CD40 stimulates a "feed-forward" NF-κB-driven molecular pathway that regulates IFN-ß expression in carcinoma cells.

IFN-ß and the CD40L (CD154) share important roles in the antiviral and antitumor immune responses. In this study, we show that CD40 receptor occupancy results in IFN-ß upregulation through an unconventional "feed-forward" mechanism, which is orchestrated by canonical NF-κB and involves the sequential de novo synthesis of IFN regulatory factor (IRF)1 and Viperin (RSAD2), an IRF1 target. RelA (p65) NF-κB, IRF1, and Viperin-dependent IRF7 binding to the IFN-ß promoter largely controls its activity. However, full activation of IFN-ß also requires the parallel engagement of noncanonical NF-κB2 signaling leading to p52 recruitment to the IFN-ß promoter. These data define a novel link between CD40 signaling and IFN-ß expression and provide a telling example of how signal propagation can be exploited to ensure efficient regulation of gene expression.

Population-ethnic group specific genome variation allele frequency data: a querying and visualization journey.

National/ethnic mutation databases aim to document the genetic heterogeneity in various populations and ethnic groups worldwide. We have previously reported the development and upgrade of FINDbase (www.findbase.org), a database recording causative mutations and pharmacogenomic marker allele frequencies in various populations around the globe. Although this database has recently been upgraded, we continuously try to enhance its functionality by providing more advanced visualization tools that would further assist effective data querying and comparisons. We are currently experimenting in various visualization techniques on the existing FINDbase causative mutation data collection aiming to provide a dynamic research tool for the worldwide scientific community. We have developed an interactive web-based application for population-based mutation data retrieval. It supports sophisticated data exploration allowing users to apply advanced filtering criteria upon a set of multiple views of the underlying data collection and enables browsing the relationships between individual datasets in a novel and meaningful way.

Tropism of Coxsackie virus A9 depends on the +1 position of the RGD (arginine- glycine- aspartic acid) motif found at the C' terminus of its VP1 capsid protein.

An understanding of how viruses interact with their receptors is vital as this step is a major determinant of host susceptibility and disease. The enterovirus coxsackievirus A9 (CVA9) is an important pathogen responsible for respiratory infections, myocarditis, infections of the central nervous system, chronic dilated cardiomyopathy and possibly type I diabetes. CVA9 harbours an integrin- recognition motif, RGD (Arg-Gly-Asp), in the capsid protein VP1 and this motif is believed to be primarily responsible for binding to integrins αvß6 and/or αvß3 during cell entry. Despite the consistent conservation of RGD-flanking amino acids in multiple RGD-containing picornaviruses, the significance of these amino acids to cell tropism has not been thoroughly investigated. In this study we used 10 CVA9 mutants and a panel of cells to analyse cell tropism. We showed that CVA9 infection proceeds by either an RGD- dependent or an apparently RGD- independent pathway. Differences in the amino acid found at the +1 position of the RGD motif affect the cell tropism of CVA9 when an RGD- dependent pathway is used. Naturally occurring CVA9 isolates have either the sequence RGDM and RGDL and we found that the corresponding viruses in our panel infected cells most efficiently. There was also a strong selection pressure for RGDL in adaptation experiments. However, there was also an unexpected selection of an RGDL variant in an apparently RGD- independent cell line. There was also no simple relationship between infection of cells and expression of integrins αvß3 and αvß6. The results obtained have greatly improved our understanding of how CVA9 infects cells. This will be useful in the design of antivirus drugs and also gives a framework for the modification of CVA9 or other RGD containing picornaviruses for specific targeting of cancer cells for oncolytic therapy.

Translational regulation and deregulation in erythropoiesis.

Translational regulation plays a critical role in erythropoiesis, as it reflects the translational needs of enucleated mature erythroid cells in the absence of transcription and the large translational demands of balanced globin chain synthesis during erythroid maturation. In addition, red blood cells need to respond quickly to changes in their environment and the demands of the organism. Translational regulation occurs at several levels in erythroid cells, including the differential utilization of upstream open reading frames during differentiation and in response to signaling and the employment of RNA-binding proteins in an erythroid cell-specific fashion. Translation initiation is a critical juncture for translational regulation in response to environmental signals such as heme and iron availability, whereas regulatory mechanisms for ribosome recycling are consistent with recent observations highlighting the importance of maintaining adequate ribosome levels in differentiating erythroid cells. Translational deregulation in erythroid cells leads to disease associated with ineffective erythropoiesis, further highlighting the pivotal role translational regulation in erythropoiesis plays in human physiology and homeostasis. Overall, erythropoiesis has served as a unique model that has provided invaluable insight into translational regulation.

CSF levels of apolipoprotein C1 and autotaxin found to associate with neuropathic pain and fibromyalgia.

OBJECTIVE: Neuropathic pain and fibromyalgia are two common and poorly understood chronic pain conditions that lack satisfactory treatments, cause substantial suffering and societal costs. Today, there are no biological markers on which to base chronic pain diagnoses, treatment choices or to understand the pathophysiology of pain for the individual patient. This study aimed to investigate cerebrospinal fluid (CSF) protein profiles potentially associated with fibromyalgia and neuropathic pain. METHODS: CSF samples were collected from 25 patients with neuropathic pain (two independent sets, n=14 patients for discovery, and n=11 for verification), 40 patients with fibromyalgia and 134 controls without neurological disease from two different populations. CSF protein profiling of 55 proteins was performed using antibody suspension bead array technology. RESULTS: We found increased levels of apolipoprotein C1 (APOC1) in CSF of neuropathic pain patients compared to controls and there was a trend for increased levels also in fibromyalgia patients. In addition, levels of ectonucleotide pyrophosphatase family member 2 (ENPP2, also referred to as autotaxin) were increased in the CSF of fibromyalgia patients compared to all other groups including patients with neuropathic pain. CONCLUSION: The increased levels of APOC1 and ENPP2 found in neuropathic pain and fibromyalgia patients may shed light on the underlying mechanisms of these conditions. Further investigation is required to elucidate their role in maintaining pain and other main symptoms of these disorders.

Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles.

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Mammalian expression vectors for metabolic biotinylation tandem affinity tagging by co-expression in cis of a mammalian codon-optimized BirA biotin ligase.

ΟBJECTIVE: To construct mammalian expression vectors for the N- or C-terminal tagging of proteins with a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag. RESULTS: We constructed and tested by transient transfections mammalian expression vectors for the co-expression from a single plasmid of N- or C-terminally tagged proteins bearing a tandem affinity tag comprised of the biotinylatable Avi tag and of a triple FLAG tag separated by a tobacco etch virus (TEV) protease cleavage site, together with a mammalian codon-optimized BirA biotin ligase fused to green fluorescent protein. We also describe platform vectors for the N- or C-terminal AVI-TEV-FLAG tagging of any complementary DNA of choice. These vectors offer versatility and efficiency in the application of metabolic biotinylation tandem affinity tagging of nuclear proteins in mammalian cells.

Semaphorin-7a reverses the ERF-induced inhibition of EMT in Ras-dependent mouse mammary epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is a key process in cancer progression and metastasis, requiring cooperation of the epidermal growth factor/Ras with the transforming growth factor-ß (TGF-ß) signaling pathway in a multistep process. The molecular mechanisms by which Ras signaling contributes to EMT, however, remain elusive to a large extent. We therefore examined the transcriptional repressor Ets2-repressor factor (ERF)-a bona fide Ras-extracellular signal-regulated kinase/mitogen-activated protein kinase effector-for its ability to interfere with TGF-ß-induced EMT in mammary epithelial cells (EpH4) expressing oncogenic Ras (EpRas). ERF-overexpressing EpRas cells failed to undergo TGF-ß-induced EMT, formed three-dimensional tubular structures in collagen gels, and retained expression of epithelial markers. Transcriptome analysis indicated that TGF-ß signaling through Smads was mostly unaffected, and ERF suppressed the TGF-ß-induced EMT via Semaphorin-7a repression. Forced expression of Semaphorin-7a in ERF-overexpressing EpRas cells reestablished their ability to undergo EMT. In contrast, inhibition of Semaphorin-7a in the parental EpRas cells inhibited their ability to undergo TGF-ß-induced EMT. Our data suggest that oncogenic Ras may play an additional role in EMT via the ERF, regulating Semaphorin-7a and providing a new interconnection between the Ras- and the TGF-ß-signaling pathways.

The death domain kinase RIP1 links the immunoregulatory CD40 receptor to apoptotic signaling in carcinomas.

CD40, a tumor necrosis factor (TNF) receptor family member, is widely recognized for its prominent role in the antitumor immune response. The immunostimulatory effects of CD40 ligation on malignant cells can be switched to apoptosis upon disruption of survival signals transduced by the binding of the adaptor protein TRAF6 to CD40. Apoptosis induction requires a TRAF2-interacting CD40 motif but is initiated within a cytosolic death-inducing signaling complex after mobilization of receptor-bound TRAF2 to the cytoplasm. We demonstrate that receptor-interacting protein 1 (RIP1) is an integral component of this complex and is required for CD40 ligand-induced caspase-8 activation and tumor cell killing. Degradation of the RIP1 K63 ubiquitin ligases cIAP1/2 amplifies the CD40-mediated cytotoxic effect, whereas inhibition of CYLD, a RIP1 K63 deubiquitinating enzyme, reduces it. This two-step mechanism of apoptosis induction expands our appreciation of commonalities in apoptosis regulatory pathways across the TNF receptor superfamily and provides a telling example of how TNF family receptors usurp alternative programs to fulfill distinct cellular functions.

Multi-platform data integration in microarray analysis.

An increasing number of studies have profiled gene expressions in tumor specimens using distinct microarray platforms and analysis techniques. One challenging task is to develop robust statistical models in order to integrate multi-platform findings. We compare some methodologies on the field with respect to estrogen receptor (ER) status, and focus on a unified-among-platforms scale implemented by Shen et al. in 2004, which is based on a Bayesian mixture model. Under this scale, we study the ER intensity similarities between four breast cancer datasets derived from various platforms. We evaluate our results with an independent dataset in terms of ER sample classification, given the derived gene ER signatures of the integrated data. We found that integrated multi-platform gene signatures and fold-change variability similarities between different platform measurements can assist the statistical analysis of independent microarray datasets in terms of ER classification.

Detection of DNA mutations using a capacitive micro-membrane array.

The detection of DNA hybridization using capacitive readout and a biosensor array of ultrathin Si membranes is presented. The biosensor exploits the ability of the ultrathin membranes to deflect upon surface stress variations caused by biological interactions. Probe DNA molecules are immobilized on the membrane surface and the surface stress variations during hybridization with their complementary strands force the membrane to deflect and effectively change the capacitance between the flexible membrane and the fixed substrate. The sensor array comprises 256 such sensing sites thus allowing the concurrent sensing of multiple DNA mutations. The biosensor and its performance for the detection of complementary DNA strands are demonstrated using beta-thalassemia oligonucleotides. The experimental results show that the presented sensors are able to detect DNA hybridization and to discriminate single nucleotide mismatches.