Brain microvascular endothelial cells (BMECs) are the primary component of the blood‑brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)‑1, ZO‑2 and ZO‑3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol‑induced loss of BBB structure remain unknown. It has been hypothesized that long‑term exposure to ethanol reduces the expression of claudin‑5, occludin and ZO‑1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin‑5, occludin and ZO‑1 protein and mRNA was measured using western blot analysis and reverse transcription‑quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα‑specific inhibitor, safingol (10 µmol/l), the expression of claudin‑5, occludin, ZO‑1 and phosphorylated (p)‑PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin‑5, occludin and ZO‑1 protein decreased, while the expression of claudin‑5, occludin and ZO‑1 mRNA increased. Exposure to ethanol significantly increased the expression of p‑PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin‑5, occludin and ZO‑1 protein was significantly decreased when compared with the control. By contrast, the expression of p‑PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long‑term exposure to ethanol downregulates the expression of claudin‑5, occludin and ZO‑1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα.
The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) in conjunction with pericytes and astrocytes (1). The BBB strictly regulates the passage of ions, molecules, leukocytes and nutrients in and out of the brain, and serves an important role in maintaining homeostasis of the central nervous system (2,3). BMECs are the primary component of the BBB. Tight junctions (TJs) are composed of transmembrane proteins, including claudins, occludin and intracellular proteins such as zonula occludens (ZO)-1, ZO-2 and ZO-3, which maintain the structural integrity of BMECs (4,5). Claudin-5 is expressed abundantly in the cerebral microvascular endothelial system and is essential for the assembly of TJs (6,7). Occludin maintains the tightness of TJs, and ZO proteins link claudins, occludin and adhesion molecules to the actin cytoskeleton in order to facilitate signaling between the intracellular actin cytoskeleton and extracellular binding proteins, thereby adjusting the TJ barrier function (8–10).
Ethanol alters endothelial cell function through a number of diverse mechanisms. The exposure of human dermal MECs to ethanol inhibits tumor necrosis factor-induced nuclear translocation of nuclear factor-κB, thereby suppressing endothelial cells activation (11). Ethanol activates Ca2+-activated K+ channels to improve endothelial cell function (12). Low levels of ethanol induce and sustain increased surface-localized endothelial cell fibrinolysis, thereby conferring cardioprotection by reducing the risk of thrombosis, myocardial infarction, cardiovascular mortality and morbidity (13). Chronic exposure to ethanol stimulates pulmonary artery endothelial cell nitric oxide production through phosphatidylinositol 3-kinase- and heat shock protein 90-dependent mechanisms, which contributes to ethanol-mediated susceptibility to lung injury (14). Ethanol activates BMEC myosin light chain kinase (MLCK), leading to the phosphorylation of MLC, occludin and claudin-5, and the subsequent impairment of the BBB (15). In addition, it has been demonstrated that oxidative stress stimulates inositol-1,4,5-triphosphate receptor-gated intracellular Ca2+ release, leading to the activation of MLCK and potentially protein tyrosine kinase (PTK); these alterations contribute to the loss of BBB integrity (16). Ethanol metabolites, acetaldehyde and reactive oxygen species lead to BBB injury by inducing alterations in the basement membrane and TJ proteins via the activation of PTK and matrix metalloproteinases (17). Therefore, the ethanol-mediated modulation of endothelial cell signaling pathways is complex and diverse and its mechanism of action remains unclear.
Protein kinase C (PKC) is a serine/threonine kinase present in the tissues and cells of humans and animals. The PKC family consists of several isoforms, which are classified into the following three major subfamilies: Classic (α, β and γ), atypical (δ, ε, η and θ) and novel (ξ and λ). Safingol is an optical isomer (an L-threo enantiomer) of dihydrosphingosine, which is a specific inhibitor of PKC (18). PKC serves a role in increasing and decreasing TJ permeability. PKCα, PKCβ and PKCξ isoforms are primarily expressed in vascular endothelial cells and the central nervous system, and affect the opening of TJs (19–21). Treatment with the PKC agonist, phorbol 12-myristate 13-acetate, may lead to the disintegration of TJs (22). Thrombin regulates the permeability of pulmonary MECs via the PKC signaling pathway (23). Ethanol increases the permeability of airway epithelial TJs in BEAS-2B and normal human bronchial epithelial cells by activating PKCα (24). Persistent activation of PKCα leads to loss of barrier function in the TJ complex (25). During ischemia and hypoxia reperfusion, activation of the PKC signaling molecule increases BBB permeability and decreases the expression of TJ proteins (26,27). This suggests that PKC may serve a role in the ethanol-induced alterations in TJs and thus cell permeability.
It has been hypothesized that ethanol impairs BBB structure by influencing the expression of TJ proteins via the PKCα signaling pathway in BMECs. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with ethanol, and the expression of claudin-5, occludin and ZO-1 mRNA and protein was detected. Following pretreatment of HCMEC/D3s with the PKCα-specific inhibitor safingol, alterations in the expression of claudin-5, occludin and ZO-1 protein were determined, and the PKCα phosphorylation status and its localization were detected.
Dimethyl sulfoxide (cat no. D4540) and safingol (cat no. D4681) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CellTiter 96® AQueous One Solution reagent was purchased from Promega Corporation (Madison, WI, USA). Normal donkey serum (cat no. 017-000-121) was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Rabbit anti-PKCα (cat no. sc-208) and mouse anti-β-actin (cat no. sc-47778) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti-occludin (cat no. ab31721) was purchased from Abcam (Cambridge, UK). Polyvinylidene difluoride (PVDF) membranes, rabbit anti-claudin-5 (cat no. ABT45), rabbit anti-ZO-1 (cat no. AB2272) and rabbit anti-phosphorylated (p)-PKCα (cat no. 07-790) were purchased from EMD Millipore (Billerica, MA, USA). Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (cat no. A-21206) was purchased from Invitrogen; Thermo Fisher Scientific, Inc. RNAiso Plus (cat no. D9108), PrimeScript™ RT reagent kit (cat no. DRR037) and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus; cat no. RR820A) were purchased from Takara Bio, Inc. (Otsu, Japan). Peroxidase-conjugated anti-mouse (cat no. ZB-2305) and anti-rabbit (cat no. ZB-2301) secondary antibodies were purchased from ZSGB-Bio (Beijing, China). All additional chemicals and supplies were purchased from Beyotime Institute of Biotechnology (Haimen, China).
HCMEC/D3s were purchased from BeNa Culture Collection (Beijing, China). HCMEC/D3s were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere.
The experimental groups were as follows: i) HCMEC/D3 cells were treated with 50, 100, 200 or 400 mM ethanol for 24, 48 and 72 h, and fresh ethanol-containing medium was replaced every 24 h to maintain a constant ethanol concentration; ii) when HCMEC/D3 cells had reached 80–90% confluence in normal medium, the cells were cultured in serum-free RPMI-1640 medium for 24 h prior to pretreatment with 10 µmol/l PKCα-specific inhibitor safingol for 1 h, followed by treatment with 200 mM ethanol for 48 h. Cells in the 200 mM ethanol-treated group were treated with ethanol alone, and cells in the control group were cultured in RPMI-1640 medium only.
The viability of HCMEC/D3 cells treated with or without ethanol was determined using an MTS assay. Cells were seeded in 96-well plates at a density of 4,000 cells/well in triplicate. Following 24 h culture, the medium was refreshed with RPMI-1640 and 50, 100, 200 or 400 mM ethanol was added. Culturing was then continued for 24, 48 and 72 h. CellTiter 96® AQueous One Solution reagent was added to each well and cells were incubated at 37°C for 2 h in the dark. The absorbance was read at a wavelength of 490 nm to determine the reduction of MTS by viable cells.
Total cellular protein was extracted using radioimmunoprecipitation assay buffer (cat no. P0013B; Beyotime Institute of Biotechnology) supplemented with 10 mg/ml phenylmethanesulfonyl fluoride and phosphatase inhibitors, and samples were centrifuged at 11,430 × g for 15 min at 4°C. The protein concentration of the supernatant was determined using a BCA protein assay kit. Protein samples (50 µg) was subjected to 10% SDS-PAGE and transferred to PVDF membranes. Following blocking with 8% non-fat milk in Tris-buffered saline-0.1% Tween-20 (TBST) at room temperature for 2 h, membranes were incubated at 4°C overnight with rabbit anti-claudin-5 polyclonal antibody (dilution, 1:5,000), rabbit anti-occludin polyclonal antibody (dilution, 1:2,000), rabbit anti-ZO-1 polyclonal antibody (dilution, 1:5,000), rabbit anti-PKCα polyclonal antibody (dilution, 1:800) or rabbit anti-p-PKCα polyclonal antibody (dilution, 1:500). Mouse anti-β-actin monoclonal antibody (dilution, 1:5,000) was used for relative protein quantification. Membranes were then washed three times with TBST and incubated with the corresponding peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (dilution, 1:5,000) at room temperature for 2 h. Chemiluminescence was visualized with luminol reagent, and images were captured and analyzed using an electrophoresis gel imaging analysis system (Tanon 5500; Tanon Science and Technology Co., Ltd., Shanghai, China). Band densities were analyzed semi-quantitatively using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
Total RNA was isolated from cells using RNAiso Plus according to the manufacturer's protocol. The PrimeScript™ RT reagent kit was subsequently used to synthesize cDNA in a total reaction volume of 10 µl, consisting of 2 µl PrimerScript buffer (5X), 0.5 µl PrimeScript RT Enzyme Mix I, 0.5 µl Oligo dT Primer, 0.5 µl Random hexamers, 4.5 µl RNase-free dH2O and 2 µl total RNA (0.4 µg). The cDNA was amplified by qPCR with sequence-specific primer pairs (Table I). qPCR was performed using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) and an Applied Biosystems 7500 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Samples were analyzed in triplicate. The reaction mixture (20 µl) consisted of 10 µl SYBR Premix ExTaq, 0.8 µl forward primer, 0.8 µl reverse primer, 0.4 µl Rox reference dye, 6 µl RNase-free dH2O and 2 µl cDNA. The thermal cycling parameters were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec, followed by 95°C for 15 sec, 60°C for 30 sec and 95°C for 15 sec. Relative quantification of claudin-5, occludin, ZO-1 and the β-actin endogenous reference gene was performed using the 2−∆∆Cq comparative threshold cycle method (28). The primers for claudin-5, occludin, ZO-1 and β-actin were designed using web-based Integrated DNA Technologies SciTools Real-Time qPCR software (Oligo Analyser version 3.1; idtdna.com/Scitools/Applications/RealTimePCR; Integrated DNA Technologies, Inc., Coralville, IA, USA).
Primer sequences used for reverse transcription-quantitative polymerase chain reaction.
Cells were cultured on glass cover slips in 24-well plates and incubated with RPMI-1640 medium containing 200 mM ethanol or 200 mM ethanol plus safingol for 48 h. Cells were then washed with PBS and fixed in 4% (volume/volume) paraformaldehyde for 20 min at room temperature. Following washing, cells were permeabilized in 0.5% Triton X-100, washed again and incubated with 2% normal donkey serum in PBS for 2 h at room temperature. The cells were subsequently immunostained with antibodies against PKCα (dilution, 1:100) at 4°C overnight. Following washing, the primary antibody was detected with the Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (dilution, 1:200) for 2 h at room temperature under light protection. The immunofluorescence images were viewed under a fluorescence microscope (Leica DM4000 B; Leica Microsystems GmbH, Wetzlar, Germany). Cells were incubated with rabbit non-immune IgG or PBS instead of the primary antibody for the negative control and no positive signal was detected.
All data are presented as the mean ± standard deviation, and were analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA). Comparisons among different groups were evaluated using a one-way analysis of variance with a Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.
An MTS assay was used to assess the effects of long-term ethanol exposure on cell viability. Compared with the respective controls, cell viability was slightly increased following 24, 48 and 72 h treatment with 50 mM ethanol and slightly decreased following 24 and 48 h treatment with 100 mM ethanol and following 24 h treatment with 200 mM ethanol; however, these differences were not statistically significant (Fig. 1). Cell viability was significantly decreased following 72 h of treatment with 100 mM ethanol (83.6±4.5%), following 48 and 72 h of treatment with 200 mM ethanol (75.8±3.4 and 70.4±5.3%, respectively) and following 24, 48 and 72 h of treatment with 400 mM ethanol (72.1±3.9, 55.1±4.1 and 40.6±3.9%, respectively; all P<0.05), when compared with their respective untreated controls (Fig. 1).
Cell viability following treatment with different doses of ethanol. Cell viability was determined using an MTS assay. Data are presented as the mean ± standard deviation and experiments were performed in triplicate. *P<0.05 vs. control at 24 h; #P<0.05 vs. control at 48 h; ^P<0.05 vs. control at 72 h.
Western blotting was used to evaluate the effects of long-term ethanol exposure on the expression of claudin-5, occludin and ZO-1. Overall, the protein expression levels of claudin-5, occludin and ZO-1 were decreased following treatment with all concentrations of ethanol for 24, 48 or 72 h (Fig. 2A-C).
Effect of ethanol treatment on the expression of claudin-5, occludin and ZO-1. Protein expression levels were measured using western blotting, and mRNA expression levels were determined using reverse transcription-quantitative polymerase chain reaction. (A) Western blotting analysis of claudin-5, occludin, ZO-1 and β-actin expression following ethanol treatment at (A) 24, (B) 48 and (C) 72 h. Quantitative analysis of western blotting results, which were normalized to β-actin expression at (D) 24, (E) 48 and (F) 72 h. Relative expression of claudin-5, occludin and ZO-1 mRNA at (G) 24, (H) 48 and (I) 72 h. The results (n=5) are presented as the mean ± standard deviation. *P<0.05 vs. control at 24 h; #P<0.05 vs. control at 48 h; ^P<0.05 vs. control at 72 h. ZO-1, zonula occludens-1.
Compared with their respective controls, the protein expression levels of claudin-5 and ZO-1 were significantly decreased following 48 and 72 h of treatment with 200 mM ethanol, and following 24, 48 and 72 h of treatment with 400 mM ethanol (all P<0.05; Fig. 2D-F). The protein expression of occludin was significantly decreased following 72 h treatment with 100 mM ethanol, following 48 and 72 h of treatment with 200 mM ethanol, and at 24, 48 and 72 h following treatment with 400 mM ethanol when compared with the untreated controls (all P<0.05; Fig. 2D-F). The protein expression levels of claudin-5 after 24, 48 and 72 h of treatment with 50 and 100 mM ethanol, and 24 h of 200 mM ethanol treatment were markedly decreased compared with their respective controls. The protein expression levels of occludin after 24, 48 and 72 h of treatment with 50 mM ethanol, 24 and 48 h of 100 mM ethanol treatment, and 24 h of 200 mM ethanol treatment were markedly decreased compared with their respective controls. The protein expression levels of ZO-1 after 24, 48 and 72 h of treatment with 50 and 100 mM ethanol, and 24 h of 200 mM ethanol treatment were markedly decreased compared with their respective controls. Although these protein levels decreased, the differences were not significant.
RT-qPCR analysis was employed to verify the effect of long-term ethanol exposure on TJ proteins by measuring the expression of claudin-5, occludin and ZO-1 mRNA. As demonstrated in Fig. 2G-I, the expression of claudin-5 and ZO-1 mRNA was significantly increased following 48 and 72 h of treatment with 200 mM ethanol, and following 24, 48 and 72 h of treatment with 400 mM ethanol compared with the untreated controls (all P<0.05). The expression of occludin mRNA was significantly increased following 72 h of treatment with 100 mM ethanol, following 48 and 72 h of treatment with 200 mM ethanol and following 24, 48 and 72 h of treatment with 400 mM ethanol when compared with untreated controls (all P<0.05; Fig. 2G-I). The expression of claudin-5 mRNA after 24, 48 and 72 h of treatment with 50 and 100 mM ethanol, and 24 h of 200 mM ethanol treatment was markedly decreased compared with their respective controls. The expression of occludin mRNA after 24, 48 and 72 h of treatment with 50 mM ethanol, 24 and 48 h of 100 mM ethanol treatment, and 24 h of 200 mM ethanol treatment was markedly decreased compared with their respective controls. The expression of ZO-1 mRNA at 24, 48 and 72 h of treatment with 50 and 100 mM ethanol, and 24 h of 200 mM ethanol treatment was markedly decreased compared with their respective controls. Although these protein levels decreased, the differences were not significant.
Immunofluorescence and western blotting analyses were employed to determine ethanol-induced PKCα activation by measuring PKCα and/or p-PKCα expression. Typically, PKCα is diffusely distributed throughout the cytoplasm and is localized in the nucleus in small amounts (29). Following ethanol treatment, immunofluorescence staining demonstrated that PKCα translocated from the cytoplasm to the perinuclear region and the nucleus, while safingol reversed this (Fig. 3A). The western blotting results indicated that PKCα expression in cells treated with all concentrations of ethanol was not significantly different following 48 h, where as p-PKCα expression was significantly increased in the 200 and 400 mM ethanol treatment groups compared with the control groups (both P<0.05; Fig. 3B and C).
Alterations in the expression of PKCα following ethanol treatment. PKCα expression was detected by immunofluorescence staining and western blotting analyses. (A) Representative immunofluorescence staining images of PKCα following 48 h treatment with 200 mM ethanol or 200 mM ethanol plus safingol (magnification, ×400). (B) Western blotting of PKCα and p-PKCα expression following treatment with different concentrations of ethanol for 48 h. (C) Quantitative analysis of PKCα and p-PKCα protein expression. The results (n=5) are presented as the mean ± standard deviation. *P<0.05 vs. control. p-PKCα, phosphorylated-protein kinase Cα.
The role of PKCα in the expression of TJ proteins was determined by measuring the protein expression levels of claudin-5, occludin, ZO-1 and p-PKCα following treatment with ethanol and safingol by western blotting. The expression of p-PKCα in cells treated with 200 mM ethanol was significantly increased when compared with untreated controls (P<0.05; Fig. 4A and B). By contrast, the protein expression levels of claudin-5, occludin and ZO-1 were significantly decreased when compared with controls (all P<0.05; Fig. 4C and D). In the ethanol plus safingol treatment group, the expression of p-PKCα was significantly decreased (P<0.05; Fig. 4A and B), and the expression of claudin-5, occludin and ZO-1 was significantly increased when compared with the ethanol-only treated group (all P<0.05; Fig. 4C and D).
Effect of the ethanol-induced increase in p-PKCα expression on claudin-5, occludin and ZO-1 expression. (A) Western blotting analysis of p-PKCα and β-actin protein expression. (B) Quantitative analysis of p-PKCα protein expression relative to β-actin expression. (C) Western blotting analysis of claudin-5, occludin and ZO-1 protein expression. (D) Quantitative analysis of claudin-5, occludin and ZO-1 protein expression relative to β-actin expression. The results (n=5) are presented as the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. 200 mM ethanol. p-PKCα, phosphorylated-protein kinase Cα; ZO-1, zonula occludens-1; S, safingol.
The increased permeability of the BBB as a result of BBB dysfunction is well studied in individuals with alcoholism (30) and animal models (31). A previous study treated BMECs with ethanol at various time points and demonstrated a progressive decline in transendothelial electrical resistance and an increase in monocyte migration across the BBB (15). Although the mechanisms of ethanol-induced BBB leakage are not fully understood, ethanol treatment is associated with a decrease in the expression of total occludin and claudin-5 without alterations in ZO-1 content in primary bovine BMECs (15). The results of the current study are consistent with these observations, as a reduction in occludin and claudin-5 expression in HCMEC/D3s was observed. However, although the expression of ZO-1 was not affected in this previous study, a decrease in the expression of ZO-1 was observed in HCMEC/D3 s treated with ethanol in the current study. This is likely due to differences between ethanol administration methods, including exposure time and concentration, as well as the cell type and species under investigation.
TJ proteins, claudin-5, occludin and ZO-1 are important for the formation and maintenance of a functional BBB (4,5). Tissues or cells that lose the ability to regulate the functions of TJs become more susceptible to various injuries, including inflammation and viral and bacterial infections (32,33).
The aim of the present study was to investigate the effect of ethanol on TJ proteins in HCMEC/D3s, and determine the primary signaling pathways involved. It was demonstrated that the ethanol-induced decrease in the protein expression levels of claudin-5, occludin and ZO-1 was concentration-dependent, and treatment with the PKCα-specific inhibitor, safingol, reversed these effects. These results indicate that ethanol may mediate its effects via a PKCα-dependent mechanism. The regulation of TJ proteins is not fully understood; therefore what is know about the regulation of TJs is just speculation. Due to the ability of ethanol to activate PKCα by Rho guanosine triphosphatases or filamentous actin (34,35), it has been hypothesized that PKCα is the major factor by which ethanol mediates its effects. A previous study implicated PKCα as a major component involved in increased TJ permeability (36). The observed reversal of the effects of ethanol treatment on occludin, claudin-5 and ZO-1 expression by safingol treatment in current study supports this hypothesis.
The effect of ethanol on HCMEC/D3 viability was determined using an MTS assay and the expression of claudin-5, occludin and ZO-1 was measured using western blotting and RT-qPCR analyses. The MTS assay revealed that ethanol exposure reduced HCMEC/D3 viability in a concentration- and time-dependent manner. The viability of cells in the low-dose ethanol group (50 mM) was increased when compared with the control, which may be associated with self-protection via enhanced antioxidant capacity (37,38). The aforementioned studies demonstrated that low concentrations of ethanol may reduce asymmetric dimethylarginine and increase dimethylarginine dimethylaminohydrolase activity, which lead to increased cell viability. Western blotting analysis demonstrated a significant reduction in the expression of claudin-5, occludin and ZO-1 protein in the cells 24 h after treatment with 400 mM ethanol, and 48 and 72 h after treatment with 200 and 400 mM ethanol; the protein level of occludin also significantly decreased after 72 h of treatment with 100 mM ethanol. This ethanol-induced impairment of TJ protein expression was reversed in cells pretreated with safingol. By contrast, the expression of claudin-5, occludin and ZO-1 mRNA in the cells was increased after 24 h of treatment with 400 mM ethanol, and 48 and 72 h of treatment with 200 and 400 mM ethanol; the expression of occludin mRNA was also significantly increased after 72 h of treatment with 100 mM ethanol. The decreased level of protein may induce a negative feedback loop, thus leading to increased mRNA levels or the binding of the specific protein to the relevant gene mRNA, inhibiting its translation. This may stabilize the mRNA so that the protein content is reduced, while the levels of mRNA are increased. RNA-binding proteins (RBPs) influence TJ mRNA stability and translation. CUG-binding protein 1 (CUGBP1) and T-cell intracellular antigen-related protein (TIAR) are negative regulators of RBPs. The co-localization of CUGBP1 and tagged occludin mRNA in processing bodies represses occludin translation and stabilizes the expression of its mRNA (39,40). The mRNA encoding the membrane-associated TJ protein, ZO-1, has an affinity for TIAR, which represses ZO-1 translation and stabilizes its mRNA expression (39). However, the specific mechanism of action is unclear and further studies are required. Claudin-5, occludin and ZO-1 proteins are important for TJ formation, therefore the observed decrease in the expression of TJ proteins may be associated with ethanol-induced BBB leakage.
PKCα is a typical PKC isoform primarily expressed in vascular endothelial cells and the central nervous system (41). PKCα increases and decreases TJ permeability (21). It is highly unlikely that increased PKC activity is solely due to increased protein expression (21), and additional intracellular mechanisms may be responsible for altering PKC activity. For example, activated PKC translocates from the cytoplasm to the perinuclear region and nucleus (42,43). In addition, PKC is itself phosphorylated, which indicates its activation (44). The translocation and/or phosphorylation of various PKC isoforms are important for changes in their respective activities. In the current study, immunofluorescence staining results demonstrated that PKCα translocated from the cytoplasm to the perinuclear region and nucleus in cells exposed to ethanol. In addition, western blotting analysis indicated that PKCα expression was not significantly altered and p-PKCα expression was significantly increased by ethanol treatment. These results suggest that ethanol induced an increase in PKCα activity by increasing its conversion from an inactive to an active form, rather than by increasing its expression. The PKCα inhibitor experiments indicated that PKCα was the most likely mechanism by which ethanol induces TJ impairment. Increased PKCα activity and altered claudin-5, occludin and ZO-1 protein levels were observed in ethanol-treated cells. These ethanol-induced alterations were all reversed by pretreating HCMEC/D3 s with the PKCα-specific inhibitor, safingol. The increased activity of PKCα and the associated decrease in the expression of TJ proteins, as well as the reversal of ethanol-induced alterations by PKCα inhibition, further the understanding of the regulation of ethanol-induced TJ impairment.
In conclusion, the results of the current study demonstrate that the ethanol-induced decrease in the expression of claudin-5, occludin and ZO-1 proteins and increase in PKCα activity in HCMEC/D3s, are concentration and time-dependent. In addition, pre-treatment of HCMEC/D3s with safingol prior to ethanol exposure reversed these ethanol-induced alterations. The results suggest that ethanol decreases the expression of TJ proteins in HCMEC/D3s via a PKCα-dependent mechanism. Ethanol-induced TJ impairment may lead to the loss of BBB structure and BBB dysfunction, thereby increasing the transport of ions, molecules and leukocytes in and out of the brain.
The present study was supported by the National Natural Science Foundation of China (grant no. 81172904), the Natural Science Foundation of Liaoning Province (grant no. 201102299) and the Shenyang Scientific and Technological Plan (grant no. F11-264-1-67).
Tan B, Huang H and Wang JJ: Protective effect of low concentration alcohol on human umbilicus vein endothelial cells. Chin J Cardiovasc Review. 5:370–372. 2006.
Yu, H., Wang, C., Wang, X., Wang, H., Zhang, C., You, J., Wang, P., Feng, C., Xu, G., Zhao, R., Wu, X., Zhang, G."Long‑term exposure to ethanol downregulates tight junction proteins through the protein kinase Cα signaling pathway in human cerebral microvascular endothelial cells". Experimental and Therapeutic Medicine 14.5 (2017): 4789-4796.


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Trinity's Links with Declan Ganley and the Far-Right are Morally Reprehensible
October 7, 2021 / laszlo / 1 Comment
From the lovely Cafe Tri Via in southside Dublin one can see a stretch of buildings, most of them the property of Trinity College Dublin, accumulated over the years for real estate investment. One of these, on the right, is CONNECT's headquarters, a research center for telecommunications projects with the involvement of third-level institutions across the country. It is funded by the government, headquartered by the College and influenced by over 45 industrial partners. Partners, who hold sway over the institution, like Rivada Networks, whose controversial founder Declan Ganley is involved with the right- to far-right sphere.
It is this link that has led to Rivada Networks directly funding Trinity researchers, like in the case of then-Professor Linda Doyle's and Peter Cramton's 2016 paper on wireless markets, and many more through CONNECT.
This is somewhat of an open secret amongst people familiar with academic research, but is not widely known. It is important to note, however, so as to realize that the corrupting influence of capital goes much further than Trinity's two and a half million euro investment into the armaments industry, and gets its claws much deeper in the institution. The history of CONNECT is representative of the ruling mode of production and the intensification of capitalism over the past few decades.
CONNECT used to be the CTVR, the Center for Communication and Value Chain Research of Trinity founded in 2004. It was now-Provost Linda Doyle who embarked on an ambitious project to transform it into what she envisioned to be a nation-wide multi-institutional research center. From this, CONNECT was born in 2015 as a public-private partnership, with the government's Science Foundation Ireland (SFI) and corporate players funding its 300+ researchers who come up with ideas, and third-level institutions like Trinity managing their projects and profiting from the system through getting paid for administrative expenses. Trinity has an especially important role in the project, not only in headquartering, but it also hosts its directors, one of them who was now-Provost Linda Doyle until 2018, when another professor from the College took over.
Of course, the involvement of the government who demand spin-off companies with profitability and multinationals means it has an entrepreneurial bent.
This is where Rivada Networks comes into the picture, which is one of the corporate players involved in funding research and real-world applications thereof. Mr. Ganley, its Chairman and CEO, sits on the board of CONNECT. And if by nothing else, his influence can be measured by his net worth of €455 million euros, which he amassed through a network of businesses in Ireland, the United States and Russia, primarily in the telecommunications field. In June 2021, he and then-Provost-elect Linda Doyle did an interview on CONNECT's YouTube channel, indicating his continued involvement, which becomes problematic when considering his political views, the organizations he runs and where he spends his money.
Mr. Ganley is described as one of Ireland's most prominent conservatives, and he is on the limit of political respectability at first glance. Always one step shy from explicitly engaging in the extreme right network, he works to support the Christian, right-wing and far-right forces from the background. This is done mostly through funding and donations.
In 2017, he gave the seed money for so-called 'conservative student newspaper' The Burkean, which at the time described itself as a publication of Trinity students aimed to fight the 'degeneracy' in the western world. This is, of course, a common far-right dog whistle.
To take another example, at the time of the abortion referendum in 2018, he campaigned fiercely on the 'No' side, even saying that he will 'not pay taxes that fund abortion'.
The question is, of course, where the profit motive lies and where his money is coming from. Naturally not solely from endeavours such as CONNECT's spin-off companies, Rivada Networks is supported by right-wing interests around the world. It is not a coincidence that his company employs people such as John McGuirk, the editor of far-right newspaper Gript.ie and political player who once called a pro-choice TD, Kate O'Connell, a "catty, spiteful, loathsome twit". He was communcations director for the anti-abortion "Save The 8th" campaign in 2018, and during his time at Trinity College Dublin he was "was forced to apologise after anonymous emails used to make allegations of sexual harrasment against a member of TCD's College Historical Society were traced back to him".
To find other proof for the involvement of vested interests one only has to open a newspaper. Near the end of his presidency, it was reported that Donald Trump was pressuring the Pentagon to give a no-bid contract to develop 5G spectrum to Mr. Ganley's company, 'whose prominent investors include Fox News regular Karl Rove and Peter Thiel, Trump's biggest supporter in Silicon Valley'.
This is not to mention that the directors of Rivada Networks include a former lieutenant general, two formal admirals and a former deputy-secretary of Homeland Security, who also sits on the board of LockHeed Martin, a defense contractor that Trinity coincidentally has an investment of 721,473 € in. In addition, it includes Richard B. Myers, fifteenth Chairman of the Joint Chiefs of Staff, who oversaw the 2001 invasion of Iraq by the United States. Mr. Ganley himself, through using Rivada Networks as a telecommunications supply company, tried to profit from the U.S. invasion of Iraq by securing contracts for the failed reconstruction effort in 2003.
Later, between 2004 and 2008, 'Rivada Pacific won contracts totaling $37.3 million from the U.S government.'
In this light, Mr. Ganley's former political party, the so-called Libertas, also makes sense. He set up Libertas to campaign against the ratification of the Lisbon Treaty in 2018 in alliance with national-conservative forces, on a platform that presented itself as defending the 'libertarian democracy' of the European Union (EU). However, upon closer inspection, this was a red herring. The Irish Times in 2008 reported that Mr. Ganley's aspirations came as part of his vision, written down in a paper for the Foreign Policy Research Institution (FRPI) in 2003, a neoconservative think-tank that promotes military interventionism and U.S. foreign policy goals, which at their core arise from an ever-growing need to expand the markets that capital can spread to.
In this paper, he denounced European criticism of the Iraqi invasion, and declared that the EU must cease its 'contradistinction to the United States, [that] this new Europe must be an equal partner and influence in the worldwide extension of justice and liberty,' – a goal which Libertas shamelessly promoted.
Declan Ganley's ex-Party, Libertas, also allied itself with the Simon Wiesenthal Centre, a pro-Israel organization which claimed in 2013 that BDS is a "thinly-disguised effort to coordinate and complement the violent strategy of Palestinian, Arab, and Muslim 'rejectionists' who have refused to make peace with Israel for over six decades, and to pursue a high-profile campaign composed of anti-Israel big lies to help destroy the Jewish State by any and all means"..
As such, if Trinity wants to adhere to its mission statement to promote a campus culture of 'dedication to societal reform', it should be more careful of where it gets its funding.
Article, Peace, Statement
Responsibility of the Provost's Office in Re-Timetabling (TCD)
Trinity Accommodation Rent Hike of 2.3%
Laszlo Molnarfi
Declan Ganley's ex-Party, Libertas, allied itself with the Simon Wiesenthal Centre, a pro-Israel organization which claimed in 2013 that BDS is a "thinly-disguised effort to coordinate and complement the violent strategy of Palestinian, Arab, and Muslim 'rejectionists' who have refused to make peace with Israel for over six decades, and to pursue a high-profile campaign composed of anti-Israel big lies to help destroy the Jewish State by any and all means".

redpajama_set_name: RedPajamaCommonCrawl


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Home » Route Planners Cut Fuel Usage in Freight Sector
Route Planners Cut Fuel Usage in Freight Sector
"In 2011, we achieved almost 69 percent improvement in fleet efficiency over our 2005 baseline," Wal-Mart boasted in its 2012 Global Responsibility Report. "We delivered 65 million more cases, while driving 28 million fewer miles, by increasing our pallets per trailer and better managing our routes. Our network efficiency improvement equates to avoiding nearly 41,000 metric tons of carbon dioxide emissions, the equivalent to taking 7,900 cars off the road," the company wrote.
In 2013, FedEx will have improved the fuel efficiency of its U.S. vehicle fleet by 22 percent compared with 2005, Chairman Frederick Smith has said. The company surpassed its previous goal of a 20-percent improvement by 2020 seven years early. Smith has now committed the company to an even more ambitious 30-percent target for the global vehicle fleet by the end of the decade.
Fuel conservation programs are being replicated at hundreds of firms across the transportation and logistics sector.
The price of oil and fuel volatility is definitely a driver, but not as much as the push to reduce carbon emissions," according to a report published by the University of Pennsylvania's Wharton School of Business. In practice, what started as a carbon-saving program during the 2004-2008 boom has become a cost-saving initiative in the lean years that have followed.
"Adding efficiency to any part of the supply chain produces better returns. The good thing is that much of what we do to improve fuel economy translates to the bottom line as improved profitability. Every bit of energy you save is money in your pocket," according to the Wharton report.
Technology SC Planning & Optimization Business Strategy Alignment Global Supply Chain Management Sustainability & Corporate Social Responsibility North America
KEYWORDS Arab News Business Strategy Alignment Fuel Efficiency Fuel Management Global Logistics Global Supply Chain Management global trade logistics management North America Route Planning SC Planning & Optimization Shipping Management supply chain Supply Chain Analysis & Consulting Supply Chain Efficiency Supply Chain Management: Environmental supply chain technology sustainability Technology Transportation Management
Canadian Freight Costs Decline in November, But Fuel Surcharges Continue to Rise
Road Usage Fee Plans Finalized in UK for Foreign Operators
Group Works to Set Up Guidelines on RFID Usage at Item Level in Retail


For years, we have wondered whether parrots are intelligent enough to speak. But perhaps the question is whether they are smart enough not to talk to us. This film dedicated to the nonsense poet Edward Lear, who loved parrots, shows the gentle, cruel, absurd and ineluctable relationships between people and birds.
Chitter Chatter, that's all you're good at. Said Vegetable.
An intimate film and an honest portrait, wherein Celeste Rodrigues proves her liveliness and passion, which show his fado's contemporaneity.
At the ritual celebrations of Saint Bartholomew of the Sea, disillusionment helps the feet enter the cold water. It's like a whisper, indifferent to the distorted music that echoes round the beach. Disillusionment makes these people beg to heaven what they cannot achieve on earth. How many are the dreams they dare not dream?
"Chasing after the wind" is a portrait of Gustavo, a marginal character who lives in Gethsemane, a historical neighborhood in Cartagena, Colombia. It's the contemplation of a shared impermanence, where Gethsemane's debris bears witness of the end of a community, but also of a human being.
"Baby-dance" is a method to strengthen the connection between the baby and the parent before the shared spoken language. This film dedicated to the discoveries and thinking of the philosopher Kirsti Määttänen.
Vova is a guard at a private fishing enterprise. In the Soviet days and the early 1990s he was the head of a collective fishing farm. Nowadays he's at the roadside of life, watching things go by from his vantage point – the abandoned Voyage cafe. His tales and stories make up a kaleidoscopic image of present-day Ukraine.
Seated on a podium in a cinema as if for a typical Q&A after a screening, Françoise Lebrun, actress here behind the camera (but in front of it too), begins by the end and politely warns her interlocutor: it will be a question of a work in the style of a quilt, of a crazy quilt.
Chiken wings are the typical American dish and are extremely popular across the United States. The picture tell a story of Matt Reynolds and bunch of his friends on a quest for the perfect chiken wings.
Have you ever seen a faucet for wine and liquor? Or a house in all colours of the rainbow, combining the aesthetics of ancient Greece with PVC joinery into the unique style of rocco-barocco?
First documentary about the Czech hip hop scene shows how close the Czech rap can come to humour and irony.
A documentary portrait of two women playing a very personal game with the fate of Horni Jiretin, a town endangered by the coal-mining industry.


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Mexico: Ongoing Covid-19 pandemic and government policies are hurting private businesses
Mexico: Ongoing Covid 19 pandemic and government policies are hurting private businesses
Mexico experienced a severe economic contraction of 8.5% in 2020, while a weak recovery can be expected this year.
Bankruptcies in the private sector are forecast to rise due to the Covid-19 pandemic and government policies.
Public finances deteriorated but to a lesser extent than in most Latin American countries.
Many of Mexico's states are heavily indebted and their capacity to honour payments is likely to be compromised in the coming months.
Country risk ratings remain unchanged.
Latin America's second-largest economy experienced a deep recession in 2020
Like most Latin American countries, Mexico faced a deep recession over the past year, and the latest update from the IMF (January 2021) forecast a severe contraction of -8.5% in 2020 for Mexico. Furthermore, it is worth noting that Mexico experienced a deeper contraction than the average regional GDP growth of -7.4%, while the region was the world's worst performing last year. This poor economic growth performance might come as a surprise, since Mexico – along with Brazil – implemented some of the least stringent restrictions when tackling the Covid-19 pandemic. However, alongside the ongoing health crisis and the consequential confinement measures, other factors have clearly had a negative impact on economic activity in Mexico. Firstly, the multiple changes to the rules of the game for investors has led to high uncertainty and falling investments for the country's economy, and generally, lower investments lead to lower economic growth. Examples are manifold: scrapping plans for an airport and a brewery which were partially built, cancelling electricity auctions, rewriting gas pipeline contracts, as well as new labelling requirements for processed food manufacturers. Secondly, the ongoing austerity programme in Mexico is dampening economic growth, because unlike the route taken by many other countries, the Mexican government's response to the economic slowdown has been fiscal austerity.
A weak recovery awaits for 2021
As graph 1 illustrates, an economic recovery of 4.3% and 2.5% is expected for Mexico in 2021 and 2022, respectively. This is quite weak given the deep recession last year and a weak performance compared with the world's expected average real GDP growth rate. Furthermore, this recovery will be mostly due to the impact of a sizeable stimulus package in the USA, which implies higher economic growth in the USA and better exports for Mexico and stronger remittances. That said, the outlook is clouded by the high levels of uncertainty looming due to potential new Covid-19 waves, and in early January Covid-19 cases were vastly on the rise in Mexico, indicating a second wave. Though the country has been relatively quick to start its vaccination programme (launched late December 2020), as it stands it still hasn't secured enough vaccinations for the entire population, leaving it vulnerable to more waves. On top of that, the efficacy of vaccines (against the Brazilian variant, for example), availability and the pace of rollout both in the USA and in Mexico could encounter setbacks, hurting economic activity in the coming months.
Bankruptcies in the private sector likely to rise
Measures to stem the Covid-19 outbreak, mainly involving localised lockdowns and restrictions, are hurting many businesses in Mexico. On top of that, government support for the private sector is extremely limited. Mexico has only spent an additional 0.7% of GDP on tackling the Covid-19 crisis – comprising mainly of loans to poorer households – compared to 8% of GDP in Brazil and an average of 4% of GDP for emerging markets. Furthermore, the government of President Andrés Manuel López Obrador is making decisions that could hurt the private sector even further: it favours a greater state role in the economy and won't hesitate to revise contracts and impose new rules and controls. Examples of such policy decisions include a tax collection drive targeting large companies, a plan to ban the outsourcing of jobs and a 15% minimum wage increase in 2021. For these reasons, further bankruptcies and job losses can be expected in Mexico's private sector in the coming months.
Public finances deteriorated but to a lesser extent than in most countries
Public finances have also been hurt by the pandemic, though to a much lesser extent than in most countries. According to the latest IMF report of October 2020, public debt stood at a moderate 53.7% of GDP at year-end 2019, and is likely to have risen to about 65.5% of GDP at year-end 2020, mainly as a result of severe economic contraction and depreciation of the Mexican peso (widening the fiscal deficit to about 5.8% of GDP in 2020). The public debt level is expected to remain around 65% of GDP in the coming years, an elevated but not unsustainable level, while fiscal deficits are expected to be relatively small in the coming years (-3.4% of GDP in 2021) and their impact on debt vis-à-vis GDP will be offset by the expected GDP growth. That said, payment troubles may unfold in certain state governments, given that most measures offered to date to mitigate the negative effects of Covid-19 on the private sector are being implemented directly at state government level. Many of Mexico's states, however, are heavily indebted, and their capacity to honour payments is likely to be severely compromised in the coming months.
Political risk ratings remain unchanged
Mexico's MLT political risk score has remained in category 3/7 for over a decade, and the country's modest risk rating reflects its moderate external debt and debt service ratios, small current account deficit, deteriorating but sustainable public finances, poor economic growth in recent years and the country's economic diversification, despite its great reliance on the US economy. The short-term political risk rating is in category 2/7, driven by the adequate level of foreign exchange reserves and relatively low levels of short-term external debt. Despite a challenging global environment, the outlook remains stable for both risk ratings.
Solid macroeconomic fundamentals
Important manufacturing sector
Sustainable debt dynamics
Exposed to altering conditions on financial markets
Oil production on downward trend (net fuel importer)
Corruption and rising crime rate
Controversial policy decisions are nurturing uncertainty
President Andrés Manuel López Obrador
127.6 million (2019)
GNP per capita
Income group
Upper-middle income
Main export products
Manufactures (7% of total current account receipts in 2018), private transfers (6.6% in 2019), oil (4.7% in 2019) and tourism (4.3% in 2018)
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Real estate speculation is king in New York City and its killing Queens County. This stark reality will define the primary election race for the NY City Council District 21 (CD 21) Democratic Party line in northwestern Queens. Concerns regarding the cozy cross-fertilization between the speculative real estate coalition and local elected officials will surface during the campaign. The race will revolve around the hot button issues of gentrification, immigrant displacement, and social justice; political dynamics will be shaded by questions of personal character; and the political stew will be stirred by New York's hyper-driven media market. Clearly this is a highly volatile mix. And what follows are my preliminary thoughts on how the primary race may tentatively play out.
At present, three candidates have cast their hats into the political ring: former state Senator Hiram Monserrate, state Assemblyperson Francisco Moya, and community activist Cristina Furlong. CD 21 is a working-class and populated by Latin American, Caribbean, and Asian immigrants. Furlong, as a Jackson Heights resident, will be viewed by many local voters as a politically inexperienced cultural interloper, reducing the race to two highly experienced Latino contenders with vastly differing economic and political views regarding the real estate sector.
In the head-to-head between the candidates, Moya will criticize Monserrate's problematic legal legacy and his history as renegade democratic state Senator in Albany. On the flipside, Moya will self-laud his profile as an elected official with a history of Latino community-based engagement, economic growth strategies, pro-immigration advocacy, and a repertoire of anti-Trumpian political positions. This strategic political story line aligns with the Queens democratic machine's standard public rhetoric. Consequently, the Queens Democratic Party, which fully supports Moya, will mobilize the requisite political resources to ensure the candidate's success. Thus, solidifying the machine's political control and reinforcing Moya's script as the unblemished liberal knight who fights the good fight.
Monserrate will design his campaign as an insurgency against entrenched political and economic interests. The emphasis will be on predatory real estate speculation, the explosion of mega projects, and how certain Latino elected officials collude with real estate developers. This will open the door to a pointed criticism of the growth coalition that has fast-tracked unsustainable levels of inequality across the borough.
Monserrate will connect the dots linking Moya and local Latino elected officials with large scale real estate capital, gentrification, and the expulsion of economically vulnerable residents and immigrant businesses from the district. He'll voice the claim that "I will be fighting for the community and not for big capital" and "I'll be ten times more tenacious then I ever was in the past." These are fighting words. Yet, what remains to be seen is if he can achieve the political redemption essential for reviving his public profile.
For Moya and Monserrate, win or lose, the lines are sharply drawn. It's evident that we are living in turbulent times. Our institutions are outmoded and in crisis. Long held conventions are falling by the wayside. What does this mean for the near future? It's still too early to say. But, continued silence in the face of deepening social injustice is unacceptable and unsustainable.
Arturo Ignacio Sánchez, Ph.D. is an urban planner and a member of Community Board 3, Queens. He has taught at Barnard College, City University of New York, Columbia University, Cornell University, New York University, Pratt Institute, and various Latin American universities.


En español | Need a kickoff for that fitness regimen you've been planning to begin? You don't have to spend a fortune to get a jump-start. Wellness vacations come in all shapes and sizes and can be much more affordable than a fancy spa.
For the past quarter of a century, the Tennessee Fitness Spa in Waynesboro has offered opportunity for those seeking to drop pounds, tone up or shed unhealthy habits such as smoking. You'll be assessed when you arrive — and through your stay — and you can participate in six hours of classes every day except Sunday. Programs and lectures are available, too. Special weeks focus on cardio, guided meditation, strength training, cooking and more. Rates, including all meals and most activities, are low for a destination spa. They start at about $675 per person for three nights, and $700 and higher per person for a week. Hook up your RV, and pay less.
"Taking the waters" was all the rage in 19th- and early 20th-century U.S. and Europe — and the custom gave rise to the modern concept of the spa. Back then, doctors dispatched patients to take treatments on Bathhouse Row in Hot Springs. Since 1912, the majestic, pillared Buckstaff Bathhouse Company has helped clients to ease the pain of arthritis and other ailments, or to ease the mind and soul by simply relaxing. Other spas dot the city. Stay at a hotel with a bathhouse for less than $100 a night or in a historic bed-and-breakfast.
There's no shortage of fitness-themed cruises, but if you set sail Oct. 2-8 in the Caribbean aboard the Carnival Splendor with a group of health-minded folk, you'll get a big bang for your buck. Book through the group Moves 2 Inspire Fitness; rates start at about $600 for a six-night trip. Zumba and boot-camp workouts will help work off those all-you-can-eat meals and free room service. Your group will enjoy private receptions and theme parties between calls at ports in Jamaica, Grand Cayman Island and Mexico. You'll also be able to join fellow passengers for shore excursions and other shipboard activities.
Walking vacations are a wonderful way to tone up as you explore a new country. U.S.-based Breakaway Adventures offers dozens of choices, including a popular seven-night outing named the White Villages of Andalusia Walk. You'll stay in a 200-year-old olive mill that's been converted into a luxury hotel, trek through the Spanish countryside and explore the region's picturesque villages. Lodging, guide, all meals and wine (but not airfare) are included in rates that begin at about $1,500 a person.
Award-winning New Life Hiking Spa in Killington offers mini-stays as well as longer programs. They include workouts, hikes in the Vermont wilderness, nutrition and wellness classes, plus a soothing massage or two. Accommodations are cozy, country-inn style. The price tag is about $230 daily per person, including all meals and a roster of activities.
Escape everyday stresses, shed pounds and build endurance while backpacking in locales ranging from the Grand Canyon and Great Smoky Mountains to West Virginia's Monongahela National Forest. Fitpacking's weight-loss backpacking adventures are labeled according to difficulty and typically involve a hotel stay at the beginning and end, with camping in between. Costs start at about $1,300 a person for a weeklong trip, including lodging, guides and all meals. Half-week and day trips are offered, too. Fitpacking says the typical participant drops five pounds a week.
Spend a weekend at the McLean Meditation Institute in Sedona, and you'll learn techniques for deep relaxation and how to quiet a racing mind. Rates starting at less than $600 for two include a hotel in mystical Red Rock country, private instruction for two, a book on how to transform your life via meditation and a guided-meditation CD. A yoga class is also offered.
This California desert oasis — favored by Hollywood's elite — doesn't have to be costly. Weekday rates off-season and during the summer at the Spring Resort & Spa in Desert Hot Springs are quite reasonable. Indulge in two spa treatments — including deep-tissue massages, facials, scrubs and wraps — per person during your stay. Luxuriate in warm mineral-water pools for free. Rates (including breakfast, lodging and treatments) start at about $750 for two for two nights.
This women's-only five-night retreat in the stunning desert of New Mexico focuses on the mind and body. Learn secrets of Native American healers, soak in the soothing Ojo Caliente Mineral Springs, take yoga classes and optional hikes, and stay in upscale accommodations. There's time to shop for crafts in town, too. This getaway from Road Scholar (formerly Elderhostel) is offered multiple times a year and starts at about $1,300, including lodging, lectures, field trips and most meals. (Cyclists can check out Road Scholar's $1,000 tour of the C&O Canal from Washington, D.C., to Cumberland, Md.).
If you're wont to make up your own fitness menu, multiple outdoor adventures await independent travelers to Alabama on snow-white beaches and in coastal marshes. Go kayaking in bays alongside dolphins, herons and egrets. Take hikes in 7,000-acre Bon Secour National Wildlife Refuge in Gulf Shores, where you can also join in guided bird-watching outings, dune walks and "night prowls." Bike on miles of trails in nearby Gulf State Park, and see alligators and tortoises. You can camp or rent a comfy cabin, starting at about $100 nightly with a senior discount.
Rancho La Puerta, just across the border from San Diego in Tecate, Mexico, attracts the rich and famous. Plan a splurge, and join them. Guests stay in rustic, elegant cottages decorated with Mexican tile and art, begin their days with desert or mountain hikes and can pick from a half-dozen exercise options every hour throughout the day. The food and spa are world class, as are the experts brought in to lecture during the evening. Butthe retreat, which turns 76 this year, occasionally offers three-night stays starting at about $500 a person daily, all inclusive, as well as specials during slow weeks. It's not cheap, but Rancho's many repeat visitors say a stay is life-changing.


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Padal Petra Sthalam
Koilvenni Vennikarumbeshwarar Temple – Diabetic Sweet Spot
August 16, 2022 August 16, 2022 Ensures3 Comments
On our repeated pilgrimages to Kumbakonam region, a visit to Kovilvenni a town near Needamangalam in Thiruvarur District about 24 km away from Thanjavur, was missed due to some reasons or the other. When we went to Kumbakonam Navagraha circuit in December 2021, we had resolved that we had to have a darshan in this temple at any cost. But then Arulmigu Soundara Nayagi sameta Shri Vennikarumbeshwararar blessings was not forthcoming as we had to rush back to Bengaluru. Finally on 14th August 2022, our wishes fructified and By Shiva! What an amazing time was in the offing. It only goes on to prove, it is His time and call not ours anyway. The highlight for us, after Darshan of the Bhagwan, was very learned and knowledgeable Chief Archaka Shri Prabhakar Shivacharya AVL – his affable, patient explanation of the sthala purana (Historical Significance of the Shrine).
Koilvenni Arulmigu Karumbeshwarar Temple
The temple dedicated to Shiva, as the Moolavar (Presiding deity), manifested as Shri Vennikarumbeswarar. Upadevata is His consort, Parvati, known as Arulmigu Soundaranayagi. The historical name of the place is Tiruvenni. It is one of the shrines of the 275 Paadal Petra Sthalams – Shiva Sthalams glorified in the early medieval Tevaram poems by Tamil Saivite Nayanars Tirugnanasambandar and Tirunavukkarasar. Koilvenni Arulmigu Karumbeshwarar Temple in 102nd of Paadal Petra Sthalams on the Southern bank of River Kaveri. He is also known as Venni Nathar, Rasapureeswarar, Triambakanathar It is believed that patients suffering from any ailments especially diabetes will be cured of the disease if they make an offering of sugar to the main deity at this temple. "Karumbu" in Tamizh means sugarcane and the main linga is in the form of a bunch of sugarcane stems tied together.
Sthala Purana or Historical Significance
In ancient times, the sthala vruksham was Nandivardhanam, a flower white in colour and hence called Venni or Nandivardhanam Flower (Pinwheel Flower or Crepe Jasmine). This is one of the favourite flowers of Bhagawan Shiva and used in His worship.
Venni Flower (Tabernaemontana divaricata) Pinwheel Flower/Crepe Jasmine
This place was also surrounded by dense Sugarcane fields. In Tamizh Sugarcane is called Karumbu. King Musukunda Chola Chakravarthy was a Chola ruler who ruled the parts of Chola Kingdom from Aarur (Thiruvarur). He was a great devotee of Bhagawan Shiva. He also had renovated the Thiruvarur Sri Thyagarajaperumal temple, after which he was passing by Venniyur with his troops. and when he overheard two Sages arguing between them on the sthala Vruksha of the place – one saying it was "Sugarcane" and the other claiming that it was "Venni". Not wanting to take any sides, fearing curse of the other sage, King asked them why this argument about the Temple Tree, when there is neither a temple nor a Vigraha of Bhagawan Shiva. They hear an Akashavani (A voice from the Skies) which was that of Bhagawan Shiva saying that He should be named after Sugarcane (Karumbu) and the Temple Tree or Sthala Vruksha will be Venni. He also says that He is present in a self manifested form (Swayambhu), in a form that has seen all the Yugas (Krita or Satya Yuga, Treta Yuga, Dwapara Yuga, and Kali Yuga), whose stem looks like a bunch of Sugar cane tied together, a form that has been worshipped by Devas and Rishis over the yugas. He continues saying He be found deep inside the earth below the very place the argument was going on, bring it up, build a shrine and perform Kumbhabhishekam. It is also said in Kanda Puranam that the wedding invitation of Devasena – Subramanya was sent to this place.
King then uses his men to dig the field and he finds the Shiva Linga whose stem part was spread deeper and deeper and he was lost how to bring it up. Once again Bhagwan Shiva gives him a solution to tie it up at the stem and lift it up. The height of this Linga pratishthe from Surya Teertham towards the East is 36 feet, signifying surpassing of 36 tatvas or elements. This incident is believed to have happened 5000 years ago. After the historic "Venni war" the Chola King , Karikala Chola rebuilt and renovated this temple.
Swayambhu Shri Vennikarumbeshwarar
This place which gained prominence even in the Sangam times, had found significance in the songs of Thirugnanasambandar and Thirunavukkarasar. Sundarar had hailed the Lord here as the one who heals diseases, in his song " Kshetra Kovai." So, the Lord of this temple, " Sri Venni Karumbeswarar," remains the Lord who heals all the diseases , especially diabetes, of his devotees. Thirunavukkarasu had sung in praise of the Lord in his " Thiruthandakam" , singing the history of the Lord's marriage in Mayiladuthurai.
The Goddess of this place is Shri Soundaranayaki. True to her name, this Goddess is amazingly beautiful, and showers her blessings. It is believed that if ladies whose Seemanta (Valai Kappu) ceremony is over visit Her to seek Her blessings and tie bangles in front of the Goddess, for having a safe delivery.
Shri Soundaranayaki
The holy waters of this place, " Surya Pushkarani" and " Chandra Pushkarani," have the power to heal all diseases.
Surya Teertham
The Main Temple, Shri Vennikarumbeshwarar faces East and the Surya Teertham is right opposite to Him. Shri Soundara Nayaki faces South in this Temple in standing position with four hands, one of which shows the Abhaya Hastham. Surya Theertham, is where Surya worshipped the Lord. Since it is an East facing Lingam, the rays of the Sun fall on Him on 2nd, 3rd and 4th days of Tamil month Panguni (Mid of March) during Sun rise. Ambal Soundara Nayaki is in a south facing shrine. The shrine of Nataraja is in between the shrines of the Lord and the Mother.
Panoramic Shot with Surya Teertham and Main Gopuram of the Temple
Sri Ikshupureeswarar (another name of the presiding Deity, who is self – manifested remains with so much greatness , is also called" Sri Venninathar " and " Sri Venni Karumbeswarar." ( The word " Ikshu" means sugarcane.)
Other deities in the temple include Murugan,Narthana Vinayagar, Gajalaxmi, Bairavar and Navagraha. Goshta moorthies include Narthana Vinayagar, Dakshinamoorthy, Lingothbavar and Durga.
The sanctorum is constructed in such a way that the outer mandapam looks like a moat. The Peetham or the Base of the Shiva Linga is Square as against Round that we usually find elsewhere just as the form of Linga is square and not round. In the recent past, this temple saw extensive renovation, done by Nattukottai Chettiar community.
The main offering here is Rava and Sugar. After the Sankalpam and offering at His feet, Rava and Sugar is mixed and strewn around the temple. The ants eat the Sugar and only Rava is left behind. This is said to cure Devotees of Diabetes. Once they are cured, Bhaktas return back and perform a Ghee Abhisheka to Bhagawan Shiva.
Since there are not any shops around, it is better to carry all Puja offerings when we go to the temple. Typically we will need to carry Betel Leaves, Betel Nuts, Some fruits, Coconuts, Ghee and Flowers, apart from about a kg each Rava and Sugar.
Driving Directions to the Temple
https://goo.gl/maps/U3fJaFr9iyP6qVnv8
Temple Opening Hours:
8:00am to 12 noon; 4pm to 8pm
Chief Archaka Shri Prabhakar Shivacharya AVL Contact
Requesting Bhaktas to donate for Conduct of Abhishekas on Pradoshas (2 per month) which costs about ₹1501 per Pradosham or ₹3001 a month
Also requesting Bhaktas to donate liberally for maintenance and renovation of the temple. Please contact Chief Archaka on the number given before for the same.
Signing off with the link to Sthala Purana (in Tamizh) recorded by me on 14th August 20222 explained beautifully by Shri Shri Prabhakar Shivacharya AVL
PS: If there are any errors please leave it in the comment and I shall edit this article after verification.
Sharing some more pictures clicked
Culture & Festivals, Gem that is Bharat, Kshetras and LegendsChandra Teertham, Diabetes, Koilvenni, Kumbakonam, Needamangalam, Padal Petra Sthalam, Rasapureeswarar, Soundanayagi, Surya Teertham, Tiruvenni, Triambakanathar, Venni Nathar


S LINE PETROL A4. This car is in Excellent Condition. It comes with Excellent Service History. It also comes with Long MOT, NEW SERVICE, 3 MONTHS WARRANTY, 2 KEYS & 12 MONTHS AA BREAKDOWN COVER for added peace of mind. HPI CLEAR with no hidden past. PX CONSIDER.


If this shows to be effective I wonder how quickly it could be approved as an MD treatment since tadlafil is already on the market?
With spring in the air and all the neighborhood kids outside playing we see Jack tiring out well before his friends and needing to come inside to rest. It would great if this drug could succeed at giving him a little more energy and protect his muscles at the same time!
After reading the article on this drug I went on to the company's website to read on the drug.It looks safe enough to try at 5mg daily.My son is 10 year's old and walking well and I want to do all possible until a proper treatment arrives.My hope is that treatments are not too far may be 2012.


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Bootleg alcohol poisons 40 people within 3 days in Iran
22 October 2018 12:40 (UTC+04:00)
Baku, Azerbaijan, Oct. 22
By Elnur Baghishov - Trend:
Starting from October 19, 40 people have applied to the treatment centers of Iran's Bandar Abbas city due to alcohol poisoning, said Fatima Nowroozian, head of the Department of Public Relations and International Affairs of Iran's Hormozgan University of Medical Sciences, IRNA reported.
She said that those who apply as a result of poisoning are aged 15-50.
So far, one person has died and two have been placed in the intensive care unit, she added.
She noted that 19 people underwent dialysis due to kidney dysfunction related to alcohol poisoning during this period, adding that 18 patients were discharged from hospital after the treatment.
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Iran 15:19
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Mint provides cooling qualities and it is thus used in many cosmetic products, including: mouthwashes, ointments, toothpaste, bubble baths etc.
Mint is a very simple plant to grow. It has specific root system, called runners since it grows new leaves and you can plant along their roots as they grow. Make sure to plant the mint leaves in separate pots, since they tend to overtake plenty of space once they start to grow.
You can use mint seeds or just replant an existing leaf. The second option works better and faster. To do this, take a water-retaining container that is 12 to 16 inches wide, potting soil, and mint plant. Make sure the mint gets a lot of sunlight and keep watering it so it doesn't dry off. Trim the mint plant to 6 inches in height- this way, it will create bush and won't extend in another direction. Remember to trim the mint's flower buds so the plant stays in place.


To choose a right school and program, student needs to consider carefully about what student wants to accomplish from study abroad. The most basic things to concern are which country to go, whether student has any relatives who are living in that country, designated city, foundation of the institution (affiliated with a college education or private), ideal program, ideal period of study, budget, ideal residence, etc.
Student needs to consider about the reason of improving the language skill, what student wants to accomplish while studying abroad, the location of school and compatibility to be helpful to choose an institution and program.
You need to think about whether the goal is improving your foreign language skills or getting experiences from traveling. Then decide how many hours you would spend for language study based on purpose of study. Also, need to consider about if you're going to prepare an exam or just willing to improve your language skills.
To find an appropriate school, student needs to think about these points.
①	Programs that school is providing; Speaking program, short-term program, intensive program, business program, program to apply for admission to a school of higher grade, examination prep program.
⑤	Faculty's native language and accent, pronunciation difference.
⑥	Students' nationalities and rate.
You need to make sure if it's short-term or long-term program. Also, need to check daily, weekly class hours and the date of programs starts fits in your plan. In case of changing plans, you need to make sure about the ability of changing programs while attending school.
You need to find a school with affordable budget, and if insurance, fee of room and board, fee of textbook are included in the tuition. Also, need to find out the policy of refund and fees in case of cancelation of admission or drop the class.
Since location of the school to apply affects to expenses and living life, student needs to consider these few things.
① Big city or country side.
② Transportation for school and shopping.
⑥ The weather of the area.
Depends on residence place, living expenses can be changed. Therefore student needs to consider few things.
① Institution's availability of accommodation and type of buildings.
② Cost of different type of residence buildings.
③ Ability of host family.
④ Location of on-campus residence building.
⑥ School's support of choosing residence.
Depends on what kind of support school can offer, students can get various opportunities. Therefore it's good to looking for any activities from off-campus and local community. Also looking for ability of international student counseling would be helpful as well.
You might need to get visa to study abroad so you need to find out a type of visa to get, documents for visa interview. Also, it'd be easier to process visa procedure if you find re places which provide assistants for visa.
Make sure if you need medical report and documents of health insurance to register for school and also looking for information about school health insurance.


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Q: What document.body is equal to with equal type and equal value comparsion?

document.getElementById("demo").innerHTML = document.body
//returns [object HTMLBodyElement]

If i compare this way: document.body == "[object HTMLBodyElement]" returns true. But if i compare with === i think the type is not equal. If i try this way: document.body === [HTMLBodyElement] returns false. Or if this way: document.body === HTMLBodyElement also returns false. What is the value of x if i want this to return true: document.body === x

A: Why would you compare object of html to a string ?
document.body is an object.
if you want to compare it you need to compare it either with similar object

let body=document.body
console.log(document.body===body)
console.log(typeof document.body=="object");
console.log(document.body == "[object HTMLBodyElement]"&& typeof document.body==="object")

redpajama_set_name: RedPajamaStackExchange


Exam supports can only be provided to students who inform the Disability Officer of their needs. Students are advised to register with the Disability & Learning Support Service (DLSS) early in the semester, in order to ensure appropriate exam arrangements are put in place. The DLSS liaise with the Examinations Office to organise additional exam supports for end of semester exams.
Students who have continuous assessments, in-class and in-house exams need to give at least one week's notice to the DLSS if they require reasonable accommodations for these exams.
While every effort will be made to provide the necessary facilities, it may not be possible to meet all requirements in every case. Specific exam supports will be discussed individually with each student and form part of the needs assessment. Students must have adequate working knowledge of any facility/technology they request to use in an exam.
Use of PC or laptop and printing facilities in AT centre with AT trainer on duty.
Use of assistive technology such as Dragon Naturally Speaking or Texthelp Read & Write.
Exam paper in an alternative format (e.g. audio format – MP3 player; electronic format – speech to text software or text enlargement software; or enlarged text paper).
Use of coloured examination paper.
Extra time (10 minutes per hour).
Marking guidelines (advice for examiners when marking papers).
Rest periods (provided for candidates who require breaks during the exam, e.g. students who have mobility difficulties; concentration difficulties; or who suffer from chronic pain.


A 1 kg ball is thrown straight up into the air with a velocity of 25 m/sec. There is a constant air resistance force of 0.5 Newtons acting against the ball the entire time. What is the maximum height the ball reaches relative to where the ball was released?
First, we need to find the force acting on the ball during its upward flight. This is done by using Netwon's 2nd Law. Fnet = Fgravity + Fair --> Fnet = m*g + 0.5N --> Fnet = 1kg*(-9.81 m/s^2) + 0.5N --> Fnet = -9.31N We can use the net force acting on the ball to find the net acceleration of the ball using F = m*a. F = m*a --> a = F/m --> a = -9.31 m/s^2 Now we can use the conservation of energy to relate the initial and final potential and kinetic energy of the ball. We need this relationship because the potential energy term includes final height which is what we are solving for. 1/2*m*(v_initial)^2 + m*g*h_initial = 1/2*m*(v_final)^2 + m*g*h_final We know that the initial height is 0 meters and the final velocity is 0 m/s (when the ball is at its peak height). Therefore we can simplify our expression. 1/2*m*(v_initial)^2 + m*g*0 = 1/2*m*0^2 + m*g*h_final --> 1/2*m*(v_initial)^2 = m*g*h_final --> h_final = 1/2*(v_initial^2)/g We know the initial velocity (25 m/s) and we calculated g to be 9.31 m/s^2. So, h_final = 1/2*(25 m/s)^2/(9.31 m/s^2) --> h_final = 33.57 meters Therefore, the maximum height reached relative to where the ball was thrown is 33.57 meters. Check: For the final expression used, let's do a unit analysis to make sure the units are what they are supposed to be. We can ignore all the scalar values here. [m] = [m/s]^2/[m/s^2] --> [m] = [m], we can see that the units match.
In general, which is more likely to undergo larger roof accelerations during an earthquake, a 4 story building with stiff concrete shear walls or a long, 8 story, slender tower built with steel moment frames?
The 4 story building with stiff concrete shear walls should undergo larger roof accelerations. Such a building would have a much larger stiffness than a slender tower built with moment frames due to the building being shorter and the use of concrete shear walls instead of steel moment frames. Although not given, the masses of the two structures would be more comparable than the stiffnesses of the structures. This is because concrete shear walls have much more mass than steel moment frames, and it was also given that the 8 story building is slender whereas, in general, 4 story buildings with shear walls tend to not be slender. So even though there are twice as many stories, the shorter building has a much higher mass per floor than the slender building. Since the natural period of a structure is equal to square root of the mass over the stiffness, we can see that the natural period of the shorter building is going to be smaller than that of the slender building. Buildings with shorter periods undergo larger accelerations and smaller deflections than buildings with longer periods. Therefore, the 4 story building will have larger roof accelerations during an earthquake.
Divide $100 among four people so that the second will have twice as much as the first, the third will have $5 less than the second, and the fourth will receive $20.
We know that the sum of money that the four people receive is $100. From there we can make an algebraic expression relating all four people and setting it equal to the total of $100. We will let the amount the first person receives be "x". The second person will receive twice as much as the first so they will receive "2x" dollars. The third person receives $5 less than the second person, so they will receive "2x-5" dollars. Finally, what the fourth person receives is known to be $20. Now we can assemble the problem knowing that the sum of 4 people is equal to 100. x + 2x + (2x-5) + 20 = 100 --> 5x = 85 --> x = 17 So know we know the first person receives x dollars or $17, the second person receives 2x dollars or $34, the third person receives 2x-5 dollars or $29, and the fourth person receives $20. Check: $17 + $34 + $29 + $20 = $100 --> $100 = $100.


History and literature of jazz music, including African roots, developments and aspects of style periods, and the contemporary state of jazz. Intended for music majors. Listening, analysis and a term paper.
Prerequisites: MUS 211 ; MUS 351A , MUS 351B .