The eukaryotic nucleus is bounded by two lipid bilayer membranes that join to form pores that mediate traffic of soluble proteins both into and out of the nuclear space[@b1]. Extracellular proteins and surface receptors can also traffic into the nucleus, where they can exert potent effects on gene expression and alter cell function[@b2][@b3][@b4][@b5][@b6][@b7]. Transport of these key extracellular factors into the nucleus is therefore a critical regulator of cellular responses to external cues, but the mechanisms that transfer them into the nucleoplasm remain poorly defined.

Following endocytosis, extracellular proteins can be directed to the recycling endosomes, endolysosomes or Golgi apparatus. The bacterial protein Pseudomonas exotoxin A (PE) binds to the cell surface receptor LRP1 for internalization before trafficking from endosomes to the Golgi, then transport to the endoplasmic reticulum (ER) where it is translocated to the cytosol[@b8]. We previously determined that PE trafficking from endosomes to the Golgi is dependent on the SNARE protein STX16 (refs [@b9], [@b10]). Transfer to the ER is dependent on the KDEL-R and ERGIC2 proteins, while translocation to the cytosol is mediated by the Sec61 translocon[@b11]. Cell surface receptors have been proposed to follow a similar route, although it has not been clearly established and a requirement for trafficking through the Golgi has yet to be demonstrated.

Here we describe a fourth endosomal route that transports cell surface receptors to the nucleoplasm through docking and membrane fusion of a population of endosomes with the nuclear envelope.

Results
=======

PE vesicles are closely associated with the nuclear envelope
------------------------------------------------------------

While tracking the transport of fluorescently labelled PE in the osteosarcoma cell line MG63, we not only detected the typical Golgi localization associated with this protein but also observed a recurrent and consistent association of toxin-labelled vesicular structures with the nucleus ([Fig. 1a](#f1){ref-type="fig"} and [Supplementary Fig. 1a](#S1){ref-type="supplementary-material"}). By using deconvolution microscopy and three-dimensional (3D) modelling together with 'super-resolution\' structured illumination microscopy (SIM), we observed that the PE+ vesicular structures were located either at the top or bottom of the nucleus in close association with the nuclear envelope and lamin sheet ([Fig. 1b--d](#f1){ref-type="fig"} and [Supplementary Movie 1](#S1){ref-type="supplementary-material"}). The ER marker protein disulfide isomerase (PDI) was excluded from the areas above and below the nucleus, suggesting that the space between nuclear envelope and cell membrane is limited. By extension, it suggests that an active mechanism is driving the accumulation of these PE-laden vesicles in this location ([Fig. 1a](#f1){ref-type="fig"}).

Nucleus-associated PE vesicles do not require Golgi traffic
-----------------------------------------------------------

Nuclear envelope-associated vesicles containing fluorescent PE could be detected as early as 15 min after protein addition to MG63 cells, although staining intensity was relatively weak at this early time point. After 1 h PE exposure, toxin-laden vesicles were clearly visible in association with the nuclear envelope in the majority of cells, and with only trace staining in the Golgi area, suggesting that toxin accumulation did not involve transit via the Golgi apparatus. Accordingly, cells pre-treated with the Golgi blocker Brefeldin A for 30 min before toxin exposure still displayed numerous PE+ vesicles associated with the nuclear envelope ([Fig. 2a--c](#f2){ref-type="fig"}).

Further analysis revealed that PE+ vesicles partially co-localized with the early endosome antigen 1 (EEA1; [Fig. 2d](#f2){ref-type="fig"}). In contrast, there was no co-localization of PE with the late endosome marker Rab7, lysosomal marker Lamp1 or secretory pathway marker ERGIC53 ([Fig. 2e](#f2){ref-type="fig"} and [Supplementary Fig. 2a,b](#S1){ref-type="supplementary-material"}). Taken together, these data strongly suggested that the PE-laden vesicles detected in association with the nuclear envelope are derived from early endosomes (hereafter referred to as nuclear envelope-associated endosomes (NAE)).

NAE display fusion events and progressive loss of content
---------------------------------------------------------

PE-positive NAE appeared within 10 min of incubation with PE and disappeared in a few hours: after a PE pulse of 1 h, the nuclear-associated PE signal decreased rapidly over the next 3 h ([Supplementary Fig. 2c](#S1){ref-type="supplementary-material"}). Using live-cell imaging, we observed that NAE were largely static and exhibited an average velocity at least threefold lower than that of their cytoplasmic counterparts ([Fig. 3a,b](#f3){ref-type="fig"} and [Supplementary Movie 2](#S1){ref-type="supplementary-material"}). We also detected a limited number of nucleus-associated vesicles that rapidly trafficked over the nucleus before abruptly stopping and becoming immobile, suggesting that NAE are not spatially constrained but are instead docked on the nuclear envelope. We next monitored PE-exposed cells using images captured every 5 s for a total duration of 3 h after addition of toxin, during which time there was a marked decrease in the number of NAE detected per nucleus, despite the fact that these structures could not be observed to exit the nuclear area ([Fig. 3c,d](#f3){ref-type="fig"}). Subsequent analyses revealed that NAE moving independently would frequently make contact with one another and become indistinguishable thereafter, accompanied by an increase in fluorescence intensity to a level roughly equivalent to the sum of the parent vesicles, strongly suggesting that NAE can readily undergo fusion events ([Fig. 3e,f](#f3){ref-type="fig"} and [Supplementary Movie 3](#S1){ref-type="supplementary-material"}).

Individual NAE frequently displayed a progressive decrease in PE fluorescence signal while some adjacent NAE exhibited stable fluorescence signals throughout the observation period, suggesting selective loss of vesicular cargo rather than a general photobleaching effect ([Fig. 3h](#f3){ref-type="fig"}, [Supplementary Fig. 3a,b](#S1){ref-type="supplementary-material"} and [Supplementary Movie 4](#S1){ref-type="supplementary-material"}). The rate of PE fluorescence decrease was uniform and comparable between NAE ([Fig. 3h](#f3){ref-type="fig"}), and often lasted tens of minutes up to 1 h ([Fig. 3f](#f3){ref-type="fig"} and [Supplementary Fig. 3a,b](#S1){ref-type="supplementary-material"}). In an analysis of 35 different NAE, 22 displayed a clear decrease in fluorescence signal ([Supplementary Fig. 3a,b](#S1){ref-type="supplementary-material"}). Since these vesicles appeared to be docked on the nuclear envelope, we postulated that NAE can undergo partial fusion with the envelope and progressively discharge their content into the nuclear envelope. Indeed, partial fusion of endosomes with the plasma membrane has been recently reported by other investigators[@b12]. We next sought to determine whether NAE co-localize with nuclear pores markers; however, we were unable to detect any co-localization ([Supplementary Fig. 2d](#S1){ref-type="supplementary-material"}).

NAE content is translocated to the nucleoplasm
----------------------------------------------

We next proceeded to assess NAE by electron microscopy (EM), which identified these structures as being in close proximity to and occasionally merging with the nuclear envelope membranes ([Fig. 4a--c](#f4){ref-type="fig"} and [Supplementary Fig. 4c](#S1){ref-type="supplementary-material"}). Furthermore, PE could be detected in the lumen of the nuclear envelope and was also present on the nucleoplasmic side of the membrane ([Fig. 4a--c](#f4){ref-type="fig"} and [Supplementary Fig. 4a--d](#S1){ref-type="supplementary-material"}). This nucleoplasmic PE staining was even more pronounced when visualized using ultra-small gold particles with silver enhancement labelling. Quantification indicated that PE staining density was about twice as high in nucleoplasm than in the cytosol, consistent with the idea that PE translocates directly from NAE to the nucleoplasm ([Fig. 4d](#f4){ref-type="fig"}). Together, these observations suggested that PE+ NAE fuse with the nuclear envelope to transfer their content into the nucleus.

To confirm our EM results, we next incubated MG63 cells with biotinylated PE for 1 h then performed subcellular fractionation experiments to separate nuclei from the cellular membranes and cytosolic fractions. Using this approach, we were able to confirm that although the majority of PE partitioned with the membrane fraction, a significant pool of protein could also be detected in the nuclear fraction. To exclude the possibility that contamination with NAE contributed to PE detection in the nuclear fraction, we next generated a HeLa cell line that stably expressed a chimeric avidin sequence featuring a nuclear localization signal as well as Green Fluorescent Protein (GFP) and a FLAG tag (hereafter referred to as nuclear trap (Nu-T); [Fig. 4f,g](#f4){ref-type="fig"}). Biotinylated PE could be readily detected interacting with Nu-T in the nucleus of HeLa cells just 1 h after protein exposure ([Fig. 4h](#f4){ref-type="fig"}), but not in wild-type (WT) cells incubated with biotinylated PE and mixed with Nu-T immediately before cell lysis and immunoprecipitation ([Supplementary Fig. 6b](#S1){ref-type="supplementary-material"}). Pre-treatment with the Golgi blocker Brefeldin A was unable to abolish the nuclear localization of PE, further indicating that trafficking of this protein to the nucleus is independent of the Golgi apparatus ([Fig. 4e](#f4){ref-type="fig"} and [Supplementary Fig. 6c](#S1){ref-type="supplementary-material"}). To evaluate the kinetics of PE transfer to the nucleus, we next lysed Nu-T cells at different time points after exposure to PE. A faint nuclear signal could be detected as soon as 10 min after PE addition, followed by a progressive increase over the following 80 min ([Fig. 4i](#f4){ref-type="fig"}). Together, these data suggest that PE translocation to the nucleus commences soon after the first vesicles are detected near the envelope and does not require transit through the Golgi.

PE nuclear translocation is SUN1/2 and Sec61 dependent
------------------------------------------------------

We next used Nu-T cells together with a targeted RNA interference screening approach to identify the genes that mediated nuclear translocation of PE, including proteins involved in nuclear envelope regulation, as well as components of the translocon complex ([Fig. 5a](#f5){ref-type="fig"} and [Supplementary Fig. 5](#S1){ref-type="supplementary-material"}). The complete list of 24 genes screened in these experiments is provided in [Supplementary Table 1](#S1){ref-type="supplementary-material"}. Depletion efficiency was not tested for all target proteins (see [Supplementary Fig. 6d](#S1){ref-type="supplementary-material"} for expression levels of a subset of target proteins). STX16 and ERGIC2, which are known to be required for PE intoxication in HeLa cells, were used as negative controls ([Fig. 5b](#f5){ref-type="fig"})[@b10]. In contrast, depletion of the nuclear envelope proteins SUN1 and SUN2 potently impaired PE translocation to the nucleus ([Fig. 5b](#f5){ref-type="fig"}), and this effect could be successfully replicated using three or more different short interfering RNAs (siRNAs) for each protein ([Supplementary Fig. 6f](#S1){ref-type="supplementary-material"}), without adversely affecting the nuclear localization of Nu-T ([Supplementary Fig. 6g](#S1){ref-type="supplementary-material"}).

The SUN1 and SUN2 proteins are embedded in the inner membrane of the nuclear envelope and protrude into the lumen, while also interacting with Nesprin proteins in the outer membrane to form a bridge between these layers[@b13]. Knockdown of the four Nesprin genes described to date, either alone or in combination, was unable to substantially restrict PE translocation to the nucleus[@b14] ([Supplementary Fig. 6h](#S1){ref-type="supplementary-material"}). However, since the individual depletion of Nesprin 3 and Nesprin 4, as well as several double knockdowns induced toxicity, it is not possible to exclude a role for these molecules in nuclear import of PE.

Other hits were three genes that encode the heterotrimeric Sec61 translocon complex, which forms a channel in membranes. Depletion of each of the Sec61 subunits, alpha1 (A1), beta (B) and gamma (G), resulted in a similar drastic reduction of PE translocation to the nucleus ([Fig. 5b](#f5){ref-type="fig"}). We confirmed these results by using several different siRNAs against Sec61B subunits, each of which replicated the restriction of PE translocation to the nucleus ([Supplementary Fig. 6i](#S1){ref-type="supplementary-material"}), while preserving localization of Nu-T ([Supplementary Fig. 6j](#S1){ref-type="supplementary-material"}). Using super-resolution microscopy, we could detect Sec61 in the nuclear envelope of MG63 cells ([Supplementary Fig. 6k](#S1){ref-type="supplementary-material"}). Furthermore, a recent report has shown that a fraction of Sec61 is localized at the inner nuclear membrane[@b15]. Taken together, these data strongly suggest that Sec61 mediates PE translocation across the inner nuclear membrane.

We next sought to confirm these results in a system that did not depend on Nu-T system. We therefore performed siRNA knockdown of Sec61B or SUN2 in HeLa cells and MG63 cells before PE exposure, then carried out cellular fractionation as before, and confirmed depletion of Sec61B or SUN2 using western blotting ([Supplementary Fig. 6l](#S1){ref-type="supplementary-material"}). Fractionation efficiency was determined by blotting for tubulin, PDI and histone H1 ([Fig. 5c](#f5){ref-type="fig"}). In the absence of either Sec61B or SUN2, the nuclear fraction of PE staining was significantly reduced, whereas the membrane-bound fraction was unaffected ([Fig. 5d,e](#f5){ref-type="fig"} and [Supplementary Fig. 6m](#S1){ref-type="supplementary-material"}).

PE binds to the cell surface receptor LRP1 (ref. [@b8]). In MG63 cells, we found LRP1 staining to co-localize with PE in NAE structures and to be significantly enriched in the nucleus ([Supplementary Fig. 6a](#S1){ref-type="supplementary-material"}). Furthermore, LRP1 could be detected in a nuclear fraction ([Supplementary Fig. 7b](#S1){ref-type="supplementary-material"}). When Sec61B or SUN2 were depleted, this nuclear fraction was significantly reduced ([Supplementary Fig. 7c,d](#S1){ref-type="supplementary-material"}), indicating that PE and LRP1 follow the same pathway into the nucleus.

To test whether Epidermal Growth Factor Receptor (EGFR) also accumulates in the nucleus following the same pathway, MG63 cells were incubated with PE for 1 h and stained for EGFR, revealing co-localization in NAE ([Fig. 5f](#f5){ref-type="fig"}). While EGFR nuclear immunofluorescence staining was not as prominent as for LRP1, subcellular fractionation revealed a nuclear fraction ([Fig. 5g](#f5){ref-type="fig"}). This fraction was reduced in cells depleted of Sec61B or SUN2, while total EGFR was not affected ([Fig. 5h--j](#f5){ref-type="fig"}). The reduction was comparable to that for PE in a similar experiment ([Fig. 5e](#f5){ref-type="fig"}).

Discussion
==========

Overall, our results indicate that PE and other cell surface molecules can follow at least two intracellular routes: one leading to the cytosol by way of the Golgi and ER, and one to the nucleoplasm via NAE ([Fig. 5k](#f5){ref-type="fig"}). The first route appears to require a few hours and is clearly important for inhibition of cytoplasmic ribosomal activity by PE, as demonstrated by the protective effect of ERGIC2 and STX16 depletion[@b10]. The second route leads to delivery of PE into the nucleoplasm within 1 h. The existence of this second route is further substantiated by the fact that nuclear PE tends to be unprocessed (that is, full length), whereas cytosolic PE is mostly cleaved. Indeed, PE is cleaved by Furin in the Trans Golgi Network (TGN), which promotes its toxic activity[@b16]. Furthermore, we could not detect any interaction of PE with KPNB1 (importin beta 1), a key factor for transport through the nuclear pore ([Supplementary Fig. 7g](#S1){ref-type="supplementary-material"}). This data further support the fact that cytosolic PE is not actively transported into the nucleus. On the other hand, full-size PE is presumably unable to diffuse freely through the nuclear pores due to its size. Therefore, nuclear PE may not be able to inhibit cytoplasmic ribosomes, explaining perhaps why the second route is not very efficient for intoxication.

On the other hand, this trafficking route is probably used by various endogenous proteins such as LRP1 and EGFR, and could explain the nuclear accumulation of various growth factors and receptors previously reported[@b5]. NAE represent a novel delivery route that permits cell surface proteins and extracellular molecules to access the genome and alter cellular function. The NAE pathway may therefore represent an alternative mechanism by which external stimuli can influence cellular activity independently of the conventional signalling cascades that operate in the cytosol. This route of surface protein transport into the nucleus is likely a tightly regulated event rather than a constitutive process. In addition, this pathway could represent an interesting route for delivery of therapeutic agents directly into the nucleus.

Methods
=======

Gene synthesis and cloning
--------------------------

The sequence of the Nu-T NLS-GFP-Avidin-Flag was synthesized and cloned in a pDONR221 vector using gene synthesis (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA). The sequence was then transferred using a pLenti6.3-DEST vector with the Gateway system.

Cell line culture and stable transduction
-----------------------------------------

All cell lines come from ATCC. Lentiviruses were generated according to the manufacturer\'s instructions (Invitrogen). WT HeLa cells were transduced to stably express Nu-T. All cell lines (HeLa WT, HeLa Nu-T, MG63 and A431) were maintained in high-glucose Dulbecco\'s modified Eagle\'s medium supplemented with 10% fetal calf serum at 37 °C in a 10% CO~2~ incubator. Cells infected with GFP virus were subjected to fluorescence-activated cell sorting sorting to enrich for GFP+ cells before rapid expansion and freezing. All experiments were performed on cells passaged fewer than 10 times after thawing.

Fluorescent PE uptake
---------------------

PE (\#341215, Merck Millipore, Darmstadt, Germany) was labelled with AnaTag Hilyte Fluor 488 according to the manufacturer\'s instructions (\#72048, AnaSpec, Fremont, CA). Typically, MG63 cells were seeded 2 days before incubation with ∼250 μg ml^−1^ fluorescent PE for 60 min (concentrations as low as 5 μg ml^−1^ produced similar images).

Immunofluorescence
------------------

Cells were seeded onto glass coverslips in 24-well dishes (Thermo Fisher Scientific) and incubated at 37 °C, 10% CO~2~ for 8--16 h. In some experiments, the cells were incubated with 10 μg ml^−1^ Bredfedin A for 30 min both before and during PE intoxication. All subsequent steps were performed at room temperature. Cells were washed with Dulbecco\'s Phosphate Buffer Saline (D-PBS), fixed for 15 min using 4% paraformaldehyde in D-PBS, washed with D-PBS, then permeabilized and blocked with 0.2% Triton X-100, 2% Foetal Bovine Serum (FBS) in D-PBS for 15 min before antibody staining (EEA1 (\#C45B10, 1:300, Cell Signaling Technology, Beverly, MA), EGFR (\#2232L, 1:200, Cell Signaling Technology), ERGIC53 (\#E1031, 1:200, Sigma-Aldrich, St. Louis, MO), giantin (\#ab24586, 1:1,000, Abcam, Cambridge, UK), Lamin a/c (\#ab8984, 1/500, Abcam), Lamp1 (\#ab24170, 1:300, Abcam), LRP1 (\#L2420, 1:200, Sigma-Aldrich), NPC (\#ab24609, 1:100, Abcam), PDI (\#RL77, 1:500, Abcam), Rab7 (\#ab137029, 1:300, Abcam), Sec61B (\# ab78276, 1:200, Abcam), SUN1 (\#HPA008461, 1:300, Sigma-Aldrich) and SUN2 (\# HPA001209, 1:300, Sigma-Aldrich)). Cells were washed three times using 2% Foetal Bovine Serum in D-PBS and then subsequently stained for 45 min with secondary Alexa Fluor-conjugated antibodies and Hoechst (Invitrogen). Cells were mounted onto glass slides using FluorSave (Merck Millipore).

Scanning confocal microscopy
----------------------------

An Olympus FV1000 upright confocal microscope (Olympus Microscopes, Essex, UK) equipped with 405, 488, 561 and 647 nm lasers for excitation and spectral/band-pass emission filters was used for acquisition of confocal images and construction of z-stacks. An Olympus Plan Apo × 60/1.35 oil immersion objective lens was used. The confocal pinhole was set to 1 Airy unit. For z-stacks, the z-spacing was fixed to meet the Nyquist criteria. Figure preparation was carried out using Fiji software[@b17].

3D-SIM and wide-field deconvolution
-----------------------------------

A DeltaVision OMX v4 Blaze microscope (Applied Precision, GE Healthcare, Issaquah, WA), equipped with 488 and 568 nm lasers (3D-SIM), a solid-state illuminator (WF) for excitation, and BGR filter drawer was used (emission wavelengths 436/31 for 4,6-diamidino-2-phenylindole, 528/48 for Alexa 488 and 609/37 for Alexa 568). An Olympus Plan Apochromat × 100/1.4 Point Spread Function (PSF) oil immersion objective lens was used with liquid-cooled Photometrics Evolve EM-CCD cameras (Photometrics, Tucson, AZ) for each channel. Fifteen images per section per channel were acquired (made up of three rotations and five phase movements of the diffraction grating) at a z-spacing of 0.125 μm (refs [@b18], [@b19]). Structured illumination reconstruction or deconvolution followed by alignment was conducted using the SoftWorX (Applied Precision) programme with figure preparation in Imaris (Andor-Bitplane, Zurich) and Fiji software[@b17]. Imaris (Andor-Bitplane, Zurich) was used for 3D rendering and to generate the animation. DeltaVision OMX images were directly imported, and serial *XY* and *XZ* planes were produced using the Orthogonal Slicer tool. The 3D modelization was created using the Surpass tool, with maximum display intensity for Lamin and the Spots tool (intensity and size threshold) to enable visualization of PE.

Spinning disk live-cell confocal microscopy
-------------------------------------------

Live-cell imaging was performed using a motorized Nikon Ti inverted microscope (Nikon, Tokyo, Japan) equipped with a Plan Apo × 60/1.4 oil immersion objective lens, a 491-nm laser (FRAP-3D laser launch; Photometrics) and CSU-22 scanning head (Yokogawa Electric Corp., Tokyo, Japan), Evolve EM-CCD camera (Photometrics) and the Nikon Perfect Focus System. Cells were maintained at 37 °C in a 5% CO~2~ humidified environment using an on-stage incubator (Chamlide, Live Cell Instrument, Seoul, South Korea). Time-lapse imaging was controlled using MetaMorph software (100 ms exposure, EM-gain 100, 15% laser power, Molecular Devices, LLC, Sunnyvale, CA). The image analysis was completed using Fiji software (ImageJ, National Institutes of Health (NIH), Bethesda, MD).

Electron microscopy
-------------------

MG63 cells were grown on coverslips and exposed to 750 μg ml^−1^ biotinylated PE for 60 min before fixation with 4% paraformaldehyde, 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 day, and then stored at 4 °C. The coverslips were rinsed in ice-cold 0.1 M phosphate buffer (pH 7.4) and then in 0.1 M sodium cacodylate trihydrate (pH 7.4). After fixation, the samples were exposed to osmium fixative solution (1% OsO~4~+1.5% K~3~Fe(CN)~6~ in 0.1 M sodium cacodylate trihydrate, pH 7.4) for 1 h at room temperature. Samples were then washed in dH~2~O and dehydrated through an ascending ethanol series. Infiltration was continued using Epon 812 resin in 100% ethanol and then fresh Epon 812 resin overnight. Finally, the samples were embedded in Epon 812 fresh resin and polymerized at 56 °C for 24 h. Ultrathin sections (∼70 nm) were cut on an ultra-microtome with a diamond knife (Diatome Inc., Bern, Switzerland) and carefully positioned on 100 mesh Nickel grids (Electron Microscopy Systems, Hatfield, PA). The sections were supported by formvar and carbon.

Immunostaining was performed following a protocol modified from Hearn *et al.*[@b20]. Grids with sections (with or without biotinylated PE) were incubated with 1% periodic acid for 10 min and then rinsed in dH~2~O followed by PBS (pH 7.4). The grids were pre-incubated on a drop of 0.05 M PBS (pH 7.4), with 0.15% glycine, 0.2% Cold Water Fish Gelatin (CWFG) and 1% bovine serum albumin for 1 h and then placed on a drop of 6-nm gold-streptavidin antibody (1:50) or ultra-small gold-streptavidin antibody (1:50) in the same solution without bovine serum albumin overnight at 4 °C (Electron Microscopy Systems). The following day, the grids were washed in PBS and incubated with 1% glutaraldehyde in dH~2~O, followed by a wash with dH~2~O. Finally, ultra-small gold labelling was subjected to silver enhancement using GoldEnhance EM kits according to the manufacturer\'s instructions (Nanoprobes, Yaphank, NY). As an additional control, some grids of cells (with or without exposure to biotinylated PE) were incubated in the same way but without the antibody.

Ultrathin sections were stained for 5 min in 5% uranylacetate in dH~2~O followed by Reynolds lead citrate for 2 min and examined under a JEM-2200FS (Jeol USA, Peabody, MA) at 100 kV. Immunogold staining was considered adequate in the absence of labelling inside mitochondria. All controls were negative for immunogold labelling.

Cell fractionation
------------------

Cells were seeded at the desired densities in six-well dishes (Thermo Fisher Scientific) and incubated at 37 °C, 10% CO~2~ for 8--16 h. PE toxin or Epidermal Growth Factor (EGF) biotinylation were performed using a DSB-X Biotin Protein labelling Kit (\#D-20655, Molecular Probes, Thermo Fisher Scientific), according to the manufacturer\'s protocol. Cells were incubated for 60 min with PE at a final concentration of 5 μg ml^−1^ for HeLa cells and 3 μg ml^−1^ for MG63 cells. A431 cells were stimulated 30 min with EGF 50 ng ml^−1^. In some cases, the cells were incubated with 10 μg ml^−1^ Bredfedin A for 30 min both before and during PE intoxication. Cells were fractionated with Cell Surface Protein Isolation Kit (\#89881, Thermo Fisher Scientific) according to the manufacturer\'s protocol. Membranes were incubated with actin (\#3280, 1:5,000, Abcam), β-tubulin (\#ab6046, 1:5,000 Abcam), EEA1 (\#E7659, 1:1,000, Sigma-Aldrich), EGFR (\#2232L, 1:1,000, Cell Signaling Technology), histone H1 (\#sc-10806, 1:1,000, Santa Cruz), PDI (\#RL77, 1:15,000, Abcam), LRP1 (\#L2420, 1:1,000, Sigma-Aldrich), Rab7 (\#ab137029, 1:1,000, Abcam), Sec61B (\#ab78276, 1:5,000, Abcam), streptavidin-horseradish peroxidase (HRP; \#21130, 1:10,000, Thermo Fisher Scientific) and SUN2 (\# HPA001209, 1:2,000, Sigma-Aldrich). The western blot quantification was done using Fiji software package (ImageJ, NIH).

Nu-T pulldown assay
-------------------

Cells were seeded at the desired densities in six-well dishes (Thermo Fisher Scientific) and incubated at 37 °C, 10% CO~2~ for 16 h. Biotinylated PE toxin was then added to HeLa NLS_GFP_Avidin_Flag or HeLa WT cells for 60 min at a final concentration of 5 μg ml^−1^. Cells were incubated with 10 μg ml^−1^ Bredfedin A for 30 min both before and during PE intoxication. Cells were then washed and lysed for 30 min in 500 μl lysis buffer (Tris 50 mM (pH 8.0), 170 mM NaCl and 0.5% NP-40, together with 7.5 mM biotin to prevent post-lysis interactions) and complete protease inhibitor (Roche Applied Science, Mannheim, Germany) before being centrifuged for 30 min at 16,000*g* and 4 °C. The supernatant was incubated overnight with 15 μl anti-FLAG M2 Magnetic Beads (\#m8823, Sigma-Aldrich). After three washes, the beads were suspended in 40 μl of *×* 2 SDS loading buffer (\#161-0737, Bio-Rad, Hercules, CA) supplemented with β-mercaptoethanol, followed by boiling at 95 °C for 5 min. Samples were resolved by SDS--polyacrylamide gel electrophoresis using bis-tris 4--12% NuPage gels as per the manufacturer\'s instructions (\#NP036BOX, Life Technologies, Thermo Fisher Scientific). The samples were then transferred onto nitrocellulose membranes and blocked using 3% milk dissolved in Tris Buffered Salin Tween (TBS-T) (50 mM Tris (pH 8.0, 4 °C), 150 mM NaCl and 0.1% Tween-20) for 15 min at room temperature. Membranes were incubated with streptavidin-HRP (\#21130, 1:10,000, Thermo Fisher Scientific), Flag-HRP (\#a8592, 1:10,000, Sigma-Aldrich) or actin (\#3280, 1:5,000, Abcam). For actin antibody staining, membranes were washed with TBS-T and then incubated with secondary HRP-conjugated antibodies (GE Healthcare). Membranes were then washed with TBS-T before ECL exposure. Uncropped western blot are presented in [Supplementary Fig. 8](#S1){ref-type="supplementary-material"}.

Gene knockdown
--------------

Knockdown assays were performed in six-well dishes (Thermo Fisher Scientific). Reverse transfection was performed using 3 μl of 50 μM siRNA (Thermo Fisher Scientific) per well, together with 180 μl Opti-MEM (Invitrogen) and 20 μl HiPerFect (\#301707, Qiagen, Hilden, Germany). After incubation for 20 min to allow complex formation, 300,000 HeLa NLS_GFP_Avidin_Flag cells, MG63 cells or A431 cells were added to each well in a final volume of 3 ml for a 50 nM siRNA. The cells were then incubated for 72--96 h at 37 °C in a 10% CO~2~ incubator.

Additional information
======================

**How to cite this article:** Chaumet, A. *et al.* Nuclear envelope-associated endosomes deliver surface proteins to the nucleus. *Nat. Commun.* 6:8218 doi: 10.1038/ncomms9218 (2015).

Supplementary Material {#S1}
======================

###### Supplementary Information

Supplementary Figures 1-8 and Supplementary Table 1

###### Supplementary Movie 1

Z-stack and its orthogonal view of confocal images of PElabeled structures and Lamin A/C staining, plus a 3D rendering of nucleus-associated vesicles containing PE.

###### Supplementary Movie 2

Live imaging of Alexa Fluor 488-PE loaded cells every 0.5 sec over a period of 5 min. Three vesicles are tracked in green (nucleus), red and blue (cytoplasm).

###### Supplementary Movie 3

Live imaging of Alexa Fluor 488-PE loaded cells every 5 sec over three hours. Movie corresponds to Figure 3C, E.

###### Supplementary Movie 4

Live imaging of Alexa Fluor 488-PE loaded cells every 0.5 sec over 15 minutes. Movie corresponds to Figure 3G.

We thank Simon Chu for its technical contribution, Dr Alessandra Calvi (Dr Brian Burkes\'s lab) for SUN2-GFP plasmid and members of F.B. lab for discussion. Dr Neil McCarthy of Insight Editing London provided writing assistance.

**Author contributions** A.C. and F.B. designed the study, performed experiments and wrote the manuscript. G.D.W. performed light and N.V.G. electron microscopy. S.H.S. performed screening experiments. K.M.T. established stable cell lines.

![PE-containing vesicles are closely associated with the nuclear envelope.\
(**a**) Confocal imaging of PE+ vesicular structures in MG63 cells stained with the ER marker PDI and the DNA intercalating dye 4,6-diamidino-2-phenylindole (scale bar, 25 μm). Image is representative of 10 independent experiments. (**b**) Deconvolution microscopy image demonstrating close association of PE+ structures with the nuclear envelope as revealed by fluorescent staining of the envelope components lamin A and C (scale bar, 5 μm). (**c**) Z/X reconstruction image demonstrating close association of PE+ structures with the lamin sheet (scale bar, 5 μm). (**d**) 3D-SIM super-resolution microscopy image of PE and lamin A/C (scale bar, 1 μm). Image is representative of three independent experiments.](ncomms9218-f1){#f1}

![Vesicles associated with the nuclear envelope are derived from early endosomes.\
(**a**) Cells pre-treated with Brefeldin A (BFA) or dimethylsulfoxide (DMSO) both before and during incubation with Alexa 488-PE were stained for giantin (scale bar, 5 μm). (**b**) Percentage of cells displaying NAE structures. Representative of three independent experiments (error bars indicate s.d.). (**c**) Average number of NAE structures by nuclei. Representative of three independent experiments (error bars indicate s.d.). (**d**) Co-labelling of PE with early endosomal marker EEA1. (**e**) Co-labelling with the late endosomal marker Rab7 (scale bar, 10 μm, × 5 image: scale bar, 2 μm). Data shown in **c** and **d** are from three experiments.](ncomms9218-f2){#f2}

![NAE display low motility, frequent fusion events and progressive loss of vesicular content.\
(**a**) MG63 cells were incubated for 1 h with Alexa 488-conjugated PE and then imaged every 0.5 s for a total duration of 5 min. (**b**) Quantification of vesicle velocity. (**c**) NAE number over time in an individual live cell. (**d**) NAE frequency in six individual cells. (**e**) Apparent NAE fusion event. (**f**) Increase in PE fluorescence intensity consistent with NAE fusion. Images were taken every 5 s for a total duration of 3 h. (**g**,**h**) Decreasing fluorescence intensity of PE-laden NAEs over time suggesting progressive loss of vesicular contents (scale bar, 10 μm). Images and data are representative of six independent experiments.](ncomms9218-f3){#f3}

![PE localizes in the nuclear envelope lumen and nucleoplasm.\
(**a**) MG63 cells intoxicated for 1 h with biotinylated PE and stained with gold-labelled streptavidin (scale bar, 100 nm). (**b**) Schematic of image shown in **a** with gold particles indicated by arrows. (**c**) Number of gold particles per cell in different compartments. (**d**) Number of gold particles per μm^2^ in the cytosol and nucleus. Representative of two independent experiments (error bar indicates s.d.). (**e**) Fractionation of cells intoxicated with biotinylated PE for 1 h with (right panel) or without (left panel) BFA pre-treatment (C, cytosolic fraction; M, membrane fraction; N, nuclear fraction). (**f**,**g**) Nu-T construct and its nuclear localization (scale bar, 25 μm). (**h**) Biotinylated PE detection after pulldown of Nu-T ([Supplementary Fig. 5a,b](#S1){ref-type="supplementary-material"}). (**i**) Kinetics of PE pulldown. (**e**,**h**,**i**) Representative of at least five independent experiments. Cy, cytosol; GA, Golgi apparatus; NE, nuclear envelope; Nu, nucleus; MT, mitochondria; V, vesicles.](ncomms9218-f4){#f4}

![PE requires SUN1/2 and the translocon complex for transport to the nucleoplasm.\
(**a**) Flowchart and (**b**) results of siRNA screening; PE and Nu-T detection as in [Fig. 4](#f4){ref-type="fig"}. Representative of five different replicates. (**c**--**e**) MG63 cell fractionation after Sec61B or SUN2 knockdown. (**c**) Fractionation controls (as in [Fig. 4](#f4){ref-type="fig"}). (**d**) PE subcellular distribution in MG63 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. (**e**) Quantification of PE in nuclear fraction after Sec61B and SUN2 knockdown. Error bars at s.d. *t*-Test type 2, two tails. (**f**) Co-labelling of PE with EGFR. Nucleus is delimited by yellow circle. Scale bar, 10 μm. (**g**) A431 cells fractionation. (**h**) Input loading control of A431 protein extract. (**i**) EGFR nuclear distribution in A431 cells after Sec61B and SUN2 knockdown. Representative of three independent repeats. (**j**) Quantification of EGFR in nuclear fraction. Error bars at s.d. *t*-Test type 2, two tails. (**k**) Two intracellular routes of cargos. After endocytosis, NAE dock on the nucleus, discharging their content into the nucleoplasm in a SUN1/2- and Sec61 translocon-dependent manner.](ncomms9218-f5){#f5}


Q:

How can I access shared folders on an Ubuntu host and a Windows XP guest?

I have installed VirtualBox 4.1.18 on Ubuntu 12.10 and I'm hosting Windows XP.
I want to share a folder between Ubuntu and Windows XP.
I have installed guest additions on the guest machine, but when I'm trying to access the shared folder, I can't find them anywhere.
So, where can I access those shared folders?

A:

At least for Ubuntu 15.04 host system and Windows Server 2012 R2 and VirtualBox 4.3.32_Ubuntu r104062, the following works. I assume, it works the same way with other versions of Windows as guest:

Boot the Guest operating system in VirtualBox.
Select Devices > Shared Folders in the VirtualBox menu
Choose the Add button. Select the host folder you want to share
Select Auto Mount
Reboot your guest system
Now you will find your shared folder mapped as network drive on Windows. No need to fiddle around with manually booting something on Linux or Windows command line as described in this guide.

A:

Have you read this guide:
Shared folders
The process of creating a shared folder is well explainded in this guide.
In short:
1) In your VM properties set a shared folder on host machine.
2) Boot the guest machine and run the following command:
net use x: \\vboxsvr\share

where x - is the name of the drive with virtual folded. You may leave it as is, and share - is the name of the folder you've created on host machine.


---
author:
- |
    L. Coffey,\
    Physics Department,\
    Illinois Institute of Technology,\
    Chicago, Illinois 60616
title: 'Effect of a Pseudogap in Strong Coupling d-Wave Superconductivity.'
---

[**Abstract**]{}

A phenomenological strong coupling model that has been used to analyze superconducting-insulator-superconducting (SIS) break junction experiments on optimal to overdoped Bi$_{2}$Sr$_{2}$CaCu$_{2}$O$_{8}$ (Bi-2212) is modified to include a pseudogap. The calculated density of states and SIS conductances are compared with experimental data on the underdoped phase of Bi-2212.

The electronic properties of the copper oxides display evidence for two energy gaps: the superconducting gap, and the pseudogap. The latter leads to a reduction in the normal state density of states about the Fermi energy, and manifests itself in various normal state thermal and transport properties [@Tim]. In addition, both normal and superconducting properties of the copper oxides depend on carrier doping. The superconducting transition temperature $T_{C}$ varies strongly with hole doping, increasing from zero in the overdoped phase, passing through a maximum at optimal doping, and decreasing to zero in the underdoped phase. The curve describing this doping dependence is usually called the superconducting dome. The temperature, usually denoted by $T^{*}$, below which the pseudogap is non zero, also depends on doping. The interplay between the pseudogap and the superconducting gap is currently an active area of study with several ideas being pursued to characterize the nature of the combined pseudogap/superconducting phase. [@Huf; @Tmr]\
\
Point contact tunneling (PCT) and scanning tunneling microscopy (STM) experiments provide evidence for both gaps in the electronic spectrum of the copper oxide superconductors. The hole doped material Bi$_{2}$Sr$_{2}$CaCu$_{2}$O$_{8}$ (Bi-2212) has been studied extensively with these techniques, and this superconductor will be referred to in the present work.\
\
In PCT[@JZ], the energy of the peaks in the conductance (dI/dV) curves of superconducting-insulator-superconducting (SIS) break junctions in Bi-2212 crystals yield superconducting gap values which correlate with the doping dependence of the superconducting transition temperature T$_{C}$ for optimal to overdoped samples. The measured superconducting gaps can also be understood semi-quantitatively using a strong coupling superconductivity model [@LC]. However, in underdoped Bi-2212 crystals, the gap values measured from the SIS conductance dI/dV peaks continue to increase to large values even as the T$_{C}$ values decrease to zero. This suggests that the superconducting gap is not solely determining the dI/dV peak positions, but that the pseudogap is being measured in this doping range. Furthermore because the measured pseudogap increases while at the same time T$_{C}$ is decreasing to zero, one interpetation of the underdoped SIS measurements is that the pseudogap is not a precursor superconducting pairing phenomenom, but is instead a competing effect.\
\
In STM experiments [@Psh; @Boy; @Fuj], the measured conductance (dI/dV) is closely related to the superconducting density of states. In the optimal to overdoped Bi-2212 samples, the STM measurements reveal a conductance with a single peak, from which the value of the superconducting gap can be estimated. In the underdoped regime, two features are seen in the dI/dV curves: a low energy shoulder-like feature which is likely associated with the superconducting gap, and a high energy peak associated with the pseudogap, similar to the SIS measurements.\
\
In the SIS measurements, the dip above the main dI/dV peak, which has been modelled as evidence of the superconducting pairing mechanism [@LC], disappears in increasingly underdoped samples[@JZ]. This behavior is also seen in some STM experiments[@Psh].\
\
Angle Resolved Photoemission Spectroscopy (ARPES) has provided information on the properties of both the superconducting gap and pseudogap along the Bi-2212 Fermi surface [@Yosh; @Kondo]. One interpetation of these ARPES measurements leads to a pseudogap which has a maximum at the Fermi surface antinodal point (the location of the maximum of the superconducting d-wave gap), and which decreases to zero at a point between the antinodal point and the d-wave superconducting gap node. In the normal state, this leads to a pseudogap state involving a gapless arc of states along the Fermi surface, centered about the nodal point of the d-wave superconducting gap (which develops below T$_{C}$). The resulting normal state density of states displays a reduction in states around the Fermi energy similar to experiment. Another interpetation of ARPES measurements suggests the existence of pockets on the Fermi surface in the pseudogap (underdoped) state [@Pdj]. This supports a theoretical model[@Yrz] based on the RVB theory which explains the pseudogap regime as arising from a complex reconstruction of the Fermi surface involving the development of electron and hole pockets at the antinodal and nodal regions respectively. The shrinking of the hole pockets with underdoping causes increasing pseudogap behavior in transport and thermal properties[@Carb1]. The interpetation of the ARPES and STM experiments is an area of active research[@Koh; @Pdc].\
\
Numerical results are presented here for the density of states, and the SIS conductance, in which a pseudogap of non-superconducting origin is included in the strong coupling model that has been used to describe the optimal to overdoped regime of Bi-2212[@LC]. Extending this model into the underdoped phase of Bi-2212 is achieved via the magnitude chosen for the pseudogap in the calculation, assuming it to be directly determined by the doping level, and to increase from zero at optimal doping as the superconductor becomes increasingly underdoped. An analogous connection between doping and the pseudogap magnitude is also incorporated in other work[@Yrz]. In that approach, the pseudogap has a doping dependence given by $0.3t(1-x/0.2)$ where $t$ is the tight binding hopping parameter, $x$ is the hole doping, and $x=0.2$ is used to denote optimal doping from which the pseudogap is assumed to start from zero (see figure 2(e) [@Yrz]).\
\
The phenomenological strong coupling model used in this work captures many experimentally measured features of tunneling data in the superconducting state of Bi-2212 over the whole doping range from under to over doped. The success of the present approach depends on the form of the pairing spectral function, or [*pairing glue*]{}, used in the calculations. This is discussed in the following section of this manuscript.\
\
The equations are solved self consistently for the resulting superconducting gap and density of states. An increasing pseudogap suppresses the superconducting gap to zero, and leads to features in the density of states and trends in the SIS conductance curves that are similar to the STM and PCT experiments.\

\
\
The strong coupling model equations to be solved self-consistently are given by\
$$\begin{aligned}
\Delta(\omega) &=& \frac{1}{Z(\omega)}\displaystyle\int_{0}^{\omega_{c}}d\nu
\int_{0}^{2 \pi} \frac{d \phi}{2 \pi} c_{\Delta} {\rm Re} \bigg\{  \frac{\Delta(\nu) {\rm cos}^{2}(2 \phi)}{[ \nu^{2} - \Delta^{2}(\nu) {\rm cos}^{2}(2 \phi) - \Delta_{PG}^{2}(\phi)]^{1/2}}\bigg\} \nonumber \\
&\times&
\displaystyle\int_{0}^{\omega_{c}}d\omega ' F(\omega ')
\bigg[ \frac{1}{\omega + \omega ' + \nu + i\delta} - \frac{1}{\omega - \omega ' - \nu +i\delta}\bigg] \nonumber \\\end{aligned}$$ and\
\
$$\begin{aligned}
[1-Z(\omega)]\omega &=& \displaystyle\int_{0}^{\infty}d\nu \int_{0}^{2 \pi} \frac{d \phi}{2 \pi}
c_{Z} {\rm Re} \bigg \{ \frac{\nu}{[ \nu^{2} - \Delta^{2}(\nu){\rm cos}^{2}(2 \phi) - \Delta_{PG}^{2}(\phi) ]^{1/2}} \bigg \} \nonumber \\
&\times&
\displaystyle\int_{0}^{\omega_{max}} d\omega ' F(\omega ')
 \bigg [ \frac{1}{\omega + \omega ' + \nu + i\delta}
  + \frac{1}{\omega - \omega ' -\nu + i\delta}\bigg ] \nonumber \\
 \nonumber \\\end{aligned}$$

Equations (1) and (2) are derived from the standard Eliashberg type equations using a phenomenological [*pairing glue*]{} given by $$[c_{Z} \; + \; c_{\Delta} {\rm cos}(2(\phi \; - \; \phi^{'}))]F(\omega)$$ This function replaces the standard $\alpha^{2}F(\omega)$ phonon spectral weight in conventional Eliashberg theory. In applying the present model to high temperature superconductivity, $F(\omega)$ in equation (3) describes the frequency dependent spectral weight of spin fluctuations that are assumed to be the origin of superconducting pairing. The $F(\omega)$ used for the present work are shown in figure (1)

![The [*pairing glue*]{} spectral weight $F(\omega)$ for different pseudogap values $\Delta_{0}^{PG}$ indicated on the figure.[]{data-label=""}](Figspec.eps)

The prominent peak in $F(\omega)$ is the spin resonance mode measured in inelastic neutron scattering (INS) which emerges on entering the superconducting state in Bi-2212 [@Brgs].The position in energy of this peak in meV sets the energy scale, and magnitude in meV of quantities such as the superconducting gap, in the calculation. The ${\rm cos}(2(\phi \; - \; \phi^{'})$ factor leads to a d-wave symmetry superconducting gap described by $$\Delta_{SC}(\nu,\phi) \; = \; \Delta(\nu) {\rm cos}(2 \phi)$$ $\phi$ denotes the angular position on the Fermi surface. The function $\Delta(\nu)$ is a complex function of frequency determined by the self consistent solution of the equations (1) and (2).\
\
The $\phi$ dependent pseudogap $\Delta_{PG}(\phi)$ is modelled with an angle dependent function which is a maximum at the antinode ($\phi = 0$), decreasing to $0$ at an angle $\phi_{C}$ before the d-wave node at $\phi= \pi/4$ is reached $$\Delta_{PG}(\phi) \; = \; \Delta_{0}^{PG}(1 - (\phi/\phi_{C})^{2}) \; \; \; 0 \; < \; \phi \; < \; \phi_{C}$$

The value of $\Delta_{0}^{PG}$ is a real valued input parameter. $\phi_{C}$ is fixed at 0.45 radians. Figure (2) depicts both the angular dependence of both gaps on the Fermi surface.

![Angle dependence ($\phi$) of the d-wave and pseudogap along the Fermi surface.[]{data-label=""}](Figgap.eps)

The resulting $\Delta_{SC}(\nu,\phi)$ are used to calculate the density of states using $$N(\nu) \; = \; \int_{0}^{2 \pi} \frac{d \phi}{2 \pi}
{\rm Re} \bigg \{ \frac{\nu}{[ \nu^{2} - \Delta^{2}(\nu){\rm cos}^{2}(2 \phi) - \Delta_{PG}^{2}(\phi) ]^{1/2}} \bigg \}$$ The SIS current-voltage curve is calculated the usual way, using a tunnelling density of states $N^{T}(\nu)$ that includes a directional tunneling matrix [@LC] $$I(eV) \; = \; \int^{\infty}_{-\infty} d \nu N^{T}(\nu +eV)N^{T}(\nu)[f( \nu )-
f( \nu +eV)]$$ The role of the tunneling directionality factor is required to generate the large peak height to high bias background that is actually seen in the SIS break junction experiments. The SIS conductance curves (dI/dV) are obtained by numerical differentiation.\

\
\
Figures (3) and (4) show weak coupling calculations of the density of states, and the corresponding SIS conductance (dI/dV), for the case of a combined d-wave superconducting gap and pseudogap. The superconducting gap function $\Delta(\nu)$ is set equal to a real constant $\Delta_{0}$ in equations (6) and (7). The values chosen for $\Delta_{0}$ and the pseudogap parameter $\Delta _{0}^{PG}$ (see equation (5)) are indicated on the figures.\
\
For $\Delta_{0} > \Delta_{0}^{PG}$, the presence of a non-zero pseudogap results in a small break in slope at an energy below the main peaks in both figure (3) and (4). Therefore, for this case, there is a small impact due to the presence of a pseudogap on the shape of the density of states, or the SIS conductance. When $\Delta_{0}  <  \Delta_{0}^{PG}$, a [*shoulder*]{} feature is visible in the density of states at an energy below that of the main peak in figure (3). This is the first noticeable evidence in the density of states indicating the presence of two gaps. The location of the main peaks in both density of states and SIS conductance in figures (3) and (4) are determined by the factor $( (\Delta_{0})^{2} + (\Delta_{0}^{PG})^{2})^{1/2}$. Therefore, identifying the energy of either the superconducting gap, or the pseudogap, with the energy of the peak is correct only in the special cases of zero superconducting gap or zero pseudogap. When both gaps co-exist, the peak is determined by a combination of both gap values. This would be true not only for the present model for incorporating the pseudogap but also in other models [@Tmr]. Note that the [*shoulder*]{} that develops below the main SIS peak in figure (4) is unrelated to a similar [*shoulder*]{} feature in the density of states seen in figure (3). This can be seen clearly in figure (5) where the conductance (dI/dV) from equation (7) is shown for a pure pseudogap case (with $\Delta_{0}^{PG} = 1.7$). The energy of the [*shoulder*]{} feature in figure (5) is at half the energy of the main peak (located at $2\Delta_{0}^{PG} $). The [*shoulder*]{} feature in figures (4) and (5) arises from the angular dependence of the pseudogap (see equation (5)). Similarly, there is also a small break in the slope of the SIS dI/dV for a pure superconducting d-wave case at the energy of half the main SIS peak. Weak coupling calculations[@Carb2] of the density of states using the electron/hole pocket model [@Tmr] display similar [*shoulder/small peak*]{} features.\
\

![Weak coupling density of states[]{data-label=""}](Figscpgdos.eps)

![Weak coupling SIS conductance dI/dV []{data-label=""}](FigSISwc.eps)

![Conductance dI/dV for pure pseudogap state using eqn (7) ](FigSISPG.eps)

Strong coupling results are shown in figures (6), (7) and (8) for the superconducting gap, the density of states, and the SIS conductance using equations (1) and (2) for a range of values of the pseudogap parameter $\Delta_{0}^{PG}$.\
\
In the present work, the pseudogap $\Delta_{0}^{PG}$ is assumed to start at zero at optimal doping in the case of Bi-2212, and to increase monotonically upon moving into the underdoped regime. Figure (6) shows the effect of the increasing pseudogap on the superconducting gap. The superconducting gap being referred to here is obtained from the usual strong coupling criterion as the energy $E$ where $E= {\rm Real} \Delta(E)$.\
The starting case $\Delta_{0}^{PG}=0$, along with the $F(\omega)$ for $\Delta_{0}^{PG}=0$ shown in Figure (1), was used previously to describe optimal doped Bi-2212 [@LC]. In this case, the position of the peak in the $F(\omega)$ of figure (1) corresponds to 40meV which is the value approximately of the spin resonance mode energy detected by INS in Bi-2212 [@Brgs]. The shape of $F(\omega)$, and the values of $c_{S}=0.14$ and $c_{\Delta}=0.8$, yield a superconducting gap of slightly less than 40meV for this optimally doped case in agreement with SIS measurements [@JZ].\
\
The superconducting gap is suppressed to zero when the pseudogap reaches 1.7 in the units shown in figure (6). Again, applying this to Bi-2212, where 40 meV corresponds to unity in figure (6), this would mean a pseudogap of about 70meV coincides with the suppression of $T_{C}$, and the corresponding superconducting gap, to zero, consistent with experimental results[@Huf].

![Superconducting gap versus increasing pseudogap $\Delta_{0}^{PG}$ (eqn (5))[]{data-label=""}](FigscpgNw.eps)

The results for increasing pseudogap $\Delta_{0}^{PG}$ shown in figures (6), (7) and (8) are calculated using the $F(\omega)$ of figure (1). The coupling constants are kept at the constant values $c_{S}=0.14$ and $c_{\Delta}=0.8$ throughout the calculations The upward shift in energy of the main peak in $F(\omega)$ spectral weight, as shown in figure (1) is similar to theoretical results for the spin fluctuation Im$\chi(\omega)$ in numerical studies on the Hubbard model [@Trem]. (see figure (1)(b) and accompanying discussion in reference (18)).\
Figure (7) shows the evolution of the density of states as the superconductor evolves from the optimally doped case ($\Delta_{0}^{PG}=0$) to complete suppression of superconductivity ($\Delta_{0}^{PG}=1.7\simeq 70$meV (Bi-2212)). As the superconductivity is suppressed, the peak in the density of states moves to higher energy, driven by the increasing pseudogap energy. A [*shoulder*]{} feature develops at low energy, the position of which is related to the magnitude of the self consistently generated superconducting gap. The position of this feature is not at the same energy as the superconducting gap, however. The dip feature gradually weakens, and disappears in the pure pseudogap state. These trends are similar to the STM experiments.\
\
Figure (8) shows the SIS conductance curves corresponding to figure (7). The main SIS peak increases with energy, driven by the increasing pseudogap, and also broadens significantly. The dip feature above the main peak weakens, and is not present in the pure pseudogap conductance. This latter behavior is seen in SIS experiments [@JZ] in the large pseudogap (very underdoped) state. This indicates that the physics of the pseudogap formation is not coupled to the physical mechanism causing the dip feature in the superconducting state, which in the latter case is the spin resonance mode. The absence of the dip feature in the SIS experimental measurements also lends support to the present model where a frequency independent pseudogap, defined in equation (5), is used in the model. The [*shoulder*]{} feature that is present in the curves in figure(8) is not seen in the experimental SIS peaks [@JZ] possibly due to the significant broadening of the experimental peaks.\
\

![Strong coupling density of states (eqn (6))[]{data-label=""}](FigDosN1.eps)

![Conductance dI/dV (eqn (7)) corresponding to Figure (7)](FigSISsc4.eps)

Figure (9) shows a plot for comparison of the superconducting gap magnitude calculated from the strong coupling equations (see figure (6)) and the gap magnitude obtained from taking the energy of the main peak in the SIS conductance curves (figure (8)) and dividing by two. The two curves diverge from each other as the pseudogap increasingly determines the peak position in figure (8) for larger pseudogap magnitudes (deeper into the underdoped region).\
\
[**Summary**]{}\
\
A strong coupling model, which has been used previously to analyze SIS break junction experiments on optimal to overdoped Bi-2212 [@LC], is modified to include a pseudogap in the electronic spectrum. The resulting density of states and SIS conductance curves compare favorably with experimental measurements on underdoped Bi-2212. Underdoping is simulated in the calculation by increasing the magnitude of the input pseudogap, assuming that the pseudogap increases as doping is decreased in the underdoped state.

![Comparison of strong coupling superconducting gap (red curve) (plotted in Figure (6)) and half the energy of the SIS peak from Figure (8) (green curve)](Figpeak.eps)

[99]{}

T. Timusk and B. Statt, Rep. Prog. Phys. [**62**]{}, 61 (1999).

S. Hufner, M.A. Hossain, A. Damascelli and G.A. Sawatsky, Rep. Prog. Phys. [**71**]{} 062501 (2008)

T.M. Rice, K. Yang, and F.C. Zhang, arxiv.org/pdf/1109.0632.pdf

J. F. Zasadzinski, L. Ozyuzer, N. Miyakawa, K. E. Gray, D.G. Hinks, and C. Kendziora, Phys. Rev. Lett. [**87**]{}, 067005 (2001). J. F. Zasadzinski et al (unpublished)

O. Ahmadi, L. Coffey, J. F. Zasadzinski, N. Miyakawa and L. Ozyuzer, Phys. Rev. Lett. [**106**]{}, 167005 (2011)

A. Pushp, C.V. Parker, A.N. Pasupathy, K.K. Gomes, S. Ono, J. Wen, Z. Xu, G. Gu and A. Yazdani, Science, [**324**]{}, 1689 (2009).

M. C. Boyer, W.D. Wise, K. Chatterjee, M. Yi, K. Kondo, T. Takeuchi, H. Ikuta and E.W. Hudson, Nature Physics, [**3**]{} 802 (2007)

K. Fujita, A. R. Schmidt, E. Kim, M.J. Lawler, D.H. Lee, J.C. Davis, H. Eisaki and S. Uchida, J. Phys Soc. Jpn. [**81**]{}, 011005 (2012)

T. Yoshida, M. Hashimoto, I. M. Vishik, Z.X. Shen and A. Fujimori, J. Phys. Soc. Japan, [**81**]{}, 011006 (2012)

T. Kondo, R. Khasanov, T. Takeuchi, J. Schmalian, and A. Kaminski, Nature, [**457**]{}, 296 (2009)

H.B. Yang, J. D. Rameau, Z.H. Pan, G.D. Gu, P.D. Johnson, H. Claus, D. G. Hinks and T.E. Kidel, Phys. Rev. Lett., [**107**]{} 047003 (2011)

K. Y. Yang, T.M. Rice, and F.C. Zhang, Phys. Rev. B. [**73**]{} 174501 (2006)

P. E. C. Ashby and J. P. Carbotte, Phys. Rev. B [**87**]{} 014514 (2013)

Y. Kohsaka, C. Taylor, P. Wahl, A. Schmidt, J. Lee, K. Fujita, J.W. Alldredge, J. Lee, K. McElroy, H. Eisaki, S. Uchida, P.H. Lee and J.C. Davis, Nature, [**454**]{}, 1072 (2008).

P. D.C. King, J.A. Rosen, W. Meevasana, A. Tamai, E. Rozbicki, R. Comin, G. Levy, D. Fournier, Y. Yoshida, H. Eisaki, K.M. Shen, N.J.C. Ingle, A. Damascelli and F. Baumberger, Phys. Rev. Lett, [**106**]{}, 127005 (2011). arxiv.org/pdf/1105.1862.pdf

H.He, Y. Sidis, P. Bourges, G.D. Gu, A. Ivanov, N. Koshizuka, B. Liang, C. T. Lin, L.P. Regnault, E. Schoenherr, and B. Keimer, Phys. Rev. Lett. [**86**]{} 1610 (2001)

A. J. H. Borne, J. P. Carbotte and E.J. Nicol, Phys. Rev. B [**82**]{}, 024521 (2010)

B. Kyung, D. Senechal, A.M.S. Tremblay, Phys. Rev. B [**80**]{}, 205109 (2009).


======================
Customizing CKAN's CSS
======================

.. seealso::

   There's nothing special about CSS in CKAN, once you've got started with
   editing CSS in CKAN (by following the tutorial below), then you just use the
   usual tools and techniques to explore and hack the CSS. We recommend using
   your browser's web development tools to explore and experiment with the CSS,
   then using any good text editor to edit your extension's CSS files as
   needed. For example:

   `Firefox developer tools <https://developer.mozilla.org/en-US/docs/Tools>`_
     These include a Page Inspector and a Style Editor

   `Firebug <https://www.getfirebug.com/>`_
     Another web development toolkit for Firefox

   `Chrome developer tools <https://developers.google.com/chrome-developer-tools/>`_
     Tools for inspecting and editing CSS in Google Chrome

   `Mozilla Developer Network's CSS section <https://developer.mozilla.org/en-US/docs/Web/CSS>`_
     A good collection of CSS documentation and tutorials

Extensions can add their own CSS files to modify or extend CKAN's default CSS.
Create an ``example_theme.css`` file in your extension's ``public`` directory::

    ckanext-example_theme/
      ckanext/
        example_theme/
          public/
            example_theme.css

Add this CSS into the ``example_theme.css`` file, to change the color of CKAN's
"account masthead" (the bar across the top of the site that shows the logged-in
user's account info):

.. literalinclude:: /../ckanext/example_theme_docs/v13_custom_css/public/example_theme.css
   :language: css

If you restart the development web server  you should be able to open this file
at http://127.0.0.1:5000/example_theme.css in a web browser.

To make CKAN use our custom CSS we need to override the ``base.html`` template,
this is the base template which the templates for all CKAN pages extend, so if
we include a CSS file in this base template then the file will be included in
every page of your CKAN site. Create the file::

    ckanext-example_theme/
      ckanext/
        example_theme/
          templates/
            base.html

and put this Jinja code in it:

.. literalinclude:: /../ckanext/example_theme_docs/v13_custom_css/templates/base.html
   :language: django

The default ``base.html`` template defines a ``styles`` block which can be
extended to link to custom CSS files (any code in the styles block will appear
in the ``<head>`` of the HTML page).

Restart the development web server and reload the CKAN page in your browser,
and you should see the background color of the account masthead change:

.. image:: /images/custom-css.png
   :alt: The account masthead with some custom CSS.

This custom color should appear on all pages of your CKAN site.

Now that we have CKAN using our CSS file, we can add more CSS rules to the file
and customize CKAN's CSS as much as we want.
Let's add a bit more code to our ``example_theme.css`` file. This CSS
implements a partial imitation of the `datahub.io <http://datahub.io/>`_ theme
(circa 2013):

.. image:: /images/more-custom-css.png
   :alt: A partial imitation of the datahub.io theme, circa 2013.

.. literalinclude:: /../ckanext/example_theme_docs/v14_more_custom_css/public/example_theme.css
   :language: css


His stats aren't eye popping -- 23-of-42 passing for 270 yards, one touchdown and one interception -- but the young quarterback was able to stretch the defense and made some pinpoint throws when he needed them late, including the go-ahead touchdown in the final minutes of regulation. Given than his No. 1 receiver didn't help him out until the final drive of the fourth quarter (Demaryius Thomas: one catch on 12 targets), it's a wonder Osweiler played as well as he did.


HelenOS 0.8 - return_0e
http://www.helenos.org/wiki/ReleaseNotes/0.8.0
======
jayalpha
Lot of new stuff coming up. Also see:

[https://harvey-os.org/](https://harvey-os.org/)

[https://www.redox-os.org/](https://www.redox-os.org/)

~~~
synchronise
Just had a look over Harvey OS, looks like a pretty neat direction to take
Plan 9.

But it got me wondering, is there any shared collaberation between any of the
next-gen 'Plan 9-like' OS's like 9atom, 9front and Harvey OS?

Also, it looks as though Harvey OS is only using Slack for its discussion
forum, which is a bit odd for an open source project.

~~~
aeneasmackenzie
9front views HarveyOS with scorn and I don't think 9atom is developed.

~~~
em-bee
why is that? got any references?

~~~
aeneasmackenzie
HarveyOS is "plan 9 with gcc" but 9front doesn't like gnu stuff. See cat-v,
suckless, and their IRC channel.

~~~
em-bee
ah, yes, i can see the problem.

------
jacques_chester
For those who, like me, wondered "what's HelenOS?" and got little idea from
release notes:
[http://www.helenos.org/wiki/About](http://www.helenos.org/wiki/About)

~~~
warent
Tiny nitpick but the screenshot showing an application window rotated ~45
degrees is a little terrifying. Why would that be allowed?

~~~
reaperducer
You assume that everyone has their monitors the way you do.

At work, I have two large 16:9 monitors rotated in portrait orientation
(tall), so this would be useful to me.

Currently macOS only allows 90-degree rotation increments. This allows you to
have your monitor in portrait orientation, or mounted from the ceiling or a
shelf, hanging upside down so there's nothing taking up space on the desk in
front of you.

I've been in a couple of events in the field where having arbitrary
application window rotation would have been nice because there was no place
flat to put the monitors.

~~~
nolok
> Currently macOS only allows 90-degree rotation increments.

Windows has supported landscape/portrait switching since forever, I think it
was already there in windows XP almost two decades ago, if not it was in 7 for
sure (and it's in 10 since I use it right now).

~~~
reaperducer
_Windows has supported landscape /portrait switching since forever, I think it
was already there in windows XP almost two decades ago, if not it was in 7 for
sure (and it's in 10 since I use it right now)._

I wasn't trying to make a comparison to Windows, or start an OS war. I'm not
sure why you even brought up Windows, since this discussion is about HelenOS.

But for what it's worth, Macs have supported portrait mode since 1989†, back
when Microsoft was still pushing Windows 2.

†
[https://everymac.com/monitors/apple/classic_monitors/specs/a...](https://everymac.com/monitors/apple/classic_monitors/specs/apple_mac_portrait.html)

~~~
nolok
My bad, for some reason I read "only macOS" instead of "macOS only"

------
ianai
I like the concept. It sounds like a modern take on some of the plan9 ideas.
Decoupling of system components into components that work together through a
message passing framework.

~~~
jpm_sd
Sounds a bit like QNX actually

~~~
xyproto
Is QNX used anywhere? Sounds interesting.

~~~
nopetrain
Lots of infotainment in cars use QNX, along with aviation.

~~~
jpm_sd
Also medical devices

------
karmakaze
Is there a 'distrowatch' for OSes other than Linux/BSD-based? I can't keep up
with them all anymore.

~~~
protomyth
osnews.com was once pretty good, but I haven't read it in a while and they did
have some recent problems.

~~~
SyneRyder
Yikes, it does seem like OSnews have had a rocky few days. (TLDR: _" It
certainly seems like we’ve had a breach."_)

[https://www.osnews.com/story/128924/what-happened-
here/](https://www.osnews.com/story/128924/what-happened-here/)

To their credit, it looks like they're now trying to find ways to run the site
profitably, so they can pay people to maintain the site part/full-time instead
of relying on volunteer help.

------
dang
From 2017:
[https://news.ycombinator.com/item?id=15046249](https://news.ycombinator.com/item?id=15046249)

------
rcarmo
Interesting. Anyone got this running on a Raspberry Pi?

------
born2discover
How is it different from [Minix3][1]? It seems to accomplish the exactly same
goal.

[1]([https://www.minix3.org/](https://www.minix3.org/))

~~~
adamnemecek
You mean because they are both microkernels? In that sense windows and Linux
accomplish the same goal as well.

------
flag_bcz_mad
What kinds of tasks are slower on a micro-kernel? Are there use-cases where a
micro-kernel is just as fast as a monolithic?

~~~
Tuna-Fish
> What kinds of tasks are slower on a micro-kernel?

Crossing from usermode to kernel is slower, and generally doing complex things
in kernel is much slower. (Where complex means cross-cutting concerns, so
multiple different kernel processes need to be involved.)

> Are there use-cases where a micro-kernel is just as fast as a monolithic?

Anything that doesn't really involve the kernel much at all, and things that
take a lot of time in kernel but do so in one part of it with no need for
input from other parts.

Generally, when you go for a microkernel you need to accept that it will be
slower than monolithic. It might still be worth it for the advantages of
microkernels, namely being easier to verify, understand and secure.


I grew up with television. I never remember not owning a color television. We had cable for most of my life. I remember looking forward to weekends at my sister's house where they had HBO and we chowed McDonalds to movies featuring nudity and profanity. Good times. When I bought my own home, my first call was to the cable provider. When they told me installation would be $5000 (in 1990), I installed an antenna. At that time, Pats home games were generally blacked out, so everyone [who cared] came to my home on Sundays as I could pull in a Maine station that was outside the blackout area.

When my first born started kindergarten, we decided it would disadvantage him to not know what Sponge Bob Square Pants was all about, so we hired Dish. Dish was fine, but as we started paying separately for phone, local long distance, long distance, 'technology', high speed internet, and cell phones, the cost of communications and entertained topped $300/month at my house. Comcast consolidated everything except my cell service for $99.99/month...for 24 months.

At the end of the 24 months, my bill increased dramatically. I offered them $149.99 for my package if they guaranteed the price for life. They countered with a bare bones package for 6 months for $99.99. I took this as a challenge. I immediately purchased an OOMA Hub and Scout VOIP hub. It cost me $205, but home phone has been free since October of 2009. So, $1.80/month for unlimited local and long distance calling. I called Comcast and told them I wanted to drop phone service (on my bill at $50/month) and they took it off, but the savings was offset by loss of my triple play discount. In January of 2010, I purchased a DB8 antenna from Amazon for $90.22. When we lost power and cable for a week in February, I plugged in my generator and hooked up my new antenna. We watched the winter Olympics via an antenna. Good stuff. When cable came back, I left my television on the antenna. At the same time, Comcast began their transition to digital. We had digital televisions, but they started moving the 'good stuff' to channels that required a box. Local broadcast television was SD without a box. Within a few weeks, both of my kids asked to have their televisions put on the antenna. The wife was a holdout -- we had a DVR in the living room and food network was the background music of her life. By April, she had warmed up to the PBS Create channel and we decided to fire Comcast.

Fairpoint offered me unlimited internet for $49.99/month guaranteed for life. So, we were saving $100/month off the bare bones post-promotion Comcast price. In the first year, I spent $1000 on five DTVPal DVRs. I broke even in the first year, but had DVRs in all three bedrooms, the living room, and the kitchen. DVRs would be my biggest expense going forward. I've spent about $3000 on DVRs over nine years -- $333/yr, $28/mo, $5,56/mo/set.

When we fired Comcast, we did not think we needed a DVR. The one DVR in the house was loaded with unwatched episodes of Bonnie Hunt and Who Wants to be a Millionaire. It did not take long for us to fully appreciate the DVR -- pausing for dinner or a phone call, rewinding after a snooze or to re-see something that could not be unseen. Putting a DVR on each television dramatically enhanced the television viewing experience. I am very happy with my DVR+ DVRs and TiVos, but I just bought a Recast and a Tablo TV DVR.

Right now, I have DirecTV at a steeply discounted price as a consequence of having an obscenely expensive cell phone plan. I'm halfway through the promotion and will not likely have the expensive phone plan after that, but, for now, I have a lot of channels including HBO. I still mostly watch broadcast television and my kids mostly watch Netflix.

If you want to know what I have spent or played with over the nine years, take a look at this page...

So, I saved $11,400 and spent $6,000 on infrastructure and toys netting about $50 per month in savings. Not much savings really, but I would not trade what I have for what I had. In fact, once when I was researching Comcast prices for a blog post, my wife walked in and let me know she was not willing to give up the antenna for Comcast.

So, what exactly do you get with an antenna? That depends on your location and your antenna. I recommend people interested in broadcast television start at http://tvfool.com/. A report for you address or coordinates will help you understand how easy or difficult antenna reception will be at your home. Google the wikipedia page for each station you are likely to receive to see if there are sub-channels. Pick and point an antenna and you are good to go.

If you want help analyzing a TVFool report, selecting an antenna, or cutting the cord, PM me or respond to this post.

Most people want to know what I would buy if I were cutting the cord right at this moment. For me, right now, I think I would get an Amazon Recast and a bunch of Fire TV Sticks. In fact, I have done that. It's not for everyone, but it works for me. I have had Amazon Prime for some time. Mostly for shipping, but I enjoy the streaming media and use a photo library as the background for my Amazon Shows. I have a Recast, so I am able to watch television on my Shows (I have seven) or any device that is attached to a Fire TV Stick. The Recast only supports two concurrent streams, but that is plenty as my kids tend to watch Netflix. Some things I like about the Prime solution...

Let's say your family enjoys some of the cable channels, network programming, and HBO for Game of Thrones. You get a Recast for $200, get a pair of 4k Fire TV Sticks for $100, pay $50/month for an ISP, $119/year for Prime, and $15/month for HBO. $300 up front and $75/month. Add Philo TV for $16/month for a $91 'skinny' cable killer.

Most of the cord cutters I know simply run a cable from an antenna to a digital television. That is FREE TV. Respect! If you are thinking along those lines, consider a TCL Roku television. These are the best televisions for cord cutters. They get guide data from the broadcast stream (PSIP) so there is no need for an ISP or phone line to get updated guide data. If you plug a USB stick into one of these, you can pause, rewind, and fast forward through four hours of programming. If the USB stick is loaded with pictures, music, or videos, you can play those. If you have come to rely on a smart phone for online activity and are thinking you need to keep your high speed internet for streaming entertainment, think again.

I own five TiVo Roamio OTAs. Awesome set top box. I paid $300 for my first three with Lifetime and $200 for the last two. Great set top box. The Mini is the best implementation of remote access I have seen. I wish I could recommend a TiVo, but the only OTA TiVo is out of stock and $500 with 'All In' service plan. Add a Mini for a second set and you are in almost $700! As much as I paid for my last three TiVos combined. Keep an eye on Woot for deals on the Roamio/OTA and previous gen Minis.

What are your thoughts on Roku vs. Fire Sticks? I have both.

I have side-loaded Kodi onto the Fire Stick to get the "free everything", but the trouble I encountered with that is it is more of a hobby. It constantly broke, or it would lag. It was difficult to watch sometimes from all of the buffering. I would tweak it to get it working good, then a few days later I would need to tweak it again. It seemed every few months I would have to try a different app. It was too difficult to keep going.

For everyone (most people) who will be using it without side-loading anything, the Fire Stick seems to be rather limited. It streams from Amazon great. You can do Netflix on it. There are a handful of other apps you can easily load. That's about it.

The Roku is a different animal. It has many free channels you can easily load. It even has "hidden" channels that you can research if you are interested in "more". There doesn't seem to be any legal issues with any of the channels I have dealt with, unlike the side-loaded apps on the Fire Stick.

The Roku has the Roku channel. It also has Netflix, Hulu, HBO, ESPN, and, yes, even Amazon. It has the typical subscription channels. It even offers a few exclusive ones. My wife appreciates TFC, one of the exclusive channels. So with the Roku, you can get EVERYTHING you can with the Fire Stick, plus much, much more without the need to constantly tweak it. It just works.

I have both. I don't love either. Roku has too many products and too few programmers. Once a device falls from Flagship status, it is an afterthought. I have found that every new software update leads to a period of instability. Roku makes things worse by forcing updates. I like the current 4K Fire TV stick. They got CEC right and the stick is fast. Still don't love it.

I have side-loaded Kodi onto the Fire Stick to get the "free everything", but the trouble I encountered with that is it is more of a hobby. It constantly broke, or it would lag. It was difficult to watch sometimes from all of the buffering. I would tweak it to get it working good, then a few days later I would need to tweak it again. It seemed every few months I would have to try a different app. It was too difficult to keep going.

Amen. My opinion on OTT all around is that everything is always a little bit broken. Great for hobbyists, but low WAF.

For everyone (most people) who will be using it without side-loading anything, the Fire Stick seems to be rather limited. It streams from Amazon great. You can do Netflix on it. There are a handful of other apps you can easily load. That's about it.

I'm preparing a blog post on this idea. Nothing in the Amazon ecosystem thrills me, but I like the whole of it a lot. I'll link to the post when it goes up.

The Roku is a different animal. It has many free channels you can easily load. It even has "hidden" channels that you can research if you are interested in "more". There doesn't seem to be any legal issues with any of the channels I have dealt with, unlike the side-loaded apps on the Fire Stick.

The Roku has the Roku channel. It also has Netflix, Hulu, HBO, ESPN, and, yes, even Amazon. It has the typical subscription channels. It even offers a few exclusive ones. My wife appreciates TFC, one of the exclusive channels. So with the Roku, you can get EVERYTHING you can with the Fire Stick, plus much, much more without the need to constantly tweak it. It just works.

I like the Roku channel. It doesn't have local channels or a DVR. With the Recast and the ability to integrate Cinemax ($9.99/mo), HBO ($14.99/mo), Showtime ($8.99/mo), and Starz ($8.99/mo) plus many other channels Amazon is really providing a la carte options. Vue and Philo skinny cable packages can be integrated into the Fire TV grid style guide as well.

What are your thoughts?

I'm no Roku fan. (Been banned from their forums four times, as I recall.) It's strength (independence) it also it's weakness. Things could be better now. The last Roku I bought was a Roku 2XS. Had a bunch. Awesome except when they weren't. No fan of Amazon either, but for $400 up front and $70/month, you can build something special with the Prime Ecosystem.

Bring a pot of salted water to a boil in a large saucepan while you peel and cut the potatoes. Blanch the potatoes in the boiling salted water for 4 minutes while you pre-heat the air fryer to 400ºF. Strain the potatoes and rinse them with cold water. Dry them well with a clean kitchen towel.

Toss the dried potato sticks gently with the oil and place them in the air fryer basket. Air-fry for 25 minutes, shaking the basket a few times while the fries cook to help them brown evenly.

While the fries are cooking, combine the ingredients to make the Cajun seasoning. With 5 minutes left on the air fryer, sprinkle the potatoes with the Cajun seasoning and drizzle in a little more oil. Toss evenly to coat the fries and continue to air-fry for the final minutes.

To make the Creole Dipping Sauce, combine all the sauce ingredients in a small bowl and mix well. Serve the Cajun Fries hot with Creole Dipping sauce on the side.

Wow, I'm honored!
I received this via the My Chart app from one of the nurses at my reconstructive surgeon's office. I knew my surgeon was really pleased with my results via keto, but didn't expect to become a mentor for others.

"Good Morning Pam,

Dr. Colohan has a new patient who would like to speak with another patient about their experience with DIEP reconstruction and starting the Ketogenic diet. Would you be willing to share your experience with another patient? If so, please let us know if you would prefer email or a call.

Checking in for May. Only 17 days of school left then I'm off till August!

On another happy note, my oldest son left his job at a small-town police department and accepted a deputy position with the Hancock County, Ohio sheriff's department! The pride (and worry) I feel for this young man is unimaginable!

I completely understand.... our daughter is graduating from her police academy this month! It is different when it is your parent or spouse, your child though that brings a whole new level of prayer!

Down to 36 members after alirwin1985 dropped out last month.

May is the month I missed last year..... but, I'm here this year had to set a reminder Why are my May's always so busy? lol

Most people want to know what I would buy if I were cutting the cord right at this moment. For me, right now, I think I would get an Amazon Recast and a bunch of Fire TV Sticks. In fact, I have done that. It's not for everyone, but it works for me. I have had Amazon Prime for some time. Mostly for shipping, but I enjoy the streaming media and use a photo library as the background for my Amazon Shows. I have a Recast, so I am able to watch television on my Shows (I have seven) or any device that is attached to a Fire TV Stick. The Recast only supports two concurrent streams, but that is plenty as my kids tend to watch Netflix. Some things I like about the Prime solution...

Let's say your family enjoys some of the cable channels, network programming, and HBO for Game of Thrones. You get a Recast for $200, get a pair of 4k Fire TV Sticks for $100, pay $50/month for an ISP, $119/year for Prime, and $15/month for HBO. $300 up front and $75/month. Add Philo TV for $16/month for a $91 'skinny' cable killer.

Over the weekend, I added Philo and Starz (Prime Channel) just to see what Amazon would do...

Each channel's programming is added to my Live Channels EPG. I have TV in a room not near network or cable. I had a TiVo Mini installed using Powerline adapters. That worked fine, but I was able to unplug everything and replace it with a Fire TV Stick and that works better. For a lot less money. I was able to power the stick using a usb port on the tv, so there is not even a cord running to the power outlet.

I have both. I don't love either. Roku has too many products and too few programmers. Once a device falls from Flagship status, it is an afterthought. I have found that every new software update leads to a period of instability. Roku makes things worse by forcing updates. I like the current 4K Fire TV stick. They got CEC right and the stick is fast. Still don't love it.

So far, I have not experienced those issues. It could be that mine are still fairly new. My coworker turned me towards Roku. He has had several for several years, but he has never mentioned instability with them. Perhaps Roku is making them unstable on purpose...to get you to upgrade.

I'm preparing a blog post on this idea. Nothing in the Amazon ecosystem thrills me, but I like the whole of it a lot. I'll link to the post when it goes up.

Interesting read! Fire Sticks do seem to be a cheaper option and will integrate in with more stuff.

I like the Roku channel. It doesn't have local channels or a DVR. With the Recast and the ability to integrate Cinemax ($9.99/mo), HBO ($14.99/mo), Showtime ($8.99/mo), and Starz ($8.99/mo) plus many other channels Amazon is really providing a la carte options. Vue and Philo skinny cable packages can be integrated into the Fire TV grid style guide as well.

I'm no Roku fan. (Been banned from their forums four times, as I recall.) It's strength (independence) it also it's weakness. Things could be better now. The last Roku I bought was a Roku 2XS. Had a bunch. Awesome except when they weren't. No fan of Amazon either, but for $400 up front and $70/month, you can build something special with the Prime Ecosystem.

So far, I haven't had any issues with my Rokus. The Fire Stick was different. I purchased it several years ago (I believe it is a 3) and every time I have reset it, I have had trouble getting the remote to reconnect. When I first purchased it, I called Amazon about to return it. Shortly after they answered, the remote finally connected. I'm sure the rep thought I was not very tech-savvy, but I had just did the exact same steps multiple times to get it to go. It didn't matter if I put fresh batteries in and got up close. Perhaps I got one that skipped by QC, but it left a bad taste in my mouth.

He has had several for several years, but he has never mentioned instability with them. Perhaps Roku is making them unstable on purpose...to get you to upgrade.

No. The problem is that they target their code to their current top model and shoehorn the code onto the other devices. If you spend much time in their forums, you would be aware as it is often talked about. The Roku 2XS seemed to be particularly susceptible. I have a couple TCL Roku TVs and I love them. I use them as regular televisions and do not have internet active, so I am not vulnerable to updates.


Reduction of circular RNA expression associated with human retinoblastoma.
This study investigated the profile of circular RNA (circRNA) expression and its epigenetic role associated with human retinoblastoma (RB). Twelve paired primary samples from un-treated RB patients (primary RB samples and corresponding adjacent normal retinal samples) and eight recurrent RB samples from RB patients having recurrence after treatment were collected. Ribosomal-RNA depleted sequencing was performed in four paired primary samples. Quantitative polymerase chain reaction was conducted to validate circRNA and mRNA expression in the other eight paired primary samples and in eight recurrent RB samples. Bioinformatic analysis was applied to predict the oncological signal pathways and the binding microRNA (miRNA) of circRNA. As a result, a total of 47640 circRNAs were identified by RNA-sequencing, with a lower abundance of circRNAs in primary RB samples relative to matched normal retinal samples [22366 (47%) versus 37161 (78%), P <0.001]. Among the 11887 overlapping circRNAs in both RB and normal retinal samples, 550 circRNAs were downregulated and seven were upregulated in primary RB samples compared to normal retinal samples. The host genes of the differentially expressed circRNAs were associated with chromatin modification. TET1-has_circ_0093996 (ten-eleven translocation-1), whose host gene TET1 participates in chromatin modifying, was downregulated in both primary and recurrent RB samples. Programmed cell death 4 （PDCD4) was also downregulated in both primary and recurrent RB samples. We used bioinformatic tools to construct a complete regulatory axis including TET1-has_circ_0093996-miR-183-PDCD4 that regulates RB pathogenesis. In conclusion, circRNA expression is downregulated in RB tumor, which suggests epigenetic regulation of RB pathogenesis by circRNAs.


"I consider asbestos to be very much alive. ... We lost under the most extraordinary circumstances," Specter, the chairman of the Senate Judiciary Committee and primary sponsor of the bill, told reporters.

But analysts argue that an uncommon alliance of liberals and fiscal conservatives are likely to continue to stand in the way of meaningful action on the hard-fought legislation for at least the remainder of the year -- a disappointment for manufacturers facing asbestos-related lawsuits and a modest plus for property and casualty insurers.

The Tuesday night vote was indeed dramatic. Supporters of the legislation needed to muster a 60-vote supermajority to overcome a budget-related objection by Sen. John Ensign, R-Nev.

As the roll call wound down, supporters had 59 votes, while opponents numbered 40. But Democratic Sen. Daniel Inouye of Hawaii, who Specter says had promised to vote to overrule the objection, wasn't on the floor, having returned home to attend to his ailing wife.

After it became clear that Inouye wouldn't show, Senate Majority Leader Bill Frist, R-Tenn., changed his vote to "no," a purely parliamentary move that allows him to call up the bill again at a later date.

Another vote

Specter, R-Pa., said that Frist indicated in a Wednesday morning meeting that he was willing to revisit the bill.

"We talked about bringing it up again and having another vote," Specter said. The senator noted, however, that with the Senate now debating an extension of the Patriot Act, the asbestos bill won't be revisited until lawmakers return from next week's Presidents Day recess, at the earliest.

Meanwhile, analysts argue that the bill, which faced opposition from a mix of liberals and fiscal conservatives, is too controversial and complicated to be accommodated in an election-year Senate session.

"We believe the extremely close vote highlights the strong opposition to the trust concept, and would make it difficult for [Frist] to find the political fortitude to spend additional time on this measure, given that other priorities lie ahead," wrote Crystal Skinner, an analyst with Susquehanna Financial Group.

The legislation, authored by Specter and Sen. Patrick Leahy, D-Vt., is designed to halt future asbestos litigation. Instead, firms facing potential asbestos liability would be required to make contributions to a $140 billion compensation fund that would then award payouts to sufferers of asbestos-related illnesses.

The fund would be administered by the Labor Department, but would be filled solely through compulsory corporate contributions.

Alternative approach being discussed

The Senate has spent three years battling over how to best respond to an explosion of asbestos-related litigation. Dozens of U.S. companies who used the once-popular fire-retardant fiber in a range of products and processes have been forced into bankruptcy by litigation. Asbestos has been determined to be a carcinogen.

Big manufacturers, along with influential business groups such as the National Association of Manufacturers, have backed the trust-fund concept.

The bill's demise would be a victory, however, for property and casualty insurers, who feared that the legislation would allow claimants to pursue a payout from the trust fund as well as from insurance companies.

Such a fear was well-founded, according to Donald Light, senior analyst with Celent, a financial research firm.

"The bill in the form that was defeated would have left the door open to double-dipping," he said. But the bigger question for insurers and their lobbyists, he added, is: "What comes next?"

Light added that he sees "only a small chance" that Specter's bill will return to the floor.

The 40 senators who voted to block the bill Tuesday night represented a combination of liberals, who feared that the fund would let some asbestos users off easy while denying victims of their legal rights, and fiscal conservatives, who worried that taxpayers would be forced to pony up if the trust fund was unable to cover future claims.

Meanwhile, Sen. John Cornyn, R-Texas, and Assistant Minority Leader Richard Durbin, D-Ill., have said that they're willing to work together to come up with an alternative approach that would keep asbestos cases in the courts, but would tighten the medical criteria for eligible patients.

Tightening the criteria would address complaints by defendants who argue that many asbestos cases are brought by plaintiffs with dubious medical claims.

Specter reiterated his opposition to a medical-criteria bill, arguing that it would do nothing to compensate victims whose former employers have gone bankrupt, or who became ill after being exposed to asbestos during military service.

But Susquehanna's Skinner said that efforts to craft a medical-criteria bill along the lines of similar state-level efforts is likely to deepen the divide over the proposed trust fund.

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British police said they had made two further significant arrests in the investigation into the attack on London's parliament and gave the birth name of the man behind the assault as Adrian Russell Ajao.

Britain's top anti-terrorism officer Mark Rowley said police had nine people in custody after the attack on Wednesday which killed five people including the assailant.

Police had said the man behind the attack was British-born Muslim covert Khalid Masood and that they were trying to establish if others had directed him.

Antwerp driver charged

Belgian authorities have charged a Tunisian man with terror-related offences after he allegedly drove his car at high speed around a busy shopping area in Antwerp. The Federal prosecutor’s office said on Friday that Mohamed R., 39, was charged with “attempted murder in a terror context”,and gun offences. After driving in the area around the Meir shopping road on Thursday, he was arrested at the port docks. No one was injured. Authorities found knives, a shotgun and a gas can containing an unknown liquid inside the car. Despite reports that people had to jump out ofthe way of the vehicle, several shop owners in the main Meir shopping area contacted by the Associated Press said they heard or saw nothing unusual. An official at the prosecutor’s office said the suspect had been “under the influence”.