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It has been shown previously that the transcription factor NF-κB and its inhibitor IκBα shuttle constitutively between cytosol and nucleus. Moreover, we have recently demonstrated nucleocytoplasmic shuttling of the NF-κB-inducing kinase NIK, a component of the NF-κB pathway, which is essential for lymph node development and B-cell function. Here we show that nuclear NIK also occurs in nucleoli and that this localization is mediated by a stretch of basic amino acids in the N-terminal part of the protein (R143-K-K-R-K-K-K149). This motif is necessary and sufficient for nucleolar localization of NIK, as judged by nuclear localization of mutant versions of the full-length protein and the fact that coupling of these seven amino acids to GFP also leads to accumulation in nucleoli. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) approaches, we demonstrate a dynamic distribution between nucleoli and nucleoplasm and a high mobility of NIK in both compartments. Together with the nuclear export signal in the C-terminal portion of NIK that we have also characterized in detail, the nuclear/nucleolar targeting signals of NIK mediate dynamic circulation of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartments. We demonstrate that nuclear NIK is capable of activating NF-κB and that this effect is diminished by nucleolar localization. Thus, subcellular distribution of NIK to different compartments might be a means of regulating the function of this kinase.
Transcriptional activators of the nuclear factor-κB (NF-κB)/Rel family have a central role in the inducible expression of a large number of genes involved in inflammation, host defense, cell survival and proliferation (de Martin et al., 2000; de Martin et al., 1999; Ghosh and Karin, 2002). NF-κB can be activated by many distinct stimuli, including pro-inflammatory cytokines such as TNFα or IL-1, but also physical stress such as UV- or γ-irradiation, reactive oxygen intermediates and virus-derived dsDNAs or RNAs. The activating signals are transduced by signaling cascades that converge on the IκB kinases (IKKs) that phosphorylate the NF-κB inhibitory molecules, including members of the IκB family and inhibitory domains of NF-κB precursors, leading to their ubiquitination and subsequent degradation by the proteasome. NF-κB is then freed and its steady state localization is shifted to the nucleus where it binds to specific sequences on chromosomal DNA. Upstream of the IKKs, several proteins have been identified that are thought to convey signals from receptor complexes. Amongst these are several members of the MAP 3kinase (MAP3K) family of kinases, including MEKK1 and the NF-κB-inducing kinase, NIK. Originally NIK was identified as a binding partner for the tumor necrosis factor (TNF) receptor associated factor 2 (TRAF2) and therefore thought to be the kinase transducing signals from this central inflammatory receptor (Malinin et al., 1997). However, genetic data from mutant mice showed NIK to be involved in alternative NF-κB signaling pathways: naturally occurring mutant mice with the alymphyoplasia (aly/aly) mutation, which lack lymph nodes and Peyer's patches, were shown to contain a point mutation in the C-terminal part of NIK (Shinkura et al., 1999). This mutation (G855R) is thought to disrupt interaction of NIK with an upstream member of the TRAF family. A complete disruption of the NIK gene (Nik–/–) leads to the same phenotype, which is reminiscent of lymphotoxin β receptor knockout mice [Ltβr–/– (Futterer et al., 1998)]. Indeed, cells from Nik–/– mice showed normal response to TNFα or IL1 treatment, while NF-κB activation in reaction to LTβ was impaired (Yin et al., 2001). Since aly/aly mice, show a cell-autonomous defect in B-cell function in addition to the LTβR–/– phenotype (Karrer et al., 2000; Yamada et al., 2000), at least one other ligand/receptor pair is involved in NIK signaling and was identified to be the TNF receptor family member CD40 (Coope et al., 2002). Moreover, similar defects as in aly/aly and Nik–/– mutants have been observed in Nf-κb2–/– mice. This member of the NF-κB family of transcriptional activators is generated by proteolytical processing of the p100 precursor by the proteasome, giving rise to the mature NF-kB2/p52 protein. Indeed, introduction of the aly mutation into NIK blocks its ability to promote p100 processing in transfected cells (Xiao et al., 2001), an effect which seems to be mediated by the IKK1 subunit of the IKK complex. IKK1 is the IKK subunit that is preferentially phosphorylated by NIK (Ling et al., 1998) and triggers proteasome-mediated processing of p100 to p52, which can then undergo nuclear translocation (Senftleben et al., 2001). Thus, NIK, IKK1 and p100/p52 seem to be components of a NF-κB activation pathway which relies on regulated NF-κB2 processing rather than IκB degradation and is necessary for secondary lymphoid organ development and B cell maturation.
Recently, components of the NF-κB signaling pathway, in particular NF-κB (p65/RelA) itself, IκBα, NIK and IKK1 have been shown to undergo nucleocytoplasmic shuttling in non-activated cells (Birbach et al., 2002; Huang et al., 2000; Johnson et al., 1999; Tam et al., 2000). Thus, these signaling molecules that cycle between the cytosol and the nucleus may provide a means to ensure basal transcriptional activity under resting conditions, but could also facilitate initiation of the activation cascade upon stimulation inside the nucleus.
The nucleus is now known to be a highly dynamic organelle containing distinct subcompartments (Carmo-Fonseca, 2002; Dundr and Misteli, 2001). Although these substructures are not defined by membranes, they have to be regarded as distinct compartments as they can be morphologically identified by light and electron microscopy, or they can be biochemically isolated and enriched, and contain a defining subset of resident proteins. The nucleolus is probably the best characterized of these nuclear subcompartments: it forms around the nucleolar organizer regions that contain the genes coding for ribosomal DNA. Its main function is the complex process of ribosome biogenesis, yet there is increasing evidence that it is also involved in other processes such as maturation of some tRNAs, assembly of ribonucleoprotein machines and regulatory functions by nucleolar retention of proteins [reviewed in (Dundr and Misteli, 2001)]. Localization of proteins to the nucleolus can be viewed in contrast to nuclear targeting, which involves transport across the nuclear pore complex. In case of a subnuclear structure such as the nucleolus, proteins would probably accumulate in a steady-state compartment by interaction with local high-affinity binding sites (Misteli, 2001). Thus, domains necessary for proper localization inside the nucleolus may be seen as retention signals rather than as classical transport signals.
In this study we have examined nucleocytoplasmic shuttling of the NF-κB-inducing kinase NIK. We find that inhibition of nuclear export leads to accumulation of NIK in nuclei and nucleoli. Partial mutation of a putative nuclear localization signal (NLS) in the N-terminal part of the protein results in exclusion of NIK from the nucleoli, yet the protein can still enter the nucleus. However, a complete mutation of the putative NLS confirms its identity as a nuclear targeting signal and shows that the motifs for nuclear and nucleolar localization of NIK are directly linked. This amino acid stretch is necessary and sufficient for nucleolar targeting and together with the C-terminal nuclear export signal (NES), which we define by further point mutation analysis, is responsible for the dynamic exchange of NIK between subcellular compartments. Furthermore, we demonstrate rapid kinetics of this shuttling process by fluorescence loss in photobleaching (FLIP) techniques and the dynamics of the protein in all three compartments by fluorescence recovery after photobleaching (FRAP) approaches.
Materials and Methods
Constructs used in this study were based on the YFP-NIK or YFP-NIK(1-794) and YFP-NIK(1-769) plasmids described previously (Birbach et al., 2002). Point mutations were introduced using the QuikChange kit (Stratagene) and the following sense primers: 5′-GGA AAG CGT CGC AGC AAA GCC GCT GCA GCT GCA AAG AAG AAG AGC TCA AAG TCC C-3′ (143-6A), 5′-GGA AAG CGT CGC AGC AAA GCC GCT GCA GCT GCA GCT GCA GCT AGC TCA AAG TCC C-3′ (143-9A), 5′-CTC TCG TGC CTC AGC ATC GAC AGC GCT TCC GCT TCG GAT GAC AGT GAG-3′ (L803A_L805A), 5′-CTC TCG TGC CTC AGC GCT GAC AGC GCT TCC GCT TCG GAT GAC AGT GAG-3′ (I800A_L803A_L805A), 5′-GAG CAG GAG CAA ATT GCC TCG TGC GCA AGC GCT GAC AGC GCT-3′ (NESmut, based on prior mutation of I800, L803, L805). Antisense primers had respective reverse complementary sequences. C-and N-terminal fusions of the nucleolar targeting signal to GFP, were cloned by inserting a double stranded oligonucleotide coding for the amino acids R-K-K-R-K-K-K into BglII and HindIII sites of the vectors pEGFP-C1 and pEGFP-N1, respectively (NIK-NLS-for: AGA TCT AGA ATG AGA AAG AAG CGA AAA AAG A, containing BglII, XbaI sites and a start codon and NIK-NLs-rev: AAG CTT CTT TTT TCG CTT CTT TCT CAT TCT A containing a HindIII site).
CFP-L23 was constructed by replacing the GFP-tag of a GFP-L23 construct obtained from Nabel Yaseen, group G. Blobel (Yaseen and Blobel, 1997) with CFP from the vector pECFP-C1 (Clontech).
Cell culture and transfections
Cell culture of 293 and HeLa cells was described previously (Schmid et al., 2000) as was culturing of endothelial cells (Stehlik et al., 1998) and transient transfections with Lipofectamine-Plus™ (Invitrogen) (Birbach et al., 2002). The breast cancer cell line MB-MDA-231 was cultured in RPMI supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 U/ml) at 37°C in a humidified, 5% CO2 atmosphere. Transfections with calcium-DNA precipitates were done essentially according to a well established protocol (Webster and Perkins, 1999) with some minor modifications. In brief, cells were seeded at a density of about 40 000 per cm2 one day before transfection. DNA (2-4 μg/10 cm2) was diluted in a total of 62 μl distilled sterile water, 9 μl of sterile 2 M calcium chloride solution were added and the solution was gently mixed. 71 μl of sterile Hepes-buffered saline solution (280 mM NaCl, 1.5 mM Na2HPO4.7H2O, 50 mM Hepes, titrated exactly to pH 7.05) was added dropwise with gentle mixing in between and incubated for 2 minutes to allow formation of the DNA-calcium precipitate. The precipitate was then added dropwise to the cells spreading the whole surface of the cell culture. The volumes are given for 10 cm2 of cell culture and were adjusted accordingly for smaller surface areas. For live cell microscopy, cells were seeded on 15 mm round glass coverslips before transfection. In comparison to Lipofectamine-Plus™, transfections with calcium-DNA precipitates often resulted in more moderate, physiological expression levels.
Fluorescence microscopy, fluorescence recovery after photobleaching and fluorescence loss in photobleaching
Immunofluorescence staining of endogenous NIK was done with paraformaldehyde-fixed cells that were permeabilized with 0.5% Triton X-100 in PBS using anti-NIK antibodies (clone H-248 from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and counterstaining with DAPI to stain DNA.
Conventional epifluorescence microscopy with a Nikon Diaphot inverted microscope equipped with a CCD camera and digital image analysis was as described previously (Birbach et al., 2002). Confocal laser scanning microscopy was performed with Zeiss LSM510 equipment. CFP and YFP images were captured sequentially in the multitrack mode using the 458 nm and the 514 nm line of the Argon laser for CFP and YFP, respectively, and a 458/514 nm beamsplitter. CFP was detected with a bandpass 475-525 nm filter and YFP with a longpass 530 nm filter.
FRAP analysis was carried out with living cells grown on round coverslips. Cells were transfected with various YFP fusion proteins and mounted on an incubation chamber filled with medium 1 day after transfection. Cells were examined with a 100× oil immersion objective on a Zeiss LSM510 confocal microscope and circular regions of interest were specified for bleaching and scanning (bleach regions). Time series were taken with one scan before bleaching, about 70 iterations of bleaching with 100% laser power of the 514 nm line followed by 50-100 scans of the bleach region (with about 4% of laser power). To obtain a good time resolution just the bleach region was scanned instead of the whole cell and an averaging of two or four scans was applied to reduce the electronic noise and improve the quantification. An image of the whole cell was taken before and after the FRAP time series in order to calculate the loss in total fluorescence of the compartment investigated (which was in the range of 5-10% for cytosolic or nuclear proteins, but sometimes higher than 50% for nucleolar proteins). This overall loss in fluorescence was taken into consideration when calculating the actual fluorescence recovery and correct plateau values defining the fraction of mobile versus immobile molecules (Reits and Neefjes, 2001; White and Stelzer, 1999). Mean fluorescence intensities of the bleach region over time were measured using ScionImage™ 4.0.2 (the Microsoft Windows equivalent to NIH Image) and the values were exported to Microsoft Excel™ to calculate the FRAP curves (with correction for total fluorescence loss). These corrected values were copied to GraphPad Prism™ 3.02 for non-linear regression analysis using the single exponential association algorithm: y=spa×(1–e–kx)+bottom – with `bottom' being the relative fluorescence intensity immediately after the bleaching (before recovery), `span' representing the extent of fluorescence recovery and k defining the rate of recovery. Data were weighted by 1/y2 to minimize the relative distances squared (which improves the curve fitting for the initial phase of the fluorescence recovery). The half-life of fluorescence recovery t1/2 is defined by 0.69/k and the plateau, which represents the percentage of mobile molecules is given by span + bottom. For a valid comparison of different FRAP experiments, the calculated half-lives of fluorescence recovery were normalized to the bleached area.
FLIP (fluorescence loss in photobleaching) was applied to determine whether YFP-fusion proteins of NIK variants distribute dynamically between two different compartments, such as nucleus and cytosol or nucleoli and nucleoplasm. For this purpose, a rather large bleaching area was specified for one compartment (e.g. the cytosol) – an image was taken of the whole cell before the bleaching, followed by bleaching with full laser power at 514 nm for about 30 seconds and capturing of another image. This sequence was repeated for about 10 minutes. A loss in nuclear fluorescence upon bleaching of a region in the cytosol indicates export of fluorescent proteins from the nucleus or shuttling between nucleus and cytosol. Similarly, a loss in nucleolar fluorescence upon bleaching of a nucleoplasmic region indicates a dynamic distribution between nucleoli and nucleoplasm.
Reporter gene assay
293 cells were transfected in 12-well plates by the calcium DNA precipitation method as described above. Different variants of NIK (0.5 μg each) were co-transfected with a luciferase reporter construct containing 5 NF-κB binding sites (0.5 μg) and an NF-κB independent β-galactosidase construct (0.5 μg) under the control of a constitutive ubiquitin-promoter for normalization. Cells were lysed with the reporter lysis buffer of a Luciferase Assay System™ (Promega) and luciferase activity was measured according to the manufacturer's protocol using a Berthold single tube luminometer.
β-Galactosidase activity for normalization was measured with chlorophenol red-β-D-galactopyranoside as substrate and photometric detection at 570 nm using a microtiter plate reader.
Nuclear localization of NIK and accumulation in nucleoli
In a previous report we demonstrated that NF-κB inducing kinase, NIK, and one of its major substrates, IκB kinase 1 (IKK1), continuously shuttle between the cytosol and the nucleus. This was demonstrated with YFP-tagged fusion proteins which acted very similarly or identically to the wild-type proteins in NF-κB reporter gene assays indicating functional integrity of YFP-NIK (Birbach et al., 2002).
While NIK is shuttling between cytosol and nucleus, its steady state localization is in many cell types in the cytosol. However, investigating certain breast cancer cells we found that endogenous NIK localizes primarily to the nucleus emphasizing that nuclear NIK might have important functional roles (Fig. 1A).
To further investigate nuclear NIK and the regulation and function of the nucleocytoplasmic shuttling, we aimed to characterize the nuclear localization sequence and the role of nuclear NIK in NF-κB activation in more detail. Interestingly, blockade of nuclear export with leptomycin B promoted not only nuclear localization of YFP-NIK, but also an accumulation in large subcompartments within the nucleus. This was also observed for truncation mutants lacking the C-terminal nuclear export signal that we identified previously (Birbach et al., 2002) (Fig. 1B). These subnuclear structures were thought to be nucleoli, from their appearance in light microscopy, size and number. To confirm this assumption, we carried out cotransfections of YFP-NIK plasmids with a CFP-L23 construct, because L23 is a ribosomal protein that localizes to nucleoli upon overexpression (Gleizes et al., 2001). Fluorescence microscopy using distinct filter sets for CFP and YFP showed clear colocalization of YFP-NIK truncations and CFP-L23 (Fig. 1C). Thus, YFP-NIK lacking its nuclear export sequence or accumulating in the nucleus after blockade of nuclear export by leptomycin B is concentrated in nucleoli, with another fraction being present in the nucleoplasm. Interestingly, nucleolar localization is sometimes also observed for endogenous NIK in the breast cancer cells that exhibited a predominant nuclear localization (Fig. 1D).
The putative N-terminal NLS contains a nucleolar retention signal
We had previously reported the identification of a classical nuclear localization signal in NIK based on sequence analysis (Birbach et al., 2002). To demonstrate the functionality of the NLS motif, we introduced point mutations into this stretch of basic amino acids. First, we mutated the initial four basic residues, which most closely resembled the classical nuclear localization sequence of SV40 large T antigen to neutral alanines. Unexpectedly, microscopy of YFP-fusion proteins of NIK 1-794 truncations carrying this mutation (denoted 143-6A), still showed the protein to be almost exclusively present in the nucleus. However, staining was restricted to the nucleoplasm, with nucleoli devoid of fluorescence (Fig. 2A). This shows that the basic stretch between amino acids 143 and 146 of NIK is essential for nucleolar localization of the protein.
We then wanted to know whether the identified motif of seven basic amino acids in NIK is sufficient to target any protein to nuclei and nucleoli. We therefore fused the sequence R-K-K-R-K-K-K to GFP, either at the N terminus or the C terminus. As shown in Fig. 2B, both of these fusion proteins accumulated in nucleoli, with the C-terminal fusion being somewhat more effective. GFP molecules containing the basic stretch fused to the N terminus, although also localizing to nucleoli, had a slightly higher fluorescence in the cytosol than in the nucleoplasm, indicating that the N-terminal motif is a weaker signal for the nuclear import machinery. The nucleolar accumulation of these GFP-mutants is in striking contrast to GFP alone, which is always diffusely spread throughout the cell (data not shown). Thus, the nuclear/nucleolar retention signal that we have identified in NIK is sufficient to target a protein without an inherent localization sequence such as GFP to nucleoli.
The leucine-rich C-terminal NES at position 795-805 counteracts nuclear localization by the N-terminal NLS
We have previously demonstrated the presence of a leucine-rich nuclear export signal between amino acids 795 and 805 in NIK using truncation mutants of the kinase (Birbach et al., 2002). Another putative NES is present between amino acids 770 and 779 and may theoretically further contribute to the nuclear export of the kinase. Truncation mutants of NIK containing the first putative NES, but not the second localize predominantly to the nucleus indicating that this sequence is not strong enough to counteract the NLS and is not able to shift the steady state localization to the cytosol. However, a subtle contribution to nuclear export cannot be ruled out from these data. To test whether the first putative NES contributes to nuclear export, we applied a confocal microscopy based method, in which one compartment of the cell (the cytosol) is repetitively bleached, and the overall fluorescence is monitored between the bleaching cycles. A loss in fluorescence in another, non-bleached compartment (e.g. the nucleus) means a dynamic shuttling of fluorescent molecules between the bleached and the non-bleached regions. We applied this method (fluorescence loss in photobleaching, FLIP) to YFP-NIK(1-794), which contains the first potential NES, and to a truncation mutant YFP-NIK(1-769), which does not contain this putative NES. Both mutants localize predominantly to the nucleus with little fluorescence in the cytoplasm. FLIP experiments with bleaching of the pool of cytoplasmic fluorophores showed that nuclear fluorescence intensity remained constant over several minutes for both truncation mutants while cytosolic fluorescence decreased steadily (Fig. 3). This illustrates that these fusion proteins do not shuttle out of the nucleus, as nuclear fluorescence would constantly decrease in case of a dynamic equilibrium with the cytosol. Thus, the putative first NES that was predicted by a computer search for positions 770-779 present in YFP-NIK(1-794) is not active and does not contribute to nuclear export of the protein. To also verify the NES at position 795-805 for full length NIK, we created point mutation constructs of NIK in which leucines and isoleucines in the NES were replaced by alanines. Mutation of leucines 803 and 805 (L803A_L805A) shifted a significant amount of YFP-NIK fluorescence to the nucleus, while additional alteration of isoleucine 800 (I800A_L803A_L805A) did not have a significant supplementary effect. Mutation of all leucines and isoleucines between positions 795 and 805 (L795A_L798A_I800A_L803A_L805A, also called NESmut) resulted in most of the YFP-NIK present localizing within the nucleus (Fig. 4A). Cotransfection studies with CFP-L23 confirmed that most of the fusion proteins carrying the NES mutation accumulated in nucleoli (Fig. 4B). Consequently, additional mutation of the nucleolar retention signal (amino acids 143-146), as described above, resulted in staining of the nucleoplasm with nucleoli being omitted (Fig. 4C). In order to verify that the remaining three basic amino acids at position 147-149 promote nuclear localization, we went on to exchange all seven consecutive basic amino acids between positions 143 and 149 with neutral alanines. Introduction of these mutations into a nuclear YFP-NIK chimera with mutated NES resulted in cytoplasmic localization of the fusion protein (Fig. 4D). This shows that the NLS of NIK is located at position 147-149 and is directly attached to the nucleolar retention signal at position 143-146.
Taken together, these data confirm the identity of the C-terminal NES and the N-terminal NLS of NIK and demonstrate the distribution of the protein between the cytoplasmic, nucleoplasmic and nucleolar compartment.
Dynamic redistribution of NIK between nucleoli and nucleoplasm
Based on our observation of the nucleolar accumulation of NIK, we wanted to investigate whether this nucleolar pool is in a dynamic equilibrium with NIK in the nucleoplasm. To answer this question we performed FLIP experiments similar to those described above. In this case we bleached inside the nucleus in regions not covered by nucleoli. As shown in Fig. 5, FLIP analysis revealed a decrease in the fluorescence of both the nucleoplasm and the nucleolus. The decline of YFP signal in the nucleolar compartment showed some delay to the fluorescence loss in the nucleoplasm and exhibited a longer half-life as compared to the nucleus. For NIK(1-769) the half-life of fluorescence loss in the nucleus was about 2.9 minutes with the half-life of nucleolar NIK fluorescence in the range of 6.9 minutes. For NIK(1-794) the data were very similar with nuclear and nucleolar half-lives of 2.1 and 6.1 minutes, respectively. These data indicate a rapid exchange of protein between nucleolar and nucleoplasmic compartments.
Dynamics and mobility of NIK in cytosol, nucleus and nucleoli
To gain further insight into diffusion kinetics and mobility of NIK in the different cellular compartments, we performed studies on fluorescence recovery after photobleaching (FRAP) for cytosolic, nuclear or nucleolar variants of YFP-NIK. This kind of assay was done with living cells grown on coverslips and mounted on a chamber filled with medium. The cells were imaged by confocal laser scanning microscopy usually at about 4% laser intensity with the 514 nm line of the Argon laser. Bleaching and scanning of a region in the compartment of interest was done as described in the Materials and Methods. Fluorescence recovery in the bleaching region resulting from diffusion of molecules from outside was monitored and quantified with appropriate control and compensation algorithms that account for the total loss in fluorescence in the course of bleaching and scanning. Half-life values of fluorescence recovery were normalized to the area of the bleached circle in order to compare the values for the different compartments. Interestingly, these analyses indicated that cytosolic NIK exhibits the lowest proportion of mobile molecules (54.1±2.4%) as compared to nuclear NIK with (82.4±7.2%) and nucleolar NIK with (80.7±13.1%) (Fig. 6A). These data indicate that cytosolic NIK binds to some immobile elements such as cytoskeletal structures in the cytosol, whereas a significantly higher percentage is mobile in nucleoplasm and nucleoli. The halftime of fluorescence recovery was in the range of 1.6 seconds for cytosolic NIK, while nuclear and nucleolar NIK had half times of recovery in the range of 0.8-0.9 seconds. Normalizing the halftimes of recovery with the area bleached indicated that NIK is most mobile in the nucleoplasm (with t1/2/μm2 of 0.129±0.046 seconds), while in the cytosol the normalized halftime of recovery was 0.424±0.094 seconds. Nucleolar NIK seemed to be present in two different states with respect to mobility, with one population exhibiting a normalized halftime of 1.14±0.15 seconds and another fraction with a value of 6.75±0.5 seconds, indicating association with elements of low mobility. We aimed to compare the mobility of NIK in the nucleolus with other GFP-tagged molecules localizing to nucleoli. Therefore, we performed FRAP experiments with GFP containing the 7 amino acid nuclear/nucleolar localization sequence of NIK (RKKRKKK-GFP) – as a rather inert marker localizing to nucleoli, and we also did FRAP experiments with the GFP-chimera of the ribosomal protein L23, a molecule that accumulates in nucleoli in the course of ribosome biogenesis. The nucleolar GFP version showed very fast diffusion kinetics with a half time of about 0.3 seconds and a mobile fraction of nearly 100% (Fig. 6B). However, normalization of the halftime with the bleached area revealed a value of 0.818±0.174 seconds/μm2, which is significantly slower than wild-type GFP in the cytosol, indicating that the basic residues mediate an association with molecules of low mobility. Nucleolar GFP-L23 showed slower diffusion with a halftime of fluorescence recovery in the range of 2.4 seconds and an area-normalized halftime of 2.37±0.27 seconds/μm2.
Localization of NIK in nucleoli results in a reduced NF-κB activation potential
An important question is whether the different localizations of NIK – as a molecule that shuttles between cytosol, nucleoplasm and nucleoli, is relevant for its function in the NF-κB signaling pathway. We addressed this question by using reporter gene assays with a NF-κB-dependent luciferase reporter that was co-transfected with NIK variants that exhibit different steady state localizations. The highest NF-κB activation potential was observed for the NIK variant that had both the nuclear localization sequence and the functional nuclear export sequence mutated – therefore a variant of NIK that is localized primarily in the cytosol without being able to shuttle efficiently between cytosol and nucleus (Fig. 7). Wild-type NIK had an NF-κB activation potential, which was similar to the variant of NIK that localized predominantly to the nucleus but not to nucleoli (the form of NIK in which both the NES and the nucleolar targeting sequence were mutated). Strikingly, this nuclear/non-nucleolar form of NIK had a much higher tendency to activate NF-κB than the nuclear form of NIK, which also shows nucleolar accumulation. This form was actually the variant of NIK with the least NF-κB induction potential amongst the different NIK constructs tested. It is worth noting that interaction domains of NIK with its upstream activator, Traf2, and its downstream effector, IKK1, both lie in the C-terminal domain of the protein (Malinin et al., 1997; Lin et al., 1998). Thus, differences in the activation potential between nucleolar and non-nucleolar variants of NIK described here are not due to a change in binding affinity to these partners. These data indicate that nucleolar NIK is sequestered from the other components of the NF-κB signaling pathway and suggest an inhibitory role for the nucleolar localization of NIK with respect to NF-κB activation.
We have investigated the dynamic subcellular localization of the NF-κB-inducing kinase NIK, which shuttles constitutively between cytoplasm, nucleoplasm and nucleoli. Nucleolar retention depends on a stretch of four basic amino acids (RKKR) in the N-terminal half of the protein, which is directly linked to a nuclear targeting sequence (KKK) (Fig. 8). The nucleolus is a structure assembled around clusters of repeated ribosomal genes (rDNA) coding for ribosomal RNA. In addition to nucleic acids, the nucleolus also contains a large number of proteins, which have recently been characterized in HeLa cells by a proteomic approach (Andersen et al., 2002). Consequently, a protein found in the nucleolus can be targeted there by contact with either DNA, RNA or protein. One may assume that a stretch of basic residues offers the opportunity for an interaction with phosphate groups in nucleic acids. In fact, the nucleolar retention signal of human T cell leukemia virus type I protein p27x-III and the HIV-Tat protein are also highly positively charged (Siomi et al., 1990; Siomi et al., 1988) and it was shown that arginine-rich basic domains bind to RNA in nucleoli (Calnan et al., 1991; Gustafson et al., 1998). Thus one possibility for nucleolar accumulation of NIK is an interaction with structural motifs in rRNA. Interestingly, our FRAP data of normalized halftimes indicated two different states of NIK in the nucleolus, with one having a mobility similar to the RKKRKKK-tagged GFP, and another one being significantly less mobile. Given the known characteristics of basic amino acid stretches to bind to RNA, it appears plausible that nucleolar GFP, as well as the more mobile fraction of nucleolar NIK bind to rRNA, while the less mobile fraction of nucleolar NIK additionally binds to other nucleolar structures.
It is an interesting phenomenon that a significant number of signaling molecules including p65 NF-κB, IκBα, Smad proteins and many others contain both nuclear localization and nuclear export sequences that counteract each other (Arenzana-Seisdedos et al., 1997; Gama-Carvalho and Carmo-Fonseca, 2001; Harhaj and Sun, 1999; Huang et al., 2000; Johnson et al., 1999; Reguly and Wrana, 2003). Signaling molecules such as transcription factors, which have roles both in the nucleus and in the cytosol, clearly require nuclear import and nuclear export mechanisms. However, more and more proteins without an obvious nuclear function are being found to shuttle between cytosol and nucleus (Gama-Carvalho and Carmo-Fonseca, 2001). Most of these proteins contain both NLS and NES domains, raising the question of how these opposing localization mechanisms are balanced and dynamically regulated. Several possibilities appear significant for regulating the equilibrium between counteracting localization signals. First, one signal can be structurally exposed and more easily accessible to the transport machinery, e.g. the NES in NIK in contrast to its NLS. Second, modifications such as phosphorylations can change this balance. Such a mechanism seems likely for IKK1, the signaling kinase downstream of NIK, which shuttles between cytosol and nucleus, as it was shown recently that its steady state localization shifts to the nucleus after addition of TNFα, i.e. after phosphorylation of its activation loop (Anest et al., 2003). The NLS of IKK1 has not yet been identified, although our own data (Birbach et al., 2002) suggest that the lysine residue at position 44, which is at the active site of the enzyme, appears to be part of a non-classical NLS. Assuming that a phosphorylation at the activation loop would expose the active site of the enzyme, it seems plausible that activation via TNFα would then also expose a domain that supports nuclear import and shifts the steady state towards the nucleus. In principle, a protein modification such as phosphorylation can facilitate nuclear import, but might also have the opposite effect. Computer analysis (Blom et al., 1999) of the human NIK sequence indicates that serines in close proximity to the nuclear/nucleolar retention signals at position 143-149 (including serines 150, 151) are likely to be phosphorylation sites. In this case negative charges introduced by phosphorylation events might interfere with nuclear targeting or nucleolar sequestration mediated by basic amino acids. Thus phosphorylation events are potential mechanisms controlling subcellular or subnuclear localizations of NIK by other kinases. It is important to note that a predominant localization in a specific compartment at steady state does not mean that the protein is acting exclusively there. Since there is a dynamic distribution between the different compartments, those molecules that are at a different location than the predominant one might still have important biological functions. Thus various cellular signaling events might not only alter the steady state distribution of molecules, but also affect transport rates into or out of specific compartments. In this work, we have examined kinetic properties of protein exchange between compartments by means of photobleaching techniques using GFP chimeras. Similar approaches have been undertaken by other groups (Cole et al., 1996; Kimura and Cook, 2001; Phair and Misteli, 2000), underlining the usefulness and applicability of this method. Our measurements on fluorescence loss in nucleoli upon photobleaching of the nucleoplasm imply a rather fast and efficient distribution of NIK between these two compartments. This is in line with other reports demonstrating rapid and significant cycling of various proteins involved in rRNA transcription and processing between nucleoli and the nucleoplasm (Chen and Huang, 2001). Besides the kinetics of distribution between distinct compartments, the mobility and diffusion dynamics of signaling molecules within a given compartment is an important parameter influencing their biological activity. In that respect, the high mobility of NIK in the nucleoplasm and the high fraction of mobile molecules in nucleoli are quite remarkable. The lowest percentage of mobile molecules as judged from the plateau of the fluorescence recovery was observed in the cytosol, which might indicate that NIK has a tendency to bind to cytoskeletal elements probably via bridging molecules, or it might be part of signal tranduction scaffolding complexes. Similar associations with the cytoskeleton were also reported for other signaling kinases, such as MEKK1 (Christerson et al., 1999).
Obviously, an important question that remains to be clarified is what is the function of NIK in the nucleus and moreover in the nucleolus? Apparently a central role of nuclear NIK is to activate IKK1, which is also shuttling between cytosol and nucleus (Birbach et al., 2002) – and which seems to be important in activating non-classical NF-κB pathways (NIK-IKK1–IκBα–NF-κB or NIK–IKK1–NF-κB2). It is striking that these pathways seem to be crucial in B-cells in which the nucleus constitutes the major part of the cell body. In this case, localization or sequestration of NIK in nucleoli might have important regulatory roles for NF-κB signaling and B-cell biology. Our findings that endogenous NIK is primarily nuclear in certain breast cancer cells where it also sometimes accumulates in nucleoli might suggest a special role of nuclear NIK for an elevated constitutive NF-κB activity in these cells, which might causally contribute to the malignancy. Another potential role for nuclear NIK might be related to the recently identified function of IKK1 in histone phosphorylation. It was shown that nuclear IKK1 can upregulate NF-κB target genes also by binding to promoter elements, direct phosphorylation of histone H3 and subsequent facilitation of the transcriptional process (Anest et al., 2003; Yamamoto et al., 2003). Very recently, another nuclear function was defined for NIK and IKK1, that is direct activation of p65 NF-κB via phosphorylation of serine-536 on the transactivation domain (Jiang et al., 2003). All these nuclear functions of IKK1 and NIK might be regulated at least in part by the distribution of NIK between nucleoli and nucleoplasm. A well-known example of a signaling pathway regulated by nucleolar sequestration is that of the p53 tumor-suppressor gene product, as its main regulator, Mdm2, is controlled by nucleolar retention (Lohrum et al., 2000; Weber et al., 1999). Similarly, our data indicate that nucleolar sequestration might influence the NF-κB-inducing activity of NIK. In reporter gene assays, we found that nuclear NIK variants can upregulate NF-κB-dependent gene expression and that this ability is negatively regulated by nucleolar localization, as mutation of the nucleolar targeting sequence enhanced the NF-κB activation potential. This is in line with a report showing that deletion of the N-terminal part of NIK, containing the basic amino acids that we identified as a nuclear/nucleolar localization signal, augments NF-κB activation, which led to the concept of a `negative-regulatory domain' in the N-terminal region of NIK (Xiao and Sun, 2000). However, spatial sequestration alone is probably not sufficient to explain the reduction of NF-κB activation by nucleolar localization, because nucleolar NIK shuttled rather efficiently to the nucleoplasm. Thus additional mechanisms such as interactions with other signaling molecules or phosphorylation events might be effective in regulating the activity of nuclear NIK by increasing or decreasing the fraction of NIK present in nucleoli.
In conclusion, we consider nuclear NIK crucial for various nuclear functions of IKK1 in the course of cytokine-induced gene expression and NF-κB activation in a cell type-specific manner and we suggest that nucleolar localization of NIK might be a mechanism for regulating these activities. Certainly further studies will be necessary to elucidate the different roles of NIK in cytosol, nucleus and nucleoli and the mechanisms that balance the functions in different compartments.
We gratefully acknowledge the assistance of Peter Gold, Eva Hörandner and Hannah Neumeier. We are also thankful for support from the Max Kade Foundation and Yale University in the final stage of the project, providing funding and equipment during a sabbatical by J.S. with Sankar Ghosh's group at the Immunobiology Department of Yale University Medical School. Finally, we thank Nabel Yaseen and Günther Blobel for providing the GFP-L23 construct.
- Accepted March 15, 2004.
- © The Company of Biologists Limited 2004


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Buddhism has long since held sway in Sri Lanka. It is one of the few places in the world where Theravada Buddhism is practiced. The 'Sangha' or clergy has played a prominent role in the history of my country, be it by advising my countrymen in choosing wise/ morally responsible Kings, opposing invaders (there are instances where they left robes to fight wars) or educating the people on the path to enlightenment; the most prominent role of them all.
For the purposes of reading this article at least, it is important to understand Buddhism, and the mentality of a majority of Sri Lankan Buddhists. The Sangha is one of the three main pillars of Buddhism, and is supposed to guide us on our spiritual path to enlightenment (nirvana), while working on reaching enlightenment themselves. Enlightenment is the ultimate achievement for Buddhists, since after reaching it we will have no rebirth, and hence, no more suffering.
As our spiritual guides, the Sangha hold immense respect among lay Buddhists (Buddhists who are not clergy). In the past, while fulfilling the duties of the clergy, they were also the educators of the village society and unofficial advisers to the headmen of the village (the Arachchi). Since they were among the most educated, they were also extremely respected and their recommendations were usually followed. However, make no mistake; they were respected because of their wisdom and education but at no point would they 'rule' land/ people. The Sangha depends (or should depend) on the lay Buddhists to provide them with food and provisions. True Buddhist clergy are supposed to go door to door to ask for food in exchange for sermons 'Pidu singha vadeema'. This would serve a double purpose of reducing pride/hubris in the monk while educating people the path to Enlightenment. The monk uses the time saved by not having to create his/ her own food to engage in meditation and reach Enlightenment. This is supposed to be the balanced relationship between Buddhist practitioners and monks.
Some of the Sangha today however, are a far cry from what the Buddha intended them to be. Whereas the purpose of donning the saffron robe is to abandon all material things and to remove all attachments to concentrate on meditation, many of the clergy today seem to have conveniently forgotten all of this. They own car dealerships, lucrative businesses and are involved in politics. It is safe to say that spiritual enlightenment is the farthest from most of their thoughts.
The newest scourge is an activist group called 'Bodu Bala Sena' (the army of Buddhism). Apart from just being racist and intolerant of religions, they are led by some militant Buddhist monks. Now, anyone who knows even the slightest bit of Buddhism will know that the two previous statements are a direct contradiction to everything Buddhism teaches. The Buddha never counseled military means to achieve any goal; Buddhism preaches non-violence and tolerance of others, no matter what. The very name 'Bodu Bala Sena' contradicts itself since Buddhism has never ever raised an army in its name or gone to war to spread its beliefs. With the same reasoning, militant Buddhist monks are a contradiction that cannot exist. The clergy are supposed to be disciplined with word and action, and there are even laws for monks that forbid even violent limb movement! This discipline is sadly lacking with some clergy today.
Recently, the leader – I refuse to recognize him as a monk – of the Bodu Bala Sena, was arrested on charges of contempt of court. He surrendered, but it was interesting watching the proceeding arrest. A throng of people had gathered to see this happening, monks as well as lay supporters. The mood was tense, and the leader actually made a small speech while in custody trying to calm them down saying that the matter was now in the courts. However, mob mentality had now taken over, and they were yelling and screaming at the leader himself. The police eventually took him into court, where he was remanded without bail and, like every other Sri Lankan politician, promptly fell sick and was transferred to the hospital.
The scene outside the court after the judgement was made public brings disrepute to all Buddhists, monks in particular. A cordon of policemen stood outside the gate of the court with riot shields. A group of monks were shoving these policemen around, yelling and screaming 'api yanna nevei awe' (we didn't come here to leave) trying to get into the court. The funny thing is, as they shove and get violent with these policemen, they are also screaming 'hamuduruwanta atha thiyanne na' (don't you dare lay a hand on priests). These were simply thugs who were using the respect shown toward priests to their advantage. There were other scenes where the priests were jumping on a bus, and laying their heads next to the tires of the bus, daring the drivers to run them over. Our spiritual guides indeed.
To dissect these actions one by one, first of all, pushing shoving screaming are all against the discipline that Buddhism asks monks to practice (as well as lay practitioners actually). One of the main teachings and first steps towards the path of enlightenment is 'Seela' (discipline). Clergy are supposed to set an example, as well as maintaining a higher amount of discipline. Secondly, you cannot behave like a thug and demand the level of respect a true Buddhist monk deserves. A thug does not deserve that respect; and these monks were acting exactly like that. The police in this case, showed an admirable amount of restraint in not retaliating and by just defending themselves using a minimum amount of force. Thirdly, our lives are supposed to be precious since we can use it to achieve enlightenment for the benefit of ourselves and others. If the monks were trying to show how brave they are, they failed miserably; they knew no one was going to run them over. Everyone present, and everyone in Sri Lanka knew that. Even misbehaving monks are shown respect; in this case, more respect than they deserved.
At the risk of repeating myself, true Buddhism does not condone violence of any kind. Racism, anger is abhorrent to it, only patience and tolerance should be practiced. Some of these monks may think that they are protecting Buddhism or the Sangha by the comments they make and their actions (although I have no idea how they can have made that leap), but in reality they are just dragging it into disrepute. The majority of Buddhists will say that these men do not represent Buddhism and the public actions of a few misbehaving monks should not be a reflection of what true Buddhism is.
These monks should not be tolerated, or given the respect usually given to clergymen. They should be disrobed and exiled from the priesthood. That is why the introduction of the new Theravada Bikkhu Bill, enabling the leaders of the priesthood in Sri Lanka (the Mahanayakas) to remove monks engaging in disreputable practices, has been long awaited. Obviously, it is also opposed by the clergymen with lucrative practices to lose, but if they are so invested in their businesses, what are they doing as clergymen? The very reason to become a monk is to remove attachment and focus on spiritual development on the path to enlightenment.
Buddhist lay practitioners have an important role to play in this. It's been long said with regard to misbehaving monks that we respect/ tolerate them because 'api siurata wadinne' (we respect/ worship the robe), not necessarily the person wearing it. This mentality should be changed. We should respect the robe only if the person wearing it is worthy of our respect. If they are engaged in practices unworthy of a monk, they should be disrobed and banned from the priesthood. They are free to leave as well; Buddhism does not force them to be monks! The number of monks may be diminished in numbers because of this action, but it will be pure, and will be deserving of the respect accorded to them. The Sangha was created to help relieve mankind from suffering, it is time that it focused on that.

subset: commoncrawl


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|Posted: April 14, 2009
SAFENANO team completes global review of nanomaterial EHS research
|(Nanowerk News) EMERGNANO the first global review of active research into the environment, health and safety risks of nanotechnology has been published today by Defra, the UK Government Department for Food & Rural Affairs.
|The review, led by the SAFENANO initiative at the Institute of Occupational Medicine in Edinburgh, UK provides a unique identification and analysis of research carried out worldwide on nanotechnology safety, including that relating to hazard, exposure, risk assessment & regulation.
|EMERGNANO identified more than 670 projects from around the world, and after careful selection assessed more than 260 unique, relevant projects completed, close to completion or in progress since 2004. The final report provides a comprehensive listing of projects, alongside detailed evaluation of their outputs. It also provides an assessment of the extent to which these projects contribute towards meeting the 18 Research Objectives (ROs) for nanotechnology risks laid out by the UK Nanotechnology Research Coordination Group (NRCG), and highlights the gaps still remaining.
|SAFENANO Director Rob Aitken, who led the team appointed to produce EMERGNANO, said "EMERGNANO was a huge undertaking carried out in a very short timescale. It provides a snapshot of worldwide research in this area, both completed and in progress, together with a unique evaluation of the contribution that this research will make to the research objectives identified by Defra In terms of numbers of studies identified, we found a widespread imbalance in the work being carried out across the main thematic areas within the eighteen research objectives, Aitken continued There are also large regional variations in the type of study funded in different countries or economic areas, perhaps reflecting different national priorities or capabilities. However, EMERGNANO represents the best available picture of current strategic research.
|EMERGNANOs assessment reveals some important progress made across the four main thematic areas of characterisation, exposure, toxicology and ecotoxicology spanned by the 18 ROs. This includes:
|Completing such a wide-spanning appraisal of studies was not however without its difficulties. A disappointing aspect was that we were unable to identify significant output from many of the studies identified, including some which had already been completed Aitken commented.
|In addition to mapping those projects identified against the 18 NRGC ROs, EMERGNANO provided a prospective appraisal on new data which may trigger further consideration of the need for regulation of nanomaterials; an assessment of the possibility of a qualitative risk assessment; and consideration of whether there is sufficient information to invoke the precautionary principle for one or more nanomaterials.
|However the study found a lack of relevant data on which such judgements could be made. After assessing study quality and completeness, we did not identify a sufficient body of evidence to make a qualitative risk assessment feasible for any category of nanomaterial Aitken added.
|EMERGNANO was undertaken by a multidisciplinary group of expert scientists, chosen from leading research institutes in the UK and beyond, to ensure full coverage of the scientific disciplines represented within the scope of nanotechnology risk research. Institutes involved include: SAFENANO, Institute of Occupational Medicine, Edinburgh Napier University, Edinburgh University, Central Science Laboratories, Cranfield University, University of Leeds & the Woodrow Wilson International Center for Scholars. Recommendations made within the report were also supported by a workshop and dialogue held to reach a consensus view about the remaining gaps and future priorities.
|Whilst EMERGNANO highlights some of the key progress made to date within nanotechnology risk research, it is clear nonetheless that major gaps in the knowledge base still remain. While incremental progress is being made in some areas, the programme of research activity has yet to deliver step changes in the knowledge base on these issues. Overall EMERGNANO highlights the need for a more strategic co-ordinated research agenda to be developed, funded and implemented Aitken concluded.
|Source: SAFENANO
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FREDERICKSBURG, VA and BARCELONA, Spain, -- QRC Technologies, a systems integrator specializing in mobile cellular measurement products, announced at Mobile World Congress 2013 the launch of its Wide Band Transcorder (WBT), a self-contained radio frequency (RF) recording and playback system that helps commercial enterprises and government organizations more quickly and cost-effectively address various RF problems.
Exploding consumer demand for smartphones, tablets, TVs, cars and other wirelessly connected devices is putting increasing pressure on the radio spectrum available for consumer use. Broadcasters, broadband providers, commercial entities and governments are challenged with operating within finite RF spectrum, while tackling major threats such as growing interference. At the same time, limited resources are forcing organizations to tackle spectrum-related challenges more cost effectively.
"Fixing RF issues and challenges such as interference or equipment malfunction is generally easy; it's reproducing the issue on-demand that can be difficult, time-consuming and costly," said Tom Callahan, General Manager and CTO, QRC Technologies. "Other RF recording devices on the market today are designed for lab environments and can be expensive, cumbersome and difficult to operate. The affordable and portable WBT requires virtually no training and takes the guesswork out of capturing RF issues and environments, so organizations can better focus on business and customer needs."
The WBT is ideal for a wide range of applications and industries including reference signal recording and playback, signal analysis, electronic warfare, market penetration, interference analysis, training, receiver test and design, security, competitive analysis, surveillance, spectrum allocation, repeater, IED profiling, RADAR testing and other capabilities. Sample use cases can be found at WBT.QrcTech.com and include, but are not limited to, industries such as:
- Telecommunications—Pinpoint the root cause of dropped calls and enhance the user experience.
- Capital Equipment Optimization—Record complex waveforms or special test cases from other equipment and/or simulation and signal processing tools and replay them at an unlimited number of locations.
- Government—Identify commercial and military radar interference to keep troops safe and to simulate real world situations in a controlled test environment.
The WBT allows users to record and analyze in real-time 2x25 MHz Blocks of RF spectrum from 50 MHz to 4.4 GHz and play it back into other devices for repeated analysis. WBT contains an embedded GPS disciplined oscillator (GPSDO), Gigabit Ethernet and eSATAp ports, removable CFAST flash card and two bays that utilize widely commercially available 2.5 in. solid-state disk drives for continuous recording.
Benefits of the WBT include:
- Compact Size and Light Weight—Unlike competitive solutions that require extensive rack space, the WBT is about the size of a laptop bag, weighs fewer than 10 lb. and consumes fewer than 70 W, making it portable for staff on the go. An optional light weight power pack provides more than two hours of fully untethered use.
- Intuitive Interface—The touch screen features a one-button record/playback interface that requires minimal training. Virtually any technician can quickly gather data from the field to replay in the lab, cutting down travel time for costly engineering staff.
- Unlimited Recording on Inexpensive Media—The operator can easily insert additional recording media into the two drive bays indefinitely without shutdown.
- Lower Cost of Ownership—Traditional systems often require three or more components, numerous interconnects and training. The WBT is physically stackable and remote controllable by other WBT units, thus lowering the cost of recording signal bandwidths larger than 50 MHz.
A live WBT demonstration can be experienced at Mobile World Congress 2013 at QRC Stand 7B60. Pricing for the WBT begins at $59,995 and is available for order immediately for July shipping.
About QRC Technologies
QRC Technologies (QRC) specializes in RF Test and Measurement products, especially for cellular communications. QRC develops and produces survey tools for cellular communication systems, phone based measurement, stimulation tools, forensic tools, and integrated RF recording and playback equipment.
QRC was founded in 1987 and has been serving as a systems integrator since its inception, and in the last decade has expanded with a portfolio of RF Test and Measurement products. The company has over 50 employees in three offices, one in Gaithersburg, MD, one in Stafford, VA, and the world headquarters located in Fredericksburg, VA.


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| 8.6°C Belfast
The pound continued its slide against the dollar and euro on Friday after a shock opinion poll showed that Labour continues to eat into Theresa May's lead.
Sterling extended losses against the greenback, falling 0.5% to 1.28 US dollars having risen above 1.30 just days earlier.
Against the euro, the pound slumped 0.4% to 1.14 euro.
The Conservatives' advantage over Labour has narrowed to just five points in the first opinion poll taken since Monday night's suicide bomb attack in Manchester.
A YouGov survey put the Conservatives down a point on the previous week on 43% and Labour up three on 38%, with Liberal Democrats up one on 10% and Ukip up one on 4%.
Craig Erlam, senior market analyst at Oanda, said: "Sterling fell again overnight after a poll showed the gap between Theresa May's Conservatives and Jeremy Corbyn's Labour party has narrowed again.
"Coming on the back of losses yesterday, it's turning into a rotten end to the week for the pound. What's more, with it having broken below 1.29 against the dollar overnight, it could be facing further misery in the near-term."
The pound has plummeted since Britain voted to quit the European Union as confidence in the country's economic prowess has evaporated.
Yesterday, official figures showed that the economy suffered an even deeper slowdown at the start of the year as the services sector came under pressure and inflation dealt a blow to household spending.
The Office for National Statistics (ONS) said gross domestic product (GDP) grew by 0.2% in the first quarter of 2017, revising down the figure from its initial estimate of 0.3%.
Mr Erlam added: "The pair (pound and dollar) had been grinding higher for the last month and while it did manage to briefly penetrate 1.30 on a few occasions, the moves were always lacking conviction which suggested markets weren't happy at these levels.
"The break of 1.29 may well be the trigger for a correction in the pair, with 1.28 and 1.2750 being notable levels below."


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Let's Be Less Stupid: An Attempt to Maintain My Mental Faculties
Somebody had fun composing the memoir-slash-cognitive practicum Let's Be Less Stupid: An Attempt to Maintain My Mental Faculties (Riverhead), and her name is Patricia Marx, a former SNL writer now at the New Yorker. Smarten up with Marx's quizzes, brainteasers, anecdotes, and self-help guides while giving your funny bone a witty workout.
Top-shelf contemporary tales of all stripes—from neorealism to sophisticated horror to metaphysical hijinks—by Zadie Smith, Mary Gaitskill, Yiyun Li, Anthony Doerr, and 28 other scribes seduce us, and then some, in the New American Stories anthology (Vintage). Writes editor Ben Marcus, "When I want to be ambushed, captured, thrust into a strange and vivid world...I read short stories"—like this stellar selection.
Naomi Jackson's debut, The Star Side of Bird Hill (Penguin), moves forcefully between Barbados and Brooklyn in a plangent coming-of-age tale about two sisters torn between their absent father's reappearance and their strong, matriarchal island family.
This article originally appeared in the July 2015 issue of ELLE.


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Oslo/Fusion Film Festival was founded in 1990 and is Norway's only film festival dedicated to stories of and by the LGBTQ+ community, and is also one of Norway's longest running film festivals. It is still the largest festival dedicated to promote diversity, equality and education through entertainment, with film screenings, panel discussions and related events.
Film is a powerful medium that can entertain, educate and create change, all at the same time. We look for new perspectives and reflections in stories from our personal or shared lives, and human experiences; films that explore and celebrate diversity in identity, gender, cultural background, and sexuality, or challenge norms and common perceptions in our global society.
The festival venue is at the Norwegian Film Institute's Cinemateket. It has two theaters, both with state-of-the-art projection and the latest Sony Atmos sound system.
Accepted formats for screenings are DCP (2K/4K), 3D, 70mm, 35mm, Betacam, HDcam, and high res files. (Blu-ray or DVD, backup only).
Jury Awards:
- Best Narrative Feature
- Best Documentary Feature
- Best Short Film
Queer Youth Award:
- Best Feature Film (narrative or documentary)
Please read the festival description - a film must in some way relate to our mission in order to be considered.
- Films must be completed after January 1, 2019.
- Please make sure to choose the appropriate category - add comments if necessary.
- In the form, we kindly ask you to say a few words about how your film meets our criteria. This will help us in speeding up the selection process, so please take a few seconds to type in a keyword or two.
- The submission fee is currently USD 20 for short films and USD 40 for feature length films.


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Future Audi cars will call for help if you get sick at the wheel
Audi has used its presence at CES to outline plans to become your personal health manager by connecting your smart devices to its in-car system in order to help you cope better with stressful driving.
Audi says it will combine vital stats from your smart watch, like heart rate and skin temperature, with information that it monitors, such as how you're driving, weather and traffic, then change the car's internal environment to help you stay calm.
That could be things like giving you a seat massage, changing the temperature or adjusting the entertainment.
Audi will even offer in-car stress-management tutorials, including breathing exercises, or suggest taking a break at the nearest rest stop.
Although this is yet to be incorporated into Audi cars, further on the horizon are plans to incorporate biometric data with safety functions, with your car able to perform emergency procedures and even call for help if you're unwell at the wheel.
I bet you never knew your car cared so much.
➤ Audi Fit Driver [via Auto Blog]


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Leider ist diese Seite zur Zeit nur auf Englisch verfügbar.
2 thoughts on "ScanSpeaker"
Hallo Matthias,
i^ve done this crossover and was very happy, but build it with the SS-9700 and and 2x 8545, second with a 6,8mH coil.
Now i have this combination with the 9900 and will try it with your crossover again.
Are or has there been any update. The original Crossover in the box (Audiodata elance) make it sound a little dull to my ears.
Kind regards, Michael


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Gastelum: It's not hype anymore: Te'o deserves the Heisman (Oct. 29)
Andrew Gastleum | Monday, October 29, 2012
NORMAN, Okla. – You know something special when you see it.
What we saw in primetime at Memorial Stadium, that was it.
A special victory at a special time, led by a special player.
For Manti Te'o, this is a special year. So it should be speculation no more: He deserves the Heisman.
This is a Notre Dame team that is 8-0, the only team in the country with four wins over top-25 teams – including two road wins over top-10 teams.
This is a Notre Dame team that was expected to struggle with a first-year quarterback and the toughest schedule in college football.
This is a Notre Dame team with one of the country's top defenses, giving up less than 10 points per game with only one rushing touchdown allowed since last November.
This is Te'o's team.
Most Heisman candidates represent their respective teams.
Yet this Irish team represents Te'o, the best compliment a player can ask for. His incredible work ethic, his levelheadedness and his whole-is-greater-than-the-sum-of-its-parts mentality all shine through this team, from the veteran offensive line returning four starters to the young secondary with three players converted from offense.
This linebacker's influence can be heard, seen and felt on the field, even when he's not on it. The last time college football saw a player like that, Tim Tebow told the world no one would work as hard as he would for the rest of the season.
And on the biggest stage of his legendary career Saturday night, Te'o's legend grew.
When the Notre Dame offense could not sustain a drive of more than two minutes in the first quarter, Te'o recorded eight tackles and gave the offense a chance to find itself without having to play catch up. With one of those first-quarter tackles, Te'o recorded his 400th career tackle, just another testament to one of the greatest collegiate careers in Notre Dame history.
He finished the first half with 11 tackles – two for a loss – and his first sack of the season. That massive second-quarter sack by Te'o sent the up-tempo Oklahoma offense to the sidelines with its first three-and-out, right after it seemed to drive down the field with ease in the first quarter.
And with his sprawling fourth-quarter interception, Te'o silenced any chance of a Landry Jones-led comeback and sealed the victory, putting him tied for second in the entire country with five interceptions.
He hasn't thrown for four passing touchdowns and 300 yards. He hasn't even scored a touchdown at all. And yet he can change the course of an entire game from the side of the ball that prevents points, not scores them.
Since the preseason, Marcus Lattimore, Matt Barkley, E.J. Manuel, Geno Smith and now Kansas State quarterback Collin Klein have all taken their turns atop the presumptive Heisman list.
Since his inspired performance at Michigan State in Week 3, Te'o has remained near the top of that list and improved his status while watching the frontrunner crash out on weekly basis.
While one loss can ruin the Heisman hopes of Klein – as it did to Barkley, Manuel and Smith – a loss for Te'o will not muffle the Heisman noise. He simply means too much to the sport.
And as we've seen with Robert Griffin III, Mark Ingram and Tebow, it's not just about the stat sheet.
"He represents all of the things the Heisman trophy espouses: integrity, character, a great football player," Irish coach Brian Kelly said postgame.
The way he inspired an island, a university and a nation through personal tragedy showed the character of a true champion. The stories of helping a young and dying cancer patient and her family showed the integrity of a true hero. The answer to the following question showed the linebacker's heart.
"Have you been able to comprehend the number of people that you have affected in a positive manner?" I asked Te'o after the game.
His response was swift.
"No, not really," he replied. "For me, I'm just trying to have an impact on as many people as possible. If that's one, if that's 10 or 20 I don't really know. As long as I change somebody's life, I would consider that a success for me."
His words read of honor, humility and sacrifice, but all I read was Heisman.
Contact Andrew Gastelum at [email protected]
The views expressed in this column are those of the author and not necessarily those of The Observer.


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California Musical Theatre is showing Beauty and the Beast now through March 17 as part of its Broadway Series. We have season tickets, which I've mentioned before, and I love getting to see these shows. I've always liked the Beauty and the Beast story - so I'll be interested to see the this performance. Hopefully I can make it up all those stairs without getting too tired (this baby is getting bigger and bigger - just like she's supposed to!)
If you're a fan of Crab Feeds, check out Sacramento Magazine's list of Crab Feeds in the area and find a good cause to help out. There are a few this week - so check it out!
Want to celebrate St. Patty's Day with some wine? Check out the celebration at Lava Cap for some wine and cheese pairing - both Saturday (March 16) and Sunday (March 17.)
Enjoy your week!
Around Town: Week of March 10
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This article is all about Bisphenol A controversy a deadly illusion. Bisphenol A is synthetic chemical used in the manufacture of polycarbonate plastic.
While doing my homework on polycarbonate plastic and plastic recycling, I came across several online discussions about Bisphenol A or BPA. There has been a lot of concern about this chemical in recent years due to the adverse findings of scientists about it and the "controversy" raging on in the scientific community about Bisphenol A's harmful effects on humans.
Bisphenol A, what is it?
Bisphenol A is the synthetic chemical used in the manufacture of polycarbonate plastic and epoxy resin. As polycarbonate plastic or epoxy resin compound, bisphenol A is used in a wide array of applications such as plastic water bottles (Nalgene), food can linings, infant feeding bottles, food and beverage containers, cell phone casing, compact disks, bottle tops, and dental sealants. It's everywhere!
Polycarbonate plastic is labelled with plastic recycle symbol number 7. It must be noted, however, that not all plastic products labelled number 7 are polycarbonate plastic. Please see Recycling Symbol for more information.
Leaching Bisphenol A
People are constantly exposed to or ingest varying levels of bisphenol a from soup cans, infant formula cans, baby feeding bottles, canned food, bottled beverages, etc. Bisphenol A is found to migrate from the polycarbonate plastic container or from the can lining to food, soup, powdered milk, etc. The most rapid leaching of bisphenol A to food has been observed to occur at higher temperatures, like a hot baby milk preparation or heated soup and canned food.
According to the Environmental Working Group, a Washington DC-based independent research organization who spearheaded the study of bisphenol A exposure in the US:
Of all foods tested, chicken soup, infant formula, and ravioli had BPA levels of highest concern. Just one to three servings of foods with these concentrations could expose a woman or child to BPA at levels that caused serious adverse effects in animal tests.
For 1 in 10 cans of all food tested, and 1 in 3 cans of infant formula, a single serving contained enough BPA to expose a woman or infant to BPA levels more than 200 times the government's traditional safe level of exposure for industrial chemicals.
Bisphenol A, what's the danger?
Mounting evidence from peer-reviewed scientific experiments conducted on laboratory animals suggest that Bisphenol A in low doses, can act as a hormone disruptor. According to Dr.Frederick Vom Saal, a developmental biologist at the University of Missouri:
These hormones control the development of the brain, the reproductive system and many other systems in the developing fetus…The most harm is to the unborn or newborn child.
According to the Environmental Working Group's report on Bisphenol A:
As of December 2004, 94 of 115 peer-reviewed studies had confirmed BPA's toxicity at low levels of exposure. At some of the very lowest doses the chemical causes permanent alterations of breast and prostate cells that precede cancer, insulin resistance (a hallmark trait of Type II diabetes), and chromosomal damage linked to recurrent miscarriage and a wide range of birth defects including Down's syndrome (vom Saal and Hughes 2005). Few chemicals have been found to consistently display such a diverse range of harm at such low doses.
Bisphenol A, what's the controversy?
Controversy comes into the picture when valid and credible objections coming from independent sectors, are raised. Dr. Steven G. Hentges, executive director of the Polycarbonate Business Unit at the American Plastics Council contests the scientific findings that tagged bisphenol A as a hormonal disruptor:
If you look at all the data together, you don't find a consistent pattern of effects that are characteristic of an estrogenic chemical.
A study funded by the Society of the Plastics Industry (July 2002) Toxicological Sciences that explored the effects of low doses on three generations of rats found no effect on reproduction or development.
Is there controversy, really?
Clearly, "independent" is not an adjective you can use on people like Hentges. These studies sponsored by the plastics industry are "profoundly flawed and in some cases exhibit outright fraud," says Dr. Vom Saal. In a paper published in 2005, Dr. Vom Saal exposed this pattern of fraudulent results by comparing 11 plastic industry-sponsored studies (all of which found no ill-effects from bisphenol A ingestion) to government-funded low-dose bisphenol A studies, 104 of which or 90% of the total number found harmful effects of bispenol A on humans. Asserts Dr. Vom Saal:
Among people who have actually read this literature there is no debate, just an illusion of controversy.
In the meantime, evidence continues to mount proving that Bisphenol A is bad news for us and our children. Perhaps the most potent blow on the plastics industry's position is the National Toxicology Program's "Draft Brief on Bisphenol A (BPA)" released last April 14, 2008 which found that the "possibility that Bisphenol A may alter human development can not be dismissed." This is a strong statement and a reversal to its 2001 position on the subject.
Scientists involved in the various researches on Bisphenol A have urged for action to be taken now. Dr. Ana Soto, a professor and researcher at Tufts University School of Medicine in Boston, who has found that BPA alters mammary-gland development in mice, in an interview with The Green Guide, said:
Science is not in the business of demonstrating anything beyond a shadow of a doubt…We cannot wait that long to discover whether this chemical is harming human reproduction and development.
Apparently, the plastics industry has found a way to game the system. By providing "evidence" from their own paid hacks, they've effectively tied up the system into countless hours of debate on the issue in an "illusion of controversy." For us ordinary consumers and parents, its a deadly illusion. As regulatory agencies like the FDA pussyfoots on the issue, plastic manufacturers who continue to use Bisphenol A are laughing all the way to the bank. In the meantime, the chemical continues to wreak untold havoc on our health and the health and future of our children.
Next topic: Alternatives to Bisphenol A-polycarbonate plastic
Update: Added CDC fact sheet (pdf) to references.
Sources and References:
- Environmental Working Group. "Bisphenol A: Toxic Plastics Chemical in Canned Food"
- National Toxicology Program (NTP) Department of Health and Human Resources
Draft Brief on Bisphenol A (BPA)
- National Geographic's Green Guide. "The Bisphenol-A Debate: A Suspect Chemical in Plastic Bottles and Cans" by Catherine Zandonella, M.P.H
- Environmental Defense, Canada: Bisphenol A Fact Sheet
- Centers for Diseases Control (CDC) – "Spotlight on Bisphenol A" (bisphenol A fact sheet – pdf)


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Dear Ones,
Last week, I took a trip to Sutter Urgent Care in Santa Rosa with my hubby who was three weeks into what seemed like walking pneumonia, but really it was the onset of bronchitis.
When we arrived, there was no wait. The woman behind the desk was as sweet as lemon meringue pie and even the waiting room felt pretty darn good.
While I sat in one of the comfortable chairs next to my beloved while he filled out the necessary forms, something on the right side of me quickly caught my eye.
I turned my head and there it was—the latest issue of People Magazine [with Oprah on the cover] neatly stacked on top of several other magazines.
I quickly grabbed it like a drug addict guiltily sneaking a quick hit, and soaked up the pages like a sponge recently filled with dish soap.
I did this for fifteen seconds and then I put it down.
You see, People Magazine is my crack.
Truthfully, I'm fascinated with the dresses celebrities wear to their fancy parties and other stories… and I admit, I've gone through binges where I'll simply lose track of time while I look at the magazine on my iPhone 6 Plus.
And then suddenly, I'll notice my husband has been standing over me calling my name several times and I've been completely zoned out in the People land.
Here's the thing…
While I oftentimes love to just veg out on People and other celebrity magazines, I never feel better after I do.
It's kind of like the way I feel about french fries. I absolutely love eating them and then afterwards, I feel like I need a good long nap to recover.
These days, I'm happy to share that I'm a recovering celebrity magazine addict. Is that in the DMS-5?
In the past, whenever I've had to wait for a doctor's appointment in the reception area and ended up reading People Magazine or its comparable, I have felt worse.
Instead of positively distracting me [as many design elements in your waiting room can], I feel my pulse elevated and I tend to worry more.
Once, I spent so much time reading the magazine while waiting for my appointment, I almost had to cancel my visit. I was that stressed out.
Okay, so I know I might be an extreme example, but I can't imagine this happens only to me.
As a matter of fact, I know it doesn't. Other people I know experience the same thing.
My big message for today?
The activities you offer your peeps in your waiting room are just as important as what you show on your walls.
Make sure that whatever you place in your waiting room or treatment room(s) evokes a sense of well-being and induces an optimistic, positive feeling among your patients and clients.
For example, adult coloring books calm the mind and relax the body. If you're a Veterinarian, include animal coloring books. If you're a Chiropractor, include coloring books about the back.
As clinical psychologist Scott M. Bea, Psy.D. says, "Coloring in an adult coloring book is very much like a meditative exercise that takes us out of ourselves."
And then there's this:
"Mindfulness art therapy has been shown to reduce physical and emotional distress and has been a helpful coping tool for people with conditions, including depression, anxiety, addictions and trauma." [Source: Cleveland Clinic, Health Essentials]
So, next time you or your team is hanging out in the waiting room, check out what magazines you offer to your peeps and maybe, just maybe, you'll remember this article and my addiction story.
With love in recovery,


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Saturday, January 28, 2006
Guided By Voices: Plantations Of Pale Pink (1996)
Plantations Of Pale Pink is a vinyl only EP which came out in 1996, so I'm guessing it is extremely hard to find now. However, like Tigerbomb, its contents are found on Demons And Painkillers from the Hardcore UFOs boxset. I am quite fond of this little EP, and usually listen to it by itself and not as a part of Demons and Painkillers. Though the songs themselves are not in any way spectacular, the EP as a whole is quite consistent, especially compared to similarly out-of-print EP Tigerbomb...
Posted by The Rock Robot at 12:53 PM 4 comments:
Saturday, January 21, 2006
Guided By Voices: Tigerbomb (1995)
I'm in a bit of a bitter mood as I'm watching my favorite hockey team, the Toronto Maple Leafs, getting smoked by the Ottawa Senators 7-0 (and there's still lots of time for the Senators to add some more pain)...alright, focus. I do not actually own this rare 7" (nor any other vinyl actually), however I do have access to it through my Hardcore UFOs box set. However, to keep things in perspective, I'll try to review releases individually. This means that my future Hardcore UFOs box set review of Demons & Painkillers will contain links to other reviews (since the disc is made up of out-of-print material). Tigerbomb is likely best known for its inclusion of two 7" alternate takes on GBV hits from the Alien Lanes era; My Valuable Hunting Knife and Game Of Pricks. Whereas this version of My Valuable Hunting Knife fails to capture the magic of what made the original from Alien Lanes so excellent, the slick production and added intro make Tigerbomb's Game Of Pricks the ultimate version...
Posted by The Rock Robot at 7:30 PM 6 comments:
Guided By Voices: An Earful O'Wax (1993)
I do not personally have this record, though I thought I should let you know about it because it is a greatest hits compilation of everything from Forever Since Breakfast (1986) through Same Place The Fy Got Smashed (1990). Other than glaring ommisions Land of Danger, The Tumblers, and A Portrait Destroyed By Fire, this is one hell of a collection, and an excellent portrait of early Guided By Voices. I'm assuming it is also extremely hard to find nowadays, especially since it was made in Germany. However, for someone just getting into the Box boxset, it is a virtual guide to its contents...
Posted by The Rock Robot at 4:57 PM 4 comments:
Wednesday, January 18, 2006
Guided By Voices: Tonics And Twisted Chasers (1996)
Originally a fan club vinyl release, Tonics And Twisted Chasers was re-released with five extra tracks in 1997, and can still be purchased from Rockathon Records. This album probably has more in common with Airport 5 than it does with Guided By Voices, since it is a collaboration between Robert Pollard and Tobin Sprout. I see it as sort of a minimalistic GBV, which makes great use of drum machines and some keyboards. As for its importance in a GBV collection, I would say it is a requirement. The album is nowhere near as rocking as other GBV albums, but instead is full of beautiful and simple melodies...
Posted by The Rock Robot at 5:39 PM 2 comments:
Sunday, January 15, 2006
Guided By Voices: Under The Bushes Under The Stars (1996)
Fans of Guided By Voices put their support behind various albums when concerning the greatest GBV album of all time. Vampire On Titus, Propeller, Alien Lanes, Bee Thousand, and sometimes even Mag Earwhig! all have their supporters. I am not alone when I throw my support behind the perfect Under The Bushes Under The Stars. From the opening onslaught of Man Call Aerodynamics, to the acoustic romp finisher Take To The Sky, and stops of genius in between (too many to list, but we'll get to them), Under The Bushes Under The Stars is a clinic of great rock musical writing. This is a hooked-filled dream, and full of the mid-fi pop gems which GBV is known for. Move over Bee Thousand...
Posted by The Rock Robot at 10:39 AM 12 comments:
Thursday, January 12, 2006
Guided By Voices: Sunfish Holy Breakfast (1996)
1996 was probably the most prolific year of Robert Pollard's career. With a live album and three full length releases (two Guided By Voices, one solo), perhaps it was possible for this EP to to be forgotten from amongst that group. However, Sunfish Holy Breakfast rates amongst GBV's best EPs (of course under Fast Japanese Spin Cycle and the excellent Hold on Hope). The bad news is that at this moment, Sunfish Holy Breakfast is out of print. I was able to find a used copy at Amazon for just over $10 after shipping and everything. Other copies are around, with some going for around $30-$50. However, if you can get your hands on it, you will be rewarding with some pretty cool tunes...
Posted by The Rock Robot at 4:36 PM 7 comments:
Monday, January 09, 2006
Slower Pace - Starting Today
Hey everyone. Thank you all for the feedback which I have received on this site. Starting today, I'm going to slow down the posting a bit as I've just started my 7th and final year of post-secondary. I hope to post at least 3 or 4 GBV/Pollard entries a week from here on in instead of the past pace of 1 a day which I have been doing. So if you come by one day, and the site wasn't updated since the prior day, just be patient, a new post will be coming.
Posted by The Rock Robot at 6:31 PM No comments:
Robert Pollard: Not In My Airforce (1996)
Alright, I get to go through my first Robert Pollard solo album for this guide! What is the difference between a solo Robert Pollard album and a Guided By Voices release? For the most part I do not really see a difference. Perhaps on future solo Pollard releases like Fiction Man and Motel of Fools, it is a more experimental aspect which makes the largest difference. However, just the first four tracks alone on Not In My Airforce rate among Pollard's finest (not to mention Chance to Buy an Island hiding out at track nine). Maybe it is the group of acoustic tracks tacked on to the end of Not in My Airforce which make this a more experimental outing. With Sunfish Holy Breakfast, Tonics and Twisted Chasers, and the phenomenal Under the Bushes, Under the Stars all released in 1996, how did Pollard have time to create this solo release? And more importantly, how did he manage to fill it with such prime music...
Posted by The Rock Robot at 6:15 PM 13 comments:
Labels: Reviews, Robert Pollard
Sunday, January 08, 2006
Guided By Voices: Jellyfish Reflector (1996)
You can still get a hold of this live CD from Luna. This was recorded live at the Patio Club in early 1996, before Under the Bushes, Under the Stars came out. As far as live albums go, the sound quality on Jellyfish Reflector is excellent. One problem though, My Valuable Hunting Knife, Matter Eater Lad, and I Am A Scientist are not actually on the CD, though the back cover of the disc case says they are. There are 30 songs played on this live CD, the first 12 of which are from the upcoming (at the time) Under the Bushes, Under the Stars. One of the finest joys when listening to this disc is not hearing the cheers as the opening riffs to Cut-Out Witch and The Official Ironman Rally Song are played, due to the audience not yet knowing the songs. However, they are listening to what will soon be legends in the canon of GBV...
Posted by The Rock Robot at 12:38 AM 2 comments:
Labels: Guided By Voices, Live, Reviews
Saturday, January 07, 2006
I would like to thank all those at Disarm the Settlers for their feedback on this site, as well as David from LargeHeartedBoy, and Rich from RobertPollard.net, for linking gbvguide. You guys all rock, and it is an honor to be linked to from such great sites (and to chat with a wonderful group of people at DTS).
Posted by The Rock Robot at 12:58 AM No comments:
Guided By Voices: Alien Lanes (1995)
Essential, absolutely essential. From the opening bassline of A Salty Salute (GBV's drinking song), through my favorite Guided By Voices track of all time (My Valuable Hunting Knife), and up to the brilliant closer (Alright), Alien Lanes is pure rock n' roll gold. There are 28 tracks on this disc, most just over a minute in length, all tempting you to pull out the old air guitar and dance around in your kitchen. As Propeller, Vampire on Titus, and Bee Thousand all have a legitimate claim to the greatest GBV album, Alien Lanes can be added to that mix...
Posted by The Rock Robot at 12:06 AM 17 comments:
Friday, January 06, 2006
Guided By Voices: Forever Since Breakfast (1986)
So like I've said many times before, I do not purchase vinyl because A) it is hard to come by where I'm from, and B) I would have nothing to play it on. Therefore, I am overjoyed that Guided By Voices' first EP, Forever Since Breakfast, came with the Hardcore UFO's box set. We're going backwards a bit to 1986. GBV may sound a lot different back in the 80's compared to their classic 90's sound, but one fact remains, they rocked from the very beginning...
Posted by The Rock Robot at 6:46 AM 4 comments:
Thursday, January 05, 2006
Guided By Voices: Box (1995)
Simply put, if you do not own the individual albums contained in Box, then it is an essential purchase for the GBV fan (and you can't get King Shit and the Golden Boys anywhere else). It includes the first four Guided By Voices' albums, but not the first EP (that is available in the Hardcore UFO's box set), and a rarities disc. Instead of providing a review, here are my reviews for the individual CD's found in Box:
1987 - Devil Between My Toes
1987 - Sandbox
1989 - Self-Inflicted Aerial Nostalgia
1990 - Same Place the Fly Got Smashed
1995 - King Shit and the Golden Boys (rarities)
1987 - Devil Between My Toes
1987 - Sandbox
1989 - Self-Inflicted Aerial Nostalgia
1990 - Same Place the Fly Got Smashed
1995 - King Shit and the Golden Boys (rarities)
Posted by The Rock Robot at 11:40 AM 2 comments:
Labels: Box Sets, Guided By Voices, Reviews
Guided By Voices: King Shit and the Golden Boys (1995)
King Shit and the Golden Boys is a rarities disc that comes with the Box box set. It contains two tracks recorded in 1988, five recorded in 1991, and twelve from 1993. Therefore, a large group of these were recorded during the same sessions in which Bee Thousand songs were done. This rarities disc for the most part plays as a cohesive album, and a fairly decent one at that. Highlights include Fantasy Creeps and Sopor Joe from Back to Saturn X Report (Propeller), and less refined versions of Don't Stop Now and Postal Blowfish. However, there are some other gems scattered about on this disc...
Posted by The Rock Robot at 11:38 AM 9 comments:
Wednesday, January 04, 2006
Guided By Voices: I Am A Scientist (1994)
I Am A Scientist may be the most famous song in the entire Guided By Voices catalog. I am of the belief that the song deserves that particular credit. The version on Bee Thousand is a lo-fi masterpiece, and an anthem for the entire genre. The reason why you must have this EP is that the version on this disc is a heavier and higher quality version. I would love to hear what people believe to be the best version of I Am A Scientist; the lo-fi and low key Bee Thousand version, or this heavy version which GBV plays live often. Oh, and by the way, Do The Earth rocks hard also...
Posted by The Rock Robot at 11:37 AM 3 comments:
Labels: Guided By Voices, Reviews, Single
Tuesday, January 03, 2006
Guided By Voices: Get Out Of My Stations (1994)
As far as GBV EP's go, this one is quite lo-fi. The highlight for me at the time of purchase was the live version of Weed King which is attached at the end of newer pressings. I did not yet have Propeller, so this was my one and only version of Weed King at the time. However, other to mention that there are live versions of Motor Away, Hot Freaks, Weed King, and Postal Blowfish attached to the end of this EP on later pressings, I will concentrate on the EP proper for this review. Overall, Get Out Of My Stations is a fine EP, which has a couple of toss offs, though nothing I would put on a Best Of Guided By Voices compilation. A song or two may make my playlist though...
Posted by The Rock Robot at 11:32 AM 5 comments:
Monday, January 02, 2006
Guided By Voices: Bee Thousand (1994)
I should first note that this is an entry in my Great Albums series as well as entry in my GBV Guide. Bee Thousand is (arguably) the best album of Robert Pollard's career. It is the best selling, and countless fans claim it as scripture. It may be one of the best entry-points for a new fan as it is a great example of Guided By Voices' lo-fi aesthetic. From the dive in sound volume in Hardcore UFO's to the what-the-hell-is-that-sound in Demons Are Real, Bee Thousand may well be the ultimate lo-fi album. There is not a bad track on this one (as almost the entire album will make my best of GBV playlist/box set). Indeed, Robert Pollard was channelling the power of the gods of rock when coming up with these tunes. Bee Thousand is the most necessary purchase for anyone who dares say that he/she likes Guided By Voices...
Posted by The Rock Robot at 10:56 AM 17 comments:
Sunday, January 01, 2006
Radio Free Burke's One-Disc GBV Compilation
After I have finished creating my ultimate GBV/Pollard playlist/boxset, I may attempt something similar to what Radio Free Burke has done, which is create a one-disc GBV compilation. Here is the tracklisting of his Guided By Voices compilation (note: Radio Free Burke has not included any solo Robert Pollard or side projects, as they will have their own one-disc compilation which I hope to post here as well). You can check out the original Disarm the Settler's thread here where the idea was discussed, and also check out Radio Free Burke's blogs A CD A Day and Safe European Blog after checking out the list...
Posted by The Rock Robot at 1:00 PM 2 comments:
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Chelsea manager Antonio Conte refused to be drawn on speculation linking his club with a late move for Everton winger Ross Barkley, reveals the Express.
The 23-year-old has been tipped for a summer transfer to Stamford Bridge, with the Telegraph reporting that the two clubs will sit down after the match on Sunday to thrash out a deal for the England international, who scored five goals for the Toffees last term.
Chelsea's Italian manager is reported to be interested in adding more additions to his squad before the transfer window comes to an end on Thursday. Yet, when asked about the possibility of signing Ross Barkley, the Blues manager refused to comment.
"I'm sorry I can't answer that question", Conte said in a press conference attended by the Express.
"To speak about players who play for another club is not right or respectful to the other club – or my players."
Barkley is currently out injured and was unavailable for selection against Chelsea on Sunday as Everton visited Stamford Bridge.
If Chelsea fail to land the Everton player, Conte will look to sign Danny Drinkwater or Alex Oxlade-Chamberlain, who have both been linked with a move to London by the Telegraph.


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Elecard Audio Decoder is an application designed to provide support to users who want to work with audio streams and decode them better. The utility does not have an interface on its own, but it is easily recognized by some features that it adds to various audio players available on one's computer. The tool is designed to enhance the audio quality of DirectShow filters.
The tool can work with various audio file formats in order to drastically enhance their quality and users will be able to experience this quality depending on their sound system. A surround system will perform much better when users listen to songs that have been decoded using this tool. There is the possibility to even further enhance the capabilities of the app by having a dedicated sound card installed on the PC.