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Making patients and families safer
We advocate for game-changing policy changes to strengthen transparency, disclosure and engagement — the indispensable pillars of any enduring relationship of trust in healthcare systems.
We apply our extensive knowledge base of patient and family narratives shared with us for close to a decade to build innovative patient safety tools and practical engagement solutions.
We work with the media and in the political arena to challenge harmful barriers that keep patients and families silent and prevent meaningful accountability, like healthcare's infamous deny and defend culture, the taxpayer-funded Canadian Medical Protective Association and insider bias that frequently thwarts true patient and family partnership in their healthcare experiences.
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The same authentic voice that defined our work in patient safety from the beginning is now heard in our game-changing approaches to mental health issues, to suicide prevention and to the elimination of gender-related healthcare barriers that place too many women at risk.
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The Center for Patient [icon name="icon-medkit"] Protection, now a part of PatientProtection.Healthcare, works to improve patient safety, reduce medical harms in the hospital setting and produce better mental health outcomes. Recognized around the world for its impassioned voice in patient safety and the collaboration of patients and families in healthcare policy, we are the oldest fully independent patient-centered advocacy and the first to offer an online outreach and advocacy clinic to support patients and families facing the trauma of medical errors.
Medical errors in the hospital setting are the third leading cause of death in the United States and Canada. They leave millions injured and emotionally traumatized, and add unnecessary billions of dollars to the cost of our healthcare systems. The cost of medical harm in Ontario alone is estimated at more than one billion dollars each year. Unaddressed mental health issues, and under-funded mental health care, and failures to adopt mitigation measures to prevent suicide, also carry an incalculable cost in human suffering. Gender-related barriers to healthcare place the lives of millions of women at risk every year. These are the realities that inform our mission.
Preparing for the CTV National News segment that introduced Canada to the use of a three-digit suicide prevention lifeline.
We advocate — typically for the most vulnerable and frequently forgotten in our healthcare and mental health systems — for greater transparency to empower patients and families in their decisions, champion innovations in mental health care and suicide prevention like the 9-8-8 lifeline, campaign to remove gender barriers in healthcare through our Safer Women's Health Initiative™ and our call for national action by the Canadian government to address the health consequences of gender-based violence and sexual harassment. In all these things, we build on our extensive knowledge base of patient- and survivor-created lived experiences to assist providers in the delivery of safer outcomes and patients and families in achieving them.
We were the first to bring to public attention critical issues that adversely affect patients and families in their hospital experiences and proposed concrete solutions to address them, such as the mandatory reporting of medical errors, ending taxpayer support for the physician liability scheme of the Canadian Medical Protective Association, providing patients free access to their medical records and strengthening safety protocols for obtaining do not resuscitate (DNR) orders. The Center for Patient Protection conceived, designed and developed numerous patient safety tools based on the lived experiences of thousands of patients and families, such as our groundbreaking 3Rs for Safer Care and our unique patient and family engagement trigger tool. We were the first to publicly identify medical errors as the third leading cause of death in Canada years before it was recognized by mainstream healthcare and patient safety communities. Our revelations about abuses involving the drug Seroquel resulted in enhanced reporting of its use and changed practices that reduced administration of the drug in long-term care settings.
In mental health, we introduced Canada to the idea of a three-digit emotional crisis/suicide prevention lifeline using the number 9-8-8. Following a lengthy campaign and numerous national news segments covering our efforts, the House of Commons voted to endorse the proposal in December of 2020. We were among the first to call upon the federal government to up its game in responding to the mental-health needs of Canadians during the first wave of the pandemic. We were also the first to propose the creation of a federal minister for mental health. Those efforts resulted in the creation of a federally-sponsored wellness portal, and, more recently, the appointment of Canada's first federal minister of mental health and addiction.
The Center for Patient Protection was founded by Kathleen Finlay following a life-altering family experience with the devastation of medical errors. After being documented in Medscape and in The Huffington Post, her story has been in hundreds of blogs, journals, law pages and websites throughout the world. Kathleen is frequently interviewed by major media platforms around the world, including CTV News and CBC News and The Wall Street Journal. She is a regular contributor to leading op-ed columns.
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By giving voice to the needs of patients and families, and especially, the most vulnerable among us, and by taking a strong role advocating for patient safety innovations and policy reforms, we're helping to save lives, reduce emotional trauma, cut the costs of medical errors and effect positive outcomes for all the participants of our healthcare systems.
Every day, our site is consulted by top healthcare providers, policy makers, healthcare media, and, of course, patients and families around the world. We are proud that iThe Center for Patient Protection and PatientProtection.Healthcare have become valued as one of the most respected clinics of its kind and a valued advocacy for those seeking safer healthcare.


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Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides
Fabrice Dumas,
Affiliations Cell Biophysics Laboratory, Lincoln's Inn Fields Laboratories, Cancer Research UK, London, United Kingdom, CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France, Université de Toulouse, UPS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France
Richard D. Byrne,
Affiliation Cell Biophysics Laboratory, Lincoln's Inn Fields Laboratories, Cancer Research UK, London, United Kingdom
Ben Vincent,
Affiliation Department of Biology, Amherst College, Amherst, Massachusetts, United States of America
Tina M. C. Hobday,
Dominic L. Poccia ,
* E-mail: banafshe.larijani@cancer.org.uk (BL); dlpoccia@amherst.edu (DLP)
Banafshé Larijani
Dominic L. Poccia,
Fabrice Dumas Richard D. Byrne ... Banafshé Larijani
Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.
Citation: Dumas F, Byrne RD, Vincent B, Hobday TMC, Poccia DL, Larijani B (2010) Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides. PLoS ONE 5(8): e12208. https://doi.org/10.1371/journal.pone.0012208
Editor: Richard Steinhardt, University of California, Berkeley, United States of America
Received: June 14, 2010; Accepted: July 20, 2010; Published: August 17, 2010
Copyright: © 2010 Dumas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the core funding of Cancer Research UK and Amherst College FRAP award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Membrane fusion is required for many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis. Historically, the role of lipids and lipid modifications in fusion has been based on model membranes in which an intermediate or hemifusion state, promoted by the localised reorganisation of lipids of negative curvature, leads to a transient fusion pore and eventually to complete fusion [1]. Recent work has integrated roles for both protein signalling and lipid modification in natural membrane fusion [1], [2], [3], [4], [5].
We have isolated a natural membrane vesicle fraction (MV1) from cytoplasm of fertilised oocytes. This membrane population consists of >50% phosphoinositides, is >100-fold enriched in a phosphatidylinositol-specific phospholipase C (PI-PLCγ) and is essential for membrane fusion leading to nuclear envelope formation [3], [6]. Using cell-free oocyte extracts to assemble nuclear envelopes from fusion of discrete membrane vesicle populations [7], we have shown that the early signalling events involve activation of a tyrosine kinase [8] which in turn activates PLCγ in MV1 [3]. Subsequent formation of diacylglycerol (DAG) alters the lamellar structure of these precursor membranes, facilitating their fusion with the endoplasmic reticulum (ER)-derived membranes that contribute most of the nuclear envelope [3], [5], [9].
Here we show, using FRET by two-photon FLIM and three dimensional reconstructions of immobilised nuclei, that the two pole regions to which MV1 membrane vesicles bind are the sites of initiation of fusion with adjacent (ER) membranes, and that further fusion propagates away from the poles to complete enclosure of the chromatin. Using inhibitors, we show that this process is dependent on PtdIns(4,5)P2, PLC and GTPase activity. We discuss how spatial control of membrane fusion may be regulated by regional binding of a potentially fusogenic membrane vesicle population and the novel consequences of such a mechanism.
Buffer and reagents
Lytechinus pictus sea urchins were purchased from Marinus (Long Beach, CA), 4-heptadecyl-7-hydroxycoumarin, BODIPY-C12 and DiIC12, from Invitrogen, U73122 and U73343 from Calbiochem, GTPγ[S] (guanosine 5-[γ-thio]triphosphate) from Sigma, and caged-GTP from Jena Bioscience. Recombinant SKIP proteins (Skeletal muscle and Kidney enriched Inositol Phosphatase, a phosphoinositide 5-phosphatase) were a generous gift from E. Rosivatz and R. Woscholski. Nuclear preparation buffer (SXN), TN (Tris/NaCl buffer) and egg lysis buffer [10] were prepared as described previously [11]. DABCO antifade was from Sigma and prepared at 2.5% (w/v) in LB. The ATP-generating system (ATP-GS) is 1 mM ATP, 20 mM creatine phosphate and 1 mg/ml creatine kinase in LB.
Nuclei and egg extracts
Isolation and permeabilisation of sperm nuclei were adapted from methods described previously [7], [12]. Nuclei demembranated with 0.1% Triton X-100 were resuspended in freezing buffer [SXN supplemented with 0.16% (w/v) BSA and 16.5% (v/v) glycerol], frozen in liquid nitrogen and stored at −80°C. S10 cytoplasmic (G1 phase) extracts from eggs at 10 min post fertilization, membrane vesicles (MVs) and subfractions MV1 and MV2, and 150,000 g supernantant cytosolic egg extracts (S150) were prepared as previously described [13]
Fluorescent labelling
Demembranated nuclei were incubated 1 hour at 4°C with 5 mM hydroxycoumarin in TN buffer to label nuclear envelope remnants (NERs). Nuclei were then collected by centrifugation (1000 g, 2 min.). Stock solutions of fluorescent probes were prepared in Wesson Oil (BODIPY-C12, 20 mM) or in MeOH (DiIC12, 10 mM). The amount of lipid was measured by phosphorus titration before adding the fluorophores in order to ensure that the probe/lipid ratio in the resulting vesicles was less than 1 mole %. MV0, MV1 or MV2 vesicles were mixed with fluorescent probes and vortexed for 5 min at room temperature. The samples were then centrifuged at 100,000 g for 30 min to remove the non-inserted fluorescent probes and resuspended in S150 cytosol.
Binding and fusion assays
To a 1.5 ml Eppendorf tube, 10 µl of BODIPY-C12 labelled MVs, 10 µl of diIC12 labelled MVs in S150, 1.2 µl of ATP-generating system and 2 µl of demembranated sperm nuclei were added. The mixture was incubated at room temperature for 1 h. The unbound vesicles were removed by centrifugation through 0.5 M sucrose (1000 g, 3 min.) and the purified nuclei with bound vesicles were suspended in 4 µl S150 cytosolic egg extract supplemented with 1 mM caged-GTP, 1 µl of 2.5% DABCO and 5 µl 1% low melting point agarose at 30°C. The mixture was immediately mounted on a Mattek® dish and viewed under a 100X oil-immersion objective. Binding was confirmed by surface coating of fluorescent membranes on nuclei. Fusion was triggered by UV (Hg lamp) illumination of the sample for 2 seconds inducing the photoactivation of the GTP. Lifetime of the BODIPY-C12 (donor) was measured before (t = 0) and after GTP activation (t = 5, 15, 30, 45 and 60 minutes). Hydroxycoumarin fluorescence of NERs was recorded during activation by the Hg source. U73122 (30 µM), U73343 (30 µM) and GTPγ[S] (2 mM) inhibitors were applied just before mounting the samples on Mattek® dishes and fusion was initiated 15 min after according to Byrne et al (2005) [14]. Caged GTP was used in all experiments except GTPγ[S] assays. Alternatively, MVs were incubated with SKIP purified protein for 1 hour at room temperature prior to binding. The activity of the purified proteins was checked according to Schmid et al. (2004) [15]. Each experiment was repeated a minimum of three times (n = 3). The images are a representative of one experiment.
Fluorescence lifetime imaging microscopy (FLIM)
All FLIM measurements were undertaken with a modified TE 2000-E inverted microscope. Fluorescence lifetime measurements were performed with an SPC 830 time-correlated single photon counting (TCSPC) electronic card (Becker and Hickl, Germany). A mode-locked tuneable Ti-sapphire laser (Mira 900; Coherent) pumped by a solid-state diode laser (Verdi; Coherent) was used. For two-photon excitation of BODIPY-C12, the laser was tuned at 890 nm and pumped at 6W. The Ti-sapphire laser generates 125-fs pulses with a repetition rate of 76.26 MHz and an average power output of 450 mW. The laser beam was focused with a 100X oil immersion objective lens (Nikon). Fluorescence was detected through the same objective in a descanned configuration with a fast photomultiplier (Hamamatsu 7400) after filtering with a bandpass filter (510–610 nm, Chroma Technology Corp). Acquisition times of the order of 60 s at low excitation power were used to achieve sufficient photon statistics for fitting (i.e. 100–10000 photons per pixel), while avoiding either pulse pile-up or photobleaching. Epifluorescence intensity images of both donor and acceptor were acquired with the mercury lamp source of the TE 2000-E microscope and fluorescence detected by a cooled CCD camera (Hamamatsu ORCA-ER). The cubes set in the TE 2000-E microscope turret were FITC (Nikon Ltd.) for BODIPY-C12 and G-2A (Nikon Ltd.) for the diIC12.
The cell-free assay to assemble nuclear membranes consists of a cytoplasmic extract of fertilised eggs (S10) and sperm nuclei demembranated with 0.1% Triton X-100, which leaves remnants of the sperm nuclear envelope at the tip and base of the nucleus. MV1 binds exclusively to these regions [7], [16], Supporting Information (Fig S1). The majority of bound vesicles however are derived from the ER (MV2), which bind over the entire surface, not just at the poles [17].
Taking advantage of this difference, we initially labelled two sets of total membrane vesicles from S10 (MV0s), one with a donor fluorophore (BODIPY-C12) and the other with an acceptor fluorophore (DiIC12). The characterisation of these is described in MethodsS1. Nuclear envelope remnants (NER) were labelled with hydroxycoumarin (arrows, Fig. 1A). Since FRET is only possible between molecules that are in close proximity (1–10 nm), it is a reliable indicator of membrane fusion which permits the donor and acceptor to interact within a common continuous bilayer.
Figure 1. GTP-induced fusion is bi-polarised.
(A) S10s containing total MVs (MV0) were independently labelled with either BODIPY-C12 (donor) or diIC12 (acceptor) and mixed together. Sperm nuclei and ATP-GS (ATP) were added. Nuclear envelope remnants of the nuclei were pre-labelled with hydroxycoumarin. Epifluorescence patterns of labelled nuclei with bound MVs were visualised by phase contrast and two-photon fluorescence microscopy using a 100X objective. MVs were bound around the entire periphery of the nucleus. The nuclear envelope remnants mark the former apex and base of the sperm nucleus (white arrowheads). Fluorescence lifetime of BODIPY was measured before (t = 0) and after (t = 5, 15, 30, 45 and 60 minutes) the induction of NE formation by photo activation of caged-GTP. (B) Quantification of FRET FLIM images. For analyses, nuclei were divided in four quadrants: p1 and p2 correspond to the poles of the nuclei that include NER while e1 and e2 correspond to the equatorial regions. The averaged mean lifetime was for each quadrant was plotted for each time point showing that MVs fusion is initiated in the polar quadrants and propagates toward the equator. Errors bars correspond to the standard deviation from 7 independent experiments.
The nuclei were mounted on a cover slip with low-melting agarose to prevent movement prior to initiation of membrane fusion and confocal imaging. To accurately set the time of initiation, 2 mM caged GTP was included prior to embedding in agarose and after photo-activation by a UV source, fusion kinetics were assessed by the decrease in lifetime of the donor.
A reference point at t = 0 was taken in the absence of GTP (Supporting Information Movie S1). By 5 minutes post-activation, initiation of FRET occurred in the regions of the NERs or poles (arrows) and proceeded laterally into the regions occupied only by the ER-derived vesicles. At 15 minutes the progression of the FRET signal can be seen in Supporting Information Movie S2. By 45–60 minutes a maximum FRET signal was attained around the entire nucleus as a complete envelope was formed by successive vesicle fusions, The time for enclosure is consistent with previous determinations [3]. Fig. 1B plots donor lifetime in the polar and equatorial quadrants as the reaction proceeds. By 5 minutes the donor lifetime decreased from 3.2 to 2.5±0.15 ns at the poles, remaining unchanged in the equatorial regions. Upon completion of fusion the entire nuclear envelope had a lifetime of 2.5±0.15 ns and a FRET efficiency of 0.7. Thus MV2 vesicle fusion apparently does not proceed unless adjacent to previously fused vesicles.
To visualise the polarised progression of fusion in three dimensions, confocal Z-stacks were obtained and a 3-D representation was reconstructed. Fig. 2 shows initiation sites of fusion in the middle set of stacks containing the NERs and the polarised progression of fusion to almost entirely envelop the spherical nucleus by 30 minutes (Supplementary Information Movie S3).
Figure 2. 3D view of nuclei visualised by FLIM.
(A) Stack measurements of a nucleus: the first image corresponds to the fluorescence lifetime of the BODIPY measured at the top of a nucleus 15 minutes after photo-activation of caged GTP. Ten successive layers of the same nucleus were obtained. For each layer the focus of the objective was moved 0.25 µm along the Z-axis. Since the acquisition of one image lasts for 1 minute, the last image corresponding to the bottom of the nucleus was measured 25 minutes after the induction of NE formation. (B) 3D reconstructions from the Z-stacks of the same nucleus to form fluorescence lifetime 3D views. The indicated times correspond to the time elapsed after photo activation when the first image of each stack was measured.
Substitution of non-hydrolysable GTPγ-S inhibited fusion (Fig. S2). To show that the GTP-initiated fusion was effected by the hydrolysis of PtdIns(4,5)P2, the PLC inhibitor U73122 was included. Fig. 3 shows that inhibition of PLC prevented the decrease of the donor lifetime, which remained at 3.3±0.16 ns.
Figure 3. Inhibition of PLC prevents membrane fusion.
The same experiment as in Fig. 2 was carried out in the presence of 30 µM U73122, a specific PLC inhibitor. The images (A) and lifetime graph (B) show complete inhibition of MV fusion. Data representative of 3 independent experiments
The reactions presented thus far measure fusion between all nuclear bound vesicles (MV0) which include the minor population MV1 and the major population from ER (MV2). To show that initiation of membrane fusion at the poles results from PLCγ hydrolysis of PtdIns(4,5)P2 in the bound MV1 vesicles, we purified both MV1 and MV2, which were separately labelled with either the donor or acceptor fluorophore. Fig. 4A shows MV1 binding at the poles and MV2 over the rest of the chromatin surface. Fusion of MV1 with MV2 was initiated by GTP hydrolysis from the MV1 region (decrease of donor lifetime from 3.3 ns to 2.4 ns). By 15 minutes the fusion wave started to spread laterally to MV2. When MV1 was pre-treated with the PLC inhibitor U73122, membrane fusion was blocked (Fig. 4B). The inactive U73343 analogue did not prevent fusion (Fig. S4).
Figure 4. DAG is required for vectorial progression of fusion.
(A) The same experiments as in Fig. 2 were performed using non-ER vesicles (MV1) labelled with BODIPY-C12 and ER vesicles (MV2) labelled with diIC12. Membrane fusion induces both a decrease of the lifetime and a spreading of BODIPY-C12 all around the nucleus. (B) Same experiment as in Fig. 4A carried out in the presence of 30 µM of U73122, indicating that fusion of the non-ER with the ER membranes requires PLC activity. (C) Same experiment as Fig. 4A using SKIP pre-treated MV1 vesicles. Dephosphorylation of PtdIns(4,5)P2 to PtdIns(4)P inhibits fusion.
If MV1 was pre-treated with a recombinant phosphoinositide 5-phosphatase (SKIP) to deplete the substrate for PLC, membrane fusion was also prevented (Fig. 4C). SKIP dephosphorylates PtdIns(4,5)P2 to PtdIns(4)P which is not recognised by PLCγ and thus DAG cannot be produced. We show in Fig 4C a sperm nucleus which was not completely decondensed so its conical shape clearly defines the apical and basal poles. Pre-treatment of MV0 with SKIP also inhibited membrane fusion as expected (Fig. S3A). In a control parallel reaction, denatured SKIP failed to block membrane fusion and nuclear envelope formation (Fig. S3B).
Inhibition results are summarised in Table 1. These results strongly support our model of nuclear envelope formation involving GTP regulation of PLC hydrolysis of PtdIns(4,5)P2 [3], [4], [5] and add for the first time confirmation of the prediction of bipolarised spatial control of fusion initiation.
Table 1. Summary of membrane fusion effectors and inhibitors.
In this paper we provide evidence that fusion initiation leading to nuclear envelope formation is GTP triggered, and requires PtdIns(4,5)P2 and PLC activity in the MV1 fraction. Fusion is propagated bi-directionally from the sites of initiation and involves fusion of MV1 with MV2 as well as successive fusions with more MV2 to complete NE formation in the cell-free assay. The properties of the non-ER derived MV1 therefore are consistent with its role as a potentially fusogenic vesicle population regulated by GTP hydrolysis and resulting in heterotypic fusion with other vesicles by localised production of DAG.
These results lead to several questions: what is the origin of the polarisation, how is fusion propagated after initiation, and is the mechanism for fusion a general one? We discuss and offer speculation on these questions in turn below.
Since non-ER derived MV1 is a major source of the fusogenic DAG (and location of PLCγ in these cells), its restricted binding to the apex and base of the sperm nucleus lead to bi-directionality of fusion. The specificity of binding to the NER regions is likely to result from non-random packing of chromosomes in the sperm nucleus. MV1 binding requires NERs [18] which themselves have an unusual lipid composition and physical properties [19]. We have shown regions with similar properties present in the sperm nuclei of a wide range of animals suggesting they serve as nuclear membrane organising regions [20]. It remains to be demonstrated that each chromosome contains one or a few such structures which might also be used during mitotic NE reassembly.
Our data show that the mechanism of propagation of fusion commences from the polar MV1vesicles, which fuse with ER-derived vesicles. Subsequently ER vesicles fuse with one another towards the equatorial regions. The nuclear envelope precursor vesicles in vitro and in vivo are approximately 0.5 µm and the nucleus about 4 µm in diameter. Surface area calculations suggest that minimally 125 vesicles must fuse to envelop the nucleus with two bilayers [21]. If each NE were initiated by a single MV1 vesicle at the poles, the lipid contribution of MV1 to the completed nuclear envelope could be <1% of the total. Since nearly all PLCγ is associated with MV1 and is >100-fold enriched in MV1 as well as its substrate PtdIns(4,5)P2 [3], it is likely that DAG formed in this compartment rapidly diffuses into the ER-derived membranes with successive fusions, continually lowering the DAG concentration until it is below the 4% estimated from synthetic systems to be required for fusion [22]. Due to this dilution, eventually the fusogenicity of the forming envelope towards successive vesicles would likely decrease, and the process might become self-limiting. To determine if a decrease in the rate of successive fusions accompanies propagation requires a much a higher resolution method than used here.
The non-ER derived MV1 vesicle fraction enriched in PLCγ is found in vesicles in the cortex of oocytes [3] whereas the ER in vivo is usually a continuous membrane structure [23] that is of necessity vesiculated during preparation of cell extracts. In vivo, it is likely that tubules or continuous sheets of ER envelop the chromosomes at telophase. Thus the role of the non-ER fusogenic MV1 vesicles would be to seal gaps in the enveloping ER [5]. Local DAG concentrations could decline through dilution, chemical modification or both, returning the membrane to a non-fusigenic state.
A complete description of the spatial rearrangements of MV1 during the cell cycle has yet to be made. It is however clear that there are many more non-ER vesicles enriched in PLCγ than are necessary to facilitate NE formation [21]. It will therefore be of interest to determine whether such potentially fusogenic vesicles are mobilised to participate in other membrane fusion events during mitosis or interphase [24]. A novel aspect of this mechanism is that the specificity of localisation of DAG would depend on its delivery through the recruitment of potentially fusogenic vesicles to the sites of fusion rather than by its generation within a domain of one or both partner membranes to be fused.
We have shown that membrane fusion can involve a vectorial progression with a specific origin and direction. This vectorial progression is dependent on the formation of localised DAG derived from polyphosphoinositide modification in one of the partner membranes. The interplay of localised lipids and protein recruitment will be of importance to explore in a variety of natural membrane fusions.
Methods S1.
(0.10 MB DOC)
Figure S1.
Confocal images of sperm nuclei, nuclear envelope remnants and bound MVs. (A) Input sperm nuclei (extracted with 0.1% Triton X-100). DNA stained with propidium iodide [23] and detergent resistant membranes of the nuclear envelope remnants stained with diOC6 located at the acrosomal and centriolar fossa regions (apex and base) of the undecondensed nucleus. (B) Decondensed sperm nucleus with bound membrane vesicles from fertilised egg extract (S10). DNA stained with Hoechst 33334 (blue) and membranes stained with diOC6 (green). Nuclei incubated in S10 with ATP have decondensed chromatin and bound vesicles. (C) Nuclei with bound vesicles separated from unbound vesicles remaining in the S10 following purification through 0.5 M sucrose. The majority of bound vesicles are from the ER-derived population (MV2), which binds over the entire surface. The binding of non-ER derived membrane vesicle (MV1) occurs only in the regions of the NERs.
(2.26 MB EPS)
GTPγS does not induce fusion. The same experiment presented in Figure 1 was carried out using GTPγS instead of caged GTP. Under these conditions the lifetime of the donor remains constant indicating that there is no fusion of the labelled vesicles.
Pre-treatment of MVs with SKIP inhibits the fusion process. (A) Total S10 membranes (MV0) were pre-treated with SKIP as described in Experimental Procedures. Pre-treated membranes were independently labelled with either BODIPYC12 (donor) or diIC12 (acceptor) and both bound in vitro to sperm nuclei with ATP-GS (ATP). MV fusion was induced by photo activation of caged GTP and assessed by FLIM measurements as presented in Figures 2 to 4. (B) As a control the same experiment as in (A) was performed with denatured SKIP (5 minutes at 100oC). Denaturation of SKIP abolished the inhibition of fusion.
U73343, an inactive analogue of U73122, does not inhibit fusion. To confirm the specificity of the U73122 inhibitor, the same experiment as presented in Figure 4 was preformed using the U73343 analogue. No inhibition of fusion was observed.
Movie S1.
3D Movie of nuclei visualised by FLIM at 0 minutes prior to activation of caged GTP.
(7.65 MB MOV)
3D Movie of nuclei visualised by FLIM at 15 minutes after photo-activation of caged GTP.
The authors would like to thank R. Woscholski and E. Rosivatz for their help with purified SKIP.
Conceived and designed the experiments: FD DP BL. Performed the experiments: FD RDB BV TMH. Analyzed the data: FD. Contributed reagents/materials/analysis tools: FD DP. Wrote the paper: FD DP BL.
1. Chernomordik LV, Kozlov MM (2008) Mechanics of membrane fusion. Nat Struct Mol Biol 15: 675–683.
2. Gruenberg J (2001) The endocytic pathway: a mosaic of domains. Nat Rev Mol Cell Biol 2: 721–730.
3. Byrne RD, Garnier-Lhomme M, Han K, Dowicki M, Michael N, et al. (2007) PLCgamma is enriched on poly-phosphoinositide-rich vesicles to control nuclear envelope assembly. Cell Signal 19: 913–922.
4. Poccia D, Larijani B (2009) Phosphatidylinositol metabolism and membrane fusion. Biochem J 418: 233–246.
5. Larijani B, Poccia DL (2009) Nuclear envelope formation: mind the gaps. Annu Rev Biophys 38: 107–124.
6. Byrne RD, Poccia DL, Larijani B (2009) Role of phospholipase C in nuclear envelope assembly. Clin Lipidol 4: 103–112.
7. Cameron LA, Poccia DL (1994) In vitro development of the sea urchin male pronucleus. Dev Biol 162: 568–578.
8. Byrne RD, Larijani B, Poccia DL (2009) Tyrosine kinase regulation of nuclear envelope assembly. Adv Enzyme Regul 49: 148–156.
9. Barona T, Byrne RD, Pettitt TR, Wakelam MJ, Larijani B, et al. (2005) Diacylglycerol induces fusion of nuclear envelope membrane precursor vesicles. J Biol Chem 280: 41171–41177.
10. Abbott B, Abbott R, Adhikari R, Ageev A, Allen B, et al. (2005) Limits on gravitational-wave emission from selected pulsars using LIGO data. Phys Rev Lett 94: 181103.
11. Collas P, Poccia D (1998) Methods for studying in vitro assembly of male pronuclei using oocyte extracts from marine invertebrates: sea urchins and surf clams. Methods Cell Biol 53: 417–452.
12. Cothren CC, Poccia DL (1993) Two steps required for male pronucleus formation in the sea urchin egg. Exp Cell Res 205: 126–133.
13. Larijani B, Barona TM, Poccia DL (2001) Role for phosphatidylinositol in nuclear envelope formation. Biochem J 356: 495–501.
14. Byrne RD, Barona TM, Garnier M, Koster G, Katan M, et al. (2005) Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes. Biochem J 387: 393–400.
15. Schmid AC, Wise HM, Mitchell CA, Nussbaum R, Woscholski R (2004) Type II phosphoinositide 5-phosphatases have unique sensitivities towards fatty acid composition and head group phosphorylation. FEBS Lett 576: 9–13.
16. Byrne RD, Zhendre V, Larijani B, Poccia DL (2009) Nuclear envelope formation in vitro: a sea urchin egg cell-free system. Methods Mol Biol 464: 207–223.
17. Collas P, Poccia D (1996) Distinct egg cytoplasmic membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro. J Cell Sci 109: 1275–1283.
18. Collas P, Poccia D (1995) Lipophilic organizing structures of sperm nuclei target membrane vesicle binding and are incorporated into the nuclear envelope. Dev Biol 169: 123–135.
19. Garnier-Lhomme M, Byrne RD, Hobday TM, Gschmeissner S, Woscholski R, et al. (2009) Nuclear envelope remnants: fluid membranes enriched in sterols and polyphosphoinositides. PLoS ONE 4: e4255.
20. Collas P, Poccia D (1996) Conserved binding recognition elements of sperm chromatin, sperm lipophilic structures and nuclear envelope precursor vesicles. Eur J Cell Biol 71: 22–32.
21. Larijani B, Poccia D (2007) Protein and lipid signaling in membrane fusion: nuclear envelope assembly. Signal Transduction 7: 142–153.
22. Villar AV, Alonso A, Goni FM (2000) Leaky vesicle fusion induced by phosphatidylinositol-specific phospholipase C: observation of mixing of vesicular inner monolayers. Biochemistry 39: 14012–14018.
23. Terasaki M, Jaffe LA (1991) Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization. J Cell Biol 114: 929–940.
24. Gould GW, Lippincott-Schwartz J (2009) New roles for endosomes: from vesicular carriers to multi-purpose platforms. Nat Rev Mol Cell Biol 10: 287–292.
Is the Subject Area "Membrane fusion" applicable to this article?
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Nuclear membrane
Is the Subject Area "Nuclear membrane" applicable to this article?
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Vesicle fusion
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Fluorescence resonance energy transfer
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redpajama_set_name: RedPajamaCommonCrawl


Gemütlichkeit for the whole family . . .
For the Family: Food and gift vendors throughout the grounds, antique car show, Wiener Takes All Dachshund races & fashion show, 5K Run and children's area. For the Adults: 3 Entertainment Areas with a great selection of German and popular music and of course, cold beverages.


The National Children's Choir of Great Britain was founded in 1998, to bring together talented young singers from throughout the United Kingdom and give them the opportunity to work together and enjoy making music under the guidance of inspiring teachers.
The choir has grown from its original membership of 38, who sang in the Millennium Youth Prom in the Albert Hall as their first concert, to more than 250 members of the Junior and Senior Choirs.  Young singers are able to join from ages 9 – 19, and each choir member is encouraged to develop their ability within a supportive environment.
The choir meets twice a year for residential courses, which give the singers the opportunity to work and live with their peers and form enduring friendships.


Today I was invited over to my friend's house to learn to make Hainan Chicken, from her friend who is Chinese/Vietnamese. The key to having the best tasting chicken, according to my friend, is to buy a free-roaming one. I soon found out that this lady ordered from a free-roaming chicken farm, and that the birds were delivered to her house this morning ALIVE. She then killed and feathered them, to use for our meal today! Here I am with the chicken. I was amazed at the large feet.
After cleaning it thoroughly, the friend, cut off the feet, and tucked them inside the body (to give it a good flavor), she bent back the wings and placed the whole chicken inside the pot to boil. After the chicken had cooked and became tender, we removed it from the pot and let cool. Use chicken broth with equal parts rice and cook rice in an asian rice cooker. For example 4 cups rice to 4 cups chicken broth, and 1-2 tsp. salt.
Chinese use every part of the bird! The gizzards and heart were chopped and stir-fried with the green vegetable side dish. A group photo, the lady next to me is the cook. We are grating fresh ginger for the dipping sauce.
Below our finished product, with some fresh cucumbers, fruit and chicken broth soup. Notice the plates of chicken, Asians simply cut the whole chicken, bones and all and place on a serving dish. Then they proceed to eat the chicken, sucking and spitting out the bones. (My contribution to the meal — in the top left hand corner — a plate of chocolate chip cookies.
TIS was invited to sponsor a booth at an Expo, this weekend. Sean, Megan and I ventured over on Saturday to support our school. However, what we had originally thought was an Expatriate Expo, turned out to be a huge event encouraging tourism in China. Large exhibit halls were filled with complex constructed, 2-story booths advertising famous Provinces throughout China. And to our BIG surprise, there was one exhibition hall filled with outdoor recreational "toys", everything from campers, to RVs to tents, white water rafts, boats, four-wheelers, pick-up trucks, and even a small 1-seater airplane! We found our school booth located in the very back of the last exhibit hall.
Megan at a booth advertising one of China's minority groups, notice the lady's decorative headpiece in the background.
I helped to set up our school booth the day before the Expo. Here is an exhibit in the making from the YunnanProvince, at about 4pm the day before opening day.
And Megan standing in front of the final product, all set up and ready by 8am the following morning. One thing China can do very well is mass setup and teardown.
This friday I had my first High School Band Concert. Although band has been tough to keep up with this year as a freshman, I have really enjoyed the challenges that have followed. The concert turned out really well and I'm proud of all that I have accomplished so far this year! I can't wait till our next concert comes in December!
Tonight we celebrated with our Malaysian friends' family for her birthday. Being away from extended family can be tough when living abroad. Rachel and I were so happy to help make Sharon's day more special by baking a 4-layer chocolate cake with buttercream filling and white chocolate espresso drizzle. (Rachel is practicing for her new cake business).
Here is Megan's special friend.
Rachel's delicious cake. Wondering about the interesting centerpiece? It's a Chinese birthday candle that opens up as a flower and shoots sparkles out of the top, singing the Happy Birthday tune (like a singing card you might buy at Hallmark).
As of late, we have all been caught up in the whirl-wind activity of a new school year! Personally, I feel like I have jumped on a boat that I am trying in vain to steer through churning waters, (Anyone else?). So what's been so crazy about the beginning of this school year? – the real question is what hasn't been crazy about the beginning of this school year?
For starters, my dad, Maddie, and I had our annual Fall Camp, a high school retreat, last week! Maddie is discovering the "joys"of a high school homework load, and I am discovering the "joys" of three AP classes.
Over in Hong Kong, Michael just returned from a church retreat which was very encouraging for him as he begins his sophomore year at Hong Kong University!
Rachel has been busy as a bee at home. She's our "sweet little fairy," baking delicious desserts, working in the blog room on her goals, and supporting us in school. Rachel too has been unusually busy this week; it seems that the news of her baking skills are getting around. This week she has baked 2 layer cakes, pavlovas (tonight to satisfy me and Maddie's meringue craving :), and biscotti. Sadly, the only above dessert we get to try is the white fluffy cookies in the oven now. The birthday cakes were made for a friend and a client!
On top of ALL of that, add the usual homework load, dentist appointments, student council work, and time spent with friends then you've about nailed down our lives as of late!
Maddie posing with her team after the Fall Camp Skits!
The 3-layer Lego cake that Rachel baked for a boy in our community turning ten. It is a vanilla cake with multi-colored layers (red, blue and green), meringue buttercream filling, and marshmallow icing on top!
Michael's Church retreat – seems like he had fun!
Since 9th grade has started life has for sure been a whirlwind. The high school fall camp was about 2 weeks ago and although my team only got 6th place out of 10 teams, we had a blast. It started with the whole high school leaving on a 6 hour bus ride to the camp site, yes the bus ride was so long and boring, but I was thankful to spend some catch-up time with Jessie.
By the time we got to the site it was dinner time so we didn't do many activities except for a night game. The next day we played a lot of team games and performed our team skit in the evening. Personally, I had the most fun on this day because the games were fun and the skit went great. Finally, the third day we packed up and bussed all the way back to TIS (our school). Although fall camp seemed to go by way too fast the time we did have together as a high school was great. Here's a photo of me with my team (I played an old lady in the skit, thus the greyish hair color).
For starters, dad, Maddie, and I had our annual Fall Camp, a high school retreat, last week! Maddie is discovering the "joys"of a high school homework load, and I am discovering the "joys" of three AP classes.
This is the first pound cake I've ever made and my first "successful" attempt at the bundt cake-shape. The dense texture of this cake was a much needed change from my usual 3-layer butter cakes. I think the best part of this baking experience was taking photos… finally, a cake that is both delicious and effortlessly beautiful. Bundt cakes are so pretty on their own without much fuss about icing colors, decoration ideas, or touch ups. I totally recommend this recipe if you're looking for something quick and relatively simple.


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Where is Florida?
Florida is situated at the south-east corner of the United States of America - USA - and is surrounded by water on three sides, making it a peninsula. It is surrounded by the Atlantic Ocean to the east and south, and the Gulf of Mexico to the west.
At the southern tip of Florida is a chain, or archipelago of islands made of sand and coral, called the Florida Keys.
What is it like in Florida?
The land in Florida is very flat and most of it lies only just above sea level. It has a tropical and subtropical climate meaning it is very warm, humid and wet almost all year round. Although it is called the 'Sunshine State', Florida is also at risk from hurricanes in summer and autumn. These are very strong storms that can create lots of damage to buildings and land.
When it rains in Florida, it rains heavily. The large amounts of rainfall collect in lakes, rivers, underground springs and swamps. This has created places such as the Everglades in the south - a wet marshy area where alligators, turtles and snakes live.
Florida's main industries are:
farming - tomatoes, strawberries and oranges are grown here
tourism - its miles of beaches, amusement parks and state parks attract over 100 million visitors each year
The Everglades is a very wet and marshy national park in South Florida.
American crocodiles are one of the more common animals found in the Everglades.
The Florida panther can also be found in the Everglades.
Oranges are one of the major crops grown in Florida. Most of the USA's oranges come from Florida!
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Sonardyne International Ltd have confirmed that this May's exhibition in Istanbul will be their first MAST in several years.
Visitors (MAST's unique composition of maritime defence/security operators, technologists, and procurement experts) will be able to research their positioning,inertial navigation,subsea communication,and sonar imaging expertise.
Sonardyne boasts a world-leading team of scientists and engineers, specialising in underwater acoustics, with over 300 man-years of experience in mine warfare, underwater imaging, navigation and underwater communications, and have successfully made the transition of bringing cost effective solutions from the offshore Oil and Gas Industry to Defence. Cost effective, reliable, modular and open architecture, their systems are in-service across the globe providing vital military capability with a COTS price tag.
No doubt their harbour security solutions alone will be of key interest to confirmed MENA/Gulf State delegates (some of whom are currently completing a major maritime defence/security requirements assessment of key ports within one of the region's major countries).


Just in time for the start of the school year, the Dallas Public Library is hosting a huge book sale at the Central Library, 1515 Young St. The sale will be held from 11 a.m. to 6 p.m. Friday, Aug. 24; 10 a.m. to 4 p.m. Saturday, Aug. 25; and 1 to 4 p.m. Sunday, Aug. 26.
The Friends of the Dallas Public Library (FODPL) is organizing and staffing the end-of-summer edition of its twice-yearly book sale. Unlike previous sales, which were only Saturdays and Sundays, the public is invited on Friday as well. Proceeds benefit the 29-branch library system and its programs, including English Language Learning, GED classes, preschool storytimes, the Mayor's Summer Reading Challenge and more.
Like a bargain warehouse store, the sale features "all you can haul" buying in bulk. A plastic grocery sack filled with sale items will cost $10, a large fabric shopping bag will cost $20 and a cardboard box full of items is $30. Patrons can buy four or fewer items for just $1 each.
FODPL is a nonprofit, nonpartisan organization that advocates for the Dallas Public Library and supports its programs. Annual memberships, which begin at $25 and are tax-deductible, are available at www.fodpl.org.


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PA 12/2019: Furnishing priority documents using the WIPO Digital Access Service (DAS) – Part II: Requesting the International Bureau to retrieve a priority document available in the DAS system
Q: I filed a national patent application with my national patent Office, and requested that Office to make the application available via DAS as a priority document for a future international application. I am now filing an international application claiming the priority of the earlier national application, and would like to request the International Bureau to retrieve the priority document from the DAS system, in order to meet the priority document requirement under PCT Rule 17.1. How do I do this, and what is the time limit for doing so?
A: It is recalled that DAS offers a secure, reliable and legally authentic electronic exchange of documents with all other Offices participating in the service, and can be used in many cases to meet priority document requirements under the PCT in accordance with PCT Rule 17.1, as well as those under other treaties and laws. Part I of this Practical Advice (published in PCT Newsletter No. 11/2019) explained how to make available to the DAS system an earlier national application, the priority of which is to be claimed in a later‑filed international application, and pointed out the situations in which it might not be possible to do this. This would be the case, in particular, if the Office of First Filing (OFF) is not a DAS depositing Office, if the earlier application does not fall under the scope of the DAS depositing Office concerned, or if the applicant does not meet the requirements of the DAS depositing Office concerned.
After requesting the OFF to make the earlier application available in DAS, you will have received an access code, which is required in order to request the International Bureau (IB) (or an Office of Second Filing (OSF)) to retrieve the priority document. The code may be provided by either the IB or the OFF (or in the case of certain Offices, the access code may be automatically made available to the applicant as part of the application process).1 Access codes issued by the IB are sent by e‑mail2 from "Noreply@wipo.int". Note that the access code of an earlier application registered in DAS can be re‑used if the priority of the earlier application is claimed in more than one later‑filed application, so there is no need to request a new access code for the same priority document.
The way in which you request the IB to retrieve the priority document from DAS for the purposes of meeting the priority requirement under PCT Rule 17.1 depends on whether you are making the request at the time of filing the international application, or after filing. The various options are described below.
At the time of filing (the preferred option):
– if you are filing using ePCT‑Filing, make the necessary indications on the "Priority Claims" screen under "Option(s) for providing the priority document to the IB" by selecting "International Bureau to obtain from a digital library (DAS)" and providing the DAS access code (you can do this for each priority document if there is more than one priority claim); ePCT will look up the priority document in DAS in real time and validate the access code against the priority claim data indicated3;
– if you file the international application using PCT‑SAFE, the priority document information page ("Details of Priority Claim of Earlier Application") enables you to request the IB to obtain the priority document from DAS, and to insert the necessary access code; or
– if you are filing using the paper version of the request form, mark the applicable check‑boxes in Box No. VI of the request form (PCT/RO/101) and indicate the access code for the priority document required (or for each priority document if there is more than one).
After filing:
– request the IB via ePCT to retrieve the priority document by:
- signing in with strong authentication and entering the corresponding access code in the online Action: "Obtain priority document from DAS"; ePCT will look up the priority document in DAS and validate the access code against the priority claim data indicated. Please note that this is the recommended method, since it is a self‑service Action that is executed automatically by the system in real time without any need to queue for manual processing by the IB;
- signing in without strong authentication and sending an ePCT Message containing the DAS access code requesting the IB to retrieve the earlier application from DAS;
- signing in without strong authentication and uploading a letter containing the DAS access code as a "General correspondence" document type requesting the IB to retrieve the priority document from DAS; or
– send a letter containing the DAS access code by mail to the IB4 requesting it to retrieve the earlier application from DAS.
Note that it is usually easier to request the IB to retrieve the priority documents from DAS at the time of filing the international application. However, if you wish to make the request later, the time limit for doing so is considered to be met if, before the date of international publication, you have:
– taken all the necessary steps with the OFF;
– made a valid request to the IB to retrieve the priority document from DAS; and
– provided the necessary access code.
Once the IB has retrieved the priority document via DAS, it will send a confirmation of retrieval to you by means of Form PCT/IB/304. If you have access rights to the international application in ePCT, you will be able to access that form immediately after is has been issued.
Please note that WIPO's DAS Applicant Portal at:
www3.wipo.int/dasapplicant/en/pages/workbench/applicant.xhtml
(accessible via your WIPO account5) provides tracking facilities to record and view details of the complete history of Offices' access to your priority documents. The portal enables you to add your priority documents to the DAS Workbench by providing the priority number, filing date and access code.
This Practical Advice concerns making a request to the IB to obtain a priority document available in the DAS system, as provided by PCT Rule 17.1(b-bis). You can also meet the priority document requirements under PCT Rule 17.1 by submitting a certified copy of the priority document to the IB or receiving Office under PCT Rule 17.1(a), or by requesting the receiving Office to prepare and transmit the priority document to the IB under PCT Rule 17.1(b)6. The latter way of meeting the priority document requirements may be preferable when filing an international application at the receiving Office where the earlier application was filed (whether as an earlier national or international application). However, such a request should be made no later than 16 months after the priority date and the receiving Office may charge a fee for this.
For further information on furnishing the priority document, please refer to the PCT Applicant's Guide, International Phase, paragraphs 5.070 to 5.070D at:
Examples of how codes are received from the European Patent Office, the Japan Patent Office and the United States Patent and Trademark Office are given in Part I of this Practical Advice.
Note that, although the IB is not authorized to give confidential information by e-mail, this restriction does not apply to the DAS access code communications.
If you are filing using a different electronic filing system, please check with the receiving Office concerned.
The mailing address of the IB is: World Intellectual Property Organization, International Bureau (PCT), 34, chemin des Colombettes, CH‑1211 Geneva 20, Switzerland.
If you do not yet have a WIPO account, you can easily create one at: https://ipportal.wipo.int/
If using ePCT-Filing, by selecting "Receiving Office to prepare and transmit to the International Bureau" on the "Priority Claims" screen; if using the paper request form (PCT/RO/101), by marking the applicable check‑boxes in Box No. VI.
XR.17 PCT The Priority Document
PA 05/2019: Possible implications of submitting informal drawings when filing the international application
PA 06/2019: The timing of the start of the international preliminary examination (following the amendment to PCT Rule 69.1(a))
PA 07/2019: Same day corrections before the International Bureau as receiving Office
PA 10/2019: Submitting declarations under PCT Rule 4.17 using ePCT, and obtaining the signatures of the inventors for the declaration of inventorship
PA 11/2019: Furnishing priority documents using the WIPO Digital Access Service (DAS) – Part I: Requesting an Office of First Filing to make a priority document available in the DAS system


Why should you never let your children do anything that makes you dislike them? Why did the ancient Egyptians worship the capacity to pay attention as the highest of their gods? What can we learn from the lowly lobster about standing up straight and moving forthrightly through life? Dr. Peterson discusses discipline, responsibility, and the necessity of clear, truthful thinking, distilling the discoveries of science and the lessons from the great myths of the world into 12 profound directives for living properly in today's ever-transforming world. 12 Rules for Life delves deeply into the hidden realms lurking beneath our commonplace assumptions, enlightening the mind and spirit of its readers, keeping the chaos and nihilism that threaten and tempt the modern mind firmly at bay.
Doidge, Norman,writer of foreword., Peterson, Jordan B.., Van Sciver, Ethan,illustrator.


Crkva is located in near center of Rijeka city, at the address Ružićeva street 22.
If you drive by the Istrian Y (croatian highway, section A8), after city of Matulji continue to drive by the Rijeka Bypass E61 direction Split / Zagreb / Rijeka. After the viaduct "Katarina" and tunnel "Trsat" you will reach the traffic junction "Orehovica" where you continue to drive on the road A7 / E61 and follow signs for "Rijeka istok". Take exit 9-"Draga" of the D404 "Draga / Rijeka istok / Luka". Continue driving on the D404, take the first exit "Vežica". Drive along Zdravko Kučić street. At the second intersection at the Church of St. Anne, turn left. Follow the main road down the street Tome Strižića accompanied by a Kumičićeva street that leads to the city center. You will come to the hotel Continental, which will be on your left. At the intersection there are four lanes. Two of them lead to the left, towards the center, and two lead straight. Stay in the left lane that leads straight. After 100m you will reach a railway overpass. Drive straight under it and after 200m is a shop "Plodine" with a parking where you can leave your car. Crkva is right across the parking. Welcome!
Take the highway towards city of Rijeka, and after the toll "Grobnik" continue to drive on A6. Take the first exit to the right output 8-Orehovica towards "Orehovica / Rijeka centar". Continue straight along the Grobnik road. You will pass through a short tunnel called "Banska vrata". Continue driving on the Franjo Rački street. At the first intersection, in front of you will be a railway overpass. Here, turn sharp to the right in order to arrive at Ružićeva street that runs parallel with Franjo Rački street. After 200m is a shop "Plodine" with a parking where you can leave your car. Crkva is right across the parking. Welcome!
Drive along the main road E65. Take the exit for Zagreb / Otok Cres / Otok Lošinj / Otok Krk. At the first roundabout take the first exit for the A7. Continue driving on the A7 straight to the E65. Continue straight and after the viaduct "Katarina" and tunnel "Trsat" turn right at exit 9 for D404 towards "Rijeka Istok / Luka / Draga". Continue driving on the D404. On the D404 / "Brajdica" turn right at the first intersection in the boulevard of Andrije Kačića Miošića. At the next intersection turn right onto Slavka Cindrića street and hold to the left side. After 20m turn left to Milana Smokvine Tvrdog street. At the first traffic light, continue straight to Rade Šupić street which has four lanes that lead to the left. Stay in the third lane, viewed from left to right. As you enter Josip Juraj Strossmayer street, stay in your lane which shows direction straight at the traffic light (on your left side is the Hotel Continental). Follow the lane that is pointing you straight. After 50m you will arrive at a crossing with the railway overpass in front of you. You are in the middle lane, which shows straight and left. Take straight under the overpass. After 200m is a shop "Plodine" with a parking where you can leave your car. Crkva is right across the parking. Welcome!
If you came to Rijeka, the city main promenade "Korzo" is a must see. To get to Crkva, take a walk on "Korzo" towards east. At "Zagrebačka banka" (Zagreb bank) continue straight to Ban Jelačić square. At the square go right and cross two pedestrian crossings. Then turn left and continue walking alog the "Mrtvi kanal" on which are small fishermen boats. Go over pedestrian crossing and continue right towards Hotel Continental. Left from Hotel, there are two pedestrian crossings. Go over them and again, take left turn. Go under the railway overpass and on your left there are again two pedestrian crossings. If you go over them, you will find yourself in Ružićeva street. Go right and after 200m there is a shop "Plodine". Crkva is right across the parking of the shop. Welcome!
Taxi price in city of Rijeka is really affordable. There are different taxi companies with different prices per kilometer, and the cheapest one is 20kn (2.66€) for 5km, nevermind the amount of persons (if you all have same departure spot).
You can recognize Rijeka public transport "Autotrolej" buses by their unique orange colour. Almost on every bus station and in every bus there is a route map. Get off the bus on "Fiumara" station or the nearest one, look out for the Hotel Continental and from there continue with instructions given in "BY FOOT" section.


This item is 6 kilograms or 30% of a Pantry box.
This item is 0.11 cubic feet or 2.7% of a Pantry box.
This item is 0.34 cubic feet or 8.5% of a Pantry box.
This item is 2.12 kilograms or 10.6% of a Pantry box.
This item is 0.46 kilograms or 2.3% of a Pantry box.
This item is 0.08 kilograms or 0.4% of a Pantry box.
This item is 2.14 kilograms or 10.7% of a Pantry box.
This item is 0.56 kilograms or 2.8% of a Pantry box.
This item is 0.22 kilograms or 1.1% of a Pantry box.
This item is 0.13 cubic feet or 3.2% of a Pantry box.
This item is 0.54 kilograms or 2.7% of a Pantry box.
Huggies Pure Extra Care 3*Baby Wipes.
This item is 1.3 kilograms or 6.5% of a Pantry box.
This item is 0.06 cubic feet or 1.4% of a Pantry box.
This item is 0.14 kilograms or 0.7% of a Pantry box.
This item is 0.48 kilograms or 2.4% of a Pantry box.
This item is 6.32 kilograms or 31.6% of a Pantry box.
This item is 0.36 kilograms or 1.8% of a Pantry box.
This item is 0.62 kilograms or 3.1% of a Pantry box.
This item is 0.07 cubic feet or 1.7% of a Pantry box.
This item is 0.96 kilograms or 4.8% of a Pantry box.
This item is 0.15 cubic feet or 3.8% of a Pantry box.
This item is 0.14 cubic feet or 3.4% of a Pantry box.
This item is 0.65 cubic feet or 16.3% of a Pantry box.
This item is 4.64 kilograms or 23.2% of a Pantry box.
This item is 0.29 cubic feet or 7.2% of a Pantry box.
This item is 1.28 kilograms or 6.4% of a Pantry box.
This item is 1.08 kilograms or 5.4% of a Pantry box.
This item is 0.06 kilograms or 0.3% of a Pantry box.
This item is 0.02 kilograms or 0.1% of a Pantry box.
This item is 0.17 cubic feet or 4.2% of a Pantry box.
This item is 0.1 kilograms or 0.5% of a Pantry box.
This item is 1.4 kilograms or 7% of a Pantry box.
This item is 0.58 kilograms or 2.9% of a Pantry box.
This item is 0.8 kilograms or 4% of a Pantry box.
This item is 0.17 cubic feet or 4.3% of a Pantry box.
This item is 0.3 cubic feet or 7.5% of a Pantry box.


text:
Q: vscode tasks.json transform pickfile I have next vscode tasks.json
{
"version": "2.0.0",
"tasks": [
    {
        "label": "build",
        "type": "shell",
        "command": "${userHome}/build.py",
        "args": ["param_def=${input:pickBuild}"]
    }
],
"inputs": [
    {
      "id": "pickBuild",
      "type": "command",
      "command": "extension.commandvariable.file.pickFile",
      "args": {
        "include": "**/default_*.py",
        "description": "Select build",
        "display": "fileName",
      }
    }
]}

when run:
actual: /home/user/build.py param_def=/home/user/default_android.py
expected: /home/user/build.py param_def=default_android
P.S: I have tried use extension.commandvariable.transform, but did not success

A: You can use the following config  in your tasks.json
{
"version": "2.0.0",
"tasks": [
    {
        "label": "build",
        "type": "shell",
        "command": "${userHome}/build.py",
        "args": ["param_def=${input:pickBuild}"]
    }
],
"inputs": [
    {
      "id": "pickBuild",
      "type": "command",
      "command": "extension.commandvariable.transform",
      "args": {
        "text": "${pickFile:build}",
        "find": "^.*/(.*).py",
        "replace": "$1",
        "pickFile": {
          "build": {
            "include": "**/default_*.py",
            "description": "Select build",
            "display": "fileName",
          }
        }
      }
    }
]}

redpajama_set_name: RedPajamaStackExchange


Published online November 1, 1996.
↵**Present address and address for correspondence: Dr. David S. W. Ho, University Department of Medicine, Queen Mary Hospital, Pokfulam, Hong Kong.
Objectives. This study sought to evaluate the behavior of late potentials on the body surface by signal averaging during programmed stimulation and to correlate the findings with the cycle length of induced ventricular tachycardia.
Background. Clinically relevant late potentials may be concealed within the QRS complex and may be missed by the conventional signal-averaged electrocardiogram (SAECG). In contrast, some late potentials may arise from dead-end pathways or pathways not capable of supporting sustained ventricular tachycardia (VT). It has been shown that durations of late potentials in sinus rhythm correlate poorly with VT cycle length.
Methods. Signal-averaged electrocardiography during sinus rhythm, right ventricular pacing (S1) and introduction of a right ventricular extrastimulus (S2) was performed in 95 patients: 11 patients with a structurally normal heart and no inducible VT (Group I); 44 with a previous myocardial infarction (MI) and no inducible monomorphic VT (Group II); and 40 with a previous MI and inducible monomorphic VT (Group III).
Results. The best subset of SAECG variables and the best cut points for each variable to differentiate between patients with and without VT were first established for each rhythm studied. Total duration of the filtered QRS complex (QRSD) was found to be the only independent predictor of inducibility of VT. When late potentials were defined for these criteria (QRSD ≥113, ≥178 and ≥168 ms for the SAECG during sinus rhythm, S1 and S2, respectively), there was no difference in the incidence of false positive (16% vs. 18%) or false negative (30% vs. 26%) late potentials between sinus rhythm and S1. During S2, there were significantly fewer false positive late potentials (11% vs. 16%) and fewer false negative late potentials (17% vs. 30%) than with sinus rhythm. Compared with sinus rhythm, 31% of the false positive late potentials detected during sinus rhythm were lost, whereas 43% of the false negative late potentials became detectable after S2, resulting in improved sensitivity (83% vs. 70%), specificity (89% vs. 84%) and predictive accuracy (86% vs. 77%, p < 0.05). Among the patients with VT, QRSD during S2 achieved the best correlation with VT cycle length (r = 0.74) and was the only independent predictor of VT cycle length when all SAECG variables were considered.
Conclusions. Late potentials revealed by ventricular extrastimuli but concealed during sinus rhythm may be clinically relevant and may explain some of the false negative late potentials and reduced sensitivity of the conventional SAECG in predicting VT. In contrast, those late potentials that are detected during sinus rhythm but lost after ventricular extrastimuli are often clinically irrelevant and may account for the false positive late potentials and reduced specificity of the conventional SAECG.
Revision received May 21, 1996.


text:
By John Biver Dispatches May 4, 2012
An excellent review of the book "Why the West is Best: A Muslim Apostate's Defense of Liberal Democracy":
Ibn Warraq's eloquent defense of Western civilization
By Bruce Thornton
Occasionally, the mainstream media will let slip something that reveals the incoherence of multiculturalist orthodoxy. Not long ago, the New York Times reported on an Indian casino in California that had begun purging its rolls of members deemed insufficiently Indian. At the end of the story, an official from the federal Bureau of Indian Affairs, himself an Indian, remarked: "The tribe has historically had the ability to remove people. Tolerance is a European thing brought to the country. We never tolerated things. We turned our back on people."
Such honesty about the Western origins of goods like tolerance is rare these days among the media, academic, and popular-culture purveyors of multicultural "diversity." For them, other cultures are just as good as, if not better than, the West's—but at the same time, these cultures allegedly endorse Western ideals such as tolerance, gender equality, human rights, political freedom, and the other universal boons to which people everywhere aspire.
They deem it Eurocentric or racist to assert the superiority of the West because it originated those goods, even as they castigate the West for its racist, sexist, imperialist, and colonialist crimes. But as Ibn Warraq shows in his thoughtful and compelling new book, the ideals that even multicultural relativists profess have their origin and highest development in the West.
Culture/ValuesForeign/Defense Policy
Previous articleGingrich admiration v. Gingrich Derangement Syndrome
Next articleHomos behaving badly: Bullying and hissy fits


text:
package org.instantlogic.designer.codegenerator.classmodel;

public class ValidationClassModel extends AbstractClassModel {

	public TextModel message;
	public int ruleDeductionIndex;

	public TextModel getMessage() {
		return message;
	}
	public int getRuleDeductionIndex() {
		return ruleDeductionIndex;
	}
}

redpajama_set_name: RedPajamaGithub


text:
Q: How to create multiple debian packages using the same debian directory I am packaging an application in my PC for installation/distribution purposes. I have created the package successfully using dpkg facility in ubuntu 12.04. Now I need to create multiple packages (binary package(like xxx.deb), source package(xxx.1.0-1-devel.deb)) using the same file set-up.
Debian folder contains all modified files required for packaging using dpkg-buildpackage -rfakeroot. If I need to create all .deb files using the same debian file set-up. What should I additionally do ?
additionally, below things also confused for me.
what should include -devel package ? does it only development header files or development header files and required shared libraries or all binary files including header files ?

A: I assume that your target program is a c/c++ or similar language.

*

*You can include all files in one binary packages. But still the best to separate development files in another one.
*-dev (not -devel) should include only headers, statics libs, shared libs and even binary tools if these tools only used for development.

*You can declare multiple binaries packages for single source in debian/control. The best is to learn by example, samba:
<to editors please keep these links clear, I want to mention them>
Source package: http://packages.ubuntu.com/source/focal/samba , see how many binary package built from same source.
Let choose one (main package, samba-*.deb): http://packages.ubuntu.com/focal/samba , see to right side *-debian.tar.xz. Download it then open it. Read its control file:
  Source: samba
  ...

  Package: samba
  ...

  Package: samba-libs
  ...

Each binary package has its own *.install file which list the files going to be installed and where. Example: samba.install, samba-libs.install, ..

*For libraries, static one included only in *-dev package.
Shared ones should be included in both if you have different versions that could be installed in same time.
Example, samba-dev, at the bottom of the page click on list of files and this with static libs libc6-dev.
Otherwise, put shared libs only in the regular binary package then add it as dependency for the development package.
References:

*

*Debian Policy Manual: Chapter 8 - Shared libraries

*Debian Library Packaging guide


text:
Preparation report of Arun Hydropower Project favor Chinese company
Kathmandu, Jan 23, 2018
Nepal Electricity Authority (NEA) and Changjiang Institute of Survey Planning Design and Research (CISPDR) have signed a deal for the preparation of detail project report (DPR) of the Upper Arun Hydropower Project.
Chief of Upper Arun Hydropower Project Bishow Dhoj Joshi on behalf of the NEA and CISPDR's Director Jhang Xinqiang signed the agreement.
According to Project Chief Joshi, the CISPDR shall immediately begin the DPR preparation task. And, the report should be prepared within two years and the cost of the DPR would cost Rs 1.31 billion.
For the DPR preparations, the World Bank has provided Rs 2 billion in assistance to the Government of Nepal. The issue of Upper Arun was discussed during then Prime Minister KP Sharma Oli's visit to the People's Republic of China.
The Government of Nepal has also asked the Government of China for support to develop the project. The project with the capacity of 335 Megawatt lies in Makalu Barun National Park in Sankhuwasabha district. But, Project Chief Joshi said that the project capacity could increase up to 700 megawatt thanks to technological advancement.
NEA Spokesperson Prabal Adhikari expressed the confidence that the much-talked-about project would advance with the current progress.
The NEA is the plan to make the project a reservoir-based one. Adhikari added that total cost of the project would be finalized after the DPR but the tentative cost estimation is around Rs 53.93 billion.


Kanchenjunga is the third-highest mountain in the world. It is 8586mt. high situated in eastern part of Nepal. It is located on the border of Nepal and Sikkim , about 46 miles northwest of Darjeeling. It is an enormous mountain mass, and many peaks rise from its narrow icy ridges. First climbed in 1955 by a British team including famed rock climber Joe Brown & George Band. The successful British expedition team of 1955 set the standard by stopping a few feet below of the actual summit, in honor of the local religion. This peak lives up to its reputation.
The route of North face is the safest route. This route also is not very easy. Pangpema at 5,180m, a charming base camp from where Kanchenjunga climbing starts. It is the most challenging to establish three camps on the expedition route of North ridge.
Day 01: Arrive in Kathmandu Airport & Transfer to Hotel .
Day 02: Kathmandu Sighting .
Day 03: Kathmandu free day .
Day 04: Preparation for expedition .
Day 05: Departure from Kathmandu to Biratnagar by plane.
Day 06: Biratnagar to Taplejung by flight .
Day 07: Taplejung to Mitlung .
Day 08: Mitlung to Chirwa .
Day 09: Chirwa to Sekathum .
Day 10: Sekathum to Amjilesa .
Day 11: Amjilesa to Gyabla .
Day 12: Gyabla to Ghunsa .
Day 13: Ghunsa to Khambachen .
Day 14: Khambachen to Lhonak .
Day 15: Lhonak to Pang Pema .
Day 16: Pang Pema to Kanchenjunga Base Camp .
Day 17: Rest Day Base Camp for acclimatization .
Day 18: D39 Climbing period of Kanchenjunga .
Day 40: Base Camp to Pang Pema .
Day 41: Pang Pema to Lhonak .
Day 42: Lhonak to Ghunsa .
Day 43: Ghunsa to Sinion La .
Day 44: Sinion La to Mirgin La to Tseram .
Day 45: Mirgin La -Tseram to Oktang-Torotan .
Day 46: Oktang -Torotan to Omje Khola .
Day 47: Omje Khola to Mamanke .
Day 48: Mamanke to Bhanjyang .
Day 49: Bhanjyang to Lali Kharka .
Day 50: Lali Kharka to Suketar .
Day 51: Suketar to Kathmandu.
Day 52: Rest Day Kathmandu .
Day 53: Departure to Airport.
- Trek in full board.
- Flight Kathmandu to Biratnagar to Taplejung and Suketar to Kathmandu.
- Permit Fee (Royalty) of Mt. Kanchejunga.
- All indicated visits and transportation.
- A professional guide, base camp staffs (cook, cook helpers, porters), camping gears up to base camp.
- Climbing guide for altitude.
- Reception at airport, transfers airport/hotel/airport.
- Permits and entrance fees in national parks/ conservation area projects.
- Transportation to/ from trek.
- Tourist standard hotel in Kathmandu on bed and breakfast basis.
Note:- Guide and staff stay in base camp. Only climbing staff accompany in altitude.
- All the drinks except tea or coffee in breakfast.
- Entrance fees in the monuments, museums and cities.
- High altitude food, fuel, gas after base camp.
- Evacuation and rescue / medical insurances of clients.
- All the airport taxes, customs and duties declaration of the expedition equipments.
- Satellite Phone and Walking Talking.
- Oxygen and Mask regulator.
Best Season --› Feb/Mar/Apr and Sep/Oct/Nov.


text:
Worst geek movies of the decade – CNET
Don't bother to watch these geek movies
There were some pretty awful (and great) geek movies in 2018. But what about this past decade? Using Metacritic's scoring system, we've compiled a list of the worst geek movies since 2010.
Tying for No. 48: Venom. In our review, we said this Tom Hardy-led superhero film "bites off more than it can chew."
Metascore: 35
Published: March 31, 2019 Caption:Lance CartelliPhoto:Sony Pictures
48 (tie). The Huntsman: Winter's War
The prequel and sequel to Snow White and the Huntsman is "a resoundingly unlovable movie," per the San Francisco Chronicle.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Universal Pictures
48 (tie). Mute
This Netflix film about a mute man searching for his missing girlfriend is "a slog, and a depressing one," The Atlantic said.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Netflix
48 (tie). Kin
This box-office bomb, which grossed just $10 million, is "remarkably empty of any sense of kinship," according to the Associated Press.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Lionsgate
43 (tie). Ghost Rider: Spirit of Vengeance
The sequel to Ghost Rider "seems to have been ill-conceived from the very start," according to Slant Magazine.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Columbia Pictures
43 (tie). The Mummy
The Mummy reboot, starring Tom Cruise, is "a monster fail," per Rolling Stone.
43 (tie). The Dark Tower
Based on the Stephen King novel of the same name, The Dark Tower is "a mess of a movie," according to the Arizona Republic.
43 (tie). Ice Age: Collision Course
The fifth film in the Ice Age saga, Collision Course is "lazy, scattershot and excruciatingly unfunny," the Washington Post said.
Published: March 31, 2019 Caption:Lance CartelliPhoto:20th Century Fox
43 (tie). Alice Through the Looking Glass
Johnny Depp reprises his role as the Mad Hatter in this sequel that is a "dull, formulaic theme-park ride," per the Philadelphia Inquirer.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Walt Disney Pictures
40 (tie). Sucker Punch
This Zack Snyder-directed fantasy film is "hands-down the most nightmarishly awful film of [2011]," per the Philadelphia Inquirer.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Warner Bros. Pictures
40 (tie). Jonah Hex
Based on the DC Comics character of the same name, Jonah Hex is "bracingly inept," according to LA Weekly.
40 (tie). After Earth
This post-apocalyptic sci-fi film starring Will and Jaden Smith is "a lethargic slog," said USA Today.
34 (tie). Transformers: Age of Extinction
The fourth film in the Transformers franchise is an "endless barrage of nonsense and noise," according to Entertainment Weekly.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Paramount Picture
34 (tie). Robin Hood
Nominated for three Razzies, the Robin Hood reboot "will make you side with the Sheriff of Nottingham," per Time Out.
34 (tie). Repo Men
This sci-fi action-thriller starring Jude Law is "simply a case of movie malpractice," Premiere said.
34 (tie). Legion
Legion is "a cartoonishly grim supernatural thriller," according to The AV Club.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Screen Gems
34 (tie). Warcraft
Based on the video game series of the same name, Warcraft is "a godawful mess," said Rolling Stone.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Legendary Pictures
34 (tie). Independence Day: Resurgence
The Will Smith-less Independence Day sequel is "an immaculate case-study in how far blockbusters have fallen," per IndieWire.
31 (tie). Your Highness
Rolling Stone said "nothing works" in this stoner fantasy film starring Natalie Portman and James Franco.
31 (tie). Teenage Mutant Ninja Turtles
The Teenage Mutant Ninja Turtles reboot is "utterly inessential," according to Time Out.
Published: March 31, 2019 Caption:Lance Cartelli
31 (tie). House at the End of the Street
This Jennifer Lawrence-led horror film is "a terrible little movie even by the standards of the genre," per the Boston Globe.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Paramount Pictures
29 (tie). Slender Man
This horror film, based on the character of the same name, is "flat-out dull," per the AV Club.
29 (tie). Paranormal Activity: The Ghost Dimension
The sixth and final entry in the Paranormal Activity saga "feels like the last wheeze of a played-out series," according to The Wrap.
26 (tie). Ratchet & Clank
This animated film, based on the video game of the same name, is "insufferable. And seemingly interminable," said the Seattle Times.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Gramercy Pictures
26 (tie). A.X.L.
This fantasy film about a robotic dog is "something worse than bad. It's utterly forgettable," per the AV Club.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Global Road Entertainment
26 (tie). Bright
Starring Will Smith, Bright is a "gaudy, overstuffed piece of blockbuster trash," according to RogerEbert.com.
23 (tie). Hitman: Agent 47
Based on the Hitman video game series, this reboot of the franchise is "just high-tech Swiss cheese, filled with holes and smelling like last week's refrigerator contents," said the Chicago Sun-Times.
23 (tie). 47 Ronin
Starring Keanu Reeves as a samurai (seriously), this fantasy film is "insufferably boring, culturally hegemonic and profoundly ugly," according to Film.com.
23 (tie). Season of the Witch
Nicolas Cage plays a heroic knight of the Crusades in this 2011 fantasy film that is "both overblown and undercooked," per Variety.
17 (tie). Transformers: The Last Knight
The sequel to Age of Extinction is an "incoherent mess," per RogerEbert.com.
17 (tie). The Happytime Murders
This dark comedy, set in a world with humans and puppets, "doesn't have an interesting story, and the jokes fall flat," per the San Francisco Chronicle.
Published: March 31, 2019 Caption:Lance CartelliPhoto:STX Entertainment
17 (tie). Terminal
This thriller that follows two assassins is "as soft-boiled as they come," according to Entertainment Weekly.
Published: March 31, 2019 Caption:Lance CartelliPhoto:RLJE Films
17 (tie). Pixels
This sci-fi comedy, starring Adam Sandler, is "stupid and lifeless," according to New York Magazine.
17 (tie). Flatliners
A remake of the 1990 film of the same name, Flatliners "plays like a malpractice case: a cheap horror film grafted on to an episode of House," said the Wrap.
17 (tie). Fantastic Four
"Someone forgot to put anything fantastic" into the Fantastic Four reboot, said the New York Daily News.
16. Skyline
This sci-fi disaster film is "the most boring alien-invasion movie ever," said the New York Daily News.
15. R.I.P.D.
Based on the comic Rest in Peace Department, this Ryan Reynolds-led film is a "dud that squanders a decent cast," according to the Washington Post.
14. Piranha 3DD
This sequel to Piranha 3D is "gloriously bad," per Boxoffice Magazine.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Dimension Films
11 (tie). The Roommate
This psychological thriller "sucks bad, real bad," said Rolling Stone.
11 (tie). Hansel and Gretel: Witch Hunters
This fantasy film starring Jeremy Renner as a witch hunter is "so demeaningly bad, so utterly without merit, that there is a kind of purity in its awfulness," according to the Guardian.
11 (tie). Underworld: Blood Wars
The fifth entry in the Underworld franchise is "tragically bereft of the pulp verve this nonsense needs to be tolerable," per Empire.
9 (tie). Max Steel
Based on the eponymous toy line, Max Steel is a "drearily lame time-waster," said Variety.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Open Road Films
9 (tie). Shark Night 3D
This 2011 horror film is "about as humorless — and joyless — as they come," according to the Austin Chronicle.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Rogue
7 (tie). The Last Airbender
Based on the television series Avatar: The Last Airbender, this M. Night Shyamalan-helmed fantasy film is "an agonizing experience," per the Chicago Sun-Times.
7 (tie). A Haunted House
Empire called this found-footage parody film "dreadful."
6. Replicas
Keanu Reeves stars as a neuroscientist who brings his family back to life in this 2018 thriller that is a "disaster," per We Got This Covered.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Entertainment Studios Motion Pictures
4 (tie). The Darkest Hour
This 2011 thriller is a "Razzie-worthy sci-fi zero," said Slant Magazine.
4 (tie). Vampires Suck
A parody of the Twilight saga, this 2010 horror comedy is a "sloppy, tossed-off collection of parodic gags of vampire flicks and gratuitous pop-cultural references," according to Time Out.
3. Silent Hill: Revelation 3D
The sequel to 2006's Silent Hill is "stiff, episodic and disjointed," per the AV Club.
Published: March 31, 2019 Caption:Lance CartelliPhoto:Alliance Films
2. The Emoji Movie
This animated comedy is a "soul-crushing disaster because it lacks humor, wit, ideas, visual style, compelling performances, a point of view or any other distinguishing characteristic that would make it anything but a complete waste of your time," The Wrap said.
1. Scary Movie 5
The fifth installment in the Scary Movie saga is "lazy, boring, vile and tragically unfunny attempt at a horror-film spoof," according to the Washington Post.
Worst geek movies of the decade
How to watch every Marvel Cinematic Universe film in the right order
by Richard Nieva
Galaxy S10E is the maverick Galaxy S10
Get a 4-pack of Tile Mate 2018 trackers for $40
Best seat in the house: Exploring the incredible Walt Disney Concert Hall
by Geoffrey Morrison
Pixel 3 vs. OnePlus 6T: Which Android should you buy?
by Lynn La
Tags: CNET, decade, Geek, movies, tech, tech news, technology, technology news, worst


I'm recently working on a project, in which we are provided two network one with IPv6 Connectivity only and other with IPv4 Connectivity only. These two networks are connected to each other with a switch(i.e., Cisco 4503 which also works as router) and our task is that, we have to configure that switch/router in such a way that a IPv4 host can send packet to IPv6 host and vice-verse. I have try all the tunneling, but in all of them IPv6 can only be connected to IPv6 host only not with IPv4. I need your help in this that how can i made router configuration so that it should be possible to connect two Different Host with different IP versions i.e., one with ipv4 and other with ipv6.
I also think that Gateway translation is best, but there is one more problem, that CISCO 4503 also not support This type of translation.
No, you need to either implement protocol translation of some sort such as NAT-PT, or dual stack one of the two networks. You cannot just pass IPv6 traffic into an IPv4 network or vise versa.


Checklist for preparation of audit in information technology (IT) department in a pharmaceutical facility.
1. Is the list of Software's installed in equipments and instruments is available?
2. Are the certificates for all installed software's available?
3. Are the CDs for software's available and stored properly?
4. Is the list for PCs available?
5. Is the back up of data taken as per schedule? Check the backup data randomly.
6. Is the list for password protection available?
7. Is the list for passwords approved?
8. Is the preventive maintenance program for computers available?
9. Is the virus protection software installed is of current version?
10. Is the IT policy available?


text:
CHECHNYA'S NEW GOVERNMENT.
Publication: Monitor Volume: 4 Issue: 7
January 13, 1998 08:00 AM Age: 25 years
Chechnya's acting prime minister, Shamil Basaev, has obtained preliminary approval from President Aslan Maskhadov for a list of candidates to fill key posts in the new government he is forming. In accordance with Chechnya's constitution, Maskhadov will give his final approval only after the candidates have been confirmed by parliament, but the main appointments are already clear. Virtually all of the well-known field commanders will remain in the new government, but the influence of the radicals — those who favor a hard line both domestically and in relations with Moscow — will increase.
The Ministry of Defense will be headed by Brig. Gen. Ruslan Gelaev, who was deputy premier in the old cabinet; like Basaev, he is seen as favoring an uncompromising approach toward Moscow. The Ministry of Internal Affairs will be reorganized into the Ministry of Shariah Security and will be headed by Islam Khalimov, who was deputy premier in charge of social issues in the last government. The former interior minister, Kazbek Makhashev, will become deputy premier overseeing the security forces. Shamil Basaev's brother, Shirvani Basaev, will become Minister of Fuel and Energy, with a mandate to put a stop finally to the massive plundering of petroleum.
Movladi Udugov is expected to lose his rank as deputy premier but will become Foreign Minister with greater powers than those his predecessor enjoyed; Udugov will continue to head the Chechen delegation in negotiations with the Russians. Udugov is the author of the modern concept of Chechen statehood and of the idea of creating an Islamic state in Chechnya. He is also leader of the "Islamic Nation" movement, which has as its goal the restoration of Dagestan within its "historical borders," that is, the union of Dagestan and Chechnya into a single state. (NTV, RTR, January 12)
Disagreement Over How to Treat Basaev.
CONFLICTING ASSESSMENTS OF RUSSIA'S ECON..Next
KAZAKHSTAN'S PRESIDENT VOWS TO CRACK DOW..


Armand De Brignac champagne bottle that has been exquisitely handcrafted into a stunning candle. This is the perfect gift for any champagne or candle lover! The candle is scented with pomegranate. It makes a fabulous gift. If you are interested in a custom order, please message me before purchasing and I can give you some options!
Each candle is unique – no two are the same. Because the cut bottle tops are hand polished and sanded, they are not perfectly formed but they are smooth to the touch.These can be reused time after time.


Announcement Welcome to The Golden Paw Company! We are so excited that you are here and looking to pick up something special for your pup.
Welcome to The Golden Paw Company! We are so excited that you are here and looking to pick up something special for your pup.
My dog is the cutest!!!!
Just a girl and her pup creating one-of-a-kind bandanas, so your fur babies can always feel their best!
Leah is a newlywed wife, who loves to create, sew and bring you the perfect bandana for your pup.
Remi is an 18 month old AKC Golden Retriever, who is the love of his parent's life and the inspiration behind The Golden Paw Co.