Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.
Copyright: © 2017 Cannito 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 study has been funded by: i) Associazione Italiana per la Ricerca sul Cancro (AIRC, Milano, Italy), IG-2014 grant, project code 15274, (to MP); ii) the University of Torino (Fondo di Ateneo ex 60%, to EN and MP).
Non-Alcoholic Fatty Liver Disease (NAFLD) has emerged in recent years as a major form of chronic liver disease affecting both children and adults worldwide, with a prevalence ranging from 3–15% in the general population and up to 70% among overweight individuals [1–5]. Epidemiological data indicate that 20–30% of NAFLD patients, particularly obese and/or diabetic type II and/or those affected by metabolic syndrome, can develop Non-Alcoholic Steato-Hepatitis (NASH) and fibrosis and eventually progress to cirrhosis and end-stage liver disease [1–9]. In the natural history of the disease, an increase in hepatic lipid deposit (i.e., fatty liver or steatosis) is considered a required early event and prerequisite, potentially benign, for the development of NASH [1–9]. Along these lines, a large body of literature data supports the notion that upon lipid accumulation within parenchymal cells certain lipids, in particular saturated fatty acids, can exert cyto-toxic effects also known as lipotoxicity, resulting in hepatocyte damage and in triggering inflammatory responses [10–12].
In this scenario, recent data suggest that fat-laden hepatocytes undergoing lipotoxicity may release extracellular vesicles (EVs). EVs are an heterogeneous family of small membrane vesicles released by dying or activated cells that includes exosomes (30–100 nm in diameter), released by exocytosis and microparticles or microvesicles (MVs, 100–1000 nm in diameter) [13,14]. MVs, in particular, are small vesicles surrounded by a phospholipid bilayer, generated and released through a controlled budding/blebbing of the plasma membrane . These MVs can act in an autocrine/paracrine manner carrying to surrounding cells several molecules, including surface receptors, membrane, cytosolic or even nuclear proteins, lipids and RNAs (mRNAs and microRNAs) [14–16]. These MVs, can either remain in the tissue of origin or get into the blood circulation, delivering "molecular information" to target cells by either interacting with surface receptors and/or following internalization [17–18].
Concerning liver parenchymal cells, previous studies have established that both primary hepatocytes and immortalized cells of hepatocellular origin can release both exosomes and MVs [19–22]. Furthermore, increased circulating levels of MVs are associated with liver injury in either "in vivo" models of chronic liver diseases or human blood samples from patients with NAFLD and alcohol or chronic hepatitis C related cirrhosis [19, 22–26]. With regard to NAFLD progression, we have reported that MVs are released by hepatocytes undergoing lipotoxicity in a caspase-3 dependent manner and act as pro-angiogenic and profibrogenic stimuli promoting endothelial and hepatic stellate cells activation [22,23]. In the same experimental setting a recent study has also shown that MVs released by fat-laden hepatocytes or HuH7 cells may act as pro-inflammatory stimuli on macrophages through signals operated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), expressed on the surface of these MVs . Along these lines, one of the most recently identified contributor to the cross talk between hepatocytes and inflammatory macrophages is represented by the multiprotein platform complex nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, whose activation has been suggested to play a crucial role in the progression of NAFLD . In particular, the transition from NAFLD to NASH associates with NLRP3-inflammasome activation and an increased expression of inflammasome-related components, including apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), caspase-1 (CASP‑1) and pannexin [28–32]. In addition, inflammasome activation involves not only liver innate immunity cells but also parenchymal cells [29,31,32], with studies indicating that saturated fatty acids can specifically activate the inflammasome complex in hepatocytes inducing IL-1β expression and release [29,32]. However, no study has so far investigated whether MVs released from fat-laden hepatocytes may promote NLRP3-inflammasome activation .
In the present study we provide for the first time evidence that MVs released by fat-laden cells can directly up-regulate NLRP3 inflammasome in both hepatocytes and macrophages, resulting in a significant increase in IL‑1β release.
Enhanced chemiluminescence (ECL) reagents, nitrocellulose membranes (Hybond-C extra), and secondary Cy3-conjugated antibodies were from Amersham Pharmacia Biotech Inc. (Piscataway, NJ, USA). Polyclonal antibodies against NF-kB p65 (#8242), IKKβ (#2370) and p-IKK α/β (#2697) or monoclonal antibodies against IKB (#4814) and p-IKB (#9246) were from Cell Signalling Technologies (Danvers, Massachusetts); polyclonal antibodies against IL-1β (sc-7884), caspase p10 (sc-515) and LaminA (sc-20680) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); monoclonal antibodies against NLRP3 were from Abcam (Cambridge, UK, code ab109314) or from Adipogen (Adipogen AG, Liestal, Switzerland, code AG-20B-0014-C100). Monoclonal antibodies for α-tubulin and β-actin and all other reagents of analytical grade were from Sigma Chemical Co (Sigma Aldrich Spa, Milan, Italy). HiPerfect Transfection Reagent was from Qiagen (QIAGEN S.r.l., Milan, Italy). ELISA kit for human IL-1β was from Abcam (Cambridge, UK, code ab100562).
Eight-week old male C57BL/6 mice were purchased from Charles River Laboratories (Charles River UK Ltd., Margate, UK) and fed for 4 or 8 weeks with either methionine- and choline-deficient (MCD) diet or related control diet sufficient in methionine and choline (MCS) (Laboratorio Dottori Piccioni, Gessate, Italy) as previously described . Experiments were approved by the Italian Ministry of Health and by the university commission or animal care following the criteria of the Italian National Research Council. The experimental MCD and MCS diets were also administered to C57BL/6 caspase 3 knockout (Casp3−/−) mice and related wild type littermates up to 6 weeks exactly as previously described . The latter genetically manipulated mice and their littermates were treated in compliance with the Guide for the Care and Use of Laboratory Animals (National Academy of Science,Washington, DC), and animal procedures were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.
HepG2 and THP-1 cells (American Type Culture Collection, USA) were maintained in Dulbecco's modified Eagle's medium or RPMI 1640 respectively supplemented with 10% fetal-bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 25 μg/ml amphotericin-B. To evaluate the role of LPS, palmitic acid (PA, 0,25mM) or MVs, HepG2 cells were seeded in normoxic conditions to obtain the desired sub-confluence level (65–70%) and then incubated in experimental conditions (up to 48h hours). THP-1 cells were seeded 7x105 in 35 mm petri dishes and differentiated for 48 hrs with phorbol 12-myristate 13-acetate (PMA, 50nM); after 24 hrs of incubation with fresh medium THP-1 cells were properly stimulated. mRNA, cellular extracts and the corresponding medium were tested for inflammasome components (NLRP-3 and caspase-1) and related cytokine (IL-1β). In some experiments HepG2 cells were transfected with specific siRNA for caspase-3 or NLRP3 and non-silencing siRNA (scramble, SC).
The procedures adopted in this study to isolate, purify, quantify and characterize MVs obtained from fat-laden HepG2 have been extensively described in previous studies [22,23,26]. Briefly, for MVs isolation HepG2 cells were seeded onto a 100-mm dish and cultured until they reached 80 to 85% confluency. Cells were then incubated with 0.25 mM palmitic acid (PA) in serum-free DMEM, supplemented with 1.1% penicillin and streptomycin and 1% endotoxin-free bovine serum albumin (BSA) for up to 24 hours. The uptake of FFAs from hepatocytes was evaluated with an Oil Red-O staining according to the manufacturer's instruction (see later). Control cells were incubated with the same serum-free medium supplemented with the vehicle used to dissolve PA.
MVs were then isolated by differential centrifugation and characterized essentially as previously described [22,23,26]. The isolation procedure involved the following steps: i) collected media were centrifuged at 3000 g for 15 min to remove cell debris and aggregates; ii) the supernatant from last centrifugation were then transferred to new tubes and ultra-centrifuged at 100,000 g for 90 minutes at 10° C; iii) the supernatants collected from the latter step were used as MVs-free control whereas the pelleted MVs fraction were gently re-suspended into 500 μl of PBS for flow cytometry or in 500 μl of serum-free DMEM medium and used for subsequent in vitro studies. To trace hepatocyte-derived MVs in flow cytometry and confocal analysis, the PKH26 fluorescent dye (Sigma-Aldrich) was used, according to the manufacturer's instructions and as previously described (22).
A complete characterization of MVs, including size, composition and distribution, was performed by dynamic light scattering, TEM (see here S1A Fig) and FACS as reported previously .
MVs were quantified either by the bicinchoninic acid (BCA) protein assay  or (see S1B Fig) by flow cytometry using MVs resuspended in PBS and the Calcein AM (BD Biosciences, San Jose, CA, USA)—based procedure previously described . The preparations of MVs employed in the present study were also tested for presence of NLRP3 and IL-1β protein levels and, as shown in S1C Fig, we could not find any trace of such proteins in pelleted MVs. In addition, no trace of mRNA levels for NLRP3 or IL-1β could be detected in MVs (data not shown).
RNA extraction, complementary DNA synthesis, quantitative real-time PCR (Q-PCR) reactions were performed as previously described . Human or murine NLRP3, IL1β and CASP-1 mRNA levels were measured by Q-PCR, using the SYBR® green method as described . The amplification mix was prepared using Roche LightCycler FastStart DNA MasterPLUS SYBR Green I kit following manufacturer's instructions and real-time PCR was performed using LightCycler instrument. Oligonucleotide sequence of primers used for RT-PCR were: sense, 5'-TGAAAGCTCTCCACCTCCAG-3', reverse 5'-CACGCAGGACAGGTACAGAT-3' (for human IL-1β); sense, 5'-AAGGAAGTGGACTGCGAGAA-3', reverse 5'-CCCTCGAATTTGCCATA-3' (for human NLRP3); sense, 5'-GCTTTCTGCTCTTCCACACC-3', reverse 5'-CATCTGGCTGCTCAAATGAA-3' (for human CASP-1); sense, 5'-TGCTCTTCACTGCTATCAAGCCCT-3', reverse 5'-ACAAGCCTTTGCTCCAGACCCTAT-3′ (for murine NLRP3); sense, 5'-GGAGAACCAAGCAACGACAAAATA-3', reverse 5'-TGGGGAACTCTGCAGACTCAAAC-3′ (for murine IL-1β); sense, 5′-TCCGCGGTTGAATCCTTTTCAGA-3', reverse 5′-ACCACAATTGCTGTGTGTGCGCA-3 (for murine caspase-1); sense, 5'-TGGCCCAGACCCTCACACTCAG-3', reverse, 5'-ACCCATCGGCTGGCACCACT-3' (for murine TNF-α). Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal reference and co-amplified with target samples using identical Q-PCR conditions. Samples were run in triplicate and mRNA expression was generated for each sample. Specificity of the amplified PCR products was determined by melting curve analysis and confirmed by agarose gel electrophoresis.
The siRNAs and related non-silencing controls were transfected in HepG2 cells with HiPerfect Transfection reagent (Qiagen Italia, Milano, Italy) according to manufacturer's instructions up to 48hrs (CASP3) or 72hrs (NLRP3). Transfected cells in fresh medium were then exposed to the desired experimental conditions and then harvested for sample preparation.
Total cell lysates and nuclear vs cytosolic extracts, obtained as described, [35, 36] as well as MVs preparations (the latter by resuspending pelleted MVs directly in RIPA buffer) were subjected to sodium dodecyl sulfate-polyacrylamide gel-electrophoresis on 12%, 10% or 7.5% acrylamide gels, incubated with desired primary antibodies, then with peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins in Tris-buffered saline-Tween containing 2% (w/v) non-fat dry milk and finally developed with the ECL reagents according to manufacturer's instructions. Sample loading was evaluated by reblotting the same membrane with antibodies raised against β-actin or α-tubulin or, for nuclear extracts, Lamin A.
Paraffin liver sections of specimens derived from patients with NAFLD/NASH-related advanced fibrosis or derived from either wild type C57BL/6 mice or caspase 3 -/- mice fed MCD diet or related control diet (MCS) as well as from the liver of NLRP3 +/- mice, were analyzed as previously described [33,34]. The use of human material conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved for this study by the University of Torino Bioethical Committee. Immunostaining procedure were performed, according to established procedures previously described in detail [33,37], on paraffin sections (2 μm thick), mounted on poly-L-lysine coated slides, that were incubated with the antibodies against NLRP3 (Abcam, Cambridge, UK, dilution 1:100, or from Adipogen AG, Liestal, Switzerland, dilution 1:100) and murine F4/80 (Affimetrix, Cleveland, OH, USA, dilution 1:1000) or human CD68 (Bio-Rad; Segrate—MI, dilution 1:40). After blocking endogenous peroxidase activity with 3% hydrogen peroxide and performing microwave antigen retrieval, primary antibodies were labeled by using EnVision, HRP-labeled System (DAKO) and visualized by 3'-diaminobenzidine substrate. For negative controls the primary antibodies were replaced by isotype- and concentrations-matched irrelevant antibody [33,37]. Quantification of NLRP3 positive immunostaining in murine liver was also performed by histo-morphometric analysis using a digital camera and a bright field microscope to collect images that were then analyzed by employing the ImageJ software.
Indirect immunofluorescence and confocal microscopy analysis was performed on cultured cells as previously described [35,36] by employing a primary rabbit polyclonal antibody raised against NF-kB p65 (Cell Signalling Technology, Danvers, MA, USA; diluted 1:400). Nuclei were stained using 4,6-diamino-2-phenylindole (DAPI, blue fluorescence).
Cultured cells, seeded in 6-well culture plates (105 cells/well) or on microscope slides, were exposed for 1 hour (THP-1) or 3 hrs (HepG2) to PKH26 fluorescent dye -MVs released by HepG2 treated with palmitic acid (0,25mM) as previously described . Internalization of MVs was evaluated by: a) flow cytometric analysis: cells were rapidly washed with PBS, collected by trypsinization and re-suspended in PBS for analysis. Detection of PKH26 red fluorescent die-MVs (FL2) was performed on at least 5,000 cells per sample with a FACScan using the CellQuest software (Becton-Dickinson, Milano, Italy); b) indirect immunofluorescence and confocal microscopy analysis: nuclei (blue fluorescence) were stained using 4,6-diamino-2-phenylindole (DAPI), MVs were stained with PKH26 fluorescent die (red fluorescence) and cytoskeleton were marked with Alexa Fluor 488 phalloidin (high-affinity filamentous actin, F-actin, green fluorescence) and slides were examined with an Olympus Fluoview 300 confocal laser scanning microscope.
HepG2 cells were seeded in 6-wells culture plates until reaching 80–85% of confluence and incubated with 0.25 mM palmitic acid in serum-free DMEM, supplemented with 1.1% penicillin and streptomycin and 1% endotoxin-free BSA, for up to 24 hours. Control cells were incubated with the same serum-free medium supplemented with the vehicle used to dissolve the FFAs. After treatment, cells were fixed with 4% of paraformaldehyde and the uptake of FFAs from HepG2 cells was detected by staining cells for 30 minutes with Oil-Red-O 0,5% (Sigma-Aldrich, St. Louis, MO, USA) diluted in isopropanol, according to the manufacturer's instruction. Then, bright field microscope was employed to collect images.
Plasma alanine aminotransferase (ALT) and liver triglycerides (TGs) were determined by spectrometric kits supplied by Radim S.p.A. (Pomezia, Italy) and Sigma Diagnostics (Milano, Italy), respectively. Release of IL-1β in culture medium was evaluated, according to the manufacturer's instructions by commercial enzyme-linked immunosorbent assay (ELISA) kit supplied by Abcam (Cambridge, UK).
Caspase-3/7 activity as a measure of apoptotic cell death was determined in HepG2 cells exposed to palmitic acid (0,25 mM) for 24 hrs and following the manufacturer's instructions (Apo-ONE Caspase-3/7 Homogeneous Assay, (Promega, Madison, WL, USA). After the treatment HepG2 cells were washed twice with PBS and a volume of 50 μL of homogeneous caspase-3/7 reagent was added to each well. The plate was incubated for 1 h at room temperature. After this time, fluorescence was measured in a fluorimeter at excitation 499 nm and emission 521 nm.
Viability of HepG2 cells was evaluated by the MTT colorimetric assay. HepG2 cells were seeded 1x104 in 96-wells plates for each wells and incubated with 0.25 mM palmitic acid in serum-free DMEM, supplemented with 1.1% penicillin and streptomycin and 1% endotoxin-free BSA, for up to 24 hours. Control cells were incubated with the same serum-free medium supplemented with the vehicle used to dissolve the FFAs. After treatment, 30 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5mg/ml in PBS solution) was added to each well and incubated up to 2 hrs at 37°. Then, 150 μl of DMSO was added to empty wells for 20 minutes at room temperature. After this time, fluorescence was measured in a ELISA plate reader at absorbance of 540 nm.
For cell culture experiments data in bar graphs represent means ± SEM, and were obtained from average data of at least three independent experiments. Luminograms and morphological images are representative of at least three experiments with similar results. Statistical analysis for these experiments was performed by Student's t-test or ANOVA for analysis of variance when appropriate (p < 0.05 was considered significant).
The aim of the present study has been to investigate the possible role of MVs released from fat-laden cells in promoting NLRP3 inflammasome activation in either parenchymal cells and macrophages. According to recent literature data, inflammasome activation is fully detectable in association with overt steatohepatitis in experimental murine models of progressive NASH and such an activation involves liver innate immunity cells and hepatocytes [29,31,32]. In line with these findings, our experiments using mice fed with a methionine/choline deficient (MCD) diet showed that the transcript levels for NLRP3 and IL1β were significantly up-regulated after 4 weeks of treatment (Fig 1A, Fig 1B) in parallel with an appreciable worsening of lobular inflammation and parenchymal damage (Fig 1D, Fig 1E). No appreciable changes were evident at earlier time points. Similar data were obtained by evaluating caspase 1 transcripts, with the exception of a transient increase detected at 4 days (Fig 1C).
Fig 1. In vivo time-dependent analyses of inflammasome components, inflammatory cytokines and necrosis in MCD fed mice.
"In vivo" analysis by quantitative real-time PCR (qPCR) of transcripts of inflammasome components (NLRP3 and CASP-1 A, C) and related cytokine (IL-1β, B) as well as of TNF-α (E) in WT mice fed with MCS diet or MCD diet for 4d, 2wks, 4wks and 8wks. D. Analysis of serum ALT in WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8 wks. Data in graphs are expressed as means ± SEM (n = 6 mice for any experimental group); p values are indicated and referred to mice fed the MCS control diet.
In line with these data, NLRP3 immunohistochemical analysis showed and appreciable increase in mice fed the MCD diet for 4 weeks (Fig 2A). In these animals, NLPR3 up-regulation paralleled with a progressive recruitment of hepatic macrophages as evidenced by F4/80 immunostaining (Fig 2D). This scenario is also consistent to what is occurring in liver specimens from human NASH in which NLRP3 positivity was detectable at immunohistochemistry in parenchymal cells and in CD68 positive macrophages (Fig 2F).
Fig 2. Immunohistochemistry analysis for NLRP3 and F4/80.
A,B. Immunohistochemistry analysis for NLRP3 on liver specimens from WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8wks (A) as well as WT mice and Casp 3-/- knockout mice fed for 6wks (B). C. Image analysis quantification for NLRP3 staining (Fig 2B) as evaluated with Imagej software in liver sections from WT or caspase 3 -/- mice fed a MCD diet for 6 weeks. D,E. Immunohistochemistry analysis for F4/80 on liver specimens from WT mice fed with MCS diet or MCD diet for 2wks, 4wks and 8wks (D) as well as WT mice and Casp 3-/- knockout mice fed for 6wks (E). F. Immunohistochemistry analysis for NLRP3 and CD68 on liver specimens from human NAFLD/NASH-related advanced fibrosis (F3 and F4). Original magnification as indicated.
Since in a previous study some of us reported that MVs production associated with NASH required caspase-3 activity and was appreciably decreased in caspase-3 knock out (caspase-3-/-) mice fed with the MCD diet , here we evaluated whether the lack of MVs production in caspase-3-/- animals might affect NLRP3 responses. As shown in Fig 2B, NLRP3 staining was appreciably reduced in caspase-3-/- mice as compared to WT littermates, as also confirmed by histo-morphometric analysis (Fig 2C). In a similar manner liver macrophage infiltration was significantly lower in MCD-fed caspase-3-/- mice as compared to similarly treated WT animals (Fig 2E).
These data were confirmed by histo-morphometric analysis by employing a different antibody targeting NLRP3 either in NLRP3 +/- mice (S2A Fig) as well in either wild type and Caspase 3 -/- mice fed 6 wks with MCD diet (S2B Fig).
On the basis of these observations, we then designed experiments to directly investigate the hypothesis that MVs released by fat-laden cells may affect synthesis and activity of NLRP3 inflammasome. In order to do so we employed a well established "in vitro" model to generate MVs based on the exposure of hepatocytes or immortalized cells (HepG2 or HuH7) cells to 0.25 mM palmitic acid [22,27]. In the present study, in order to maintain reproducible conditions in the different experiments, we employed human HepG2 cells.
As expected, the incubation of HepG2 cells with palmitic acid resulted in a significant intracellular accumulation of lipids, as shown by Oil-Red-O staining (Fig 3A), as well as in a conditions of progressive caspase–dependent lipoapoptosis, as evidenced by Caspase 3/7 and MTT assays (Fig 3B, Fig 3C). The culture media was used to isolate MVs released by fat laden HepG2-cells as previously described . Protein content of the preparations of MVs in these experiments gave mean routine values from control cells of 0.02–0.03 μg/μl vs values of 4.3–4.6 μg/μl from cells exposed to PA for 24 hrs.
Fig 3. In vitro experimental model of lipotoxicity.
A. Red Oil-O staining in control HepG2 cells, HepG2 cells treated with BSA 1% or HepG2 cells exposed to palmitic acid 0.25mM in BSA 1% (BSA + PA) for 24hrs. B. Detection of Caspase-3/7 Activity in HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, analyzed by using Apo-ONE Caspase-3/7 Homogeneous Assay. C. Viability of HepG2 cells treated with BSA 1% or PA 0.25mM for indicated times, evaluated by MTT assay. D,E. Analysis of internalization of MVs in HepG2 cells by (D) flow cytometry or by (E) confocal microscopy: nuclei (blue fluorescence), MVs (red fluorescence) and cytoskeleton (F-actin, green fluorescence).
Interestingly, the addition to HepG2 cells of purified MVs conjugated with the fluorescent dye PKH26 lead to an internalization (within 3 hours) of red fluorescent MVs as shown by either flow cytometry analysis (Fig 3D) or by using confocal laser microscopy (Fig 3E).
We next analysed whether HepG2 cells exposure to MVs may modulate inflammasome activation. Fig 4 shows that MVs addition to resting HepG2 cells was followed within 3 hours by a significant up-regulation of the transcripts levels for NLRP3 (Fig 4A) and Caspase-1 (Fig 4B). When analysed in Western blots MVs-treated HepG2 cells also showed time-dependent increase in NLRP3, pro-IL-1β and pro-caspase-1 proteins levels that were associated with an early detection of active caspase-1 that was detected starting from 3 hrs and detectable until 48 hrs (Fig 4C). Conversely IL-1β protein levels were consistently enhanced at 16 hrs and thereafter (Fig 4C). ELISA assay analysis of IL-1β released in the culture media revealed that MVs exposure resulted in a rather early, although limited, release of the cytokine at 3 and 6 hrs to reach a significant level at 16 hrs (Fig 4D).
Fig 4. MVs activate NLRP3 inflammasome cascade.
A, B. Quantitative real-time PCR (qPCR) analysis of inflammasome components (NLRP3 and CASP-1) in HepG2 naïve cells treated with MVs up to 24 hrs. C. Western blot analysis of activation of inflammasome pathway by evaluating NLRP3 protein levels as well as cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed to MVs for indicated times. Equal loading was confirm by re-probing the same membrane with monoclonal antibody against the house-keeping β-actin. D. ELISA assay to evaluate IL-1β release (pg/ml) in culture medium of HepG2 naïve cells exposed to MVs up to 16 hrs. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control HepG2 cells) of three independent experiments.
It is known that NLRP3 expression requires NF-kB phosphorylation and its nuclear translocation . To get more insights on the mechanisms responsible for inflammasome activation by MVs we next analysed whether MVs may be able to modulate NF-kB pathway. In our hand, HepG2 cells exposure to MVs was followed by a very early phosphorylation of both IkB kinase (IKK) and of Ikβ (Fig 5A) as well as by an increase of p65 subunit in both the cytosolic and in nuclear extracts (Fig 5B). Nuclear translocation of p65 was further confirmed by means of indirect immunofluorescence (Fig 5C).
Fig 5. MVs up-regulate NF-KB pathway.
A. Western blot analysis of activation of IKK and IKβ evaluated in total extracts of HepG2 naïve cells exposed to MVs for indicating time. Equal loading was confirmed by re-probing the same membrane with un-phosphorylated protein. B. Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed to MVs up to 16 hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). C. Immunofluorescence analysis of NF-Kb/p65 nuclear translocation in control HepG2 cells or HepG2 cells exposed to MVs for 6hrs: nuclei (blue, DAPI staining), NF-Kb/p65 (green). Original magnifications are indicated.
From the data showing that caspase-3 deficiency can prevent NLRP3 up-regulation in mice fed the MCD diet, we next decided to deplete this enzyme by means of a specific siRNA. According to previous reports , blocking caspase-3 in HepG2 cells before lipid loading (Fig 6A) resulted in a decrease in the release of MVs that (as also documented by analysis of protein content of MVs preparations from cells silenced or not for caspase 3, see Fig 6B) that was reflected by a sharp reduction in MVs internalization in naïve HepG2 cells (Fig 6C) as compared to cells exposed to non-silencing siRNA. Moreover, the relationships between MVs and NF-kB pathway was further supported by the observation that the exposure of naïve HepG2 cells to MVs from cells treated with caspase-3 siRNA resulted in a significant decrease in p65 nuclear translocation as compared to cells receiving MVs from cells treated with the non-silencing vector (Fig 6D). Accordingly, silencing of caspase-3 in the parental fat-laden cells led to a decrease in the protein levels of the inflammasome components (Fig 6E) as compared to fat-laden cells receiving the non-silencing control vector.
Fig 6. NLRP3 inflammasome activation is prevented by blocking the caspase 3 –dependent release of MVs.
A. Western blot analysis of caspase-3 in total extracts of HepG2 cells not transfected (C), transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirm by re- probing the same membrane with monoclonal antibody against the house-keeping β-actin. B. Protein quantification of preparation of MVs as obtained from control HepG2 cells (C), from HepG2 cells transfected with the non silencing RNA and then exposed to PA (NsC) and HepG2 cells silenced for caspase 3 and then exposed to PA (siCASP3). Data are expressed as μg/μl and as mean ± SD being (n = 12 for any condition). C. Flow cytometric analysis of internalization of PKH26-fluorescent die-MVs in HepG2 naïve cells. Color legend: i) black trace is related to HepG2 naïve cells treated with ultracentrifugated solution (also marked with PKH26-fluorescent die) derived from cells just exposed to BSA (used as control), ii) pink trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with non-silencing treated with palmitic acid 0,25mM for 24hrs; iii) green trace is related to HepG2 naïve cells treated with MVs derived from HepG2 cells transfected with specific siRNA for caspase-3 and treated with palmitic acid 0,25mM for 24hrs. D. Western blot analysis of NF-Kb/p65 protein levels in cytosolic and nuclear extract obtained from HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. Equal loading was confirmed by re-probing membrane with α-tubulin and Lamin-A (cytosolic and nuclear house-keeping, respectively). E. Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 naïve cells exposed or not (C) to MVs derived from HepG2 cells transfected with non-silencing siRNA (NsC) or specific siRNA for caspase-3 (siCASP3) and treated with palmitic acid 0,25mM for 24hrs. LPS 1mg/ml was used as positive control. Equal loading was confirmed by re-probing membrane with β-actin.
In order to establish whether the MVs-induced release of IL-1β was directly dependent on NLRP3 inflammasome, HepG2 cells were depleted of NLRP3 using a specific siRNA before exposure to MVs for 16 or 24 hrs. In control arm cells were exposed to a non-silencing vector. HepG2 cells silenced for NLRP3 responded to MVs by a sharp decrease in NLRP3 transcript (Fig 7A) as well as by low protein levels of activated caspase-1 (Fig 7B) and IL-1β (Fig 7C). In addition, NLRP3 silencing in HepG2 cells also resulted (Fig 7D) in a reduction of transcript levels for IL-1β (i.e., pro-IL-1β in this case).
Fig 7. Silencing of NLRP3 in target cells prevents MVs-dependent inflammasome activation.
A. Quantitative real-time PCR (qPCR) of NLRP3 mRNA in HepG2 cells transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed or not to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. LPS 1mg/ml was used as positive control. B. Quantitative real-time PCR (qPCR) of IL-1β mRNA in HepG2 cells not transfected (C) and transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed or not for 3hrs to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control HepG2 cells; # p<0.05 vs NsC+MVs) of three independent experiments. C,D. Western blot analysis of NLRP3 protein levels as well as of cleaved caspase-1 (p-10-CASP-1) and IL-1β in total extracts obtained by HepG2 cells transfected for 72hrs with non-silencing siRNA (NsC) or specific siRNA for NLRP3 (siNLRP3) and exposed for indicating time to MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs. Equal loading was confirmed by re-probing membrane with β-actin.
Finally, additional experiments were performed to verify whether MVs may be able also to promote inflammasome activation in macrophages. To this aim, we exposed macrophage differentiated from human THP-1 cells to MVs obtained from fat-laden HepG2 cells. In differentiated THP1 cells we observed a rapid (as early as after 1 hr) internalization of MVs marked with the fluorescent dye PKH26 (Fig 8A, Fig 8B) as well as an increase in transcript levels of NLRP3, Caspase-1 and IL-1β (Fig 8C–8E) accompanied by a time-dependent increase in IL-1β release in the extracellular medium (Fig 8F).
Fig 8. MVs activate NLRP3 inflammasome in THP-1 differentiate macrophages.
A,B. Analysis of internalization of MVs in THP-1 cells by confocal microscope (3hrs): nuclei (blue fluorescence), MVs (red fluorescence) and cytoskeleton (F-actin, green fluorescence) (A) or by flow cytometry (1 hr, B). C-E. Quantitative real-time PCR (qPCR) of NLRP3, caspase-1 and IL-1β mRNAs in THP-1 cells treated with MVs derived from HepG2 cells treated with PA 0.25mM for 24hrs, for indicating times. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control THP-1 cells). F. ELISA assay to evaluate IL-1β release (pg/ml) in culture medium of THP-1 differentiated macrophages exposed to MVs up to 24 hrs. Data in graphs are expressed as means ± SEM (*p< 0.05 and ** p<0.01 vs related control HepG2 cells) of three independent experiments.
Hepatocyte injury due to lipotoxicity and inflammatory infiltration by cells of innate immunity, mainly activated macrophages, are two well recognized pathological hallmarks of NASH [10–12,38]. In this scenario, multiple hits involving oxidative stress, endotoxemia, cytokines and gut microbiota have been suggested to play a role in favouring NAFLD progression to NASH [10–12,38–40]. In recent years different reports have provided evidence that during NAFLD progression hepatocyte injury induced by lipotoxicity may affect, and somehow drive, the response of surrounding cells through the release of hepatocyte-derived MVs. At present, MVs released by lipotoxic hepatocytes have been proposed to up-regulate angiogenic and pro-fibrogenic responses in endothelial cells [19,22] and hepatic stellate cells , respectively. Moreover, lipotoxicity-mediated MVs release has been recently shown to initiate a CHOP/DR5/caspase 8/caspase-3 signalling cascade leading to the release of MVs expressing on their surface TRAIL as molecule able to trigger a pro-inflammatory and NF-kB–dependent response in macrophages , thus acting act as "messengers" between injured hepatocytes and macrophages [27,41].
In the present study we provide novel evidence suggesting that MVs released by cells undergoing lipotoxicity may actively contribute to pro-inflammatory responses by activating in a paracrine way NLRP3 inflammasome in either parenchymal cells and macrophages with a mechanism that involves MVs internalization by "target" cells. This is in agreement with previous data showing that endocytosis is required for the triggering of MVs responses in endothelial and hepatic stellate cells [22,23]. The inflammasome stimulation results in caspase-1 activation and a sustained release of IL-1β, an event that can support the lobular inflammation in NASH and contributes to the maintenance of insulin resistance [42–47].
It is well known that the inflammasome represents a multimeric protein complex which senses several stimuli related to pathogen and damage-associated molecular patterns in tissues undergoing infections, cell damage or metabolic imbalances [28,48,49]. The formation of the inflammasome complex leads to the activation of caspase-1, which in turn proteolytically cleaves the precursors of pro-inflammatory cytokines IL-1β and IL-18. Inflammasome responses can also cause pyroptosis, a peculiar and rapid pro-inflammatory form of cell death relevant in the so-called "sterile inflammation" which is known to occur also in NASH . Along these lines, a recent study using mice expressing a constitutively active form of NLRP3 under the control of the endogenous NLPR3 promoter, has shown that increased NLRP3 activity results in extensive liver inflammation, hepatocyte pyroptosis, and liver fibrosis . Indeed, activation of NLRP3 inflammasome in both hepatocytes and macrophages has been detected only when NAFLD had advanced to NASH [28–33,50], a scenario that seems to be confirmed by this study showing that NLRP3 protein is overexpressed in liver biopsies from NASH patients. Furthermore, in MCD model of NASH hepatic NLRP3 and IL-1β transcript levels are both increased only in severe steatohepatitis.
To characterize the mechanisms involved in MVs—induced activation of NLRP3 inflammasome we used a well characterized "in vitro" model of MVs release by fat-laden cells. This model has been used by different laboratories in recent years [22,23,27] and is based on the exposure of HepG2 cells to palmitic acid resulting in rapid intracellular lipid accumulation that leads to the induction of caspase 3 –dependent lipoapoptosis and the release of MVs. The capacity of MVs released by cells undergoing lipotoxicity to activate NLRP3 inflammasome is supported by data indicating that activation is prevented by either blocking MVs production by silencing caspase 3 in lipotoxic cells before exposure to palmitate or by the silencing NLRP3 target cells before exposure to MVs. Furthermore, MVs promote target cells release of IL-1β in the extracellular medium. Such a release is evident in both HepG2 cells and macrophages 16 hrs after the addition of MVs, but already detectable, although at lower levels, at earlier time points. The analysis of the events triggered by MVs suggests that they induce an early transcription of NLRP3 and pro-IL-1β, likely through NF-kB–dependent signals as recently described by Hirsova et al. . However, protein analysis shows that that NLRP3 and pro-caspase 1 are already present in resting cells and that their apparent levels increase not before 6 hrs of exposure to MVs. Of interest, the levels of active caspase 1 that is responsible for the cleavage of pro-IL-1β start to increase already 1 hr after exposure to MVs. This suggests that MVs internalization may lead to the assembly and activation of NLRP3 inflammasome even before an increase in NLRP3 and pro-caspase 1 protein levels. This interpretation is in agreement with literature data stating that NLRP3 canonical inflammasome activation can occur as a two-step process, in which a first signal up-regulates NLRP3 expression through NFκB-dependent pathways , while a second signal leads to multimeric protein complex assembly and activation through distinct pathways [52–54], that involve the interaction between the danger signal(s) and the NACHT domain of NLRP3. Although in the present study we have not yet characterized the precise nature of signal(s) leading to MVs-mediate assembly/activation of inflammasome, the overall message is of potential relevance and further supports the concept that MVs released by damaged cells undergoing lipotoxicity can have a role in sustaining hepatic inflammation in NASH along with previously shown fibrogenesis and angiogenesis. Further experimental studies are needed in order to fully clarify and detail the reported relationships between the release of MVs from fat laden hepatocytes and surrounding cells in the scenario of NAFLD/NASH chronically injured microenvironment.
S1 Fig. Characterization of microvesicles (MVs) obtained from fat-laden HepG2 cells.
(A). Representative TEM micrograph of HepG2-derived MVs released after 24 hrs of 0.25 mM palmitic acid (PA) treatment. Scale bar 200 nm. (B) Flow cytometry analysis (Whisker plot) of Calcein+ MVs per mL of media isolated from fat-laden HepG2 cells at the end of 24 hrs exposure to PA. Values represent mean ± SD of 6 different MVs preparations. *P < 0.05. (C). Western blot analysis of NLRP3 and IL-1β in the following experimental condition: 1: total extract from MVs release from HepG2 cells treated for 24hrs with BSA (C-); 2: total extract from MVs release from HepG2 cells treated for 24hrs with PA 25mM; 3:total extract from control HepG2 cells; 4: total extract from HepG2 cells treated with LPS 1μg/μl for 1 hr; 5: total extract from HepG2 cells treated with PA 25mM for 24hrs. Equal loading was evaluated by re-probing the same membrane with the monoclonal antibody targeting β-actin. Red ponceau images were also included to show protein loading.
Immunohistochemistry analysis for NLRP3 on liver specimens from NLRP3 hemizygous +/-mice (A) as well as WT mice or Casp 3-/- knockout mice fed for 6wks with MCD diet (B). Original magnification as indicated. Right panels represent histomorphometric analysis (ImageJ program) that has been performed on n = 4 liver sections obtained from three different animals for each condition indicated in panels A and B in order to evaluate positive staining for NLRP3.
We would like to thank Prof. Gianluca Svegliati-Baroni (Università Politecnica delle Marche, Department of Gastroenterology, Ancona, Italy) for kindly providing us with paraffin sections obtained from the liver of NLRP3+/- mice used in immunohistochemistry.
Conceptualization: SC EM EA AF MC MP.
Formal analysis: SC EM SS E. Benetti DP.
Investigation: SC EM CB BF E. Benetti EN FC MR RF DP SS E. Bugianesi.
Project administration: MP EA MC AF.
Resources: E. Bugianesi EA SS DP AF.
Supervision: SC E. Bugianesi DP AF EA MC MP.
Writing – original draft: SC EM EA MC MP.
2. Farrell GC, Larter CZ. Non-alcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 2006;43(2 Suppl.1):S99–S112.
14. Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, et al. Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovascular Res. 2012;93:633–644.
17. Morel O, Morel N, Jesel L, Freyssinet JM, Toti F Microparticles: a critical component in the nexus between inflammation, immunity, and thrombosis. Sem Immunopathol. 2011; 33:469–486.
26. Povero D, Eguchi A, Li H, Johnson CD, Papouchado BG, Wree A, et al. Circulating extracellular vesicles with specific proteome and liver microRNAs are potential biomarkers for liver injury in experimental fatty liver disease. PlosOne. 2014; 9(12):e113651.
46. Vandanmagsar B, Youm Y-H, Ravussin A, Galgani JE, Stadler K, Mynatt RL, et al. The NLRP3 inflammasome instigate obesity-induced inflammation and insulin resistance. Nat Med. 2011;15:179–188.
48. Henao-Mejia J, Elinav E, Thaiss CA, Flavell RA. Inflammasomes and metabolic disease. Ann Rev Physiol. 2014;76:57–78.


text:
Bruce Watt, Ph.D
Vice President, Europe, India, & Australia, DDI
Bruce is passionate about helping organizations transform and grow through unleashing the potential of their people. Throughout his career working with multinational companies around the world, he has considered it a privilege to have helped thousands of leaders develop their self-insight and leadership capabilities.
Bruce joined DDI in 2000 and currently serves as Vice President for DDI's operations in Europe, India, and Australia; DDI's Global Centre of Excellence in Pittsburgh; and DDI licensee operations around the world. His team is on a quest for building stronger organizations through creating better leaders for a better future. Based mainly in London, Bruce's role requires him to be very mobile, and he loves working with his teams and clients across such a diverse landscape of regions and cultures.
Bruce's passion for football (soccer) has helped him develop a better appreciation and understanding of the world as every club tells a story about a city or region's history and culture.
Anshika Malviya
Lexter Martin

redpajama_set_name: RedPajamaCommonCrawl


Nance Home Furnishing in Ardmore, OK is an authorized dealer of Amana Products. The Amana Corporation is an American brand of household appliances. It was founded in 1934 by George Foerstner as The Electrical Equipment Co. in Middle Amana, Iowa, to manufacture commercial walk-in coolers. The business was later owned by the Amana Society and became known as Amana Refrigeration, Inc. It is now owned by Whirlpool Corporation.
So if you are looking for Amana products in Ardmore, Lone Grove, Marrietta, Healdton, Madill, Springer, Dickson, Lake Texhoma, Sulphur, Rineline and Davis, or if you have any questions about Amana products, please feel free to call us at (580) 223-3488 or simply stop by Nance Home Furnishing at any time and we would be glad to help you.


text:
North West heart disease shock figures
PEOPLE living in the North-West are more likely to die from heart disease than anywhere else in the UK, figures have shown.
8th Jul 2011, 12:17pm - 2 min read
The research, carried out by cholesterol charity Heart UK, revealed the mortality rate in the area was higher than the rest of the country with three of the five worst death rates found in the North-West.
Among the areas with the highest deaths through the disease was Blackburn with Darwen PCT while the mortality rate for the East Lancashire PCT, which includes Burnley, based on 2009 figures from the NHS Information Centre, was 102.92 people per 100,000 – significantly higher than the national average of 77.63.
The mortality rate for the North-West was 93.72 people per 100,000 with a similar pattern emerging across all northern areas compared to the south, where on the whole the mortality rate was lower.
Heart UK has now launched a "Heart Hotspots" campaign to raise awareness of the inequalities across England and encourage patients to look after their health.
However Heart UK chief executive Jules Payne said people could reduce their risk of having a heart attack or stroke through being aware of the risk factors no matter where they lived.
"There are simple changes that people can make to improve their heart health.
"Those diagnosed with heart problems should take a proactive approach towards their health - knowing their cholesterol and blood pressure numbers and weight, going for regular check-ups and speaking to their doctor if they have any concerns.
"For those with a family history of heart disease, small changes to diet and lifestyle for example can help reduce the risk of cardiovascular disease."
Professor Roger Boyle CBE, Department of Health's national director for heart disease and stroke, added: "The latest data reinforces the fact that despite recent progress in the management of cardiovascular disease there is still more that can be done to ensure people are aware of the risk factors for having a heart attack or stroke and what can be done to address these risk factors.
"The Heart Hotspots campaign is a valuable step in raising awareness and it is vital that we continue to focus on tackling the UK's leading cause of death, cardiovascular disease, and ensuring that inequalities are being addressed."
More details on the charity can be found at www.heartuk.org.uk.


Take back your weekends. We bring the food and the service trucks to the office park where you work. Currently servicing office parks in Raleigh, Durham, RTP - North Carolina. Soon MOBLZ will be in locations everywhere.


It was a typical evening. Mommy picked you and your brother up from summer day camp. I had just driven back home from work.
As I began to help Mommy prep dinner, your brother mentioned something about taking you upstairs to see something.
Ten minutes later, I looked up to see that he had brought you back down, but decked out in his Captain America mask from his Halloween costume 3 years ago, with the accompanying shield.
You didn't say a word, but I could tell it was important to you that I recognized that you were now Captain America.
Then during breakfast the next morning, I stepped into the kitchen to realize you were wearing the mask again; refusing to take it off while you ate breakfast.
A little bit later as Mommy was getting ready to leave for work, you added Mommy's slippers to your superhero outfit. It somehow made sense.
I instantly assured him that we could make our own superhero videos now if he wanted to.
But after he thought about it, your brother decided that instead of reprising the titular character of the 22 episode series, Jack-Man, he would like to try writing his own series.
So if this ends up really happening, I will be making the video, and he'll be writing and starring in it. He's also having Mommy look into buying him a lizard costume for it.
← Today is Our 10th Wedding Anniversary!


What's the best audio remix or electronic composing software for beginners?
I think Acid XPress 7.0, it's free and you can download beats and loops from their website. It might take a little getting used to but it's totally worth it.
. For capturing, mixing, &c music, it's hard to beat Audacity.
+1 for the Audacity. Any ideas on the composing?
Finale, I think, is a great notation program. There 4 versions, costing 50 bucks for the cheapest.
I never needed a synthesizer, so I couldn't really tell you which one is best. Also, before asking about software in forums, I recommend you to search around in freebyte.com. It has a bazillion links to any free (and not free) software for machines running windows.


For maximum pampering and total relaxation in the Caribbean, it's hard to beat Sandals. Their all-inclusive resorts, for couples only, are located on prime beaches in Jamaica, Antigua, St Lucia, Grenada, Barbados and the Bahamas and now boast the Caribbean's first over-the-water suites.
While we can't lay claim to inventing the all-inclusive holiday, we have spent the last 30 years perfecting it. We're proud to be family-owned too; an all-Caribbean company that takes pride in delivering great holiday experiences.
We have 16 stunning couples-only resorts on the dream Caribbean islands of St Lucia, Jamaica, Antigua, the Bahamas, Grenada and Barbados.
Every Sandals resort sits directly on a stunning beach. From Jamaica's warm reggae pulse to the Bahamas' sophisticated beat; St. Lucia's volcanic beauty to Antigua's exotic allure; the spice aromas of Grenada and Barbados' vibrancy and charm, Sandals provides the best luxury all–inclusive resorts in the Caribbean, each with its own unique Luxury Included® experience.
Each of our resorts is truly unique. You will find a range of spacious rooms and suites, some with a private pool and/or butler service, up to 16 gourmet restaurants, unlimited premium brand drinks, water sports including water-skiing and scuba-diving for certified divers and championship golf courses close by if not on site – all included, all unlimited, all the time.
We are proud to introduce the Caribbean's first ever Over-the-Water Suites, now available at Sandals Royal Caribbean and Sandals South Coast in Jamaica and Sandals Grande St Lucian. Featuring ground-breaking innovative designs that marry modern luxury and exotic palapa-style architecture, they are truly spectacular.
Relax in our Red Lane® Spas where you can choose from a host of pampering treatments in tranquil surroundings (extra cost).
Sandals LaSource is our first resort on the Spice Island of Grenada. Located on beautiful Pink Gin Beach, this resort offers three distinctive villages – Pink Gin Village, Italian Village and Tahiti Village. In the Italian Village, our luxurious Skypool Suites are even more romantic – open your bedroom doors to reveal your own infinity plunge pool overlooking the Caribbean.
Sandals Royal Barbados, our newest retreat, can be found in the spirited St Lawrence Gap area. This all-suite resort features all the luxury you would expect from a Sandals holiday, as well as a number of Sandals firsts including a rooftop pool and bar, a four-lane bowling alley and two new restaurant concepts – American Tavern and Chi Asian.
Find out more about Sandals Luxury Included holidays and our latest special offers.


text:
Q: Why am I getting a KeyError: 'scheduler' when trying to run a celery beat? I am using celery in Django to periodically schedule tasks. Here are the files:
tasks.py
@periodic_task(run_every=timedelta(minutes=1))
def query():
...

celery.py
from __future__ import absolute_import, unicode_literals

from os import environ, path

from celery import Celery
from django.conf import settings

PROJECT_NAME = path.basename(path.dirname(__file__))
environ.setdefault('DJANGO_SETTINGS_MODULE', '%s.settings' % PROJECT_NAME)

app = Celery(PROJECT_NAME)
app.config_from_object('django.conf:settings')
app.autodiscover_tasks(lambda: settings.INSTALLED_APPS)

When I run
celery -A project worker

to start the worker, that executes fine. However, when I run
celery -A project beat

to start the beat scheduler, I get the following error:
KeyError: 'scheduler'

Why would this be occurring? I am using vagrant in PyCharm and accessing the server through vagrant ssh.
Update:
If I run
celery -A project worker --beat --scheduler django --loglevel=info

I get no error but no tasks are being received by the workers. So it looks like tasks are not being received for some reason.
Also adding
app.conf.beat_schedule = {
    "query": {
        "task": "query",
        "schedule": crontab(minute="*", hour="*", day_of_month='*')
    }
}

to celery.py and adding
@app.task(name="query")

to tasks.py does not work.

A: Try adding path over the task
app.conf.beat_schedule = {

    "query": {

        "task": "query",

        "schedule": crontab(minute="*", hour="*", day_of_month='*')
    }
}

"Try adding path over the task - project_name.tasks.func_name "

redpajama_set_name: RedPajamaStackExchange


WATERVILLE, Maine -- Despite 38 saves from Colby College goalie Nina Prunster, Amherst College scored the game-winning goal at 14:53 of the second period in a 4-2 win in NESCAC women's hockey Saturday.
* After a scoreless opening period, the game got wild with five goals over a stretch of just 7 minutes, 8 seconds.
* The Mules (8-5-3 overall, 5-5 NESCAC) scored first when Aimely Michaud-Nolan kept a puck in at the blue line. Tess Dupre picked up a loose puck near the left boards and quickly found Cassidy Holzer (pictured) in the middle for a wrist shot to the right corner of the goal at 7:45.
* Emma Flynn scored at 10:55 on what turned into a three-on-one situation. Flynn, coming in from the right side, went to the upper right corner for the score. Kaitlin Hoang and Jocelyn Hunyadi had assists on the play.
* Flynn scored again at 12:55 off her own rebound. Her first shot was stopped nicely by Prunster, but the rebound came right back to Flynn for the goal. Hunyadi was credited with an assist.
* Colby tied the game at 2-2 just 17 seconds later when Sadie Kuhn came into Amherst's zone hard near the left boards. Her centering cross was redirected by Holzer for her second score.
* Just 1:47 after Colby tied the game, Sarah Wagner got the winner. Stephanie Nomicos kept in a clearing attempt by Colby and saw Wagner wide open in the middle.
* The Mammoths closed out the scoring when Mia DelRosso scored an empty net goal. Her shot came from near the face-off dot to the right of Amherst goalie Caitlin Walker.
* Walker finished with 23 saves and needed to make just two in the final period for Amherst (8-5-2, 5-3-2 NESCAC).
* Prunster made 16 of her 38 saves in the third period for Colby.


text:
tags: Billy Wilder, Buster Keaton, Cecil B. DeMille, classic film, Erich von Stroheim, film noir, Gloria Swanson, Hollywood, Nancy Olson, Sunset Boulevard, William Holden
229. Sunset Boulevard
Directed by Billy Wilder
While trying to avoid repo men, impoverished screenwriter Joe Gillis (William Holden) stumbles upon a secluded mansion. There he meets Norma Desmond (Gloria Swanson), a silent movie star whose fame has long since faded. She lives with her loyal German butler Max (Erich von Stroheim), himself a forgotten director.
Norma hires Joe to rewrite her script for Salome, insisting he live with her in the mansion until the job is completed. Gradually Joe becomes a kept man, taking advantage of Norma's desperate need to perpetuate her fantasy.
Norma visits Cecil B. DeMille at Paramount Studios, having received a call from the studio which she assumes was on his behalf.
Cecil B. DeMille: Norma, I must apologise for not calling you.
Norma Desmond: You'd better, I'm very angry.
Cecil B. DeMille: Well, you can see I'm terribly busy.
Norma Desmond: That's no excuse. You read the script, of course?
Cecil B. DeMille: Yes, I did.
Norma Desmond: Then you could have picked up the telephone yourself instead of leaving it to one of your assistants.
Cecil B. DeMille: Hmm? What assistant?
Norma Desmond: Now don't play innocent, somebody named Gordon Cole.
Cecil B. DeMille: Gordon Cole?
Norma Desmond: And if you hadn't been pretty darn interested in that script, he wouldn't have tried to get me on the telephone ten times!
Concerned, DeMille leaves Norma in the director's chair while he goes to call Gordon Cole himself. While she waits, an electrician nicknamed Hog-eye recognises Norma and turns his spotlight on her. Soon, the cast and crew of DeMille's film is swarming around Norma, delighted to see her.
Meanwhile, DeMille speaks to Gordon Cole and discovers the reason for the telephone calls; the studio wants to rent Norma's vintage automobile because it's "perfect for the new Crosby picture." DeMille returns to find the crowd surrounding Norma.
Cecil B. DeMille: Hog-eye, turn that light back where it belongs.
As the spotlight is turned away, the crowd disperses.
If you live in France, for instance, and you have written one good book, or painted one good picture, or directed one outstanding film fifty years ago and nothing else since, you are still recognized and honored accordingly. People take their hats off to you and call you "maître." They do not forget. In Hollywood — in Hollywood, you're as good as your last picture. If you didn't have one in production within the last three months, you're forgotten, no matter what you have achieved ere this.
~ Erich von Stroheim
Sunset Boulevard is the tragic tale of a forgotten actress who retreats into her own delusions of stardom, and a vicious satire of the Hollywood system that made her that way. It's narrated from beyond the grave by Joe Gillis, a cynical hack who begins and ends the film floating face down in a pool. Delightful!
Norma Desmond was a bright star in silent cinema's heyday. The advent of sound ended her career, but years in the limelight left her with a completely warped worldview (à la Michael Jackson, complete with pet monkey). No one has the heart to puncture her fantasy. "You don't yell at a sleepwalker — he may fall and break his neck."
There are eerie similarities between the characters of Norma and Max and the actors who play them. Gloria Swanson was a silent star whose popularity waned after the advent of sound. Erich von Stroheim was a celebrated director of the silent era — his films Foolish Wives (1922) and Greed (1924) are both on The List — who later became known for playing German villains in war films. Perhaps most mind-bogglingly, the film clip Norma watches of herself was actually from an unreleased film starring Swanson and directed by von Stroheim.
I always enjoy revisiting Sunset Boulevard. Its nightmarish mood and cynical William Holden wisecracks attracted me from the off. But as I learn more about film history, the context and satire resonates with me too.
Fascinating fact: David Lynch's character in Twin Peaks is named Gordon Cole, in reference to the man who tries to rent Norma Desmond's car.
Roughly third viewing
Joe Gillis has missed car payments and the finance company are after him to take the vehicle away. After a close encounter with them, he turns into the driveway of an Italianate villa (which he assumes to be empty) and parks in the garage. Within minutes he is in the company of Norma Desmond, a faded silent film star who lives with her butler and her recently deceased pet monkey. Assuming Gillis is the funeral director, she calls him inside. But his true identity soon becomes obvious, and Norma demands that he leave.
Gillis: Wait a minute, haven't I seen you before? I know your face.
Norma: Get out, or should I call my servant?
Gillis: You're Norma Desmond. You used to be in silent pictures; you used to be big.
Norma: I am big. It's the pictures that got small.
Gillis: Uh huh. I knew there was something wrong with them.
Norma: They're dead! They're finished! There was a time in this business when they had the eyes of the whole wide world. But that wasn't good enough for them, oh no. They had to have the ears of the world too. So they opened their big mouths and out came talk, talk, talk!
Gillis: That's where the popcorn business comes in. Buy yourself a bag and plug up your ears.
Norma: Look at them in the front offices, the masterminds! They took the idols and smashed them! The Fairbankses, the Gilberts, the Valentinos! And who've we got now? Some nobody.
Gillis: Don't blame me, I'm not an executive, just a writer.
Norma: You are? Writing words, words, more words. You've made a rope of words and strangled this business. But there's a microphone right there to catch the last gurgles, and Technicolor to photograph the red, swollen tongue.
In his wonderful book on early cinema, The Parade's Gone By (1968), film historian Kevin Brownlow describes the fate of silent films at the time of the book's publication:
The silent era is regarded as prehistoric, even by those who work in motion pictures. Crude, fumbling, naïve, the films exist only to be chuckled at — quaint reminders of a simple-minded past, like Victorian samplers.
Hollywood is a ruthless place. Someone can be at the top of their profession one decade and in the gutter the next. But perhaps no generation of talent have felt this as deeply as those who were at the height of their career during the silent era.
The arrival of sound was the biggest evolution the film industry has ever seen, and while some managed to continue a career in some shape or form, it left countless others on the scrapheap. Often their hard work was destroyed when studios threw away the prints they thought were no longer of any value. When silent film was finally recognised as an art form, many stars of the era had not lived to enjoy their well-deserved recognition.
I see Sunset Boulevard as their big "up yours" to Hollywood. Not all of them were bitter, and it's very probable that none of them were as melodramatic as Norma Desmond, but she still had a valid point to make about their unique experience — as seen in my essential scene.
The plot may be a little wobbly, and Gloria Swanson's acting purposely hammy, but Sunset Boulevard is a film that sent shockwaves through Tinseltown and continues to have an impact. Wilder didn't soften his blows. He exposed industry for the magical yet venomous business it is (and pissed off a lot of people while doing so) — and the silent era's Gloria Swanson, Erich von Stroheim, Cecil B. DeMille, Buster Keaton, Anna Q. Nilsson and H.B. Warner all get a chance to be part of the story that tells of the Hollywood that brought them all happiness and pain in equal measure.
from → Ally, Rachel
← Pandora's Box
Casablanca →
John permalink
This is one of my all-time favorites. There are a lot of things I find amazing about it. First, it's got a noir setting. Norma Desmond's house has wrought iron fences and stonework, and the shadows are long, and it's bleak… That's not necessarily a huge feat until you realize that this was filmed in Hollywood, California- one of the sunniest places on earth. It might as well have been Bela Lugosi's Transylvania, but the setting was Hollywood.
There's another layer here. I read something about Billy Wilder coming up with the idea when he was an aspiring, struggling screenwriter himself, living on Sunset Blvd. Apparently, the Blvd. was loaded with these old decaying mansions occupied by people like Desmond- lost, out of work, somewhat vampiritic (is that even a word?) former silent stars. So here's the youth of Hollywood, vibrant and struggling and working hard but not quite making it… juxtaposed against the decaying Hollywood guard, not quite completely useless but clearly beyond their prime and former glory.
The whole thing plays out like a really amazing Gothic horror movie, specifically aimed at Hollywood wanna-be folks. And Joe Gillis' dialogue keeps it funny and light while you're being horrified by things like monkey funerals and Norma Desmond's full-on insanity.


Is Optical Prime an exact match with the DPACK host collector? Or have there been some updates?
What is the interval length for a performance collection?
Does Optical Prime also collect server components, HW inventory, like HBA's firmware and drivers?
Optical Prime does not show disk HBA HW, firmware or drivers - make, model, CPU and memory are shown.
Will I be able to see all the applications installed on the hosts?
Is there a capability to do comparison of data gathered at different project run times on the same target server (s), ie server "X" across monthly scans capturing it over the last x number of months?
No but you can save the output of the information gathered and manually compare this to a future report.
Can customers upload their own data as with traditional DPACK host collections?
It is in the same directory as the Live Optics collector software. The standard name for this folder is "LiveOptics".
What collectors are available for Optical Prime?
Will Live Optics be available for VMware?
Will I be able to group a few Virtual machines and see the workload?
Does the VMware reporting in Optical Prime include VSAN metrics?
Can we use Optical Prime for desktop virtualization assessments?
When should I collect infomation from VMs directly rather than through vCenter?
When you want to understand the performance attributes in detail, connect to the VM directly.
What is the difference between virtual and hypervisor?
Virtualized is the virtual machine and hypervisor is the physical box running the hypervisor OS (e.g. VMWare, Hyper-V, etc).
Does the customer need VMware rights to run Optical Prime in a virtualized environment?
Does Optical Prime display 3rd party arrays information - ie show how busy disk drives are or memory or director/SP ports?
Optical Prime reports on the host operating system - the disk busy is from the application standpoint. Use Optical Prime to target servers that are connected to the 3rd party array (ie NetApp, 3Par etc), information like front end IOPS, peak and 95% IOPS, throughput at peak, R/W ratio and used/free capacity will be reported on.
What about the #GetModern Assessments?
The PSS team uses the output from Optical Prime to provide host based assessment reports.
Can you remove ip address ad server names from the Optical Prime reports?
The ability to obfuscate the server and disk names at the Live Optics collector software level. Starting Live Optics from the command prompt using the switch "/anon" will provide random server and disk names in the project created in Optical Prime.
The other way that a project can have the server and disk names changed would be from the Live Optics project. When a project is shared, there is an option to anonymize the project for the recipient. Additionally, you can also use the Publish option to generate a URL to be accessed outside of the Live Optics portal for anybody to access, regardless of access to it.
Does Optical Prime collect data from OS as well as VMware?
Please refer to the list of supported Operating Systems that Live Optics supports. It can be located at the bottom of the Live Optics collector download page (link).
Can you put a range of IP addresses in the Optical Prime collector? Is it possible to use wildcards to add servers to the Optical Prime collector?
No, Live Optics requires either the full IP address or host name of each server when configuring it for a collector run. Also, it will not scan an IP address range for any devices available. There is a bulk import function that can be used for sites with high server count to be added to Live Optics. The articles for bulk server import can be found here and here.
For the Optical Prime collector, when searching active directory does that simply help with IP address? I assume the credentials still need to be added?
What port does Optical Prime use when streaming data? Standard http/https?
Port 443 (HTTPS) is used when streaming data from the Live Optics collector back to the Live Optics servers.
How do you run Optical Prime behind firewalls etc..?
With no internet connection, you should disable the web streaming function to direct Live Optics to only create a SIOKIT file after the collector run has completed. This file will then be uploaded to the Live Optics portal to create a project for review.
Is there any time restrictions for how many days the user can wait to start the collector after it is requested?
I see there is a process for dark sites - where can I find out how that is done?
Did I miss the core count view of the processors?
How long will the project information be available in Optical Prime, (e.g. a customer uploads the data today, will it be available for a week, a month or a year?
Live Optics projects are available indefinitely on the portal.
In the "Project View", does it show 'total CPUs' and 'total CORES'? If it's not in the dashboard, is it rolled up in a report? So we don't add up manually?
How can I get support for Live Optics when it is released?
Where can I go to get trained on Live Optics?
Live Optics servers <-- Which Servers are these? Too often the connection will close after a few minutes and this FAQ doesn't provide those Informations so that a customer can do a search on his Firewall.


Lancaster office of our client seeks litigation attorney with 3-5 years of experience.
This law firm offers a wide range of services to a variety of clients. Their areas of experience include employment and labor law, wills, estates, health care, corporate law, insurance, commercial litigation, and much more. The firm is located in Pennsylvania.


text:
Padmaavat Review- Cinema express
Padmaavat Review: An embarrassing cultural aesthetic all of us could have done without
For the first time in his long career, a Sanjay Leela Bhansali film comes across as superficial, a mere costume extravaganza.
Aditya Shrikrishna
Published: 24th January 2018
Oh would you believe it, it is finally here. Sanjay Leela Bhansali's Padmavati, rechristened as Padmaavat, makes it to the big screens this week even as calls for the ban are still ringing. After committee after committee was formed to evaluate the film, censor the film, find out if it is offensive to everyone from the Rajputs to Rani Padmini's (who is a figment of imagination by the way) first cousin twice removed, the film releases after a handful of cuts and modifications. Adapted from Malik Muhammad Jayasi's 16th century poem Padmavat, everything you need to know about the film is out there and has been playing in front of your television screens, phones, WhatsApp and social media for the last 2 months. What you did not know, however, is that the Karni Sena - the safe-guarders of Rajputs' culture, women, practices like jauhar - are likely to break into a standing ovation by the time Sanjay Leela Bhansali's name appears on screen. Or did you?
Director: Sanjay Leela Bhansali
Cast: Deepika Padukone, Ranveer Singh, Shahid Kapoor
Padmavati (Deepika Padukone) gets an introduction very similar to the one the Rajput princess - Mastani - got in Bhansali's earlier film, Bajirao Mastani. We see her hunting in the woods, swords exchanged for bow & arrows, when, as part of Bhansali's inescapable symbolism, she makes a mark on Ratan Singh (Shahid Kapoor) of Mewar. In both the films, it is Bhansali's muse who gets the bigger introduction, the heroes can only dream of such a sequence. But unintentionally or by design, Bhansali loves to introduce his women in all their feisty glory and then pull the agency from under their feet. Not that the roles they go on to inhabit fail to service the film, but these introductions exist in vacuum, they are mere set pieces and the one in Bajirao Mastani turns out to be more organic in retrospect. One can, of course, argue that Bhansali's films are clever, elaborate set pieces themselves but Padmaavat will probably go down as one of his weakest films. There is a lot of dead air and Deepika Padukone is - as unforgivable as this is - given precious little to work with. Padmaavat therefore becomes more about Alauddin Khilji (Ranveer Singh) - reigning over Delhi Sultanate - who establishes a watchtower outside Chittor in his obsession with Padmavati.
Bhansali throws everything at you. With a Muslim king pitching tent outside the Helm's Deep of Rajputs, he has the Chittor populace celebrating everything from Diwali to Holi but still fails to repeat the magic of that scene in Bajirao Mastani when Bajirao returns from war and an exquisite Albela Sajan washes over a range of colours and hues. Alauddin is depicted like a ghost, his garments oversize, his hair unkempt, and when he gallops into battle, he brings the dust storm with him. He disappears into one and appears out of it with a head mounted on his spear. And that is what he expects with Padmavati too - to bring a dust storm into Chittor and walk away with the queen. A dust storm does occur when his hopes are all dashed, and he returns to Delhi, not with Padmavati but with Ratan Singh. At this point, Padmaavat plays out like a gender reversed Helen of Troy saga between the Greeks and the Trojans. It is also the point when you no longer care.
You no longer care that the gravitas one usually associates with a Bhansali film - the star-crossed lovers, the mise en scène - has spectacularly gone missing. The stakes are never high on screen - an anti-climax to the off-screen din all these months. You no longer care that Bhansali does nothing with the character of Malik Kafur (Jim Sarbh), Khilji's eunuch-general - the homosexual undertones only alluded to, ever so lightly. You no longer care that Jim Sarbh hasn't yet built on the promise of Neerja with Padmaavat and Raabta. You no longer care that it was a Brahmin who betrayed the Rajputs to Alauddin, committed something amounting to treason, and was invited by Khilji to have his circumcision done. Wonder what the Karni Sena makes of such anti-nationals. You do care about the Brahmin being instructed to bring Alauddin Khilji's food which would have made for a nice visual. You no longer care that the Muslim king is not only depicted as uncivilized but is also referred to as Yama - Hindu god of death - in the context of Satyavan-Savitri from Mahabharata, when Padmavati saves Ratan Singh from the jaws of death. For the first time in his long career, a Sanjay Leela Bhansali film comes across as superficial, a mere costume extravaganza. An embarrassing cultural aesthetic that all of us could have done without.
Sanjay Leela Bhansali Ranveer Singh Deepika Padukone Shahid Kapoor Padmavati Padmaavat


text:
/*
 * Copyright 1999-2004 The Apache Software Foundation.
 *
 * Licensed under the Apache License, Version 2.0 (the "License");
 * you may not use this file except in compliance with the License.
 * You may obtain a copy of the License at
 *
 *     http://www.apache.org/licenses/LICENSE-2.0
 *
 * Unless required by applicable law or agreed to in writing, software
 * distributed under the License is distributed on an "AS IS" BASIS,
 * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
 * See the License for the specific language governing permissions and
 * limitations under the License.
 */
/*
 * $Id: DOMSerializer.java,v 1.2.4.1 2005/09/15 08:15:15 suresh_emailid Exp $
 */
package com.sun.org.apache.xml.internal.serializer;

import java.io.IOException;

import org.w3c.dom.Node;

/**
 * Interface for a DOM serializer implementation.
 *

 * The DOMSerializer is a facet of a serializer and is obtained from the
 * asDOMSerializer() method of the Serializer interface.
 * A serializer may or may not support a DOM serializer, if it does not then the
 * return value from asDOMSerializer() is null.
 *

 * Example:
 *

 * Document     doc;
 * Serializer   ser;
 * OutputStream os;
 *
 * ser = ...;
 * os = ...;
 *
 * ser.setOutputStream( os );
 * DOMSerialzier dser = ser.asDOMSerializer();
 * dser.serialize(doc);
 *

 *
 * @see Serializer
 *
 * @xsl.usage general
 *
 */
public interface DOMSerializer
{
    /**
     * Serializes the DOM node. Throws an exception only if an I/O
     * exception occured while serializing.
     *
     * This interface is a public API.
     *
     * @param node the DOM node to serialize
     * @throws IOException if an I/O exception occured while serializing
     */
    public void serialize(Node node) throws IOException;
}

redpajama_set_name: RedPajamaGithub


Here is a wonderful Paris One Bedroom Apartment rental located in the Latin Quarter, 2 steps from Place Monge and the famous Rue Mouffetard, in the heart of the sought-after Paris student life (Pantheon, Sorbonne, Gobelins). Welcome home for new long stays in Paris. The flat is situated in an authentic building in the quiet Rue du Puits de l'Ermite, 4th floor (no lift) on a quiet courtyard and offers a very convenient plan : equiped kitchen (including a washing machine, shower room (with toilet), spacious living room and cosy bedroom. It has been decorated in an african spirit, a good way to work and relax at home according what you have to do. Enjoy your stay in Paris!
1920's Building, 4 rd floor, without Lift, 1 bedroom Appartement, furnished, Approx: 33m² / 355 Sq.Ft.


When improving looks, individuals most especially women think of fixing their hair. Surely hair is the most visible part of the body. However, making hair appealing can be restricted if you have short hair. Because of this, some women opt for hair extensions. This option can provide numerous benefits that can match your needs. Below are some of the following.
One of the main benefits women can attain when opting for extensions is they can make their hair more appealing instantly. Of course, growing hair healthily and appealing can be stressful. In addition, this task can be quite expensive since you need to make use of different products to ensure better growth. Plus, when growing hair, you need to avid certain aspects such as basking under the sun and making use of products that can irritate your hair. By opting for hair extensions, women can enjoy a better appearance which can also help increase their confidence.
The next benefit of opting for extensions is individuals can enjoy different hair styles. One of the reasons why women opt for hair extensions is to make their hair longer easily By doing so, women can access numerous hairstyles that can match their styles and complement their looks. Not to mention, when having a short hair, women have limited styles to make use of. With hair extensions, women can enjoy also enjoy adding colour and vibrancy to their hair. As a result, women can opt for the best style that can match any event.
Another benefit of making use of extensions is women can choose from different types of extension options. For instance, if women want to have a long hair for a long time, it is best to try out keratin bond. This type of hair extension can last up to six months with proper maintenance. On the other hand, women can opt for clip in extension if they want to have temporary extensions. This is option is ideal for women who will be attending to special events or meetings.
Finally, apart from improving your looks and increasing your confidence, hair extensions can also get rid of some hair issues such as split ends. Split ends are commonly caused by frequently dying hair. Fortunately, hair extensions allow these split ends to be concealed, which can help improve the appearance of hair.
Many people are using human hair wigs these days because they are available in a wide variety of products. Whatever lifestyle, budget or type of personality you have, there's always an ideal wig for you. Since there are so many wigs that are sold on the market, how would you know which among these is right for you? What you need to do is to learn about the various human wig types. There are three types of these hairpieces that you should know about and these are; the Indian Virgin hair, Indian Remy hair and the Fallen hair wigs. Let's get to know each of them.
When we talk about wigs that are made of Indian Virgin hair, this means that it has been taken from a woman's hair as a form of thanks for answered prayers. Many women prefer hairpieces which are composed of Indian hair because they are known to be sturdy, thick and adjustable. In addition, these products also have great texture which will really catch the attention of others. Why is it called virgin hair? It's because it did not undergo any form of processing and no amount of damage is present in it. You will really be impressed with its beauty and natural look.
Now let's move on to Indian Remy human hair wigs. When we say that a hair piece is made of Remy hair, it means that its cuticles are intact. The advantage of having these cuticles around is that the natural shine of the hair is maintained and the hair itself is protected. The more expensive ones have unidirectional cuticles which free the hair from tangles. You can find these products either in single drawn or double drawn strands. It has been said that the double drawn one is more expensive than the other type.
The third type of human wigs is known as the Fallen Hair. Since these products are very affordable, they are extremely popular among consumers. Unlike Remy hair, a Fallen hair wig does not have unidirectional strands of hair. Due to this reason, tangling can be a problem with this product. However, its cuticles and tips can be cut to solve this problem. You can also shave its ends to prevent tangling. Although these three types of human hair wigs have differences with one another, they still have some things in common such as; ease of maintenance, capability to be styled using chemicals or heat, bounce, natural feel and look. If you want to possess the best of them all, you should also be willing to pay a higher price.


It's in the details that BurgerIM, which started in Israel and began franchising worldwide in 2011, sets itself apart from other chains. The chain offers full-sized burgers at a quarter and third pound, as well as smaller super-sized sliders. There are eight different patties to choose from: Angus beef, Spanish beef (chorizo), dry-aged beef, lamb, grilled chicken, crispy chicken, salmon and falafel. The mini-burgers come in Angus beef, grilled or crispy chicken, lamb, turkey and veggie patties.
The more than a dozen toppings range from favorites like bacon and sauteed mushrooms to avocado, jalapenos, red cabbage and fried eggs. The restaurant has vegan and gluten-free options available as well. Sides include round fries, onion rings, sweet potato fries, salad and milkshakes. The restaurant also serves wine and beer on tap. And those drafts get filled in an unusual way, from the bottom to the top of specialized pint glasses with a magnetized bottom. It's essentially beer and a show.
The Fraiolis bring a combined more than 40 years experience running restaurants. They both served as managers for Round Table Pizza shops, him in Ceres for 27 years and her on Orangeburg Avenue for 14 years. They were also able to bring some of their past employees, as well as three of their adult sons, to work on the staff of about 30.
While the company first launched in the states touting its larger-than-a-slider mini-burgers (traditional sliders are 1 ounce, BurgerIM's are 3 ounces), it transitioned this month to offering more full-sized selections based on customer demand. The mini-burgers are still available in two, three or party-sized packs. Full-sized burgers run $6.99 to $7.99, and the mini-burgers are $6.99 for two, $9.99 for three. Party packs run $34.99 to $44.99 and can include sides and chicken wings.
The brightly accented restaurant seats around 50 people for dine-in or carry out orders. The restaurant also offers delivery through Uber Eats and GrubHub.
And then there's the name. "Burgerim" is Hebrew for "many burgers." While the unusual name is a conversation starter, people are already finding the restaurant on their own. First announced last July, the burger joint moved into the vacant former Mr. Pickle's location near the Home Depot. The west side of Modesto location sets it apart from many other chains, which tend to flock to the area around the Vintage Faire Mall or McHenry Avenue. Customers come from surrounding businesses like next door's Cycle Gear motorcycle shop.
BurgerIM is open 11 a.m. to 9 p.m. Sunday to Thursday and 11 a.m. to 10 p.m. Friday and Saturday at 1521 N Carpenter Road, Building F. For more information, call 209-579-9602 or visit www.burgerim.com.
Valley nut growers have a new nursery to check out.
Roos Tree Nursery started last year by third-generation valley farmer Tim Roos and his wife, three-time Olympian and Modesto native Suzy Powell-Roos. The wholesale nursery planted its first trees in spring 2018, and had its first sales last winter.
The nursery on Albers Road in Oakdale specializes in June Bud Walnuts (1-year-old bare root trees budded in June) and Bare Root Almonds (trees harvested while dormant for winter planting).
Roos has nearly two decades of experience in the industry, and worked for a number of years as the operations and sales manager for the Bonilla Tree Nursery in Oakdale. That nursery closed in 2017, and Roos hired eight full-time employees from Bonilla to join his team.
For more information, call 209-847-2520 or visit www.roostreenursery.com.


text:
Q: Simple Chained Select Menu bug I have a simple jQuery select menu that displays content depending on selection.
Here is my jsFiddle & my code is below.
Bug#1: When I initially select US, the State dropdown doesn't appear. I have to select UK first, then US before this kicks in. Why?
Bug#2: Right now, I'm using:
if(jQuery('#statecat').val()=="ak")
 jQuery('.ak').show();
}); - is there a more efficient way to do this, so I don't have to do this for all 50 states?
Bug#3: When I go from Alaska back to Choose a State..., the US results are blank. Why?
Many thanks for any help with this.
CODE
<form method="get" action="/" id="languageSwitch">
        <fieldset>

            <select name='cat' id='cat' class='postform' >
                <option value='0' selected='selected'>Choose a country…</option>
                <option class="level-0" value="united-kingdom">United Kingdom</option>
                <option class="level-0" value="us">US</option>
            </select>

        </fieldset>
    </form>

    <form method="get" action="/" id="stateSwitch">
        <fieldset>

            <select name='statecat' id='statecat' class='postform' >
                <option value='0' selected='selected'>Choose a state…</option>
                <option class="level-0" value="ak">Alaska</option>
                <option class="level-0" value="wy">Wyoming</option>
            </select>

        </fieldset>
    </form>

United Kingdom

US

Alaska

                Alaska Test

                Address:
Address

                Country: Alaska

                URL: http://www.somesite.co.nz

                Telephone: 01902

                Fax: 01293

Wyoming

A: The first problem of the 'state' dropdown not appearing on the first selection was because for some reason you had nested change() handlers, so that event wasn't fired until the second selection was made.
Secondly, you can shorten your code by concatenating the selector for the display div of the state chosen from the value of the dropdown like this:
$('.' + $('#statecat').val()).show();

I also included some improvements to your code, such as removing unnecessary loops and caching selectors:
$('#cat').change(function () {
    var val = $(this).val();
    $("#statecat").toggle(val == "us");

    if (val == "0")
        $('#languageSwitch').siblings('div').show();
    else {
        $('form').siblings('div').hide();
        $('.' + val).show();
    }
});

$('#statecat').change(function() {
    $('.state1').hide();
    $('.' + $(this).val()).show();
});

Updated Fiddle