The expression of OX40 and OX40L in 107 invasive ductal carcinomas (IDCa), 9 ductal carcinomas in situ (DCIS), and 31 fibroadenomas from breast tissues and its relationship with the clinical features were determined using immunohistochemistry (peroxidase-conjugated polymer method, ChemMate™ Envision™ Detection kit). The positive immunostaining rates for OX40 in IDCa, DCIS and fibroadenomas from breast tissues were 85.0%, 66.7% and 38.7% respectively, showing a significant difference in OX40 expression among IDCa, DCIS and fibroadenoma of breast (z=5.206, P=0.001). Increased staining intensity of OX40 was associated with TNM stages (z=2.112, P=0.017). Meanwhile, a relation of OX40 expression with lymph node metastatic status in IDCa was found (P=0.041). The expression of OX40L did not show any obvious difference among IDCa, DCIS and fibroadenomas from breast tissues. OX40L expression was also not related to histopathological parameters in IDCa except for progesterone receptor (PR) being positive (P=0.005). However, a high coincidental positive rate for OX40 and OX40L was observed in biopsy samples with IDCa (P=0.017, Kappa=0.231). The present results suggest that high OX40 expression may be associated with malignant transformation, progression, invasion and metastasis in breast cancer biology.

The inflammatory cytokine IL-1 induces profound changes in gene expression. This is contributed in part by activating translation of a distinct set of mRNAs, including IκBζ, as indicated by genome-wide analysis of changes in ribosomal occupancy in IL-1α-treated HeLa cells. Polysome profiling of IκBζ mRNA and reporter mRNAs carrying its 3' UTR indicated poor translation in unstimulated cells. 3' UTR-mediated translational silencing was confirmed by suppression of luciferase activity. Translational silencing was unaffected by replacing the poly(A) tail with a histone stem-loop, but lost under conditions of cap-independent internal initiation. IL-1 treatment of the cells caused profound shifts of endogenous and reporter mRNAs to polysome fractions and relieved suppression of luciferase activity. IL-1 also inhibited rapid mRNA degradation. Both translational activation and mRNA stabilization involved IRAK1 and -2 but occurred independently of the p38 MAPK pathway, which is known to target certain other post-transcriptional mechanisms. The translational silencing RNA element contains the destabilizing element but requires additional 5' sequences and is impaired by mutations that leave destabilization unaffected. These differences in function are associated with differential changes in protein binding in vitro. Thus, rapid degradation occurs independently of the translational silencing effect. The results provide evidence for a novel mode of post-transcriptional control by IL-1, which impinges on the time course and pattern of IL-1-induced gene expression.

<h4>Background</h4>Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear.<h4>Design and methods</h4>Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice.<h4>Results</h4>We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation.<h4>Conclusions</h4>The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression.

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40(+)CD44(hi)effector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti-OX40L monoclonal antibody (mAb) (alpha-OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, alpha-OX40L was added to the combined treatment protocol of LF15-0195 (LF) and anti-CD45RB (alpha-CD45RB) mAb-a protocol that induced heart allograft tolerance in non-presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor-specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs). Of note, CD25(+) T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor-primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.

The balance between descending inhibition and facilitation is thought to be disturbed in chronic pain states. Increased facilitation by spinally released serotonin has been suggested by demonstration that mechanically evoked neuronal responses of wide dynamic range neurons are inhibited by 5-HT3 receptor antagonists in rats following spinal nerve ligation (SNL) but not sham operation. Despite these physiologic data, the effects of spinal 5-HT3 receptor blockade on behavioral hypersensitivity and neurochemical alterations in spinal serotonergic system have not been thoroughly investigated following spinal nerve ligation in the rat. To test this, we acutely injected intrathecal ondansetron in rats between 14 and 30 days after SNL and assessed effects on thermal and mechanical hypersensitivity. We also determined the density of serotonergic nerve fibers, serotonin content and the levels of 5-HT3 receptors within the spinal cord at this time point. Intrathecal ondansetron (1, 3, 10, 30, and 100microg) produced no effect on behavioral measures of thermal or mechanical hypersensitivity whereas intrathecal morphine (1microg) and gabapentin (200microg) partially reversed thermal and mechanical hypersensitivity following SNL. In addition, SNL did not alter the density of serotonergic fibers or 5-HT3 receptor immunoreactivity or spinal tissue content of 5-HT within the dorsal horn. These results do not support anatomic plasticity of descending serotonergic pathways or tonic 5-HT3 receptor activity in maintaining hypersensitivity after nerve injury and in contrast to previous studies fail to demonstrate an anti-hypersensitivity effect of intrathecal injection of the 5-HT3 receptor antagonist ondansetron. Importantly, behavioral measures of mechanical hypersensitivity assess threshold responses whereas physiological studies of mechanically evoked neuronal responses involve application of suprathreshold stimuli. Thus, suprathreshold or more intense stimuli may be necessary to recruit descending serotonergic facilitatory drive required to observe the inhibitory effects of ondansetron on spinal neuronal excitability and behavioral hypersensitivity.

The proteome of the radiation- and desiccation-resistant bacterium D. radiodurans features a group of proteins that contain significant intrinsically disordered regions that are not present in non-extremophile homologues. Interestingly, this group includes a number of housekeeping and repair proteins such as DNA polymerase III, nudix hydrolase and rotamase. Here, we focus on a member of the nudix hydrolase family from D. radiodurans possessing low-complexity N- and C-terminal tails, which exhibit sequence signatures of intrinsic disorder and have unknown function. The enzyme catalyzes the hydrolysis of oxidatively damaged and mutagenic nucleotides, and it is thought to play an important role in D. radiodurans during the recovery phase after exposure to ionizing radiation or desiccation. We use molecular dynamics simulations to study the dynamics of the protein, and study its hydration free energy using the GB/SA formalism. We show that the presence of disordered tails significantly decreases the hydration free energy of the whole protein. We hypothesize that the tails increase the chances of the protein to be located in the remaining water patches in the desiccated cell, where it is protected from the desiccation effects and can function normally. We extrapolate this to other intrinsically disordered regions in proteins, and propose a novel function for them: intrinsically disordered regions increase the "surface-properties" of the folded domains they are attached to, making them on the whole more hydrophilic and potentially influencing, in this way, their localization and cellular activity.

HFE-linked hereditary hemochromatosis is a common form of iron-overload disease in European populations. We studied the role of HFE in macrophage iron metabolism. Patients under venesection treatment had higher EPO levels and drastically reduced levels of transferrin receptor (TfRC and TfR2) mRNA, and also decreased levels of HAMP mRNA in macrophages cultured in autologous serum. Macrophages from C282Y/C282Y patients cultured either in autologous serum or in FBS with or without iron supplementation, had elevated CYBRD1 (cytochrome b reductase 1), SLC40A1 (ferroportin) and FTL (ferritin L) mRNA levels. Those incubated with holo-Tf also showed lower levels of TfRC and TfR2 mRNA. Iron flux from C282Y/C282Y macrophages incubated with a low concentration of non-transferrin-bound iron (NTBI) was similar to that from wild-type macrophages, but incubation with holo-Tf or high NTBI did not trigger a continuous increase in the cytosolic calcein-chelatable iron pool in C282Y/C282Y macrophages conversely to wild-type cells. All culture conditions revealed a high level of intracellular ferritin in C282Y/C282Y macrophages compared to wild-type cells. These results suggest that the non-functional C282Y form of HFE may alter the balance between cytosolic calcein-chelatable iron and sequestered iron, thereby disrupting the iron uptake and release equilibrium in cells involved in iron storage.

<h4>Background & aims</h4>Phosphatidylethanolamine-binding protein 1 (PEBP1, also RKIP) plays a pivotal role in cancer by regulating multiple cellular signaling processes and suppressing metastasis in animal models. We examined whether PEBP1 expression in hepatocellular carcinoma (HCC) correlated with the risk of recurrence and survival after resection.<h4>Methods</h4>A randomly selected cohort of 240 Chinese HCC patients, predominantly hepatitis B related, formed the basis of the study. PEBP1 expression levels were evaluated by immunohistochemistry and real-time reverse-transcriptase PCR. Survival analysis was performed by univariate and multivariate analyses. The results were further validated in an independent series of 403 patients. The relevance of PEBP1 to phospho-ERK was determined by Western blot analysis on clinical samples and hepatoma cell lines.<h4>Results</h4>PEBP1, prevalently down-regulated in HCC, was significantly associated with tumor invasive characteristics (such as vascular invasion, lack of encapsulation, poor differentiation and large size). Both PEBP1 protein and mRNA levels were independent predictors for tumor recurrence (hazard ratio (HR) = 1.877, p=0.001; HR = 2.633, p = 0.001; respectively), and patient survival (HR = 1.796, p = 0.004; HR = 1.730, p = 0.044; respectively). The prognostic value of PEBP1 was then confirmed in the validation cohort. In addition, Western blot suggested that loss of PEBP1 led to hyperactivity of MAPK signaling.<h4>Conclusions</h4>Down-regulation of PEBP1 in HCC indicated aggressive tumor behaviors and predicted a worse clinical outcome, which may be a useful biomarker to identify the patients at high risk of post-operative recurrence.

<h4>Background/aims</h4>It has been suggested that the serotonergic systems are associated with anger and aggressive behaviors. We investigated the association between several single nucleotide polymorphisms in the serotonergic genes and anger-related personality traits.<h4>Methods</h4>A total of 228 healthy female Korean women participated in this study. All subjects were assessed with the State-Trait Anger Expression Inventory (STAXI) and were genotyped for 3 polymorphisms: serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR), tryptophan hydroxylase 1 (TPH1) A218C, and TPH2 G-703T.<h4>Results</h4>The Anger Expression-Out (AX-Out) subscale scores of the STAXI differed significantly between the genotypes for the TPH2 G-703T polymorphism (F = 4.825, p = 0.009). G/G homozygous subjects scored significantly higher on the AX-Out subscale than those with the G/T genotype. However, no significant differences were observed in the relationships between the STAXI subscale scores of subjects with other polymorphisms.<h4>Conclusions</h4>This study suggests that the TPH2 G-703T polymorphism might contribute to anger-related traits, especially to the expression of anger.

Milk thistle contains silybin, which is a potential iron chelator. We aimed to determine whether silybin reduced iron absorption in patients with hereditary haemochromatosis. In this crossover study, on three separate occasions, 10 patients who were homozygous for the C282Y mutation in the HFE gene (and fully treated) consumed a vegetarian meal containing 13.9 mg iron with: 200 ml water; 200 ml water and 140 mg silybin (Legalon Forte); or 200 ml tea. Blood was drawn once before, then 0.5, 1, 2, 3 and 4 h after the meal. Consumption of silybin with a meal resulted in a reduction in the postprandial increase in serum iron (AUC±s.e.) compared with water (silybin 1726.6±346.8 versus water 2988.8±167; P<0.05) and tea (silybin 1726.6±346.8 versus tea 2099.3±223.3; P<0.05). In conclusion, silybin has the potential to reduce iron absorption, and this deserves further investigation, as silybin could be an adjunct in the treatment of haemochromatosis.

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

<h4>Aims</h4>to characterize the transcriptome of human myometrium during spontaneous labor at term.<h4>Methods</h4>myometrium was obtained from women with (n=19) and without labor (n=20). Illumina HumanHT-12 microarrays were utilized. Moderated t-tests and false discovery rate adjustment of P-values were applied. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for a select set of differentially expressed genes in a separate set of samples. Enzyme-linked immunosorbent assay and Western blot were utilized to confirm differential protein production in a third sample set.<h4>Results</h4>1) Four hundred and seventy-one genes were differentially expressed; 2) gene ontology analysis indicated enrichment of 103 biological processes and 18 molecular functions including: a) inflammatory response; b) cytokine activity; and c) chemokine activity; 3) systems biology pathway analysis using signaling pathway impact analysis indicated six significant pathways: a) cytokine-cytokine receptor interaction; b) Jak-STAT signaling; and c) complement and coagulation cascades; d) NOD-like receptor signaling pathway; e) systemic lupus erythematosus; and f) chemokine signaling pathway; 4) qRT-PCR confirmed over-expression of prostaglandin-endoperoxide synthase-2, heparin binding epidermal growth factor (EGF)-like growth factor, chemokine C-C motif ligand 2 (CCL2/MCP1), leukocyte immunoglobulin-like receptor, subfamily A member 5, interleukin (IL)-8, IL-6, chemokine C-X-C motif ligand 6 (CXCL6/GCP2), nuclear factor of kappa light chain gene enhancer in B-cells inhibitor zeta, suppressor of cytokine signaling 3 (SOCS3) and decreased expression of FK506 binding-protein 5 and aldehyde dehydrogenase in labor; 5) IL-6, CXCL6, CCL2 and SOCS3 protein expression was significantly higher in the term labor group compared to the term not in labor group.<h4>Conclusions</h4>myometrium of women in spontaneous labor at term is characterized by a stereotypic gene expression pattern consistent with over-expression of the inflammatory response and leukocyte chemotaxis. Differential gene expression identified with microarray was confirmed with qRT-PCR using an independent set of samples. This study represents an unbiased description of the biological processes involved in spontaneous labor at term based on transcriptomics.

Reversibility of liver fibrosis or cirrhosis involves complete restoration of normal liver architecture. This phenomenon has been well documented in chronic liver diseases such as autoimmune hepatitis, biliary obstruction, hemochromatosis, nonalcoholic steatohepatitis, and viral hepatitis. There are very few reports of reversal of cirrhosis after antiviral therapy in patients with chronic hepatitis B virus (HBV) infection. We report a case of disappearance of HBV-induced liver cirrhosis after years of treatment with distinct antiviral drugs, documented by successive biopsy results. This disappearance was accompanied by normalization of platelet count, gammaglobulin titers, and radiologic findings.

<h4>Objectives</h4>We have recently found a high prevalence of non-typeable pneumococcal isolates (NTPn) circulating in day-care centers in Central Brazil, besides serotype 14 isolates. We therefore examined the genetic relationship among NTPn and serotype 14 from carriage and invasive pneumococcal isolates obtained from children attending emergency rooms enrolled in a population-based surveillance.<h4>Methods</h4>The isolates were characterized by Quellung reaction serotyping, PCR for the presence of pneumolysin and the loci for a capsule gene (cpsA) and the type 14 gene (cps14H) in all NTPn, and by multilocus sequence typing and pulsed field gel electrophoresis.<h4>Results</h4>87.2% of the isolates were clustered into nine clusters. The major cluster included 41 pneumococcal serotype 14 (28 carriage and 13 invasive isolates) and two NTPn related to the global pneumococcal clone Spain(9V)-3. Overall, 95.4% of the NTPn carriage strains were genetically related to carriage or invasive strains expressing serotype 14. A dominant NTPn lineage was found, that grouped 14 pneumococcal strains. Almost half of the multidrug-resistant isolates grouped into the NTPn cluster.<h4>Conclusion</h4>These findings provide baseline data to assess the impact of the pneumococcal vaccination on the molecular epidemiology of Streptococcus pneumoniae. Changes in frequency of NTPn isolates and also genetic changes should be carefully monitored post vaccination, to detect potential vaccine-escape or replacement disease by capsule switched strains, especially in areas where colonization with NTPn has been frequently observed.

<h4>Rationale</h4>Previous studies have shown extensive serotonergic deficits in the hippocampus of Alzheimer's disease (AD) patients. However, it is unclear whether such deficits play a role in non-cognitive, neuropsychiatric behaviors that occur frequently in AD and cause significant caregiver distress.<h4>Objectives</h4>In this study, we aimed to correlate serotonergic markers in the AD hippocampus with neuropsychiatric behaviors.<h4>Methods</h4>Using postmortem hippocampal homogenates from aged controls as well as a cohort of longitudinally assessed AD patients, measurements of 5-HT(1A) receptors, 5-HT(2A) receptors, and serotonin re-uptake (5-HTT) sites were performed by binding with (3)H-labeled 8-OH-DPAT, ketanserin, and citalopram, respectively.<h4>Results</h4>Alterations of 5-HT(1A) receptors and 5-HTT were found to be differentially involved in neuropsychiatric behaviors, with loss of 5-HT(1A) receptors specifically correlated with depressive symptoms, while 5-HTT sites were preserved or up-regulated in patients with aggressive behaviors.<h4>Conclusions</h4>Our data suggest that neuropsychiatric behaviors in AD share certain neurochemical features with psychiatric disorders like major depression and that serotonergic drugs used in psychiatric disorders may also be efficacious against behavioral symptoms in AD.

The kidney is the main physiologic source of erythropoietin (EPO) in the adult and responds to decreases in tissue oxygenation with increased EPO production. Although studies in mice with liver-specific or global gene inactivation have shown that hypoxia-inducible factor 2 (Hif-2) plays a major role in the regulation of Epo during infancy and in the adult, respectively, the contribution of renal HIF-2 signaling to systemic EPO homeostasis and the role of extrarenal HIF-2 in erythropoiesis, in the absence of kidney EPO, have not been examined directly. Here, we used Cre-loxP recombination to ablate Hif-2α in the kidney, whereas Hif-2-mediated hypoxia responses in the liver and other Epo-producing tissues remained intact. We found that the hypoxic induction of renal Epo is completely Hif-2 dependent and that, in the absence of renal Hif-2, hepatic Hif-2 takes over as the main regulator of serum Epo levels. Furthermore, we provide evidence that hepatocyte-derived Hif-2 is involved in the regulation of iron metabolism genes, supporting a role for HIF-2 in the coordination of EPO synthesis with iron homeostasis.

Within its life cycle Eimeria bovis undergoes a long lasting intracellular development into large macromeronts in endothelial cells. Since little is known about the molecular basis of E. bovis-triggered host cell regulation we applied a microarray-based approach to define transcript variation in bovine endothelial cells early after sporozoite invasion (4 h post inoculation (p.i.)), during trophozoite establishment (4 days p.i.), during early parasite proliferation (8 days p.i.) and towards macromeront maturation (14 days p.i.). E. bovis infection led to significant changes in the abundance of many host cell gene transcripts. As infection progressed, the number of regulated genes increased such that 12, 45, 175 and 1184 sequences were modulated at 4 h, 4, 8 and 14 days p.i., respectively. These genes significantly interfered with several host cell functions, networks and canonical pathways, especially those involved in cellular development, cell cycle, cell death, immune response and metabolism. The correlation between stage of infection and the number of regulated genes involved in different aspects of metabolism suggest parasite-derived exploitation of host cell nutrients. The modulation of genes involved in cell cycle arrest and host cell apoptosis corresponds to morphological in vitro findings and underline the importance of these aspects for parasite survival. Nevertheless, the increasing numbers of modulated transcripts associated with immune responses also demonstrate the defensive capacity of the endothelial host cell. Overall, this work reveals a panel of novel candidate genes involved in E. bovis-triggered host cell modulation, providing a valuable tool for future work on this topic.

A genome's ability to produce two separate sexually dimorphic phenotypes is an intriguing biological mystery. Microarray-based studies of a handful of model systems suggest that much of the mystery can be explained by sex-biased gene expression evolved in response to sexually antagonistic selection. We present the first whole-genome study of sex-biased expression in the red flour beetle, Tribolium castaneum. Tribolium is a model for the largest eukaryotic order, Coleoptera, and we show that in whole-body adults, approximately 20% of the transcriptome is differentially regulated between the sexes. Among T. castaneum, Drosophila melanogaster, and Anopheles gambiae, we identify 416 1:1:1 orthologs with conserved sex-biased expression. Overrepresented functional categories among sex-biased genes are primarily those involved in gamete production and development. The genomic distribution of sex-biased genes in T. castaneum is distinctly nonrandom, with the strongest deficit of male-biased genes on the X chromosome (9 of 793) of any species studied to date. Tribolium also shows a significant enrichment of X-linked female-biased genes (408 of 793). Our analyses suggest that the extensive female bias of Tribolium X chromosome gene expression is due to hyperexpression of X-linked genes in both males and females. We propose that the overexpression of X chromosomes in females is an evolutionary side effect of the need to dosage compensate in males and that mechanisms to reduce female X chromosome gene expression to autosomal levels are sufficient but imperfect.

Auditory neuropathy is a rare form of deafness characterized by an absent or abnormal auditory brainstem response with preservation of outer hair cell function. We have identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for autosomal dominant nonsyndromic auditory neuropathy (AUNA1), which we previously mapped to chromosome 13q21-q24. Genotyping of additional family members narrowed the interval to an 11-Mb, 3.28-cM gene-poor region containing only four genes, including DIAPH3. DNA sequencing of DIAPH3 revealed a c.-172G>A, g. 48G>A mutation in a highly conserved region of the 5' UTR. The c.-172G>A mutation occurs within a GC box sequence element and was not found in 379 controls. Using genome-wide expression arrays and quantitative RT-PCR, we demonstrate a 2- to 3-fold overexpression of DIAPH3 mRNA in lymphoblastoid cell lines from affected individuals. Likewise, a significant increase (approximately 1.5-fold) in DIAPH3 protein was found by quantitative immunoblotting of lysates from lymphoblastoid cell lines derived from affected individuals in comparison with controls. In addition, the c.-172G>A mutation is sufficient to drive overexpression of a luciferase reporter. Finally, the expression of a constitutively active form of diaphanous protein in the auditory organ of Drosophila melanogaster recapitulates the phenotype of impaired response to sound. To date, only two genes, the otoferlin gene OTOF and the pejvakin gene PJVK, are known to underlie nonsyndromic auditory neuropathy. Genetic testing for DIAPH3 may be useful for individuals with recessive as well as dominant inheritance of nonsyndromic auditory neuropathy.

Although the apoptotic role of caspases has been largely understood, accumulating evidence in Drosophila suggests that caspases also control other processes than apoptotic cell death. However, how caspases contribute to the development of the mammalian nervous system remains obscure. Here, we provide unique evidence that Apaf-1/caspase-9-mediated caspase signaling regulates the development of olfactory sensory neurons (OSNs), which includes axonal projection, synapse formation, and maturation of these neurons. This caspase signaling leads to a cleavage of Semaphorin 7A, a membrane-anchored semaphorin that is required for the proper axonal projection. Mutant mice deficient for apaf-1 or caspase-9 exhibit misrouted axons, impaired synaptic formation, and defects in the maturation of OSNs without affecting the number of these cells. Our findings suggest that Apaf-1/caspase-9-mediated nonapoptotic caspase signaling is required for the proper neural network formation during olfactory development.

Although previous studies of Huntington's disease (HD) have addressed many potential mechanisms of striatal neuron dysfunction and death, it is also known, based on clinical findings, that cortical function is dramatically disrupted in HD. With respect to disease etiology, however, the specific molecular and neuronal circuit bases for the cortical effects of mutant huntingtin (htt) have remained largely unknown. In the present work, we studied the relationship between the molecular effects of mutant htt fragments in cortical cells and the corresponding behavior of cortical neuron microcircuits by using a novel cellular model of HD. We observed that a transcript-selective diminution in activity-dependent brain-derived neurotrophic factor (BDNF) expression preceded the onset of a synaptic connectivity deficit in ex vivo cortical networks, which manifested as decreased spontaneous collective burst-firing behavior measured by multielectrode array substrates. Decreased BDNF expression was determined to be a significant contributor to network-level dysfunction, as shown by the ability of exogenous BDNF to ameliorate cortical microcircuit burst firing. The molecular determinants of the dysregulation of activity-dependent BDNF expression by mutant htt seem to be distinct from previously elucidated mechanisms, because they do not involve known neuron-restrictive silencer factor/RE1-silencing transcription factor-regulated promoter sequences but instead result from dysregulation of BDNF exon IV and VI transcription. These data elucidate a novel HD-related deficit in BDNF gene regulation as a plausible mechanism of cortical neuron hypoconnectivity and cortical function deficits in HD. Moreover, the novel model paradigm established here is well suited to further mechanistic and drug screening research applications.

Glomerular diseases account for the majority of cases with chronic renal failure. Several genes have been identified with key relevance for glomerular function. Quite a few of these genes show a specific or preferential mRNA expression in the renal glomerulus. To identify additional candidate genes involved in glomerular function in humans we generated a human renal glomerulus-enriched gene expression dataset (REGGED) by comparing gene expression profiles from human glomeruli and tubulointerstitium obtained from six transplant living donors using Affymetrix HG-U133A arrays. This analysis resulted in 677 genes with prominent overrepresentation in the glomerulus. Genes with 'a priori' known prominent glomerular expression served for validation and were all found in the novel dataset (e.g. CDKN1, DAG1, DDN, EHD3, MYH9, NES, NPHS1, NPHS2, PDPN, PLA2R1, PLCE1, PODXL, PTPRO, SYNPO, TCF21, TJP1, WT1). The mRNA expression of several novel glomerulus-enriched genes in REGGED was validated by qRT-PCR. Gene ontology and pathway analysis identified biological processes previously not reported to be of relevance in glomeruli of healthy human adult kidneys including among others axon guidance. This finding was further validated by assessing the expression of the axon guidance molecules neuritin (NRN1) and roundabout receptor ROBO1 and -2. In diabetic nephropathy, a prevalent glomerulopathy, differential regulation of glomerular ROBO2 mRNA was found.In summary, novel transcripts with predominant expression in the human glomerulus could be identified using a comparative strategy on microdissected nephrons. A systematic analysis of this glomerulus-specific gene expression dataset allows the detection of target molecules and biological processes involved in glomerular biology and renal disease.

FBXW7 is a tumor suppressor gene that induces the degradation of positive cell-cycle regulators such as c-Myc, cyclin E, c-Jun and Notch. The loss of FBXW7 promotes cell-cycle progression and cell proliferation. In the present study, we investigated the relationship between FBXW7 expression and the clinicopathological characteristics of patients with esophageal squamous cell carcinoma (ESCC). The expression of FBXW7 was quantified by real-time reverse transcription polymerase chain reaction in 43 primary ESCCs and their paired normal esophageal mucosa in patients who had not received preoperative therapy. FBXW7 expression levels were significantly correlated with the progression of the cancer and with local invasiveness. In muscle-invasive tumor cases (T2-4), lymphatic invasive tumor cases and stage II-IV cases, FBXW7 expression levels were significantly decreased (P=0.0315, P=0.0336 and P=0.0289, respectively). Decreased expression of FBXW7 was correlated with poor prognosis (P=0.0255). In conclusion, this study examined the relationship between FBXW7 expression and tumor progression in ESCC. We suggest that FBXW7 is a molecular prognostic marker and can be used to elucidate the mechanism of carcinogenesis.

The retinal homeobox (Rx/rax) gene is essential for the development of the eye. Rax is among the earliest genes expressed during eye development, beginning in the prospective eye fields in the anterior neural plate. Additionally Rax expression persists in retinal progenitor cells and in differentiated photoreceptors. We have isolated and characterized a 2.8 kb genomic DNA fragment that regulates expression of Rax in the developing and maturing retina. We have discovered and characterized cis-acting elements that function to specifically control spatial and temporal Rax expression during retinal development. We have found that the regulation of Rax2A promoter activity requires cooperative interactions between positive and negative regulatory elements. Further, a highly conserved genomic element containing SOX, OTX, and POU transcription factor binding sites is necessary but not sufficient for promoter activity in retinal progenitor or stem cells. Finally, a putative binding element for forkhead transcription factors is necessary for promoter activity and can cooperate with other cis-acting elements to drive Rax2A promoter activity.

Peroxiredoxin 6 (Prdx6) and cytosolic GSH peroxidase (GPx1), both GSH-dependent peroxidases, were compared for the effects of their knockout on injury and lipid peroxidation in: (a) lungs of mice exposed to 0.85 or 1.0atm O(2), (b) isolated perfused mouse lungs exposed to 5mM tert-butylhydroperoxide (t-BOOH) or 1mM paraquat, and (c) primary mouse pulmonary microvascular endothelial cells exposed to 50muM t-BOOH. Derangements in GPx1 null were similar or slightly greater than in wild type for all parameters in the various models of oxidant stress, whereas Prdx6 null showed markedly increased effects. GSH peroxidase activity with phosphatidylcholine hydroperoxide as substrate in GPx1-null lung homogenate was decreased only slightly vs wild type, whereas activity in Prdx6-null lungs was decreased by ~95%, indicating that Prdx6 is the major enzyme for reduction of oxidized lung phospholipids. Expression levels of oxidant-related genes measured with a PCR-based gene array indicated no significant differences between the Prdx6 and the GPx1 null except for the target genes and IL-19. Thus, Prdx6-null mice are significantly more sensitive to oxidant stress compared to GPx1 null, suggesting that scavenging of phospholipid hydroperoxides by Prdx6 plays a major role in lung antioxidant defense.

Ferritin is an acute-phase reactant that is elevated in the course of infectious, inflammatory, autoimmune, and oncological diseases and the hemophagocytic syndrome. In asymptomatic patients, isolated hyperferritinemia may be due to different causes depending on whether or not it is accompanied by iron overload. Hyperferritinemia values above 300 ng/ml and an excess of body iron levels may be indicative of hemochromatosis. However, if such values develop in the absence of iron overload, they may be secondary to hemochromatosis type 4a (ferroportin disease) or more often to hereditary hyperferritinemia-cataract syndrome (HHCS; Aguilar-Martinez et al., Am J Gastroenterol 100:1185-1194, 2005; Ferrante et al., Eur J Gastroenterol Hepatol 17:1247-1253, 2005). HHCS results from different mutations in the L-ferritin gene (FTL) on chromosome 19 (19q13.1), causing autosomal dominant transmission (Bertola et al., Curr Drug Targets Immune Endocr Metabol Disord 4:93-105, 2004). We present a child with HHCS due to the allelic variant c.-167C>T (C33T) in the iron-responsive element region of the FTL gene. When pediatricians encounter an asymptomatic patient with isolated hyperferritinemia in the absence of iron overload, they should consider the possibility of HHCS, especially if other members of the family have developed cataracts from a young age.

<h4>Objective</h4>To examine the association between maternal and fetal genetic variants and small-for-gestational-age (SGA).<h4>Methods</h4>A case-control study was conducted in patients with SGA neonates (530 maternal and 436 fetal) and controls (599 maternal and 628 fetal); 190 candidate genes and 775 SNPs were studied. Single-locus, multi-locus and haplotype association analyses were performed on maternal and fetal data with logistic regression, multifactor dimensionality reduction (MDR) analysis, and haplotype-based association with 2 and 3 marker sliding windows, respectively. Ingenuity pathway analysis (IPA) software was used to assess pathways that associate with SGA.<h4>Results</h4>The most significant single-locus association in maternal data was with a SNP in tissue inhibitor of metalloproteinase 2 (TIMP2) (rs2277698 OR = 1.71, 95% CI [1.26-2.32], p = 0.0006) while in the fetus it was with a SNP in fibronectin 1 isoform 3 preproprotein (FN1) (rs3796123, OR = 1.46, 95% CI [1.20-1.78], p = 0.0001). Both SNPs were adjusted for potential confounders (maternal body mass index and fetal sex). Haplotype analyses resulted in associations in α 1 type I collagen preproprotein (COL1A1, rs1007086-rs2141279-rs17639446, global p = 0.006) in mothers and FN1 (rs2304573-rs1250204-rs1250215, global p = 0.045) in fetuses. Multi-locus analyses with MDR identified a two SNP model with maternal variants collagen type V α 2 (COL5A2) and plasminogen activator urokinase (PLAU) predicting SGA outcome correctly 59% of the time (p = 0.035).<h4>Conclusions</h4>Genetic variants in extracellular matrix-related genes showed significant single-locus association with SGA. These data are consistent with other studies that have observed elevated circulating fibronectin concentrations in association with increased risk of SGA. The present study supports the hypothesis that DNA variants can partially explain the risk of SGA in a cohort of Hispanic women.

The aggregation of mutant polyglutamine (polyQ) proteins has sparked interest in the role of protein quality-control pathways in Huntington's disease (HD) and related polyQ disorders. Employing a novel knock-in HD mouse model, we provide in vivo evidence of early, sustained alterations of autophagy in response to mutant huntingtin (mhtt). The HdhQ200 knock-in model, derived from the selective breeding of HdhQ150 knock-in mice, manifests an accelerated and more robust phenotype than the parent line. Heterozygous HdhQ200 mice accumulate htt aggregates as cytoplasmic aggregation foci (AF) as early as 9 weeks of age and striatal neuronal intranuclear inclusions (NIIs) by 20 weeks. By 40 weeks, striatal AF are perinuclear and immunoreactive for ubiquitin and the autophagosome marker LC3. Striatal NIIs accumulate earlier in HdhQ200 mice than in HdhQ150 mice. The earlier appearance of aggregate pathology in HdhQ200 mice is paralleled by earlier and more rapidly progressive motor deficits: progressive imbalance and decreased motor coordination by 50 weeks, gait deficits by 60 weeks and gross motor impairment by 80 weeks of age. At 80 weeks, heterozygous HdhQ200 mice exhibit striatal and cortical astrogliosis and a approximately 50% reduction in striatal dopamine receptor binding. Increased LC3-II protein expression, which is noted early and sustained throughout the disease course, is paralleled by increased expression of the autophagy-related protein, p62. Early and sustained expression of autophagy-related proteins in this genetically precise mouse model of HD suggests that the alteration of autophagic flux is an important and early component of the neuronal response to mhtt.

Recent genome-wide association (GWA) studies have identified 18 genetic loci for obesity. Using directly observed and imputed GWA genotyping data on approximately 5,000 Chinese women (1996-2007), the authors evaluated 17 single nucleotide polymorphisms (SNPs) that represent 17 distinct obesity loci. Two SNPs near the BAT2 and MC4R genes and 3 SNPs within the FTO, SEC16B, and SH2B1 genes were significantly associated with body mass index (weight (kg)/height (m)(2)), body weight, and the prevalence of obesity. The per-allele increase in body mass index ranged from 0.16 units (BAT2) to 0.38 units (SH2B1). Odds ratios for obesity ranged from 1.46 (95% confidence interval (CI): 1.12, 1.92) for BAT2 to 2.16 (95% CI: 1.39, 3.37) for MC4R. A genetic risk score calculated by summing the number of risk-increasing alleles that each woman carried at these 5 loci was significantly associated with the prevalence of obesity. Women carrying 5 or more risk alleles had a 3.13-fold (95% CI: 2.06, 4.77) higher prevalence of obesity than women carrying 1 or no risk alleles. Results from this study extend some previous GWA findings to Chinese women and show the need for additional studies to identify susceptibility loci in Chinese and other Asian populations.

<h4>Motivation</h4>The vertebrate retina is a complex neuronal tissue, and its development, normal functioning and response to injury and disease is subject to a variety of genetic factors. To understand better the regulatory and functional relationships between the genes expressed within the retina, we constructed an interactive gene network of the mouse retina by applying a Bayesian statistics approach to information derived from a variety of gene expression, protein-protein interaction and gene ontology annotation databases.<h4>Results</h4>The network contains 673 retina-related genes. Most of them are obtained through manual literature-based curation, while the others are the genes preferentially expressed in the retina. These retina-related genes are linked by 3403 potential functional associations in the network. The prediction on the gene functional association using the Bayesian approach outperforms predictions using only one source of information. The network includes five major gene clusters, each enriched in different biological activities. There are several applications to this network. First, we identified approximately 50 hub genes that are predicted to play particularly important roles in the function of the retina. Some of them are not yet well studied. Second, we can predict novel gene functions using 'guilt by association' method. Third, we also predicted novel retinal disease-associated genes based on the network analysis.<h4>Availability</h4>To provide easy access to the retinal network, we constructed an interactive web tool, named MoReNet, which is available at http://bioinfo.wilmer.jhu.edu/morenet/.

Hepcidin is a liver-synthesized hormone that inhibits the cellular efflux of iron by binding to ferroportin and its subsequent degradation. The main role of hepcidin is regulation of ferroportin expression and cell membrane function. Recent studies implicate hepcidin in a variety of iron disorders in addition to its primary role in the regulation of systemic iron homeostasis. Hepcidin excess has a key pathologic role in anemia of inflammation, chronic kidney disease, and iron-refractory iron deficiency anemia, while hepcidin deficiency is responsible for most cases of familial hemochromatosis and iron-loading anemia. The most important advances on the role of hepcidin in normal and abnormal iron metabolism and the main clinical and diagnostic implications are summarized in this review.

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a CAG repeat that encodes a polyglutamine tract in ATAXIN1 (ATXN1). Molecular and genetic data indicate that SCA1 is mainly caused by a gain-of-function mechanism. However, deletion of wild-type ATXN1 enhances SCA1 pathogenesis, whereas increased levels of an evolutionarily conserved paralog of ATXN1, Ataxin 1-Like, ameliorate it. These data suggest that a partial loss of ATXN1 function contributes to SCA1. To address this possibility, we set out to determine if the SCA1 disease model (Atxn1(154Q/+) mice) and the loss of Atxn1 function model (Atxn1-/- mice) share molecular changes that could potentially contribute to SCA1 pathogenesis. To identify transcriptional changes that might result from loss of function of ATXN1 in SCA1, we performed gene expression microarray studies on cerebellar RNA from Atxn1-/- and Atxn1(154Q/+) cerebella and uncovered shared gene expression changes. We further show that mild overexpression of Ataxin-1-Like rescues several of the molecular and behavioral defects in Atxn1-/- mice. These results support a model in which Ataxin 1-Like overexpression represses SCA1 pathogenesis by compensating for a partial loss of function of Atxn1. Altogether, these data provide evidence that partial loss of Atxn1 function contributes to SCA1 pathogenesis and raise the possibility that loss-of-function mechanisms contribute to other dominantly inherited neurodegenerative diseases.

Puberty is a critical period in mesocorticolimbic dopamine (DA) system development, particularly for the medial prefrontal cortex (mPFC) projection which achieves maturity in early adulthood. The guidance cue netrin-1 organizes neuronal networks by attracting or repelling cellular processes through DCC (deleted in colorectal cancer) and UNC-5 homologue (UNC5H) receptors, respectively. We have shown that variations in netrin-1 receptor levels lead to selective reorganization of mPFC DA circuitry, and changes in DA-related behaviors, in transgenic mice and in rats. Significantly, these effects are only observed after puberty, suggesting that netrin-1 mediated effects on DA systems vary across development. Here we report on the normal expression of DCC and UNC5H in the ventral tegmental area (VTA) by DA neurons from embryonic life to adulthood, in both mice and rats. We show a dramatic and enduring pubertal change in the ratio of DCC:UNC5H receptors, reflecting a shift toward predominant UNC5H function. This shift in DCC:UNC5H ratio coincides with the pubertal emergence of UNC5H expression by VTA DA neurons. Although the distribution of DCC and UNC5H by VTA DA neurons changes during puberty, the pattern of netrin-1 immunoreactivity in these cells does not. Together, our findings suggest that DCC:UNC5H ratios in DA neurons at critical periods may have important consequences for the organization and function of mesocorticolimbic DA systems.

The mechanisms of stress tolerance in sessile animals, such as molluscs, can offer fundamental insights into the adaptation of organisms for a wide range of environmental challenges. One of the best studied processes at the molecular level relevant to stress tolerance is the heat shock response in the genus Mytilus. We focus on the upstream region of Mytilus galloprovincialis Hsp90 genes and their structural and functional associations, using comparative genomics and network inference. Sequence comparison of this region provides novel evidence that the transcription of Hsp90 is regulated via a dense region of transcription factor binding sites, also containing a region with similarity to the Gamera family of LINE-like repetitive sequences and a genus-specific element of unknown function. Furthermore, we infer a set of gene networks from tissue-specific expression data, and specifically extract an Hsp class-associated network, with 174 genes and 2,226 associations, exhibiting a complex pattern of expression across multiple tissue types. Our results (i) suggest that the heat shock response in the genus Mytilus is regulated by an unexpectedly complex upstream region, and (ii) provide new directions for the use of the heat shock process as a biosensor system for environmental monitoring.

Tight junctions are the apical cell-cell adhesion that regulate paracellular permeability and are critical for epithelial cell polarity. Molecular architecture of tight junction has been studied extensively, which has confirmed that claudin family of proteins is integral component of tight junction. Loss of cell-cell adhesion is central to the cellular transformation and acquisition of metastatic potential; however, the role of claudin family of proteins play in a series of pathophysiological events, including human carcinoma development, is only now beginning to be understood. Several claudin mouse knockout models have been generated and the diversity of phenotypes observed clearly demonstrates their important roles in the maintenance of tissue integrity in various organs and suggest that claudins also participate in cellular contexts other than tight junctions. The mechanisms of claudin regulation and their exact roles in normal physiology and disease are being elucidated, but much work remains to be done. In this review, we have discussed the conceptual framework concerning claudins and their potential implication in cancer. We predict that next several years will likely witness a boom in our understanding of the potential role of claudins in the regulation of tumorigenesis, which may, in turn, provide new approaches for the targeted therapy.

<h4>Background</h4>Synuclein gamma (SNCG), initially identified as a breast cancer specific gene, is aberrantly expressed in many different malignant tumors but rarely expressed in matched nonneoplastic adjacent tissues. In this study, we investigated the prognostic potential of SNCG in colon cancer particularly in the patients with normal carcinoembryonic antigen (CEA) levels.<h4>Methods</h4>SNCG levels were assessed immunohistochemically in cancer tissues from 229 colon adenocarcinoma patients with a mean follow-up of 44 months. Correlations between SNCG levels and clinicopathologic features, preoperative serum CEA level, and clinical outcome were analyzed statistically using SPSS.<h4>Results</h4>SNCG levels in colon adenocarcinoma were closely associated with intravascular embolus and tumor recurrence but independent of preoperative serum CEA levels. SNCG expression was an independent prognostic factor of a shorter disease-free survival (DFS) and overall survival (OS) (P < 0.0001). Multivariate analysis revealed that both tissue SNCG and serum CEA were independent prognostic factors of DFS (P = 0.001, <0.0001, respectively) for 170 patients with colon adenocarcinomas. Importantly, SNCG remained a prognostic determinant of DFS and OS (P = 0.001, 0.002) for 97 patients with normal preoperative serum CEA level.<h4>Conclusions</h4>Our results suggest for the first time that SNCG is a new independent predicator for poor prognosis in patients with colon adenocarcinoma, including those with normal CEA levels. Combination of CEA with SNCG improves prognostic evaluation for patients with colon adenocarcinoma.

<h4>Background</h4>Mutarotases are recently characterized family of enzymes that are involved in the anomeric conversions of monosaccharides. The mammalian fucose mutarotase (FucM) was reported in cultured cells to facilitate fucose utilization and incorporation into protein by glycosylation. However, the role of this enzyme in animal has not been elucidated.<h4>Results</h4>We generated a mutant mouse specifically lacking the fucose mutarotase (FucM) gene. The FucM knockout mice displayed an abnormal sexual receptivity with a drastic reduction in lordosis score, although the animals were fertile due to a rare and forced intromission by a typical male. We examined the anteroventral periventricular nucleus (AVPv) of the preoptic region in brain and found that the mutant females showed a reduction in tyrosine hydoxylase positive neurons compared to that of a normal female. Furthermore, the mutant females exhibited a masculine behavior, such as mounting to a normal female partner as well as showing a preference to female urine. We found a reduction of fucosylated serum alpha-fetoprotein (AFP) in a mutant embryo relative to that of a wild-type embryo.<h4>Conclusions</h4>The observation that FucM-/- female mouse exhibits a phenotypic similarity to a wild-type male in terms of its sexual behavior appears to be due to the neurodevelopmental changes in preoptic area of mutant brain resembling a wild-type male. Since the previous studies indicate that AFP plays a role in titrating estradiol that are required to consolidate sexual preference of female mice, we speculate that the reduced level of AFP in FucM-/- mouse, presumably resulting from the reduced fucosylation, is responsible for the male-like sexual behavior observed in the FucM knock-out mouse.

The feline leukemia virus subgroup C receptor (FLVCR) is a heme export protein that is required for proerythroblast survival and facilitates macrophage heme iron recycling. However, its mechanism of heme export and substrate specificity are uncharacterized. Using [(55)Fe]heme and the fluorescent heme analog zinc mesoporphyrin, we investigated whether export by FLVCR depends on the availability and avidity of extracellular heme-binding proteins. Export was 100-fold more efficient when the medium contained hemopexin (K(d) < 1 pm) compared with albumin (K(d) = 5 nm) at the same concentration and was not detectable when the medium lacked heme-binding proteins. Besides heme, FLVCR could export other cyclic planar porphyrins, such as protoporphyrin IX and coproporphyrin. However, FLVCR has a narrow substrate range because unconjugated bilirubin, the primary breakdown product of heme, was not transported. As neither protoporphyrin IX nor coproporphyrin export improved with extracellular hemopexin (versus albumin), our observations further suggest that hemopexin, an abundant protein with a serum concentration (6.7-25 mum) equivalent to that of the iron transport protein transferrin (22-31 mum), by accepting heme from FLVCR and targeting it to the liver, might regulate macrophage heme export and heme iron recycling in vivo. Final studies show that hemopexin directly interacts with FLVCR, which also helps explain why FLVCR, in contrast to some major facilitator superfamily members, does not function as a bidirectional gradient-dependent transporter. Together, these data argue that hemopexin has a role in assuring systemic iron balance during homeostasis in addition to its established role as a scavenger during internal bleeding or hemolysis.

Unusually for invertebrates, linguliform brachiopods employ calcium phosphate mineral in hard tissue formation, in common with the evolutionarily distant vertebrates. Using solid-state nuclear magnetic resonance spectroscopy (SSNMR) and X-ray powder diffraction, we compare the organic constitution, crystallinity and organic matrix-mineral interface of phosphatic brachiopod shells with those of vertebrate bone. In particular, the organic-mineral interfaces crucial for the stability and properties of biomineral were probed with SSNMR rotational echo double resonance (REDOR). Lingula anatina and Discinisca tenuis shell materials yield strikingly dissimilar SSNMR spectra, arguing for quite different organic constitutions. However, their fluoroapatite-like mineral is highly crystalline, unlike the poorly ordered hydroxyapatite of bone. Neither shell material shows (13)C{(31)P} REDOR effects, excluding strong physico-chemical interactions between mineral and organic matrix, unlike bone in which glycosaminoglycans and proteins are composited with mineral at sub-nanometre length scales. Differences between organic matrix of shell material from L. anatina and D. tenuis, and bone reflect evolutionary pressures from contrasting habitats and structural purposes. The absence of organic-mineral intermolecular associations in brachiopod shell argues that biomineralization follows different mechanistic pathways to bone; their details hold clues to the molecular structural evolution of phosphatic biominerals, and may provide insights into novel composite design.

DNA methylation and histone modifications have essential roles in remodeling chromatin structure of genes necessary for multi-lineage differentiation of mammary stem/progenitor cells. The role of this well-defined epigenetic programming is to heritably maintain transcriptional plasticity of these loci over multiple cell divisions in the differentiated progeny. Epigenetic events can be deregulated in progenitor cells chronically exposed to xenoestrogen or inflammatory microenvironment. In addition, epigenetically mediated silencing of genes associated with tumor suppression can take place, resulting in clonal proliferation of undifferentiated or semidifferentiated cells. Alternatively, microRNAs that negatively regulate the expression of their protein-coding targets may become epigenetically repressed, leading to oncogenic expression of these genes. Here we further discuss interactions between DNA methylation and histone modifications that have significant contributions to the differentiation of mammary stem/progenitor cells and to tumor initiation and progression.

The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.

Fourteen novel conjugates of 3,28-di-O-acylbetulins with AZT were prepared as anti-HIV agents, based on our previously reported potent anti-HIV triterpene leads, including 3-O-acyl and 3,28-di-O-acylbetulins. Nine of the conjugates (49-53, 55, 56, 59, and 60) exhibited potent anti-HIV activity at the submicromolar level, with EC(50) values ranging from 0.040 to 0.098muM in HIV-1(NL4-3) infected MT-4 cells. These compounds were equipotent or more potent than 3-O-(3',3'-dimethylsuccinyl)betulinic acid (2), which is currently in Phase IIb anti-AIDS clinical trial.

White matter lesions (WMLs), commonly seen as hyperintensities on T2-weighted MRI scans of healthy elderly individuals, are considered to be related to small vessel disease in the brain, and are often associated with subtle cognitive and functional impairments. WMLs also show a strong correlation with a wide range of neurodegenerative and neuropsychiatric disorders. Although a number of vascular risk factors for WMLs have been identified, genetic factors are also important with twin and family studies reporting high heritability. Mutations in several genes have been described that lead to monogenic disorders manifesting WMLs, such as Fabry disease and CADASIL. Because most individuals with WMLs do not have Mendelian disorders, most of the focus has been on single nucleotide polymorphisms as genetic risk markers for WMLs, either directly or through their interactions with other genes or medical risk factors. Candidate genes examined to date include those involved in cholesterol regulation and atherosclerosis, hypertension, neuronal repair, homocysteine levels, and oxidative stress pathways. In addition, although there have been a few genome-wide linkage studies, only one genome-wide association study has been performed. The majority of the genetic findings need independent replication, and studies need to be extended to other candidate genes. Collaborative efforts to examine genome-wide associations in large samples of both sexes of a broad age range using longitudinal studies are necessary. The identification of individuals genetically at risk of developing white matter lesions will have important implications for recognizing the etiology of WMLs and thereby developing clinical intervention strategies for their prevention.

<h4>Background</h4>Hyperthermia has been shown in a number of organisms to induce developmental defects as a result of changes in cell proliferation, differentiation and gene expression. In spite of this, salmon aquaculture commonly uses high water temperature to speed up developmental rate in intensive production systems, resulting in an increased frequency of skeletal deformities. In order to study the molecular pathology of vertebral deformities, Atlantic salmon was subjected to hyperthermic conditions from fertilization until after the juvenile stage.<h4>Results</h4>Fish exposed to the high temperature regime showed a markedly higher growth rate and a significant higher percentage of deformities in the spinal column than fish reared at low temperatures. By analyzing phenotypically normal spinal columns from the two temperature regimes, we found that the increased risk of developing vertebral deformities was linked to an altered gene transcription. In particular, down-regulation of extracellular matrix (ECM) genes such as col1a1, osteocalcin, osteonectin and decorin, indicated that maturation and mineralization of osteoblasts were restrained. Moreover, histological staining and in situ hybridization visualized areas with distorted chondrocytes and an increased population of hypertrophic cells. These findings were further confirmed by an up-regulation of mef2c and col10a, genes involved in chondrocyte hypertrophy.<h4>Conclusion</h4>The presented data strongly indicates that temperature induced fast growth is severely affecting gene transcription in osteoblasts and chondrocytes; hence change in the vertebral tissue structure and composition. A disrupted bone and cartilage production was detected, which most likely is involved in the higher rate of deformities developed in the high intensive group. Our results are of basic interest for bone metabolism and contribute to the understanding of the mechanisms involved in development of temperature induced vertebral pathology. The findings may further conduce to future molecular tools for assessing fish welfare in practical farming.

<h4>Background</h4>Colorectal cancer is (CRC) one of the commonest cancers and its therapy is still based on few drugs. Currently, no biological criteria are used to choose the most effective of the established drugs for treatment.<h4>Methods</h4>A panel of 77 CRC cell lines was tested for sensitivity to 5-fluorouracil (5FU) using the SRB assay. The responses were grouped into three categories and correlated with genetic changes in the cell lines.<h4>Results</h4>The strongest and most clearcut correlation was between 5-fluorouracil response and replication error status (mismatch repair deficiency). All the other significant correlations (loss of heterozygosity for DCC and mutations in TGFbIIR) are secondary to the association with replication error status.<h4>Interpretation and conclusion</h4>Our findings validate previous analyses based mainly on clinical data, and indicate that replication error status could be a useful guide to 5-fluorouracil-based CRC therapy. Essentially, all previously described correlations with 5FU response are secondary to the association with replication error status.

Haemochromatosis is predominantly associated with the HFE p.C282Y homozygous genotype, which is present in approximately 1 in 200 people of Northern European origin. However, not all p.C282Y homozygotes develop clinical features of haemochromatosis, and not all p.C282Y homozygotes even present abnormal iron parameters justifying venesection therapy. This situation was not apparent from initial genotype/phenotype correlation studies as there was a selection bias of patients. Only those patients with a significant iron burden were included in these early studies. It is now largely accepted that the p.C282Y/p.C282Y genotype is necessary for the development of HFE haemochromatosis. However, this genotype provides few clues as to why certain symptoms are associated with the disease. Expression of iron overload in people with this genotype depends on the complex interplay of environmental factors and modifier genes. In this review, we restrict our discussion to work done in humans giving examples of animal models where this has helped clarify our understanding. We discuss penetrance, explaining that this concept normally does not apply to autosomal recessive disorders, and discuss the usefulness of different biochemical markers in ascertaining iron burden. Hepcidin, a peptide synthesized primarily by the liver, has been identified as the central regulator in iron homeostasis. Consequently, understanding its regulation is the key. We conclude that the main goal now is to identify important modifiers that have a significant and unambiguous effect on iron storage.

<h4>Purpose</h4>Limbal stem cell deficiency is a challenging clinical problem and the current treatment involves replenishing the depleted limbal stem cell (LSC) pool by either limbal tissue transplantation or use of cultivated limbal epithelial cells (LEC). Our experience of cultivating the LEC on denuded human amniotic membrane using a feeder cell free method, led to identification of mesenchymal cells of limbus (MC-L), which showed phenotypic resemblance to bone marrow derived mesenchymal stem cells (MSC-BM). To understand the transcriptional profile of these cells, microarray experiments were carried out.<h4>Methods</h4>RNA was isolated from cultured LEC, MC-L and MSC-BM and microarray experiments were carried out by using Agilent chip (4x44 k). The microarray data was validated by using Realtime and semiquntitative reverse transcription polymerase chain reaction.<h4>Results</h4>The microarray analysis revealed specific gene signature of LEC and MC-L, and also their complementary role related to cytokine and growth factor profile, thus supporting the nurturing roles of the MC-L. We have also observed similar and differential gene expression between MC-L and MSC-BM.<h4>Conclusions</h4>This study represents the first extensive gene expression analysis of limbal explant culture derived epithelial and mesenchymal cells and as such reveals new insight into the biology, ontogeny, and in vivo function of these cells.

Psoriasis, as the most common inflammatory skin disorder, affects about 2-3% of the world's population. Many non-dermatological conditions have been linked with psoriasis, including cardiovascular diseases, depression, inflammatory bowel disorders, and some cancers, i.e. lung, colon and kidney cancers. Among systemic factors are endocrine and metabolic disturbances as well as many drugs. Erythrodermic psoriasis, the most severe form of the disease, is characterized by diffuse erytrema and scaling, often accompanied by fever, chills, and malaise. A 57-year-old Caucasian man was admitted for curative radiation therapy of adenocarcinoma of the prostate after 3 months of initial hormonal therapy. The management comprised the combined androgen blockade (CAB). On admission the patient reported escalation of psoriasis symptoms, which he had been treated for since 2002. Due to a mild course of the disease he had not required any systemic treatment ever before, even during aggravation periods. The last exacerbation started appearing a month after hormonal therapy implementation. The cutaneous eruptions, already existing, become larger with new foci revealing, mainly on upper and lower limbs. During radiotherapy planning, there appeared a diffuse erythema and scaling on hands and feet with accompanying pruritis. We decided to start the previously planned radiation therapy which included the prostate gland with 1.5 cm margin and provided for the total dose of 72 Gy in 36 fractions. The irradiation was conducted with the four-field technique using a megavoltage linear accelerator. During radiotherapy we photo-documented skin lesions. To our best knowledge hormone therapy (androgen deprivation) of prostate cancer patients has not been reported as an aggravating factor. Thus, the aim of our work is to present the case of a prostate cancer patient who experienced psoriasis exacerbation after implementation of hormonal blockade as a neoadjuvant oncological treatment.

HFE is a type 1 transmembrane protein that becomes N-glycosylated during transport to the cell membrane. It influences cellular iron concentrations through multiple mechanisms, including regulation of transferrin binding to transferrin receptors. The importance of glycosylation in HFE localization and function has not yet been studied. Here we employed bioinformatics to identify putative N-glycosylation sites at residues N110, N130 and N234 of the human HFE protein, and used site-directed mutagenesis to create combinations of single, double or triple mutants. Compared with the wild-type protein, which co-localizes with the type 1 transferrin receptor in the endosomal recycling compartment and on distributed punctae, the triple mutant co-localized with BiP in the endoplasmic reticulum. This was similar to the localization pattern described previously for the misfolding HFE-C282Y mutant that causes type 1 hereditary haemachromatosis. We also observed that the triple mutant was functionally deficient in beta2-microglobulin interactions and incapable of regulating transferrin binding, once again, reminiscent of the HFE-C282Y variant. Single and double mutants that undergo limited glycosylation appeared to have a mixed phenotype, with characteristics primarily of the wild-type, but also some from the glycosylation-deficient protein. Therefore, although they displayed an endosomal recycling compartment/punctate localization like the wild-type protein, many cells simultaneously displayed additional reticular localization. Furthermore, although the majority of cells expressing these single and double mutants showed decreased surface binding of transferrin, a number appeared to have lost this ability. We conclude that glycosylation is important for the normal intracellular trafficking and functional activity of HFE.

A serotonin deficiency state has been implicated in alcohol-dependent individuals' experience of obsessive-compulsive alcohol craving. Because the serotonin transporter (5-HTT) functions to remove serotonin from the synapse, it is thought that increased reuptake (indicated by the number of high-expressing L(A) alleles present in the 5-HTT gene-linked polymorphic region [5-HTTLPR] of the SLC6A4 gene) is associated with an increase in obsessive-compulsive alcohol craving. The current pilot investigation sought to explore this hypothesis by examining the extent to which obsessive-compulsive alcohol craving varies by 5-HTTLPR genotype among participants enrolled in an ongoing pharmacogenetics trial. All participants were screened with a semi-structured diagnostic interview, completed self-report measures of alcohol-related behavior, and underwent peripheral venous blood draw for DNA genotyping. Cross-sectional data obtained at baseline from 176 currently drinking alcohol-dependent individuals were analyzed using multiple regression. Preliminary findings suggest that 5-HTTLPR is not predictive of Obsessive Compulsive Drinking Scale total and factor scores. Although the 5-HTTLPR polymorphism was not related to obsessive-compulsive alcohol craving in this pilot study, additional research is needed to clarify the possible role of serotonergic mechanisms in alcohol craving.

Parental support and parental depressive feelings are found to be associated with depressive feelings in adolescent boys and girls, but results are inconsistent. In addition, the 5-HTTLPR genotype has been found to interact with environmental stressors in predicting adolescents' depressive feelings, but this has not been examined longitudinally. Therefore, the present study examined the relationships between parental support, parental depressive feelings, and adolescent depressive feelings. In addition, the relationships between the 5-HTTLPR genotype and adolescent depressive feelings were explored, as well as gene-environment interactions. Adolescents (N = 306; Girls = 53.3%; M (age T1) = 13.4) filled out questionnaires at five annual waves and provided saliva samples for DNA. Latent growth curve modelling (LGCM) was used to examine the baseline level and the change in depressive feelings over time. Maternal support was related to baseline levels of depressive feelings in girls, whereas paternal support was related to baseline levels in boys. Paternal depressive feelings were only related to boys' depressive feelings at baseline, and maternal depressive feelings were not related to any outcome measures. Furthermore, no associations were found between 5-HTTLPR genotype and adolescent depressive feelings, and no gene-environment interactions emerged. Limitations of the study and implications of the findings are discussed.

Systemic sclerosis (SSc) is an autoimmune inflammatory disease with unknown etiology characterized by microvascular injury and fibrosis of the skin and internal organs. A growing body of evidence suggests that deficiency of the transcription factor Fli1 (Friend leukemia integration-1) has a pivotal role in the pathogenesis of SSc. Fli1 is expressed in fibroblasts, endothelial cells, and immune cells, and has important roles in the activation, differentiation, development, and survival of these cells. Previous studies demonstrated that Fli1 is downregulated in SSc fibroblasts by an epigenetic mechanism and a series of experiments with Fli1-deficient animal models revealed that Fli1 deficiency in fibroblasts and endothelial cells reproduces the histopathologic features of fibrosis and vasculopathy in SSc, respectively. In this article, we review the impact of Fli1 deficiency on the pathogenesis of SSc and discuss a new therapeutic strategy for SSc by targeting the transcription factor Fli1.

The intestinal epithelium contains a rapidly proliferating and perpetually differentiating epithelium. The principal functional unit of the small intestine is the crypt-villus axis. Stem cells located in the crypts of Lieberkühn give rise to proliferating progenitor or transit amplifying cells that differentiate into the 4 major epithelial cell types. The study of adult gastrointestinal tract stem cells has progressed rapidly with the recent discovery of several putative stem cell markers. Substantial evidence suggests 2 populations of stem cells: long-term quiescent (reserved) and actively cycling (primed) stem cells. These cells are in adjoining locations and are presumably maintained by the secretion of specific proteins generated in a unique microenvironment or stem cell niche surrounding each population. The relationship between these 2 populations, as well as the cellular sources and composition of the surrounding environment, remains to be defined, and is an active area of research. In this review, we will outline progress in identifying stem cells and in defining epithelial-mesenchymal interactions in the crypt. We will summarize early advances using stem cells for therapy of gastrointestinal disorders.

<h4>Background</h4>The prevalence of primary (hereditary) hemochromatosis and secondary iron overload (hemosiderosis) is reaching epidemic levels worldwide. Iron-overload leads to excessive iron deposition in a wide variety of tissues, including the heart and endocrine tissues.<h4>Methods and results</h4>Iron-overload cardiomyopathy is the primary determinant of survival in patients with secondary iron overload, while also being a leading cause of morbidity and mortality in patients with primary hemochromatosis. Iron-induced cardiovascular injury also occurs in acute iron toxicosis (iron poisoning), myocardial ischemia-reperfusion injury, cardiomyopathy associated with Friedreich ataxia, and vascular dysfunction. The mainstay therapies for iron overload associated with primary hemochromatosis and secondary iron overload is phlebotomy and iron chelation therapy, respectively. L-type Ca(2+) channels provide a high-capacity pathway for ferrous (Fe(2+)) uptake into cardiomyocytes in iron-overload conditions; calcium channel blockers may represent a new therapeutic tool to reduce the toxic effects of excess iron.<h4>Conclusions</h4>Iron-overload cardiomyopathy is a an important and potentially reversible cause of heart failure at an international scale and involves diastolic dysfunction, increased susceptibility to arrhythmias and a late-stage dilated cardiomyopathy. The early diagnosis of iron-overload cardiomyopathy is critical since the cardiac dysfunction is reversible if effective therapy is introduced before the onset of overt heart failure.

Precise regulation of gene expression is crucial to myogenesis and is thought to require the cooperation of various transcription factors. On the basis of a bioinformatic analysis of gene regulatory sequences, we hypothesized that myogenic regulatory factors (MRFs), key regulators of skeletal myogenesis, cooperate with members of the SIX family of transcription factors, known to play important roles during embryonic skeletal myogenesis. To this day little is known regarding the exact molecular mechanism by which SIX factors regulate muscle development. We have conducted a functional genomic study of the role played by SIX1 and SIX4 during the differentiation of skeletal myoblasts, a model of adult muscle regeneration. We report that SIX factors cooperate with the members of the MRF family to activate gene expression during myogenic differentiation, and that their function is essential to this process. Our findings also support a model where SIX factors function not only 'upstream' of the MRFs during embryogenesis, but also 'in parallel' to them during myoblast differentiation. We have identified new essential nodes that depend on SIX factor function, in the myogenesis regulatory network, and have uncovered a novel way by which MRF function is modulated during differentiation.

This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication may contribute to the pathology of endometriosis. An endometrial stromal cell line (telomerase-immortalized human endometrial stromal cell [T-HESC]) was treated with macrophage-conditioned medium (CM) +/- estradiol + progesterone. Macrophages were treated without or with T-HESC CM. DNA microarray identified 716 differentially expressed genes in T-HESCs in response to factors secreted by macrophages. Upregulated genes in T-HESC included interleukin 8 (IL-8)/chemokine (C-X-C motif) ligand 8 (CXCL8), matrix metalloproteinase 3 (MMP3), phospholamban, cysteine-rich angiogenic inducer 61 (CYR61), connective tissue growth factor (CTGF), tenascin C, and nicotinamide N-methyltransferase (NNMT), whereas integrin alpha-6 was downregulated. In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by endometrial stromal cells. The data document reciprocal communication between macrophages and endometrial stromal cells and suggest that interaction with macrophages stimulates the expression of genes in endometrial stromal cells that may support the establishment of endometriosis.

<h4>Background</h4>Clinical penetrance of hereditary hemochromatosis is highly variable. We hypothesized that it might be modified by factors involved in the cellular immune response, such as toll-like receptors (TLRs) or nucleotide oligomerization domain proteins (NODs).<h4>Methods</h4>Clinical expression of hemochromatosis was assessed as a function of TLR4, TLR9, and NOD2 polymorphisms in 99 homozygous carriers of the HFE C282Y mutation with mild-to-severe iron overload.<h4>Results</h4>Thirteen (13%) of the 99 hemochromatosis patients were heterozygous for a TLR4 Asp299Gly polymorphism and 86 (87%) were TLR4 wild-type-only carriers. Clinical expression of hemochromatosis was observed more frequently in carriers of the TLR4 polymorphism (100%) than in TLR4 wild-type carriers (56%, P = 0.002). This was based on higher prevalences of liver disease (92 vs. 45%, P = 0.002) and arthropathy of metacarpophalangeal joints (69 vs. 35%, P = 0.018) in TLR4 polymorphism carriers. The finding was strengthened by the strong association of TLR4 polymorphism with liver fibrosis in the subgroup of 52 patients who underwent a liver biopsy (P = 0.011). The TLR4 polymorphism did, however, not correlate with body iron overload. The study results remained significant in multiple regression analyses after excluding possible confounding effects, such as age, sex, alcohol, or meat intake, and in the subgroup of 84 patients presenting as the first members of their families.<h4>Conclusion</h4>TLR4 Asp299Gly polymorphism modulates clinical expression in patients with hereditary hemochromatosis. The polymorphism does not correlate with iron overload suggesting that TLR4 plays a role in an inflammatory process arising from toxic effects of iron accumulation.

Adult human bone marrow-derived multipotent progenitor cells (MPCs) are able to differentiate into a variety of specialized cell types, including chondrocytes, and are considered a promising candidate cell source for use in cartilage tissue engineering. In this study, we examined the regulation of MPC chondrogenesis by mitogen-activated protein kinases in an attempt to better understand how to generate hyaline cartilage in the laboratory that more closely resembles native tissue. Specifically, we employed the high-density pellet culture model system to assess the roles of ERK5 and ERK1/2 pathway signaling in MPC chondrogenesis. Western blotting revealed that high levels of ERK5 phosphorylation correlate with low levels of MPC chondrogenesis and that as TGF-beta 3-enhanced MPC chondrogenesis proceeds, phospho-ERK5 levels steadily decline. Conversely, levels of phospho-ERK1/2 paralleled the progression of MPC chondrogenesis. siRNA-mediated knockdown of ERK5 pathway components MEK5 and ERK5 resulted in increased MPC pellet mRNA transcript levels of the cartilage-characteristic marker genes SOX9, COL2A1, AGC, L-SOX5, and SOX6, as well as enhanced accumulation of SOX9 protein, collagen type II protein, and Alcian blue-stainable proteoglycan. In contrast, knockdown of ERK1/2 pathway members MEK1 and ERK1 decreased expression of all chondrogenic markers tested. Finally, overexpression of MEK5 and ERK5 also depressed MPC chondrogenesis, as indicated by diminished activity of a co-transfected collagen II promoter-luciferase reporter construct. In conclusion, our results suggest a novel role for the ERK5 pathway as an important negative regulator of adult human MPC chondrogenesis and illustrate that the ERK5 and ERK1/2 kinase cascades play opposing roles regulating MPC cartilage formation.

<h4>Background</h4>The heptavalent pneumococcal CRM197 conjugate vaccine (PCV-7) has been incompletely studied in very-low-birth-weight (< or =1500 g) infants.<h4>Objective</h4>To assess PCV-7 immunogenicity in very-low-birth-weight, premature infants. We hypothesized that the frequency of postvaccine antibody concentrations > or =0.15 microg/mL would vary directly with birth weight.<h4>Methods</h4>This was a multicenter observational study. Infants 401 to 1500 g birth weight and <32 0/7 weeks gestation, stratified by birth weight, were enrolled from 9 National Institute of Child Health and Human Development Neonatal Research Network centers. Infants received PCV-7 at 2, 4, and 6 months after birth and had blood drawn 4 to 6 weeks following the third dose. Antibodies against the 7 vaccine serotypes were measured by enzyme-linked immunosorbent assay.<h4>Results</h4>Of 369 enrolled infants, 244 completed their primary vaccine series by 8 months and had serum obtained. Subjects were 27.8 +/- 2.2 (mean +/- standard deviation) weeks gestation and 1008 +/- 282 g birth weight. Twenty-six percent had bronchopulmonary dysplasia and 16% had received postnatal glucocorticoids. Infants 1001 to 1500 g birth weight were more likely than those 401 to 1000 g to achieve antibody concentrations > or =0.15 microg/mL against the least 2 immunogenic serotypes (6B: 96% vs. 85%, P = 0.003 and 23F: 97% vs. 88%, P = 0.009). In multiple logistic regression analysis, lower birth weight, postnatal glucocorticoid use, lower weight at blood draw, and Caucasian race were each independently associated with antibody concentrations <0.35 microg/mL against serotypes 6B and/or 23F.<h4>Conclusions</h4>When compared with larger premature infants, infants weighing < or =1000 g at birth have similar antibody responses to most, but not all, PCV-7 vaccine serotypes.

A rare variant of hepatocellular carcinoma (HCC) is encountered that produces small cirrhosis-like nodules diffusely throughout the liver (CL-HCC), instead of a larger evident mass. This pattern remains undetected as carcinoma clinically and radiographically and is unexpectedly discovered after liver transplantation in the explanted native liver. We studied 10 such cases (9 males and 1 female, age 35 to 80 y) from 4 medical centers. The pretransplant clinical, laboratory, and radiographical studies were reviewed to determine the cause and stage of liver disease, alpha-fetoprotein (AFP) levels, and detectability of a mass on imaging. All 10 cases had underlying cirrhosis of varying etiology [3 hepatitis C virus (HCV), 3 alcoholic hepatitis, 1 hepatitis B virus, 1 autoimmune, and 2 mixed HCV/alcoholic hepatitis and hemochromatosis/HCV] and underwent orthotopic liver transplantation with no preoperative clinical suspicion of HCC. Ultrasound and/or dynamic imaging showed cirrhosis and no definite HCC. AFP levels were only mildly elevated in only 3 of 10 cases (144, 150, and 252 ng/mL). Grossly, there were innumerable (from about 20 to >1000) small CL-HCC nodules (0.2 to 0.6 cm) scattered among cirrhotic nodules. Histologically, these were well or moderately differentiated HCC, often with pseudoglandular pattern, perinodular sclerotic rims, cholestasis, frequent Mallory bodies, and small vessel invasion. In addition to the usual HCC immunophenotype, CL-HCC showed frequent ubiquitin and cytoplasmic and membranous CD10 positivity, relatively low Ki-67 proliferative index and absence of AFP immunohistochemically. CL-HCC warrants recognition as a unique HCC variant that evades pretransplant detection despite massive tumor burden, mimics cirrhotic nodules, and shows some uncommon pathologic and immunophenotypical characteristics.

Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrheae, Neisseria meningitidis, and Haemophilus influenzae. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with loading of iron(III) into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate, only two steps are discernible. A stable intermediate containing iron(III) citrate-bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics and hamper iron sequestration ability. H9E, E57D, and E57Q have near native iron sequestration behavior; however, iron binding rates are altered, enabling assignment of sequential side chain interactions. Additionally, this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as "naked" or free iron as originally proposed.

Moenomycin A (MmA) belongs to a family of natural products that inhibit peptidoglycan biosynthesis by binding to the peptidoglycan glycosyltransferases, the enzymes that make the glycan chains of peptidoglycan. MmA is remarkably potent, but its clinical utility has been hampered by poor physicochemical properties. Moenomycin contains three structurally distinct regions: a pentasaccharide, a phosphoglycerate, and a C25 isoprenyl (moenocinyl) lipid tail that gives the molecule its name. The phosphoglycerate moiety links the pentasaccharide to the moenocinyl chain. This moiety contains two negatively charged groups, a phosphoryl group and a carboxylate. Both the phosphoryl group and the carboxylate have previously been implicated in target binding but the role of the carboxylate has not been explored in detail. Here we report the synthesis of six MmA analogues designed to probe the importance of the phosphoglycerate. These analogues were evaluated for antibacterial and enzyme inhibitory activity; the specific contacts between the phosphoglycerate and the protein target were assessed by X-ray crystallography in conjunction with molecular modeling. Both the phosphoryl group and the carboxylate of the phosphoglycerate chain play roles in target binding. The negative charge of the carboxylate, and not its specific structure, appears to be the critical feature in binding since replacing it with a negatively charged acylsulfonamide group produces a more active compound than replacing it with the isosteric amide. Analysis of the ligand-protein contacts suggests that the carboxylate makes a critical contact with an invariant lysine in the active site. The reported work provides information and validated computational methods critical for the design of analogues based on moenomycin scaffolds.

The consequences of dysmyelination are poorly understood and vary widely in severity. The shaking mouse, a quaking allele, is characterized by severe central nervous system (CNS) dysmyelination and demyelination, a conspicuous action tremor, and seizures in approximately 25% of animals, but with normal muscle strength and a normal lifespan. In this study we compare this mutant with other dysmyelinated mutants including the ceramide sulfotransferase deficient (CST-/-) mouse, which are more severely affected behaviorally, to determine what might underlie the differences between them with respect to behavior and longevity. Examination of the paranodal junctional region of CNS myelinated fibers shows that "transverse bands," a component of the junction, are present in nearly all shaking paranodes but in only a minority of CST-/- paranodes. The number of terminal loops that have transverse bands within a paranode and the number of transverse bands per unit length are only moderately reduced in the shaking mutant, compared with controls, but markedly reduced in CST-/- mice. Immunofluorescence studies also show that although the nodes of the shaking mutant are somewhat longer than normal, Na(+) and K(+) channels remain separated, distinguishing this mutant from CST-/- mice and others that lack transverse bands. We conclude that the essential difference between the shaking mutant and others more severely affected is the presence of transverse bands, which serve to stabilize paranodal structure over time as well as the organization of the axolemmal domains, and that differences in the prevalence of transverse bands underlie the marked differences in progressive neurological impairment and longevity among dysmyelinated mouse mutants.

A new sharply pH- and temperature-responsive hydrogel system was designed for delivering drugs to regions of local acidosis, as found in wound healing, tumor sites, or sites of ischemia. The reversible addition-fragmentation chain transfer (RAFT) polymerization technique was used to synthesize copolymers of N-isopropylacrylamide (NIPAAM) and propylacrylic acid (PAA) with feed ratios of PAA between 0 and 20 mol %. The pH-responsive viscoelastic properties of these materials as a function of pH and temperature were quantified by rheometry. At physiologic pH (7.4) and 5 wt %, the polymer did not form gels but rather remained soluble at temperatures as high as 50 degrees C. At lower pH values (pH ca. 5.5 and below), the polymer was liquid at 20 degrees C, but exhibited a sol-gel phase transformation with increasing temperature and existed as a physical gel at 37 degrees C. Incorporation of the hydrophobic monomer, butyl acrylate, into the random copolymer raised the pH of gel formation to greater than 6.0 at 37 degrees C. Drug loading studies demonstrated that p(NIPAAm-co-PAA) hydrogels are able to maintain the bioactivity of basic fibroblast growth factor following storage in hydrogel for 40 h and can provide sustained pH-dependent release of vascular endothelial growth factor over a period of at least three weeks. This hydrogel system will thus gel at controllable acidic pH values upon injection, and is designed to undergo gradual dissolution as it performs its drug delivery function and the ischemic site returns to physiological pH.

Gastric cancer has one of the highest morbidities and mortalities worldwide. Early detection is key measure to improve the outcome of gastric cancer patients. In our efforts to identify potential markers for gastric cancer detection, we coupled xenotransplantation mouse model with a plasma proteomic approach. MKN45 gastric cancer cells were subcutaneously injected into nude mice and plasma samples from mice bearing different sizes of tumors were collected and subjected to iTRAQ and mass spectrometry analysis. ITIH3 protein was found to be more highly expressed in plasma of tumor bearing mice compared to control. Subsequent screening of ITIH3 expression in 167 clinical plasma samples, including 83 cancer-free subjects and 84 gastric cancer patients, revealed higher ITIH3 level in the plasma of gastric cancer patients. A receiver operating characteristics (ROC) curve estimated a maximal sensitivity of 96% at 66% specificity for ITIH3 in gastric cancer detection. In addition, plasma from early stage gastric cancer patient has significantly (p < 0.001) higher level of ITIH3 compared to that from noncancer subject. Our data suggest that ITIH3 may be a useful biomarker for early detection of gastric cancer.

Mutant huntingtin (htt) carries an expanded polyglutamine (polyQ) repeat (> 36 glutamines) in its N-terminal region, which leads htt to become misfolded and kill neuronal cells in Huntington disease (HD). The cytotoxicity of N-terminal mutant htt fragments is evident by severe neurological phenotypes of transgenic mice that express these htt fragments. Clearance of mutant htt is primarily mediated by the ubiquitin-proteasomal sysmtem (UPS) and autophagy. However, the relative efficiency of these two systems to remove toxic forms of mutant htt has not been rigorously compared. Using cellular and mouse models of HD, we found that inhibiting the UPS leads to a greater accumulation of mutant htt than inhibiting autophagy. Moreover, N-terminal mutant htt fragments, but not full-length mutant htt, accumulate in the HD mouse brains after inhibiting the UPS. These findings suggest that the UPS is more efficient than autophagy to remove N-terminal mutant htt.

Accessory cells, which include glia and other cell types that develop in close association with neurons, have been shown to play key roles in regulating neuron development. However, the underlying molecular and cellular mechanisms remain poorly understood. A particularly intimate association between accessory cells and neurons is found in insect chordotonal organs. We have found that the cap cell, one of two accessory cells of v'ch1, a chordotonal organ in the Drosophila embryo, strongly influences the development of its associated neuron. As it projects a long dorsally directed cellular extension, the cap cell reorients the dendrite of the v'ch1 neuron and tows its cell body dorsally. Cap cell morphogenesis is regulated by Netrin-A, which is produced by epidermal cells at the destination of the cap cell process. In Netrin-A mutant embryos, the cap cell forms an aberrant, ventrally directed process. As the cap cell maintains a close physical connection with the tip of the dendrite, the latter is dragged into an abnormal position and orientation, and the neuron fails to undergo its normal dorsal migration. Misexpression of Netrin-A in oenocytes, secretory cells that lie ventral to the cap cell, leads to aberrant cap cell morphogenesis, suggesting that Netrin-A acts as an instructive cue to direct the growth of the cap cell process. The netrin receptor Frazzled is required for normal cap cell morphogenesis, and mutant rescue experiments indicate that it acts in a cell-autonomous fashion.

Cellular proliferation, differentiation, integration, and survival within the adult neural stem cell niche are altered under pathological conditions, but the molecular cues regulating the biology of this niche are mostly unknown. We examined the hippocampal neural stem cell niche in a transgenic rat model of Huntington disease. In this model, progressive cognitive deficits develop at the age of 9 months, suggesting possible hippocampal dysfunction. We found a disease-associated progressive decline in hippocampal progenitor cell proliferation accompanied by an expansion of the pool of 5-bromo-2-deoxyuridine label-retaining Sox-2-positive quiescent stem cells in the transgenic animals. Increments in quiescent stem cells occurred at the expense of cAMP-responsive element-binding protein-mediated neuronal differentiation and survival. Because elevated levels of transforming growth factor-beta1 (TGF-beta1) impair neural progenitor proliferation, we investigated hippocampal TGF-beta signaling and determined that TGF-beta1 induces the neural progenitors to exit the cell cycle. Although phospho-Smad2, an effector of TGF-beta signaling, is normally absent in subgranular stem cells, it accumulated progressively in Sox2/glial fibrillary acidic protein-expressing cells of the subgranular zone in the transgenic rats. These results indicate that alterations in neurogenesis in transgenic Huntington disease rats occur in successive phases that are associated with increasing TGF-beta signaling. Thus, TGF-beta1 signaling seems to be a crucial modulator of neurogenesis in Huntington disease and may represent a target for future therapy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complicated pathogenesis with variable presentation and disease progression. There is a critical need for a panel of biomarkers to provide clinicians and researchers with additional information. In this study, multiplex immunoassays were used to screen a number of cytokines, growth factors, and iron-related proteins. ALS patients had significantly higher plasma levels of L-ferritin and lower concentrations of transferrin when compared to healthy controls and together classified a test group of subjects with 82% accuracy. Duration of ALS symptoms correlated positively with levels of monocyte chemoattractant protein 1 (MCP-1) and negatively with levels of granulocyte-macrophage colony stimulating factor (GM-CSF). The biomarker profile suggests iron homeostasis is disrupted in ALS patients, and changes in ferritin and transferrin (Tf) appear to be indicators of ongoing inflammatory processes. The data demonstrate a plasma biomarker profile in ALS patients that may differ from published reports of cerebrospinal fluid biomarkers.

Hepatocellular carcinoma (HCC) remains a highly complex disease resistant to commonly used chemotherapy and radiotherapy. As the sixth most common cancer worldwide with the third highest mortality rate and very poorly understood molecular pathways driving hepatocarcinogenesis, new treatment strategies are urgently needed for this devastating disease. The multikinase inhibitor sorafenib was the first molecular targeted drug in HCC that led to significant survival benefit in patients with advanced tumors. It is the first drug to be considered standard of care for advanced HCC and supports the importance of molecular therapies in the treatment of this cancer. Analyses of genetic and epigenetic alterations as well as different molecular pathways involved in the development of HCC help to identify potential new druggable targets. A variety of novel compounds are already under preclinical or clinical investigation, and accumulating evidence suggests that combination therapy targeting different pathways will potentiate anti-tumoral effects and will become the future therapeutic approach. In addition the establishment of a robust molecular classification will pave the way for a more personalized treatment scheme in HCC. In this article we review the current knowledge of the molecular pathogenesis of HCC and provide an overview of molecular targeted therapies in the management of HCC.

<h4>Unlabelled</h4>High-dose (28-30 mg/kg/day) ursodeoxycholic acid (UDCA) treatment improves serum liver tests in patients with primary sclerosing cholangitis (PSC) but does not improve survival and is associated with increased rates of serious adverse events. The mechanism for the latter undesired effect remains unclear. High-dose UDCA could result in the production of hepatotoxic bile acids, such as lithocholic acid (LCA), because of limited small bowel absorption of UDCA and conversion of UDCA by bacteria in the colon. We determined the serum bile acid composition in 56 patients with PSC previously enrolled in a randomized, double-blind controlled trial of high-dose UDCA versus placebo. Samples for analysis were obtained at the baseline and at the end of treatment. The mean changes in the UDCA level (16.86 versus 0.05 micromol/L) and total bile acid level (17.21 versus -0.55 micromol/L) were significantly higher in the UDCA group (n = 29) versus the placebo group (n = 27) when pretreatment levels were compared (P < 0.0001). LCA was also markedly increased (0.22 versus 0.01 micromol/L) in the UDCA group compared to the placebo group (P = 0.001). No significant changes were detected for cholic acid, deoxycholic acid, or chenodeoxycholic acid. Patients (n = 9) in the UDCA group who reached clinical endpoints of disease progression (the development of cirrhosis, varices, liver transplantation, or death) tended to have greater increases in their posttreatment total bile acid levels (34.99 versus 9.21 micromol/L, P < 0.08) in comparison with those who did not.<h4>Conclusion</h4>High-dose UDCA treatment in PSC patients results in marked UDCA enrichment and significant expansion of the total serum bile acid pool, including LCA.

Zinc-dependent histone deacetylase 8 (HDAC8) catalyzes the removal of acetyl moieties from histone tails, and is critically involved in regulating chromatin structure and gene expression. The detailed knowledge of its catalytic process is of high importance since it has been established as a most promising target for the development of new antitumor drugs. By employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations and umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have provided further evidence against the originally proposed general acid-base catalytic pair mechanism for Zinc-dependent histone deacetylases. Instead, our results indicated that HDAC8 employs a proton-shuttle catalytic mechanism, in which a neutral His143 first serves as the general base to accept a proton from the zinc-bound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond. During the deacetylation process, the Zn(2+) ion changes its coordination mode and plays multiple catalytic roles. For the K(+) ion, which is located about 7 A from the catalytic Zn(2+) ion and conserved in class I and II HDACs, our simulations indicated that its removal would lead to the different transition state structure and a higher free energy reaction barrier for the rate-determining step. It is found that the existence of this conserved K(+) ion would enhance the substrate binding, increase the basicity of His143, strengthen the catalytic role of zinc ion, and improve the transition state stabilization by the enzyme environment.

Hyperferritinemia occurs in Gaucher disease but its clinical spectrum or its association with systemic iron overload and HFE mutations are not known. In 114 patients with Type 1 Gaucher disease, we determined serum ferritin, transferrin saturation and HFE genotype. The results were correlated with the extent of hepatosplenomegaly, overall Gaucher disease severity score index, and response to enzyme replacement therapy. In a subset of patients with radiological and/or laboratory evidence of systemic iron overload, liver biopsy was performed. There was a mean 3.7-fold elevation of serum ferritin over the upper limit of normal (ULN). Prior splenectomy was associated with most severe hyperferritinemia compared to patients with intact spleen (6.53 x ULN vs. 2.69 x ULN, P = 0.003). HFE genotyping revealed two patients homozygous for H63D mutation and 30% of patients heterozygote carriers of H63D mutation; no patients harbored C282Y mutation; there was no correlation of ferritin with HFE genotype. Ferritin level correlated with liver volume (Pearson correlation coefficient = 0.254, P = 0.035) and it was negatively correlated with hemoglobin (r = -0.315, P = 0.004); there was no relationship with other indicators of Gaucher disease activity. Enzyme replacement therapy (ERT) resulted in amelioration of hyperferritinemia: 707 +/- 898 ng/ml vs. 301 +/- 310 ng/ml (P = 0.001), transferrin saturation remained normal. Three patients were suspected of clinical iron overload, confirmed on liver biopsy. Iron accumulation was variably noted in hepatocytes and Kupffer cells. There is a high prevalence of hyperferritinemia in Type 1 Gaucher disease that is associated with indicators of disease severity, reversed by ERT and is not related to HFE mutations.

The physiological lag between blood and interstitial fluid (ISF) glucose is a major challenge for noninvasive glucose concentration measurements. This is a particular problem for spectroscopic techniques, which predominantly probe ISF glucose, creating inconsistencies in calibration, where blood glucose measurements are used as a reference. To overcome this problem, we present a dynamic concentration correction (DCC) scheme, based on the mass transfer of glucose between blood and ISF, to ensure consistency with the spectral measurements. The proposed formalism allows the transformation of glucose in the concentration domain, ensuring consistency with the acquired spectra in the calibration model. Taking Raman spectroscopy as a specific example, we demonstrate that the predicted glucose concentrations using the DCC-based calibration model closely match the measured glucose concentrations, while those generated with the conventional calibration methods show significantly larger deviations from the measured values. In addition, we provide an analytical formula for a previously unidentified source of limiting uncertainty arising in spectroscopic glucose monitoring from a lack of knowledge of glucose kinetics in prediction samples. A study with human volunteers undergoing glucose tolerance tests indicates that this lag uncertainty, which is comparable in magnitude to the uncertainty arising from noise and nonorthogonality in the spectral data set, can be reduced substantially by employing the DCC scheme in spectroscopic calibration.

<h4>Unlabelled</h4>Surveillance for hepatocellular carcinoma (HCC) in patients with cirrhosis is recommended but may not be performed. The extent and determinants of HCC surveillance are unknown. We conducted a population-based United States cohort study of patients over 65 years of age to examine use and determinants of prediagnosis surveillance in patients with HCC who were previously diagnosed with cirrhosis. Patients diagnosed with HCC during 1994-2002 were identified from the linked Surveillance, Epidemiology, and End-Results registry-Medicare databases. We identified alpha-fetoprotein (AFP) and ultrasound tests performed for HCC surveillance, and examined factors associated with surveillance. We identified 1,873 HCC patients with a prior diagnosis of cirrhosis. In the 3 years before HCC, 17% received regular surveillance and 38% received inconsistent surveillance. In a subset of 541 patients in whom cirrhosis was recorded for 3 or more years prior to HCC, only 29% received routine surveillance and 33% received inconsistent surveillance. Among all patients who received regular surveillance, approximately 52% received both AFP and ultrasound, 46% received AFP only, and 2% received ultrasound only. Patients receiving regular surveillance were more likely to have lived in urban areas and had higher incomes than those who did not receive surveillance. Before diagnosis, approximately 48% of patients were seen by a gastroenterologist/hepatologist or by a physician with an academic affiliation; they were approximately 4.5-fold and 2.8-fold, respectively, more likely to receive regular surveillance than those seen by a primary care physician only. Geographic variation in surveillance was observed and explained by patient and physician factors.<h4>Conclusion</h4>Less than 20% of patients with cirrhosis who developed HCC received regular surveillance. Gastroenterologists/hepatologists or physicians with an academic affiliation are more likely to perform surveillance.

<h4>Background</h4>Orofacial development is a multifaceted process involving precise, spatio-temporal expression of a panoply of genes. MicroRNAs (miRNAs), the largest family of noncoding RNAs involved in gene silencing, represent critical regulators of cell and tissue differentiation. MicroRNA gene expression profiling is an effective means of acquiring novel and valuable information regarding the expression and regulation of genes, under the control of miRNA, involved in mammalian orofacial development.<h4>Methods</h4>To identify differentially expressed miRNAs during mammalian orofacial ontogenesis, miRNA expression profiles from gestation day (GD) -12, -13 and -14 murine orofacial tissue were compared utilizing miRXplore microarrays from Miltenyi Biotech. Quantitative real-time PCR was utilized for validation of gene expression changes. Cluster analysis of the microarray data was conducted with the clValid R package and the UPGMA clustering method. Functional relationships between selected miRNAs were investigated using Ingenuity Pathway Analysis.<h4>Results</h4>Expression of over 26% of the 588 murine miRNA genes examined was detected in murine orofacial tissues from GD-12-GD-14. Among these expressed genes, several clusters were seen to be developmentally regulated. Differential expression of miRNAs within such clusters wereshown to target genes encoding proteins involved in cell proliferation, cell adhesion, differentiation, apoptosis and epithelial-mesenchymal transformation, all processes critical for normal orofacial development.<h4>Conclusions</h4>Using miRNA microarray technology, unique gene expression signatures of hundreds of miRNAs in embryonic orofacial tissue were defined. Gene targeting and functional analysis revealed that the expression of numerous protein-encoding genes, crucial to normal orofacial ontogeny, may be regulated by specific miRNAs.

Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

<h4>Background</h4>The significance of H63D homozygosity remains uncertain, although it is associated with a tendency for patients to develop iron overload.<h4>Aims</h4>To study the prevalence of homozygotic H63D mutation in patients with phenotypic hemochromatosis (PH) and to compare the results with those of the general population and with patients with porphyria cutanea tarda (PCT) in the Basque Country, Spain. A secondary aim was to evaluate the differences in phenotypic expression and liver injury according to different genotypes in the PH cohort.<h4>Methods</h4>Mutations of the HFE gene were obtained by polymerase chain reaction (PCR). Forty consecutive patients diagnosed with PH, 116 controls and 54 patients with PCT were included in the study. We performed liver biopsies, measured liver iron concentration (LIC), by atomic spectrophotometry, serum ferritin and transferrin saturation, and compared the histology according to the genotype.<h4>Results</h4>The H63D homozygote mutation was identified in 7.76% of the control group, in 7.50% of the PH group, and in 11.11% of patients with PCT (P > 0.05). The C282Y/C282Y mutation was present in 50% of patients with PH, and LIC was identified in 15/20. The LIC in C282Y/C282Y patients was higher than in H63D/H63D patients (P = 0.26), while H63D homozygosis caused greater iron overload in PH patients than other genotypes. All the C282Y/C282Y genotype patients had elevated serum ferritin and transferrin saturation. The H63D homozygotes had high ferritin, but two out of three had normal transferrin saturation. Six of the eight patients with high-grade fibrosis and genetic study results were found to be C282Y/C282Y.<h4>Conclusions</h4>The prevalence of H63D mutation in patients with PH in our region does not differ from that of the general Basque population.

<h4>Background</h4>Angiotensin and serotonin have been identified as inducers of cardiac hypertrophy. DNA polymorphisms at the genes encoding components of the angiotensin and serotonin systems have been associated with the risk of developing cardiovascular diseases, including left ventricular hypertrophy (LVH).<h4>Methods</h4>We genotyped five polymorphisms of the AGT, ACE, AT1R, 5-HT2A, and 5-HTT genes in 245 patients with Hypertrophic Cardiomyopathy (HCM; 205 without an identified sarcomeric gene mutation), in 145 patients with LVH secondary to hypertension, and 300 healthy controls.<h4>Results</h4>We found a significantly higher frequency of AT1R 1166 C carriers (CC+AC) among the HCM patients without sarcomeric mutations compared to controls (p = 0.015; OR = 1.56; 95%CI = 1.09-2.23). The AT1R 1166 C was also more frequent among patients who had at least one affected relative, compared to sporadic cases. This allele was also associated with higher left ventricular wall thickness in both, HCM patients with and without sarcomeric mutations.<h4>Conclusions</h4>The 1166 C AT1R allele could be a risk factor for cardiac hypertrophy in patients without sarcomeric mutations. Other variants at the AGT, ACE, 5-HT2A and 5-HTT did not contribute to the risk of cardiac hypertrophy.

Alzheimer's disease (AD) is a complex disorder with a clear genetic component. Three genes have been identified as the cause of early onset familial AD (EOAD). The most common form of the disease, late onset Alzheimer's disease (LOAD), is, however, a sporadic one presenting itself in later stages of life. The genetic component of this late onset form of AD has been the target of a large number of studies, because only one genetic risk factor (APOE4) has been consistently associated with the disease. However, technological advances allow new approaches in the study of complex disorders. In this review, we discuss the new results produced by genome wide association studies, in light of the current knowledge of the complexity of AD genetics.

Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency difference between Tibetan and Han samples, representing the fastest allele frequency change observed at any human gene to date. This SNP's association with erythrocyte abundance supports the role of EPAS1 in adaptation to hypoxia. Thus, a population genomic survey has revealed a functionally important locus in genetic adaptation to high altitude.

Subtypes of renal tumors have different genetic backgrounds, prognoses, and responses to surgical and medical treatment, and their differential diagnosis is a frequent challenge for pathologists. New biomarkers can help improve the diagnosis and hence the management of renal cancer patients. We extracted RNA from 71 formalin-fixed paraffin-embedded (FFPE) renal tumor samples and measured expression of more than 900 microRNAs using custom microarrays. Clustering revealed similarity in microRNA expression between oncocytoma and chromophobe subtypes as well as between conventional (clear-cell) and papillary tumors. By basing a classification algorithm on this structure, we followed inherent biological correlations and could achieve accurate classification using few microRNAs markers. We defined a two-step decision-tree classifier that uses expression levels of six microRNAs: the first step uses expression levels of hsa-miR-210 and hsa-miR-221 to distinguish between the two pairs of subtypes; the second step uses either hsa-miR-200c with hsa-miR-139-5p to identify oncocytoma from chromophobe, or hsa-miR-31 with hsa-miR-126 to identify conventional from papillary tumors. The classifier was tested on an independent set of FFPE tumor samples from 54 additional patients, and identified correctly 93% of the cases. Validation on qRT-PCR platform demonstrated high correlation with microarray results and accurate classification. MicroRNA expression profiling is a very effective molecular bioassay for classification of renal tumors and can offer a quantitative standardized complement to current methods of tumor classification.

<h4>Objective</h4>Increasing data support a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV-1 infection. We recently isolated a naturally occurring dimeric form of the anti-HIV-1 antibody 2G12 and found it to be significantly more potent than 2G12 monomer in neutralizing primary virus strains. However, given the unusual structure of dimeric 2G12 with two Fc regions, it was not clear whether 2G12 dimer could bind to the CD16 Fc receptor on ADCC effector cells or trigger ADCC. Here we compared the in-vitro ADCC activities of 2G12 monomer and dimer and investigated the effects of including ADCC-enhancing mutations in both forms of 2G12.<h4>Methods</h4>An in-vitro ADCC assay using target cells stably expressing gp160 was developed to evaluate the activities of 2G12 monomer and dimer with and without ADCC-enhancing mutations that increase the CD16-binding affinity of the 2G12 Fc region.<h4>Results</h4>Both 2G12 monomer and 2G12 dimer elicited ADCC, although the dimer showed increased potency [lower half-maximal concentration (EC(50))] in triggering ADCC, thus confirming its ability to bind CD16 and trigger ADCC. The ADCC-enhancing mutations improved the ADCC activity of 2G12 monomer more than 2G12 dimer such that their EC(50) values were nearly equal. However, no increase in nonspecific ADCC activity was observed using 2G12 IgGs with these mutations.<h4>Conclusion</h4>Given the likelihood that ADCC plays a role in protecting against initial infection and/or controlling chronic infection, these data suggest 2G12 dimers and/or addition of ADCC-enhancing mutations could augment the prophylactic and/or therapeutic potential of 2G12.

Previous studies have shown that pretargeting protocols, using cancer-targeting fusion proteins, composed of 4 anti-CD20 single chain Fv (scFv) fragments and streptavidin (scFv(4)-SAv), followed by a biotinylated dendrimeric N-acetyl-galactosamine blood clearing agent (CA), 1, then a radiolabeled DOTA-biotin derivative (a monobiotin), 3a, can provide effective therapy for lymphoma xenografts in mouse models. A shortcoming in this pretargeting system is that endogenous biotin may affect its efficacy in patients. To circumvent this potential problem, we investigated a pretargeting system that employs anti-CD20 scFv(4)-SAv mutant fusion proteins with radioiodinated bis-biotin derivatives. With that combination of reagents, good localization of the radiolabel to lymphoma tumor xenografts was obtained in the presence of endogenous biotin. However, the blood clearance reagents employed in the studies were ineffective, resulting in abnormally high levels of radioactivity in other tissues. Thus, in the present investigation a bis-biotin-trigalactose blood clearance reagent, 2, was designed, synthesized, and evaluated in vivo. Additionally, another DOTA-biotin derivative (a bis-biotin), 4a, was designed and synthesized, such that radiometals (e.g., (111)In, (90)Y, (177)Lu) could be used in the pretargeting protocols employing scFv(4)-SAv mutant fusion proteins. Studies in mice demonstrated that the CA 2 was more effective than CA 1 at removing [(125)I]scFv(4)-SAv-S45A mutant fusion proteins from blood. Another in vivo study compared tumor targeting and normal tissue concentrations of the new reagents (2 and [(111)In]4b) with standard reagents (1 and [(111)In]3b) used in pretargeting protocols. The study showed that lymphoma xenografts could be targeted in the presence of endogenous biotin when anti-CD20 fusion proteins containing SAv mutants (scFv(4)-SAv-S45A or scFv(4)-SAv-Y43A) were employed in combination with CA 2 and [(111)In]4b. Importantly, normal tissue concentrations of [(111)In]4b were similar to those obtained using the standard reagents (1 and [(111)In]3b), except that the blood and liver concentrations were slightly higher with the new reagents. While the reasons for the higher blood and liver concentrations are unknown, the differences in the galactose structures of the clearance agents 1 and 2 may play a role.

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Human immunodeficiency virus type 1 (HIV-1) is commonly associated with immune dysfunctions and the suppression of antigen-presenting cells. This results in immune alterations, which could lead to impaired neuronal functions, such as neuroAIDS. The neurotoxic factor kynurenine (KYN), the rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO), serotonin (5-HT), and serotonin transporter (5-HTT) may play a role in tryptophan deficiency and serotogenic dysfunction in neuroAIDS. HIV-1 transactivator regulatory protein (Tat) is known to play a major role in immune dysfunction. Previous studies suggest that HIV-1 B and C clades differentially manifest neuronal dysfunctions in the infected host. In the present study we examine the effect of HIV-1 B and C clade-derived Tat on IDO and 5-HTT gene and protein expressions by dendritic cells as studied by quantitative polymerase chain reaction (qPCR) and Western blot. In addition, the intracellular IDO expression, IDO enzyme activity, and the levels of 5-HT and KYN were also measured. Results indicate that HIV-1 clade B Tat up-regulates IDO and down-regulates 5-HTT gene and protein expressions. Further, HIV-1 clade B Tat caused a reduction of 5-HT with simultaneous increase in KYN levels as compared to HIV-1 clade C Tat. These studies suggest that HIV-1 clade B and C Tat proteins may play a differential role in the neuropathogenesis of HIV-associated dementia (HAD) or HIV-associated neurocognitive disorder (HAND).

The balance between self-renewal and differentiation of neural progenitor cells is an absolute requirement for the correct formation of the nervous system. Much is known about both the pathways involved in progenitor cell self-renewal, such as Notch signaling, and the expression of genes that initiate progenitor differentiation. However, whether these fundamental processes are mechanistically linked, and specifically how repression of progenitor self-renewal pathways occurs, is poorly understood. Nuclear factor I A (Nfia), a gene known to regulate spinal cord and neocortical development, has recently been implicated as acting downstream of Notch to initiate the expression of astrocyte-specific genes within the cortex. Here we demonstrate that, in addition to activating the expression of astrocyte-specific genes, Nfia also downregulates the activity of the Notch signaling pathway via repression of the key Notch effector Hes1. These data provide a significant conceptual advance in our understanding of neural progenitor differentiation, revealing that a single transcription factor can control both the activation of differentiation genes and the repression of the self-renewal genes, thereby acting as a pivotal regulator of the balance between progenitor and differentiated cell states.

Heparin (HP) and heparan sulfate (HS) play important roles in many biological events. Increasing evidence has shown that the biological functions of HP and HS can be critically dependent upon their precise structures, including the position of the iduronic acids and sulfation patterns. However, unraveling the HP code has been extremely challenging due to the enormous structural variations. To overcome this hurdle, we investigated the possibility of assembling a library of HP/HS oligosaccharides using a preactivation-based, one-pot glycosylation method. A major challenge in HP/HS oligosaccharide synthesis is stereoselectivity in the formation of the cis-1,4-linkages between glucosamine and the uronic acid. Through screening, suitable protective groups were identified on the matching glycosyl donor and acceptor, leading to stereospecific formation of both the cis-1,4- and trans-1,4-linkages present in HP. The protective group chemistry designed was also very flexible. From two advanced thioglycosyl disaccharide intermediates, all of the required disaccharide modules for library preparation could be generated in a divergent manner, which greatly simplified building-block preparation. Furthermore, the reactivity-independent nature of the preactivation-based, one-pot approach enabled us to mix the building blocks. This allowed rapid assembly of twelve HP/HS hexasaccharides with systematically varied and precisely controlled backbone structures in a combinatorial fashion. The speed and the high yields achieved in glycoassembly without the need to use a large excess of building blocks highlighted the advantages of our approach, which can be of general use to facilitate the study of HP/HS biology. As a proof of principle, this panel of hexasaccharides was used to probe the effect of backbone sequence on binding with the fibroblast growth factor-2 (FGF-2). A trisaccharide sequence of 2-O-sulfated iduronic acid flanked by N-sulfated glucosamines was identified to be the minimum binding motif and N-sulfation was found to be critical. This provides useful information for further development of more potent compounds towards FGF-2 binding, which can have potential applications in wound healing and anticancer therapy.

Commissural neurons project axons across the floor plate at the spinal cord ventral midline. After crossing, commissural axons turn rostrally, sort into distinct positions within the ventrolateral funiculus, and never reenter the floor plate. Robo1 and Robo2 are receptors for the midline repellents Slit1-Slit3, and upregulation of Robos in post-crossing axons allows expulsion from the floor plate and prevents recrossing. Before crossing, Robo-mediated repulsion is attenuated by the divergent family member Robo3/Rig-1. To define the relative contributions of Robo family members to commissural axon guidance in mice, we studied commissural axon trajectories in combination mutants between Robo1, Robo2, and Robo3. Our results suggest the existence of another receptor contributing to Slit repulsion because the failure of midline crossing in Robo3 mutants is rescued largely but not entirely by loss of both Robo1 and Robo2 and because axon guidance defects in mice lacking both Robo1 and Robo2 are less severe than in mice lacking all Slits. Analysis of post-crossing axon trajectories indicates that Robo1 and Robo2 collaborate to prevent axons from reentering the gray matter and projecting dorsally alongside contralateral pre-crossing axons. We also discovered a previously unappreciated division of labor between Robo1 and Robo2 in post-crossing axons. Robo2 is required for axons to project away from the floor plate into the lateral funiculus. In contrast, Robo1 prevents axonal stalling after crossing. Our results reveal specialized and complementary actions of Robo1 and Robo2 in commissural axon guidance and suggest the existence of an as yet unidentified Slit receptor.

<h4>Aim</h4>To investigate the prevalence of, and risk factors for, diabetes mellitus (DM) in Algerian patients with chronic hepatitis C virus (HCV) infection and in a control group.<h4>Methods</h4>A cross-sectional study was undertaken. A total of 416 consecutive patients with viral chronic hepatitis attending the Internal Medicine Department of the University Hospital Center Touhami Benflis in Batna [290 HCV-infected and 126 hepatitis B virus (HBV)-infected patients] were prospectively recruited.<h4>Results</h4>The prevalence of DM was higher in HCV-infected patients in comparison with HBV-infected patients (39.1% vs 5%, P < 0.0001). Among patients without cirrhosis, diabetes was more prevalent in HCV-infected patients than in HBV-infected patients (33.5% vs 4.3%, P < 0.0001). Among patients with cirrhosis, diabetes was more prevalent in HCV-infected patients, but the difference was not significant (67.4% vs 20%, P = 0.058). The logistic regression analysis showed that HCV infection [odds ratio (OR) 4.73, 95% CI: 1.7-13.2], metabolic syndrome (OR 12.35, 95% CI: 6.18-24.67), family history of diabetes (OR 3.2, 95% CI: 1.67-6.13) and increased hepatic enzymes (OR 2.22, 95% CI: 1.1-4.5) were independently related to DM in these patients.<h4>Conclusion</h4>The high prevalence of diabetes in HCV-infected patients, and its occurrence at early stages of hepatic disease, suggest that screening for glucose abnormalities should be indicated in these patients.

Protein scaffolds play an important role in signal transduction, regulating the localization of signaling components and mediating key protein interactions. Here, we report that the major binding partners of the Connector Enhancer of KSR 1 (CNK1) scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors, and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream from insulin and insulin-like growth factor 1 (IGF-1) receptors. We identified a domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins, and found that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and rescue experiments, we found that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) to generate a PIP(2)-rich microenvironment that is critical for the membrane recruitment of insulin receptor substrate 1 (IRS1) and signal transmission to the PI3K/AKT cascade. These findings identify CNK1 as a new positive regulator of insulin signaling.

Neogenin has been identified as a receptor for the neuronal axon guidance cues netrins and RGMs (repulsive guidance molecules). Here we provide evidence for neogenin in regulating endochondral bone development and BMP (bone morphogenetic protein) signaling. Neogenin-deficient mice were impaired in digit/limb development and endochondral ossification. BMP2 induction of Smad1/5/8 phosphorylation and Runx2 expression, but not noncanonical p38 MAPK activation, was reduced in chondrocytes from neogenin mutant mice. BMP receptor association with membrane microdomains, which is necessary for BMP signaling to Smad, but not p38 MAPK, was diminished in neogenin-deficient chondrocytes. Furthermore, RGMs appear to mediate neogenin interaction with BMP receptors in chondrocytes. Taken together, our results indicate that neogenin promotes chondrogenesis in vitro and in vivo, revealing an unexpected mechanism underlying neogenin regulation of BMP signaling.

Gonadotrophin-releasing hormone-1 (GnRH-1) is essential for mammalian reproduction, controlling release of gonadotrophins from the anterior pituitary. GnRH-1 neurones migrate from the nasal placode into the forebrain during development. Although first located within the nasal placode, the embryonic origin/lineage of GnRH-1 neurones is still unclear. The migration of GnRH-1 cells is the best characterised example of neurophilic/axophilic migration, with the cells using a subset of olfactory-derived vomeronasal axons as their pathway and numerous molecules to guide their movement into the forebrain. Exciting work in this area is beginning to identify intersecting pathways that orchestrate the movement of these critical neuroendocrine cells into the central nervous system, both spatially and temporally, through a diverse and changing terrain. Once within the forebrain, little is known about how the axons target the median eminence and ultimately secrete GnRH-1 in a pulsatile fashion.

<h4>Unlabelled</h4>Numerous studies have demonstrated the value of team-based care to improve blood pressure (BP) control, but there is limited information on whether these models would be adopted in diverse populations. The purpose of this study was to evaluate whether a collaborative model between physicians and pharmacists can improve BP control in multiple primary care medical offices with diverse geographic and patient characteristics and whether long-term BP control can be sustained. This study is a randomized prospective trial in 27 primary care offices first stratified by the percentage of underrepresented minorities and the level of clinical pharmacy services within the office. Each office is then randomized to either a 9- or 24-month intervention or a control group. Patients will be enrolled in this study until 2012. The results of this study should provide information on whether this model can be implemented in large numbers of diverse offices, if it is effective in diverse populations, and whether BP control can be sustained long term.<h4>Clinical trial registration</h4>URL: http://www.clinicaltrials.gov. Unique identifier: NCT00935077.

<h4>Aim</h4>To study the role of hepcidin in hereditary hyperferritinemia cataract syndrome (HHCS).<h4>Methods</h4>Six patients from two families with HHCS, confirmed by genetic analysis showing A to G mutation at position +40 in the L-ferritin gene, were recruited to undergo serum hepcidin and prohepcidin measurements using radioimmunoassay and enzyme linked immunoassay, respectively, and measurements were compared with levels in serum from 25 healthy volunteers (14 females), mean age 36 +/- 11.9 years.<h4>Results</h4>The serum hepcidin and prohepcidin levels in patients with HHCS were 19.1 +/- 18.6 and 187 +/- 120.9 ng/mL, respectively. Serum ferritin was 1716.3 +/- 376 microg/L. Liver biopsy in one patient did not show any evidence of iron overload. Serum hepcidin and prohepcidin values in healthy controls (HCs) were 15.30 +/- 15.71 and 236.88 +/- 83.68 ng/mL, respectively, while serum ferritin was 110 +/- 128.08 microg/L. There was no statistical difference in serum hepcidin level between the two cohorts (19.1 +/- 18.6 ng/mL vs 15.30 +/- 15.71 ng/mL, P = 0.612) using two-tailed t-test.<h4>Conclusion</h4>Serum hepcidin levels in HHCS patients is similar to that in HCs. Our study suggests that circulating ferritin is not a factor influencing hepcidin synthesis and does not have a role in the iron-sensing mechanism in hepatocytes.

Hand-foot-and-mouth disease (HFMD) is caused by a group of enteroviruses, most commonly coxsackievirus A 16 (CA16) and enterovirus 71 (EV71). In general, the disease is mild and self-limited except in the case of EV71 infections, which may incur serious complications. This research focused on virus characterization of HFMD cases in Thailand from 2008-2009, related clinical findings and complications of specific enterovirus subtypes. Specimens (stool, vesicle fluid, throat swab/sputum) from 48 cases were collected during 2008-2009. Reverse transcriptase-polymerase chain reaction (PCR) followed by direct sequencing and phylogenetic analysis served to detect enterovirus and determine subtype. Enterovirus was found in 58.3% (28/48) of cases, specifically EV71 (n=23), CA16 (n=4), and CA10 (n=1). Two patients infected by EV71 had brainstem encephalitis (one death). Eight patients required hospital admission due to dehydration. Of these, 3 were PCR positive for EV71, 1 for CA16, and the reminder negative. This study demonstrated EV71 as the most prevalent present cause of HFMD in Thailand in 2008-2009. Potentially fatal complications of HFMD should be taken into consideration. Surveillance of epidemiology and monitoring of disease severity should be continued, and as a prevention measure, sanitation and hygiene should be improved.

Specificity in the projections from the mammalian ventral cochlear nucleus (VCN) is essential for sound localization. Globular bushy cells project from the VCN to the medial nucleus of the trapezoid body (MNTB) on the contralateral, but not the ipsilateral, side of the brainstem, terminating in large synaptic endings known as calyces of Held. The precision in this pathway is critical for the computation of interaural intensity differences, which are used in sound localization. The mechanisms underlying the development of this projection are not completely understood. In this study, we tested the role of Eph receptor tyrosine kinases and their ephrin ligands in limiting the VCN-MNTB projection to the contralateral side. We found that mice with null mutations in EphB2 and EphB3 had normal contralateral VCN-MNTB projections, yet these projections also had significant numbers of aberrant collateral branches in the ipsilateral MNTB. These aberrant branches ended in calyceal terminations in MNTB. Similar ipsilateral projections were seen in mice with mutations in ephrin-B2. In both of these mouse lines, ipsilateral projections formed concurrently with normal contralateral projections and were not eliminated later in development. However, mice with mutations that affected only the intracellular domain of EphB2 had normal, strictly contralateral VCN-MNTB projections. Expression studies showed that EphB2 is expressed in VCN axons and ephrin-B2 is expressed in MNTB. Together, these data suggest that EphB2-ephrin-B2 reverse signaling is required to prevent the formation of ipsilateral VCN-MNTB projections and that this signaling operates non-cell autonomously.

Behavioral circadian rhythms disintegrate progressively in the R6/2 mouse model of Huntington's disease (HD), recapitulating the sleep-wake disturbance seen in HD patients. Here we show that disturbances in circadian pacemaking are not restricted to the brain, but also encompass peripheral metabolic pathways in R6/2 mice. Notably, circadian rhythms of clock-driven genes that are key metabolic outputs in the liver are abolished in vivo. This deficiency is accompanied by arrhythmic expression of the clock genes Cry1 and Dbp, and a phase-advanced Per2 cycle. Compromised circadian metabolic cycles are not, however, a consequence of deficient pacemaking intrinsic to the liver, because when cultured in vitro, R6/2 liver slices exhibit self-sustained circadian bioluminescence rhythms. We therefore propose that compromised metabolic cycles arise from an internal desynchronization secondary to altered feeding patterns and impaired circadian signaling from the central pacemaker of the suprachiasmatic nucleus (SCN). Importantly, the SCN-independent food-entrainable oscillator remains intact in R6/2 mice and, when invoked, can restore daily behavioral cycles and reverse some of the metabolic abnormalities seen in the liver. Disturbances of metabolism have long been thought to be an important feature of HD. Uncoupling liver metabolism from circadian drives will reduce metabolic efficiency and cause imbalances in metabolites known to be deleterious to brain function. Thus, even subtle imbalances in liver function may exacerbate symptoms of HD, where neurological function is already compromised.

Mutant huntingtin proteolysis mediated by various proteases plays a key role in Huntington's disease (HD) pathogenesis. In this issue of Neuron, Miller et al. have identified 11 proteases, including matrix metalloproteinases (MMPs), that when inhibited reduce huntingtin proteolysis and produce beneficial therapeutic effects. These findings provide new insights into huntingtin proteolysis and its potential as a therapeutic target.

Proteolytic cleavage of huntingtin (Htt) is known to be a key event in the pathogenesis of Huntington's disease (HD). Our understanding of proteolytic processing of Htt has thus far focused on the protease families-caspases and calpains. Identifying critical proteases involved in Htt proteolysis and toxicity using an unbiased approach has not been reported. To accomplish this, we designed a high-throughput western blot-based screen to examine the generation of the smallest N-terminal polyglutamine-containing Htt fragment. We screened 514 siRNAs targeting the repertoire of human protease genes. This screen identified 11 proteases that, when inhibited, reduced Htt fragment accumulation. Three of these belonged to the matrix metalloproteinase (MMP) family. One family member, MMP-10, directly cleaves Htt and prevents cell death when knocked down in striatal Hdh(111Q/111Q) cells. Correspondingly, MMPs are activated in HD mouse models, and loss of function of Drosophila homologs of MMPs suppresses Htt-induced neuronal dysfunction in vivo.

The present paper deals with studying ofhemostasis system indices after short-term (10-11 days) space flights as well as in the course of the experiment with a 7-day "dry" immersion. The following values were determined: activated partial thromboplastin time, prothrombin time, prothrombin index, international normalized ratio (INR), thrombin time (TT); fibrinogen, soluble fibrin-monomer complexes, D-dimer, plasminogen (PG) concentration; activity of antithrombin III (ATIII), protein C (PC), alpha2-antiplasmin (AP). In the 1st day after space flights a fibrinogen concentration was increased significantly as well as TT shortening tendency and fibrinolysis activation tendency were observed. There were no significant changes in ATI I11 and PC activities. During the immersion period there were no significant changes in investigated indices, with the exception of AP activity increase in 3rd day of experiment. In readaptation period, however, a decrease in ATIII and PC activities as well as PG concentration was observed. Hemostasis system response features after space flights and during immersion are probably due to different intensity and duration of external factors effects.

Retinoblastoma (RB) is an embryonic neoplasm of retinal origin. For many years, scientists have sought the fundamental origins of tumorigenesis, with the ultimate hope of discovering a cure. Indeed, these efforts have led to a significant understanding that multiple molecular and genetic aberrations, such as uncontrolled proliferation and the inhibition of apoptosis that contribute to the canonical characteristics of tumor biology. Despite these advances, a thorough understanding, such as the precise cells, which are the targets of neoplastic transformation, especially in solid tumors, is currently lacking. The focus of this review is to emphasize the molecular defects involved in the RB tumor progression and mechanisms associated with inhibition of tumor cell apoptotic processes. This review also discusses the importance of target molecules characterization and their potential therapeutic or prognostic use in RB disease.

The osteoconductive and possibly osteoinductive characteristics of OCP increased the interest in preparation of bone graft materials that contain OCP in its composition. Calcium phosphate cements (CPCs) were prepared using a mixture of α-tricalcium phosphate (α-TCP) and dicalcium phosphate anhydrous (DCPA), with α-TCP / DCPA molar ratio of 1/1 and distilled water or 0.5 mol / L phosphate aqueous solution (pH = 6.1 ± 0.1) as the cement liquid. Hardening time was (30 ± 1) min for the CPC mixed with water and (5 ± 1) min for the CPC mixed with phosphate solution. Diametral tensile strength (DTS), porosity (P), and phase composition (powder x-ray diffraction) were determined after the hardened specimens had been immersed in a physiological-like solution (PLS) for 1 d, 3 d, and 7 d. In CPC specimens prepared with water, calcium hydroxyapatite (HA) was formed and DTS and P were (9.03 ± 0.48) MPa and (37.05 ± 0.20) vol % after 1 d, respectively, and (9.15 ± 0.45) MPa and (37.24 ± 0.63) vol % after 3 d, respectively. In CPC specimens prepared with phosphate solution OCP and HA were formed and DTS and P were (4.38 ± 0.49) MPa and (41.44 ± 1.25) vol % after 1 d, respectively,(4.38 ± 0.29) MPa and (42.52 ± 2.15) vol % after 3 d, respectively, and (4.30 ± 0.60) MPa and (41.38 ± 1.65) vol % after 7 d, respectively. For each group DTS and P did not change with PLS immersion time. DTS was significantly higher and P was significantly lower for CPCs prepared with water. HA formation slightly increased with immersion time from 40 mass % after 1 d to 50 mass % after 3 d in CPCs prepared with water. OCP + HA formation increased with immersion time from 30 mass % after 1 d to 35 mass % after 3 d and to 45 mass % after 7 d in CPCs prepared with 0.5 mol / L phosphate solution.

Fabry disease is a rare tesaurismosis due to a deficit of the lysosomal enzyme activity of alpha-galactosidase, needed for the normal catabolism of globotriaosylceramides (GL3). Fabry cardiac involvement has several clinical manifestations: concentric left ventricular hypertrophy without left ventricular dilation and severe loss of left ventricular systolic function, mitral and aortic valvulopathy, disorders of the atrioventricular conduction or repolarization, and compromised diastolic function. Differentiating Fabry disease from similar conditions is often quite straightforward, e.g., cardiac amyloidosis is often associated with low electrocardiographic voltages, and systemic symptoms are usually associated with hemochromatosis and sarcoidosis. However, sometimes second-level (genetic analysis, alpha-galactosidase levels) or invasive investigations are required, which can include endomyocardial biopsy. Diagnostic imaging techniques have been described, but they lack specificity. Echocardiographic imaging with tissue Doppler analysis and/or strain rate analysis can allow diagnosis of Fabry disease even before left ventricular hypertrophy becomes apparent. This review illustrates the techniques for staging cardiac involvement and damage in Fabry disease and for the long-term follow-up of Fabry patients with or without cardiac involvement. Careful cardiac monitoring is especially important in elderly female carriers, who often develop renal disorders and/or left ventricular hypertrophy as the only manifestations of their late Fabry disease. In some clinical series, Fabry disease was diagnosed in 12% of women with adult-onset hypertrophic cardiomyopathy. Cardiological problems and outcomes of enzyme replacement therapy, associated with or without other cardiological treatments, are also discussed.

<h4>Background</h4>Although chronic liver disease is associated with gastroesophageal reflux disease (GERD), the impact of chronic hepatitis B virus (HBV) infection on this association remains unclear. We thus aimed to evaluate the relationship between chronic HBV infection and GERD.<h4>Methods</h4>In this prospective population-based study, 1,001 adult subjects who underwent an upper gastrointestinal endoscopic examination in a health check-up and completed a gastroesophageal reflux questionnaire were consecutively enrolled. Endoscopic findings were classified according to the Los Angeles classification. Hepatitis B surface antigen was used as a marker of HBV infection. Univariate and multivariate approaches were used to evaluate the effects of chronic HBV infection on GERD.<h4>Results</h4>Chronic HBV infection was associated with heartburn sensation [odds ratio (OR) 1.27, 95% confidence interval 1.01-1.61, P = 0.037], and erosive esophagitis (adjusted OR 1.75, 1.03-2.97, P = 0.037). Although male gender is a risk factor of erosive esophagitis, further analyses stratified by gender and aspartate aminotransferase to platelet ratio index (APRI) showed that chronic HBV infection was associated with erosive esophagitis in female subjects (adjusted OR 2.70, 1.14-6.39, P = 0.024) and those with APRI of more than 0.3 (adjusted OR 3.94, 1.73-8.96, P = 0.001). Moreover, higher serum aspartate aminotransferase (AST) and triglyceride (TG) levels were risk factors of erosive esophagitis in patients with chronic HBV infection.<h4>Conclusions</h4>Our findings indicate a close association between chronic HBV infection and GERD, especially in female subjects and those with higher APRI levels. Moreover, HBV carriers with higher AST or TG levels have higher incidence of erosive esophagitis. The interactions between chronic HBV infection and GERD need further studies.<h4>Electronic supplementary material</h4>The online version of this article (doi:10.1007/s12072-010-9184-4) contains supplementary material, which is available to authorized users.

Advances in recent years in the understanding of, and the genetic diagnosis of hereditary hemochromatosis (HH) have changed the approach to iron overload hereditary diseases. The ability to use a radiologic tool (MRI) that accurately provides liver iron concentration determination, and the presence of non-invasive serologic markers for fibrosis prediction (serum ferritin, platelet count, transaminases, etc), have diminished the need for liver biopsy for diagnosis and prognosis of this disease. Consequently, the role of liver biopsy in iron metabolism disorders is changing. Furthermore, the irruption of transient elastography to assess liver stiffness, and, more recently, the ability to determine liver fibrosis by means of MRI elastography will change this role even more, with a potential drastic decline in hepatic biopsies in years to come. This review will provide a brief summary of the different non-invasive methods available nowadays for diagnosis and prognosis in HH, and point out potential new techniques that could come about in the next years for fibrosis prediction, thus avoiding the need for liver biopsy in a greater number of patients. It is possible that liver biopsy will remain useful for the diagnosis of associated diseases, where other non-invasive means are not possible, or for those rare cases displaying discrepancies between radiological and biochemical markers.

The true prevalence of pediatric nonalcoholic fatty liver disease (NAFLD) is unknown. Challenges in determining the population prevalence of NAFLD include the type of test (and the reference intervals used to define normal and abnormal), the type of population (general population, hospital series), the demographic characteristics of the population sampled, and the nature of the study design. The natural history of pediatric NAFLD remains uncertain. The issue of when to perform a liver biopsy in children with suspected NAFLD remains controversial. Children with NAFLD but normal alanine aminotransferase are rarely investigated. However, evidence of alterations in glucose metabolism parameters should prompt a better understanding of the natural history of pediatric NAFLD not only in terms of the progression of liver disease but also regarding its potential relationship with other health outcomes such as type 2 diabetes mellitus and cardiovascular disease. This evidence could make liver biopsy mandatory in the majority of cases at risk of progressive and severe hepatic and extrahepatic disease. This conclusion, however, raises the question of the feasibility of liver biopsy assessment in an extremely large at risk population, and of the cost/effectiveness of this policy. There is a considerable, continuous interest in reliable, noninvasive alternatives that will allow the prognosis of pediatric NAFLD to be followed in large community or population-based studies.

Genetics of hypertension is complex with no known single gene playing a major role, but rather many genes each with mild effects reacting to different environmental stimuli contribute to blood pressure. The heritable component of blood pressure has been documented in familial and twin studies suggesting that 30%-50% of the variance of blood pressure readings are attributable to genetic heritability and about 50% to environmental factors. Early studies in hypertension identified specific enzymes, channels and receptors implicating sodium handling in the regulation of blood pressure including genes involved with the renin-angiotensin-aldosterone system controlling blood pressure and salt-water homeostasis, proteins in hormonal regulation of blood pressure (enzymes and receptors of the mineralo- and glucocorticoid pathways) and proteins coded by genes involved in the structure and/or regulation of vascular tone (endothelins and their receptors). The field of molecular genetics has revolutionized the study of hypertension by identifying single gene syndromes or Mendelian forms and several candidate genes for blood pressure variance. Genes have been localized to at least 20 chromosome regions. For example, recent genome-wide association studies (GWAS) of common genetic variants found 13 single nucleotide polymorphisms (SNPs) or variants in systolic and 20 for diastolic blood pressure readings representing different genes and genetic heterogeneity. Further understanding of the genetics of hypertension will require the use of advances in bioinformatics tools and genetic technology [e.g., SNP, exon and noncoding (micro) RNA arrays]. New approaches will allow for identification of not only single genes, but other interacting genes contributing to hypertension by merging multiple genetic data sets (structural and functional) from individuals with hypertension and development of new molecular targets for study and treatment.

The development of multicellular organisms is associated with extensive rearrangements of tissues and cell sheets. The driving force for these rearrangements is generated mostly by the actin cytoskeleton. In order to permit the reproducible development of a specific body plan, dynamic reorganization of the actin cytoskeleton must be precisely coordinated in space and time. GTP-exchange factors that activate small GTPases of the Rho family play an important role in this process. Here we review the role of this class of cytoskeletal regulators during important developmental processes such as epithelial morphogenesis, cytokinesis, cell migration, cell polarity, neuronal growth cone extension and phagocytosis in different model systems.

In recent years, there has been a great deal of attention toward the field of free radical chemistry. Free radicals reactive oxygen species and reactive nitrogen species are generated by our body by various endogenous systems, exposure to different physiochemical conditions or pathological states. A balance between free radicals and antioxidants is necessary for proper physiological function. If free radicals overwhelm the body's ability to regulate them, a condition known as oxidative stress ensues. Free radicals thus adversely alter lipids, proteins, and DNA and trigger a number of human diseases. Hence application of external source of antioxidants can assist in coping this oxidative stress. Synthetic antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole have recently been reported to be dangerous for human health. Thus, the search for effective, nontoxic natural compounds with antioxidative activity has been intensified in recent years. The present review provides a brief overview on oxidative stress mediated cellular damages and role of dietary antioxidants as functional foods in the management of human diseases.

1,1 '-Diethyl-2,2'-cyanine iodide (decynium22; D22) is a potent blocker of the organic cation family of transporters (EMT/OCT) known to move endogenous monoamines like dopamine and norepinephrine across cell membranes. Decynium22 is a cation with a relatively high affinity for all members of the OCT family in both human and rat cells. The mechanism through which decynium22 blocks OCT transporters are poorly understood. We tested the hypothesis that denynium22 may compete with monoamines utilizing OCT to permeate the cells. Using the ability of D22 to aggregate and produce fluorescence at 570 nm, we measured D22 uptake in cultured astrocytes. The rate of D22 uptake was strongly depressed by acid pH and by elevated external K+. The rate of uptake was similar to that displayed by 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+), a well established substrate for OCT and high-affinity Na+-dependent monoamine transporters. These data were supported by measurement of electrogenic uptake using whole cell voltage clamp recording. Decynium22 depressed norepinephrine, but not glutamate uptake. These data are also consistent with the described OCT transporter characteristics. Taken together, our results suggest that decynium22 accumulation might be a useful instrument to study monoamine transport in the brain, and particularly in astrocytes, where they may play a prominent role in monoamine uptake during brain dysfunction related to monoamines (like Parkinson disease) and drug addiction.

Do intellectual property (IP) rights on existing technologies hinder subsequent innovation? Using newly-collected data on the sequencing of the human genome by the public Human Genome Project and the private firm Celera, this paper estimates the impact of Celera's gene-level IP on subsequent scientific research and product development. Genes initially sequenced by Celera were held with IP for up to two years, but moved into the public domain once re-sequenced by the public effort. Across a range of empirical specifications, I find evidence that Celera's IP led to reductions in subsequent scientific research and product development on the order of 20 to 30 percent. Taken together, these results suggest that Celera's short-term IP had persistent negative effects on subsequent innovation relative to a counterfactual of Celera genes having always been in the public domain.