Washing scaling of GeneChip microarray expression

BACKGROUND Post-hybridization washing is an essential part of microarray experiments. Both the quality of the experimental washing protocol and adequate consideration of washing in intensity calibration ultimately affect the quality of the expression estimates extracted from the microarray intensities. RESULTS We conducted experiments on GeneChip microarrays with altered protocols for washing, scanning and staining to study the probe-level intensity changes as a function of the number of washing cycles. For calibration and analysis of the intensity data we make use of the 'hook' method which allows intensity contributions due to non-specific and specific hybridization of perfect match (PM) and mismatch (MM) probes to be disentangled in a sequence specific manner. On average, washing according to the standard protocol removes about 90% of the non-specific background and about 30-50% and less than 10% of the specific targets from the MM and PM, respectively. Analysis of the washing kinetics shows that the signal-to-noise ratio doubles roughly every ten stringent washing cycles. Washing can be characterized by time-dependent rate constants which reflect the heterogeneous character of target binding to microarray probes. We propose an empirical washing function which estimates the survival of probe bound targets. It depends on the intensity contribution due to specific and non-specific hybridization per probe which can be estimated for each probe using existing methods. The washing function allows probe intensities to be calibrated for the effect of washing. On a relative scale, proper calibration for washing markedly increases expression measures, especially in the limit of small and large values. CONCLUSIONS Washing is among the factors which potentially distort expression measures. The proposed first-order correction method allows direct implementation in existing calibration algorithms for microarray data. We provide an experimental 'washing data set' which might be used by the community for developing amendments of the washing correction.This publication is supported by the Leipzig Interdisciplinary Research Cluster of Genetic Factors, Clinical Phenotypes and Environment (LIFE Center, Universität Leipzig) and an Australian Academy of Science Visits to Europe grant. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERFD) and by means of the Free State of Saxony within the framework of the excellence initiative

Consistent model of magnetism in ferropnictides

The discovery of superconductivity in LaFeAsO introduced the ferropnictides as a major new class of superconducting compounds with critical temperatures second only to cuprates. The presence of magnetic iron makes ferropnictides radically different from cuprates. Antiferromagnetism of the parent compounds strongly suggests that superconductivity and magnetism are closely related. However, the character of magnetic interactions and spin fluctuations in ferropnictides, in spite of vigorous efforts, has until now resisted understanding within any conventional model of magnetism. Here we show that the most puzzling features can be naturally reconciled within a rather simple effective spin model with biquadratic interactions, which is consistent with electronic structure calculations. By going beyond the Heisenberg model, this description explains numerous experimentally observed properties, including the peculiarities of the spin wave spectrum, thin domain walls, crossover from first to second order phase transition under doping in some compounds, and offers new insight in the occurrence of the nematic phase above the antiferromagnetic phase transition.Comment: 5 pages, 3 figures, revtex

Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

BACKGROUND: Lipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling. PRINCIPAL FINDINGS: Immunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion. CONCLUSION: We conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles

An IP-10 (CXCL10)-derived peptide inhibits angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis. © 2012 Yates-Binder et al

Expression and Regulation of the Fkbp5 Gene in the Adult Mouse Brain

BACKGROUND: Chronic stress has been found to be a major risk factor for various human pathologies. Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, which is tightly regulated via, among others, the glucocorticoid receptor (GR). The activity of the GR is modulated by a variety of proteins, including the co-chaperone FK506 binding protein 51 (FKBP5). Although FKBP5 has been associated with risk for affective disorders and has been implicated in GR sensitivity, previous studies focused mainly on peripheral blood, while information about basal distribution and induction in the central nervous system are sparse. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we describe the basal expression pattern of Fkbp5 mRNA in the brain of adult male mice and show the induction of Fkbp5 mRNA via dexamethasone treatment or different stress paradigms. We could show that Fkbp5 is often, but not exclusively, expressed in regions also known for GR expression, for example the hippocampus. Furthermore, we were able to induce Fkbp5 expression via dexamethasone in the CA1 and DG subregions of the hippocampus, the paraventricular nucleus (PVN) and the central amygdala (CeA). Increase of Fkbp5 mRNA was also found after restrained stress and 24 hours of food deprivation in the PVN and the CeA, while in the hippocampus only food deprivation caused an increase in Fkbp5 mRNA. CONCLUSIONS/SIGNIFICANCE: Interestingly, regions with a low basal expression showed higher increase in Fkbp5 mRNA following induction than regions with high basal expression, supporting the hypothesis that GR sensitivity is, at least partly, mediated via Fkbp5. In addition, this also supports the use of Fkbp5 gene expression as a marker for GR sensitivity. In summary, we were able to give an overview of the basal expression of fkbp5 mRNA as well as to extend the findings of induction of Fkbp5 and its regulatory influence on GR sensitivity from peripheral blood to the brain

Vaccination of metastatic renal cell carcinoma patients with autologous tumour-derived vitespen vaccine: clinical findings

The aim of this study was to evaluate the clinical efficacy as determined by time to progression and response rate (RR) of autologous vitespen (formerly HSPPC-96; Oncophage, Antigenics Inc., New York, NY, USA) with and without interleukin-2 (IL-2; Proleukin: Chiron, Emoryville, CA, USA) in stage IV metastatic renal cell carcinoma (RCC) patients undergoing nephrectomy. Eighty-four patients were enrolled on study, and then underwent nephrectomy and harvest of tumour tissue for use in autologous vaccine manufacture. Initial treatment schedule started approximately 4 weeks after surgery and consisted of six injections: once weekly for 4 weeks, then two injections biweekly (vaccines administered at weeks 1, 2, 3, 4, 6, 8), followed by restaging at or around week 10. Patients who had stable or responsive disease continued to receive vaccine, with four more vaccinations biweekly (at weeks 10, 12, 14, 16). Patients who had progressive disease at week-10 evaluation received four consecutive 5-day-per-week courses of 11 × 106 U of IL-2 subcutaneously (weeks 10, 11, 12, 13), with four doses of vitespen at 2-week intervals (at weeks 10, 12, 14, 16). At the next evaluation (week 18), patients with a complete response received two further cycles of vitespen (with IL-2 if also received during prior cycle) or until vaccine supply was exhausted. Patients with stable disease or partial response repeated their prior cycle of therapy. Disease progressors who had not yet received IL-2 began IL-2 treatment, and progressors who had already received IL-2 came off study. Of 60 evaluable patients, 2 demonstrated complete response (CR), 2 showed partial response (PR), 7 showed stable disease, and 33 patients progressed. Sixteen patients had unconfirmed stable disease. Two patients who progressed on vaccine alone experienced disease stabilisation when IL-2 was added. Treatment with vitespen did not result in a discernable benefit in the majority of patients with metastatic RCC treated in this study. Use in combination with immunoregulatory agents may enhance the efficacy of vitespen

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 90 (HSP90) inhibitors have emerged as a promising class of anti-cancer drugs in both solid and hematologic malignancies. The HSP90 family includes the cytosolic HSP90 (HSP90AA1), the ER paralogue gp96 (HSP90B1) and the mitochondrial member TRAP1 (HSP90L). We evaluated the <it>in vitro </it>anti-tumor activity and mechanism of action of PU-H71, a novel purine scaffold HSP90 inhibitor in human multiple myeloma cell lines.</p> <p>Methods</p> <p>Multiple human myeloma cell lines including cells that are resistant to corticosteroids and bortezimab were treated with PU-H71, followed by analysis of cell viability, cell cycle progression and apoptosis, by flow cytometry and caspase 3 immunoblot. Induction of unfolded protein response was studied by XBP-1 s immunoblot. The role of gp96 was further assessed by small hairpin RNA knockdown of gp96 before treatment with PU-H71.</p> <p>Results</p> <p>PU-H71 has potent <it>in vitro </it>anti-myeloma activity in both drug-sensitive and drug-resistant cell lines. PU-H71 activates the unfolded protein response and induces caspase-dependent apoptosis. The stable gp96 knockdown human myeloma cell line was found to be more resistant to PU-H71 and other HSP90 inhibitors including 17-AAG and 17-DMAG, even though these cells are more sensitive to conventional anti-myeloma drugs.</p> <p>Conclusion</p> <p>We conclude that PU-H71 is a promising drug for the treatment of myeloma. Our finding further suggests that PU-H71 and the geldanamycin analogues work in part by inhibiting the endoplasmic reticulum gp96 along with the cytosolic HSP90.</p

Phylogenetic Distribution of Fungal Sterols

BACKGROUND: Ergosterol has been considered the "fungal sterol" for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation. METHODOLOGY/PRINCIPAL FINDINGS: The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Delta(5) sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Delta(5) sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28)-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis. CONCLUSIONS/SIGNIFICANCE: Five dominant end products of sterol biosynthesis (cholesterol, ergosterol, 24-methyl cholesterol, 24-ethyl cholesterol, brassicasterol), and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs (e.g., azoles) target reactions in the synthesis of ergosterol. These findings also invalidate use of ergosterol as an indicator of biomass of certain fungal taxa (e.g., Glomeromycota). Data from this study are available from the Assembling the Fungal Tree of Life (AFTOL) Structural and Biochemical Database: http://aftol.umn.edu

Cortisol secretion after adrenocorticotrophin (ACTH) and Dexamethasone tests in healthy female and male dogs

<p>Abstract</p> <p>Background</p> <p>For the conclusive diagnosis of Cushing's Syndrome, a stimulating ACTH test or a low suppressive Dexamethasone test is used. Reports in other species than the dog indicate that plasma cortisol concentration after ACTH administration is affected by gender. We investigated the effect of gender on the cortisol response to ACTH and Dexamethasone tests in dogs.</p> <p>Methods</p> <p>Seven healthy adult Cocker Spaniels (4 females and 3 males) were assigned to a two by two factorial design: 4 dogs (2 females and 2 males) received IV Dexamethasone 0.01 mg/kg, while the other 3 dogs received an IV saline solution (control group). Two weeks later the treatments were reversed. After one month, ACTH was given IV (250 μg/animal) to 4 dogs (2 female and 2 males) while the rest was treated with saline solution (control group). Cortisol concentrations were determined by a direct solid-phase radioimmunoassay and cholesterol and triglycerides by commercial kits.</p> <p>Results and Discussion</p> <p>No effect of treatment was observed in metabolite concentrations, but females presented higher cholesterol concentrations. ACTH-treated dogs showed an increase in cortisol levels in the first hour after sampling until 3 hours post injection. Cortisol concentrations in Dexamethasone-treated dogs decreased one hour post injection and remained low for 3 hours, thereafter cortisol concentrations increased. The increase in cortisol levels from one to two hours post ACTH injection was significantly higher in females than males. In Dexamethasone-treated males cortisol levels decreased one hour post injection up to 3 hours; in females the decrease was more pronounced and prolonged, up to 5 hours post injection.</p> <p>Conclusion</p> <p>We have demonstrated that cortisol response to ACTH and Dexamethasone treatment in dogs differs according to sex.</p