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Autophagy-mediated occludin degradation contributes to blood–brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models
Kyeong-A Kim1 na1,
Donghyun Kim1 na1,
Jeong-Hyeon Kim1,
Young-Jun Shin1,
Eun-Sun Kim1,
Muhammad Akram1,2,
Eun-Hye Kim1,
Arshad Majid3,
Seung-Hoon Baek4 &
Ok-Nam Bae ORCID: orcid.org/0000-0003-0583-21161
The blood–brain barrier (BBB) maintains homeostasis of the brain environment by tightly regulating the entry of substances from systemic circulation. A breach in the BBB results in increased permeability to potentially toxic substances and is an important contributor to amplification of ischemic brain damage. The precise molecular pathways that result in impairment of BBB integrity remain to be elucidated. Autophagy is a degradation pathway that clears damaged or unnecessary proteins from cells. However, excessive autophagy can lead to cellular dysfunction and death under pathological conditions.
In this study, we investigated whether autophagy is involved in BBB disruption in ischemia, using in vitro cells and in vivo rat models. We used brain endothelial bEnd.3 cells and oxygen glucose deprivation (OGD) to simulate ischemia in culture, along with a rat ischemic stroke model to evaluate the role of autophagy in BBB disruption during cerebral ischemia.
OGD 18 h induced cellular dysfunction, and increased permeability with degradation of occludin and activation of autophagy pathways in brain endothelial cells. Immunostaining revealed that occludin degradation is co-localized with ischemic autophagosomes. OGD-induced occludin degradation and permeability changes were significantly decreased by inhibition of autophagy using 3-methyladenine (3-MA). Enhanced autophagic activity and loss of occludin were also observed in brain capillaries isolated from rats with middle cerebral artery occlusion (MCAO). Intravenous administration of 3-MA inhibited these molecular changes in brain capillaries, and recovered the increased permeability as determined using Evans blue.
Our findings provide evidence that autophagy plays an important role in ischemia-induced occludin degradation and loss of BBB integrity.
A well-controlled physiological environment is required for normal brain function [1], and the entry of substances or cells circulating in the blood is strictly restricted by blood–brain barrier (BBB) [2]. Tight junctions (TJs) between brain endothelial cells are major structural and functional components of the BBB and play a crucial role in sealing the extracellular space. Damage to or loss of TJs results in a compromised BBB [3, 4]. TJs are mainly composed of transmembrane proteins such as claudins, occludin, and intracellular accessory proteins such as zonula occludens (ZOs) [5, 6]. Each TJ component has a specific role and provides a vital and important contribution to overall BBB integrity [7,8,9,10,11]. In particular, occludin enhances the stability of the TJ assembly which assists in their function as barriers [12, 13]. Several studies have reported that the alteration or decrease in occludin during cerebral ischemia results in increased BBB permeability [14, 15]. Although matrix metalloproteinase (MMP)-mediated occludin degradation has been reported as a potential pathway for TJ dysfunction, the exact molecular mechanisms underlying ischemic BBB disruption remain to be elucidated.
Autophagy is constantly occurring at a basal level in cells and is the mechanism by which organelles or proteins are degraded and recycled [15]. Autophagy involves the formation of membrane bound intracellular vacuoles called autophagosomes, which engulf the target protein or organelle [16]. These autophagosomes then fuse with intracellular lysosomes which digest target proteins by lysosomal enzymes [17]. While this pathway is a normal maintenance and recycling mechanism, recent data have suggested that autophagy also occurs during pathological conditions in which high levels of autophagy contribute to cellular dysfunction and death [18, 19]. We have recently reported on the excessive activation of autophagic activity in brain tissue during ischemic stroke in rats, and found that ischemia-induced autophagy contributed to neuronal cell death [18]. However, little is known about whether autophagy is involved in BBB disruption during ischemia.
In the present study, we used cultured brain endothelial cells and a rat middle cerebral artery occlusion model to investigate whether autophagy is involved in BBB dysfunction during ischemia. In particular, we investigated whether autophagy plays a role in injury to occludin.
Silicone-coated 4-0 monofilament nylon sutures were purchased from Doccol Co. (Redlands, CA, USA). Primary antibodies against lysosomal-associated membrane protein 1 (LAMP-1), and p62/SQSTM1, and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies against LC3B and β-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). A primary antibody against CD31 was obtained from R&D Systems (Minneapolis, MN, USA). A primary antibody against occludin and the fluorescently labeled secondary antibodies of Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit were purchased from Thermo Fisher Scientific (Rockford, IL, USA). All other chemicals were purchased from Sigma-Aldrich.
The bEnd.3 cell line, immortalized mouse brain endothelial cells, was obtained from American Type Culture Collection (Manassas, VA, USA). bEnd.3 cells were grown in DMEM (Dulbecco's Modified Eagle's Medium with 4500 mg/L d-glucose, 110 mg/L sodium pyruvate, 1.5 g/L sodium bicarbonate, and l-glutamine; Welgene, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS; Mediatech Inc., Manassas, VA, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Welgene, Korea). bEnd.3 cells were maintained in a humidified incubator at 37 °C with 5% CO2 and 95% air. All experiments were carried out when the density of cells was 90–100%.
Oxygen–glucose deprivation (OGD) exposure
bEnd.3 cells were rinsed twice with Earle's Balanced Salt Solution (EBSS; 116 mM NaCl, 5.4 mM KCL, 26.2 mM NaHCO3, 1.8 mM CaCl2, 1 mM NaH2PO4H2O, 0.8 mM MgSO4, and 0.01 mM glycine), and the media were replaced with glucose-free EBSS or EBSS supplemented with 5.5 mM glucose for OGD stimulation or control treatment, respectively. Cell plates were placed in a hypoxia chamber (Billups-Rothenberg Inc., San Diego, CA, USA), and the air was replaced with OGD gas (95% N2 and 5% CO2) by flushing. Cells were exposed to the OGD condition for 6, 12 or 18 h at 37 °C to measure the cell viability. After selecting 18 h OGD as the ischemic insult, cells were exposed to 18 h OGD for the other experiments. Oxygen depletion in the chamber was monitored using BD GasPak™ Dry Anaerobic Indicator Strips (BD, Franklin Lakes, NJ, USA). For autophagic inhibition experiments, bEnd.3 cells were treated with 3-methyladenine (3-MA; Sigma-Aldrich) for 1 h prior to OGD stimulation. The concentration of 3-MA was selected according to the previous reports [16, 20]. Treatment with 3-MA was continued during OGD exposure.
Measurement of cell viability
bEnd.3 cells were seeded on 48- or 96-well plates and incubated to confluence. To determine the effect of OGD on cell viability, cells were exposed to OGD for 18 h and then incubated with 0.5 mg/mL MTT for 2 h [21]. The supernatants were discarded and insoluble formazan was dissolved using dimethyl sulfoxide. The absorbance of the solution was measured at 570 nm using an EnSpire microplate reader (PerkinElmer, Santa Clara, CA, USA).
Determination of in vitro permeability
bEnd.3 cells were seeded on 0.4-µm Pore Polycarbonate Membrane Inserts in 6.5-mm Transwell® plates (Corning, New York, NY, USA) at a density of 2 × 104 cells/well and grown for 6 days to confluence. After OGD exposure, permeability was determined by FITC-dextran or transendothelial electrical resistance (TEER). For the FITC-dextran permeability assay, 20 µg/mL FITC-dextran (molecular weight 70 kDa, Sigma-Aldrich) in phosphate buffered saline (PBS) was added to the upper compartment while the lower compartment was filled with fresh PBS after OGD exposure. After incubation for 30 min in the dark, the transferred amount of FITC-dextran in the lower compartment, which passed through the membrane insert via damaged junctional integrity, was measured by an EnSpire microplate reader at 490-nm excitation and 520-nm emission wavelength. In measurement of TEER, which indicates the integrity of the tight junction, the resistance was measured before and right after OGD exposure using electronic circuit of EVOM2 and STX2 electrode (World Precision Instruments, Sarasota, FL, USA). All TEER data was subtracted by the value from the cell-free inserts of each treatment group.
Immunofluorescence staining
Cells were seeded at a density of 2 × 104 cells/well into a Lab-Tek™ 8-well Chambered Coverglass (Thermo Fisher Scientific, Rochester, NY, USA). When the cells reached confluence, cells were exposed to OGD for 18 h. Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (Promega, Madison, WI, USA) for 10 min, and blocked using 1% BSA (Sigma-Aldrich) in PBS. The cells were incubated with primary antibodies (anti-LC3, anti-occludin or anti-LAMP1 antibodies) diluted in 1% BSA and then further incubated with secondary antibodies (Alexa Fluor 555-conjugated anti-rabbit or Alexa Fluor 488-conjugated anti-mouse antibodies) in 1% BSA. Fluorescence images were acquired and analyzed with a K1-Fluo confocal laser scanning microscope (Nanoscope Systems, Daejeon, South Korea). To quantify the degree of co-localization of proteins, fluorescence images were analyzed with ImageJ plug-in, JACoP (Just Another Co-localization Plugin), and Pearson's correlation coefficient was calculated [22].
Total proteins from bEnd.3 cells or brain capillaries were extracted using RIPA buffer (Thermo Fisher Scientific) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Laemmli buffer containing 5% 2-mercaptoethanol was added to the protein samples, followed by heating at 95 °C for 3 min. Equal amounts of protein samples (30 μg/lane) were resolved using Tris–HCl sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a PVDF membrane (Merck Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk in Tris buffered saline with 0.1% Tween 20 (Bio-Rad, Hercules, CA, USA) (TBS-T) and then incubated with primary antibodies against LC3B, occludin, β-actin, p62/SQSTM1, or CD31 at 4 °C overnight. After washing with TBS-T, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies. Protein bands were visualized by enhanced chemiluminescence using the Clarity Western ECL Substrate (Bio-Rad), SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). Relative levels of protein expression were quantified by NIH ImageJ software and normalized to the corresponding loading controls.
Rat ischemic stroke model
For in vivo study, permanent ischemic stroke was induced in rats by intraluminal middle cerebral artery occlusion (MCAO). Experiments and procedures were performed in accordance with the institutional and international guidelines and regulations. All protocols for animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Hanyang University. Male Sprague–Dawley (SD) rats (230–300 g; Koatech, Gyeonggi, Korea) were housed in a specific-pathogen-free zone (12 h light/dark cycle, 23 °C, and 50% humidity). Rats were randomly divided into the treatment groups. To establish a rat ischemic stroke model, anesthesia was induced by isoflurane inhalation and maintained during the surgical period. Rectal temperature was monitored and maintained at 37 °C during surgery. The measurement of cerebral blood flow (CBF) was performed by laser Doppler (Perimed, North Royalton, OH, USA) before and after MCAO for 24 h. The left common carotid artery (CCA) and the external carotid artery (ECA) were carefully isolated and ligated tightly with a suture. After the ECA branch was cauterized, the internal carotid artery (ICA) was isolated and the pterygopalatine artery was coagulated. To initiate the ischemia, a silicone-coated 4-0 monofilament nylon suture (Doccol Co.) was inserted into the CCA. Approximately 18.0 mm of the suture was advanced through the ICA from the CCA bifurcation to reach the origin of the MCA [18]. The validity of the MCAO ischemic animal models was confirmed by histological analysis of brain sections stained with triphenyltetrazolium chloride (TTC; 2%), and the reduction of CBF in ischemic region. After occlusion, CBF decreased to less than 30% of baseline. For sham-operated rats, the CCA was only exposed to air.
To inhibit autophagic processes in the rats, 0.3 mg/kg of 3-MA in sterile saline was administered through a tail vein injection at 30 min prior to the initiation of MCAO. Rats in the sham-operation and MCAO groups were administered the same amount of sterile saline.
Isolation of rat brain capillaries
Rats were transcardially perfused with saline and the brain was quickly isolated. The whole hemispheres were separated and homogenized with ice-cold physiological buffer containing 4 mM KCl, 147 mM NaCl, 3 mM CaCl2, 1.2 mM MgCl2, and 15 mM HEPES, pH 7.4. The brain homogenates were centrifuged at 3500×g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended using 20% Ficoll (Sigma-Aldrich) in physiological buffer. The suspension was centrifuged at 25,000×g for 10 min at 4 °C. After removing the supernatant, the pellet was resuspended in 15% dextran (Sigma-Aldrich) and the suspension was carefully added to the same amount of 20% dextran. Then, the suspension was centrifuged at 25,000×g for 10 min at 4 °C. The supernatant was completely removed and the pellet was used for further western blot analysis. To validate the isolation of the brain capillaries from the brain homogenates, protein expression level of the molecular markers of endothelial cells and neuronal cells (CD31 and NeuN, respectively) was compared between the brain homogenates and isolated capillaries.
Evans blue in vivo permeability assay
2% Evans blue (Sigma-Aldrich, USA) in sterile saline was injected into tail vein in rats (4 mL/kg) at 20 h after MCAO. After 4 h circulation, rat was transcardially perfused with saline and the brain was quickly removed. The brain hemisphere was homogenized in 2 mL of dimethylformamide (DMF) and incubated at 55 °C for 18 h, and then homogenates were centrifuged at 10,000×g for 20 min. The absorbance of supernatant was measured at 620 nm, using an EnSpire microplate reader (PerkinElmer).
All experimental values were expressed as the mean and standard error (SEM). Statistical significance between groups was determined by the Student's t-test. In all analyses, a p value < 0.05 was considered statistically significant.
Cellular dysfunction and changes in occludin in brain endothelial cells following OGD stimulation
We used the bEnd.3 cell line as in vitro BBB model. To find an appropriate in vitro ischemic insult, cells were exposed to OGD for 6, 12 or 18 h to simulate ischemia and cell viability was determined. Cells exposed to 18 h OGD showed approximately 60% cell viability compared to control cells, suggesting that the ischemia decreased cell survival (Fig. 1a). To evaluate functional alteration by ischemic insult, bEnd.3 cells were exposed to OGD for 18 h and in vitro cellular permeability were measured (Fig. 1b, c). The relative cell permeability was determined by measuring the permeability of FITC-dextran across the cell monolayer and the transendothelial electrical resistance (TEER). The values of ischemic and control groups in both tests were calculated by normalizing to the cell-free membrane value. The permeability of cells exposed to OGD increased approximately sixfold compared to the control group (Fig. 1b) and the TEER value in OGD-exposed cells was also significantly decreased to about half of the control group (Fig. 1c)
Changes on cell viability, permeability, and occludin in brain endothelial cells following exposure to oxygen–glucose deprivation (OGD). The brain endothelial cell line bEnd.3 was exposed to OGD for 6, 12 or 18 h. a Cell viability was examined by the MTT assay. N = 3–4. b Functional changes in cellular permeability were analyzed by an in vitro FITC-dextran permeability assay (N = 5) and c transendothelial electrical resistance (TEER) measurements (N = 3) after 18 h OGD. d The protein level of occludin after 18 h OGD was determined by western blot analysis. Relative occludin levels were normalized to β-actin. N = 4. e 18 h OGD-exposed bEnd.3 cells were stained with an antibody to occludin and visualized by immunofluorescence in confocal microscope. Representative images are shown. Scale bar: 50 μm. Data were expressed as the mean ± SEM and analyzed by the Student's t-test. White arrows show occludin distribution. *p < 0.05 and **p < 0.01 vs. the control group without OGD
Next, we examined whether OGD stimulation in bEnd.3 cells affected the level of occludin, an essential component of the tight junction proteins which is important for maintaining BBB integrity. Western blot results demonstrated a significant decrease in the amount of occludin protein in bEnd.3 cells exposed to OGD compared to controls (Fig. 1d). Intracellular distribution of occludin was examined using immunofluorescence staining. In OGD-exposed cells, occludin exhibited a dotted fluorescence signal only in the cytoplasm in OGD-exposed cells, suggesting that the intracellular distribution of occludin had shifted along with the decreased cellular occludin level upon ischemic insult (Fig. 1e). These results indicate that OGD triggers occludin degradation and redistribution in brain endothelial cells, accompanied by impaired permeability.
Increased autophagy in OGD-exposed brain endothelial cells
To investigate whether autophagy is activated in bEnd.3 cells after OGD, we examined changes in the protein levels of the well-established autophagy indicators, LC3-II and p62/SQSTM1 (Fig. 2a, b). The level of LC3-II correlates with the number of autophagosomes during autophagy. The adapter protein p62/SQSTM1 binds to the LC3 on the autophagosome membrane and is degraded by the fusion of the autophagosomes with lysosomes [23]. Relative to controls, the amount of LC3-II was increased approximately sevenfold, indicating that autophagosome formation was increased in OGD-exposed bEnd.3 cells (Fig. 2a). In contrast, the amount of p62 was reduced by approximately 0.4-fold, demonstrating that autophagic flux was activated in the cells by OGD exposure (Fig. 2b). While LC3 is spread evenly within normal cells, it is observed as punctate aggregates throughout cells when autophagy is activated. To further investigate autophagosome formation and autophagic flux, the intracellular location of LC3 (autophagosomal marker) and LAMP1 (lysosomal marker) were determined by orange and green immunofluorescence staining, respectively. Both of the fluorescence signals were randomly and evenly distributed in control cells. However, in OGD-exposed cells, fluorescent orange puncta were observed along with co-localized green signals, demonstrating the fusion of autophagosomes with lysosomes (Fig. 2c). These data suggest that OGD exposure activates autophagic pathways and autophagic flux in brain endothelial cells.
Activation of autophagy in OGD-exposed brain endothelial cells. The brain endothelial cell line bEnd.3 was exposed to OGD for 18 h. The protein levels of a LC3-II and b p62/SQSTM1 were determined by western blot. Protein levels were normalized to β-actin. N = 3. c Intracellular localization of LC3 and LAMP-1 was examined in OGD-exposed bEnd.3 cells using double immunostaining. Representative images are shown. Scale bar: 50 μm, scale bar in the magnified images: 20 μm. White arrows show the co-localization of LC3 and LAMP1. Pearson's Correlation Coefficient were calculated using JACoP. Data are presented as the mean ± SEM and analyzed by the Student's t-test. *p < 0.05 vs. control
Occludin as a potential target protein of autophagic degradation in OGD-exposed brain endothelial cells
Next, we investigated whether the changes in occludin levels and localization following OGD exposure are due to autophagy. Double immunofluorescence staining for occludin and LC3 was performed to examine the association between OGD-induced autophagy and occludin degradation. In control cells, the fluorescence signals of LC3 and occludin were evenly dispersed, while both proteins were observed as punctate aggregates in cells exposed to OGD (Fig. 3). Furthermore, occludin and LC3-positive puncta were co-localized in cells after OGD exposure (Fig. 3), indicating that autophagy is a possible degradative pathway of occludin in brain endothelial cells during OGD.
Co-localization of LC3 and occludin in OGD-exposed brain endothelial cells. After exposure of bEnd.3 cells to OGD for 18 h, cells were stained for occludin and LC3 by immunofluorescence. Representative images are shown. Scale bar: 50 μm, scale bar in the magnified images: 30 μm. White arrows show the co-localization of occludin and LC3. Pearson's Correlation Coefficient were calculated using JACoP. Data are presented as the mean ± SEM and analyzed by the Student's t-test. **p < 0.01 vs. control
Restoration of OGD-induced cell damage through the inhibition of autophagy by 3-MA in brain endothelial cells
We next investigated whether OGD-induced cellular dysfunction and occludin degradation are restored by inhibiting autophagy by 3-MA, a well-established autophagy inhibitor [24, 25]. Before and during OGD exposure, cells were incubated in the presence or absence of 5 mM of 3-MA. LC3-II levels were increased by approximately sixfold in OGD-exposed cells compared to controls, but this increase was significantly attenuated through the inhibition of autophagy by 3-MA (Fig. 4a), confirming that 3-MA (5 mM) treatment inhibits OGD-triggered autophagy in bEnd.3 cells. Next, we examined whether autophagy inhibition by 3-MA restores OGD-induced cellular dysfunction. Cells exposed to OGD showed a significant decrease in cell viability, which was significantly restored by autophagy inhibition with 3-MA (Fig. 4b). Moreover, autophagy inhibition by 3-MA treatment prevented the OGD-induced degradation of occludin (Fig. 4c), resulting in attenuation of the OGD-impaired cellular permeability (Fig. 4d, e). These results demonstrate that autophagy contributes to change of cell viability, decrease in occludin level, and abnormal hyper-permeability induced by OGD in brain endothelial cells.
Role of ischemic autophagy in OGD-induced dysfunction in brain endothelial cells. bEnd.3 cells were treated with 3-MA for 1 h prior to and during the 18 h OGD exposure. a The protein amount of LC3-II was determined by western blot. β-actin was used as a loading control. N = 7. b Changes in cell viability were analyzed by the MTT assay. N = 3. c The protein levels of occludin were detected by western blot. The bands were normalized by β-actin levels. N = 3. d In vitro FITC-dextran assay and e TEER measurement were conducted to examine cellular permeability. N = 4. Data are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. OGD without 3-MA
Ischemia-induced autophagy and occludin degradation in vivo
We used rats with MCAO for a permanent ischemic stroke model to examine whether autophagy is involved in in vivo BBB dysfunction in cerebral ischemia. To confirm the ischemic insult in rats, the cerebral blood flow (CBF) changes suture was measured before and after intraluminal MCAO (Fig. 5a left), and the brain sections were stained with TTC (Fig. 5a middle). After isolation of brain capillaries from rats following ischemia, the protein expression levels of endothelial marker (CD31) and neuronal marker (Neu N) were compared (Fig. 5a right), to make sure if the separated fraction is mainly composed with endothelial cells. To determine whether autophagy is induced in the BBB during cerebral ischemia, the protein levels of LC3-I and LC3-II in brain capillaries were quantified from both the ischemia-injured (ipsilateral) and non-injured (contralateral) hemispheres. LC3-I conversion to LC3-II was significantly enhanced in ipsilateral brain capillaries isolated from MCAO-operated rats compared to that in contralateral brain capillaries or in both hemispheres from the sham-operated rats (Fig. 5b). MCAO-induced LC3 conversion in ipsilateral brain capillaries was significantly inhibited by intravenous administration of 3-MA to levels similar to that of sham-operated rats (Fig. 5b). These findings indicate that autophagy is activated in the BBB during ischemic stroke and is inhibited by intravenous 3-MA administration (0.3 mg/kg).
Autophagy and occludin degradation in brain capillaries from rats with ischemic stroke by MCAO. a–c Ischemic stroke was induced in rats by middle cerebral artery occlusion (MCAO). At 24 h after MCAO, rats were sacrificed by transcardial perfusion, and brain capillaries were isolated from both the ischemic and non-ischemic hemispheres. 3-MA (0.3 mg/kg) or sterile saline solution was administrated to rats at 30 min prior to MCAO. N = 5–9. a Cerebral blood flow (CBF) changes were monitored before and after MCAO (Right). TTC-stained brain sections confirmed the ischemic brain damage (Middle). The protein levels of endothelial and neuronal marker (CD31 and NeuN, respectively) in the whole brain homogenates and isolated brain capillary were measured (Left). b Autophagic activation in brain capillaries was examined based on the level of LC3-II. The conversion ratio of LC3-II/I was calculated. c The amount of occludin protein in the brain capillaries was detected by western blot. Protein levels were normalized with the corresponding β-actin levels. d 2% Evans blue (4 mL/kg) was intravenously injected at 20 h after MCAO. The rats were transcardially perfused to remove any remaining Evans blue in blood and sacrificed at 24 h after MCAO. The amount of Evans blue was determined in ipsilateral or contralateral hemisphere. N = 5–7. Data are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01 vs. sham-operated rats, #p < 0.05 vs. rats with MCAO without 3-MA treatment
Next, we examined occludin levels in brain capillaries to investigate whether ischemia-induced autophagy contributes to alterations in TJ proteins (Fig. 5c). The ipsilateral-to-contralateral (I/C) ratio of occludin was decreased in MCAO-operated rats to approximately half that of sham-operated rats. 3-MA administration restored the decrease in the occludin I/C ratio in MCAO rats, indicating that autophagy mediates the degradation of occludin during ischemia. These in vivo results using brain capillaries are consistent with our in vitro data. Taken together, our data from both in vitro and in vivo models demonstrate that autophagy is an important pathway responsible for occludin degradation during cerebral ischemia.
To further evaluate the effect of autophagy inhibition by 3-MA on ischemic BBB function in vivo, the integrity of BBB was determined using Evans blue leakage in rats. The extravasation of intravenously administered Evans blue to brain tissue indicates the disruption of BBB. While I/C ratio of Evans blue in MCAO-operated rats was increased following MCAO suggesting that ischemic injury increased BBB permeability, administration of 3-MA significantly reduced the increased I/C ratio of Evans blue (Fig. 5d).
When blood flow to the brain is interrupted during ischemic stroke, which is one of the leading causes of death and disability [26], the depletion of oxygen and nutrients in the ischemic area triggers a cascade of detrimental processes leading to neuronal cell death, neuro-inflammation, and cerebrovascular disruption [15, 27,28,29]. There have been extensive attempts to understand the pathophysiology of ischemic stroke [18, 28, 30,31,32]. Recently, the concept of the neurovascular unit (NVU) has been used to explain the pathophysiology of brain diseases including stroke. In the NVU, the brain is described as an integrated system with diverse cellular components, and harmonized interactions between these cells are crucial for normal brain function [33]. Of note, brain endothelial cells are the main component of the NVU. Compromise of the NVU during ischemia results in BBB disruption and the influx of inflammatory cells and substances from the systemic circulation which amplifies brain injury [34]. The elucidation the molecular mechanisms underlying ischemic BBB damage is pivotal to understand NVU impairment during ischemia.
Previous studies have focused on MMP-mediated degradation of TJs in brain endothelial cells [35, 36]. However, the precise mechanisms and pathways that ultimately lead to BBB dysfunction are largely unknown. The findings of our study show that autophagy, which plays a role in cellular maintenance in normal cells [37], might also play a role in damaging cellular components during pathological processes such as stroke. Our results demonstrated that ischemia-induced autophagy mediated the degradation of occludin and that inhibition of autophagy by 3-MA reversed occludin degradation and attenuated BBB dysfunction. In line with our observation, claudin-5 was degraded by autophagy-lysosome activation in OGD-exposed brain endothelial cells [38], and autophagic activation with BBB damage were observed in p50 knockout mice [39], suggesting that autophagy may contribute to ischemic neuronal and vascular damage. It is also demonstrated that enhanced autophagy during brain ischemia in diabetic mice might be responsible for excessive BBB disruption [40].
Contrary to our findings, Li et al. recently reported the protective potential of autophagy in brain endothelial cells and BBB dysfunction during cerebral ischemia/reperfusion (I/R) injury [41]. In their study, activation of autophagic processes using the autophagy inducers rapamycin and lithium carbonate significantly reversed BBB disruption following I/R injury, while inhibition of autophagy with 3-MA pretreatment intensified I/R-induced BBB damage. Although the specific cellular target of activated autophagy was not identified in their study, the authors concluded that autophagy protected cells against the generation of reactive oxygen species and restored the decreased level in ZO-1 levels. There are differences between the experimental models used in our study and those used by Li et al. While we used 18-h OGD in cells and 24-h MCAO in rats, they used 6-h OGD/4-h reoxygenation in cells and 2-h ischemia/22-h reperfusion in rats. Supporting our findings, a recent study by Liu et al. demonstrated that another BBB component claudin-5 is degraded by 4-h OGD, but not by 2-h OGD/2-h reoxygenation, and suggested that the degradation of claudin-5 is mediated by caveolin-1 and autophagy-lysosome activation in endothelial cells [38]. However, in vivo experiments were not performed in the study by Liu et al. We believe that these previous observations along with our current findings contribute to an integrated understanding of the role of autophagy in ischemic BBB dysfunction.
Still, our current study has limitations in terms of the lack of evidence for ischemic duration-dependent contribution of autophagy in occludin change and BBB integrity during dynamics of ischemic injury. The duration of 18 h for OGD stimulation induced cell death (Fig. 1a) and the decreased cell viability itself can possibly contribute to the increased permeability (Fig. 1b, c), not essentially mediated by autophagy-mediated occludin degradation. Although we could not exclude the cell death itself can contribute to functional alteration, the decrease of occludin protein is not non-specific phenomena accompanied by cell death. Unlike occludin, the total level of ZO-1, another important TJ protein, was not affected by 18 h OGD in our system (data not shown). Moreover, the autophagic inhibition by 3-MA reversed autophagy, occludin degradation, cellular permeability and cell death, suggesting that at least autophagy may be an important mediator of these consecutive phenomena (Fig. 4). The relationship between ischemic insult and the TJ protein expression along with the role of autophagy should be examined according to the ischemic duration also with the presence of reperfusion in the future study, to comprehensively understand the dynamics of ischemic BBB injury.
It is known that moderate autophagy plays a protective role against pathological conditions since it rapidly removes damaged cellular components which contribute to cellular dysfunction, but excessive autophagy itself can target the normal pivotal components leading to cellular damage and aggravation of the pathological processes [42,43,44]. The opposing roles of autophagy have been reported in diverse pathological conditions such as stroke, diabetes, and several neurodegenerative diseases, suggesting that autophagy is a delicate regulator for cell function, ultimately affecting cell survival and death [44,45,46]. Here, we have shown that occludin is a direct target of ischemic autophagy resulting in ischemic BBB dysfunction. However, the determination of the precise role of autophagy in each component of the BBB and its relative contribution will require further investigation. This includes the extent and time course of autophagic activation as well as the substances targeted for degradation in the absence or presence of reperfusion during ischemic stroke. It would be also worthy to elucidate if non-ischemic autophagic activation, such as pharmacologically induced by rapamycin, or pathologically induced by starvation, etc., could be involved in occludin degradation in brain endothelial cells and BBB integrity.
We have also shown that the TJ protein occludin is degraded by cytoplasmic autolysosomes, which is an important insight into the potential role of autophagy in degradation of the transmembrane cytoskeleton, suggesting that the target substrates of autophagic degradation are not limited to intracellular components. There may be intracellular trafficking signaling for the translocation of TJ proteins, which is essential for degradation of these proteins by autolysosomes. Liu et al. suggested that nitric oxide (NO) facilitated caveolin-1-mediated translocation of claudin-5 to autophagosomes in endothelial cells [38]. The molecular mechanisms that mediate occludin modification and translocation to autolysosomes during autophagy-mediated degradation during ischemia remain to be elucidated. Another interesting point is that the tight junction proteins in BBB contains oligomeric protein assembly which is pivotal for maintaining BBB integrity [47]. TJ transmembrane protein homodimers including occludin multimers are essential in TJ complex assembly. In this study, we have measured the alteration of occludin monomer (~ 52 kDa) by ischemic damage in bEND.3 cells, which was reversed by autophagic inhibition. Further studies warrant the autophagic regulation of TJ protein multimers to clarify the role of autophagy in TJ proteins and BBB integrity.
We used levels of LC3-II as a representative marker for autophagic activation. An increase in the LC3-II protein levels, which indicates an increase in autophagosomes does not always indicate normal autophagy flux. In several pathological conditions, the accumulation of autophagic vacuoles and the corresponding increase in LC3-II were often observed due to a failure of fusion between autophagosomes with lysosomes, and these phenomena were explained as abnormal autophagy rather than autophagic activation [48, 49]. We examined the formation of autolysosomes by co-localization of intracellular LAMP1, a lysosomal marker, and LC3 in order to elucidate whether increased LC3-II indicates abnormal autophagy. LC3 and LAMP1 proteins showed a co-localized intracellular distribution under OGD stimuli, indicating that autophagy had occurred rather than LC3-II protein accumulation by abnormal autophagy. In addition, the level of protein p62, an autophagy adapter protein, was decreased by OGD in endothelial cells, further supporting the occurrence of autophagy.
There has been accumulating evidence that autophagy plays important roles in ischemic BBB damage, but the exact contribution of autophagy or the target autophagic cargo still remains unclear [50]. Here, we have demonstrated that ischemia-induced autophagy occurs in brain endothelial cells in vitro and rat brain capillaries in vivo and that this mediates the degradation of occludin, a major component of TJs in the BBB. Furthermore, we showed that ischemic autophagy increased brain endothelial permeability which can be attenuated by inhibiting autophagy. Taken together, our current findings contribute to an integrated understanding of the role of autophagy in ischemic BBB dysfunction.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
OGD:
Oxygen glucose deprivation
3-MA:
3-Methyladenine
MCAO:
Middle cerebral artery occlusion
ZOs:
Zonula occludens
LAMP-1:
Lysosomal-associated membrane protein 1
CBF:
Cerebral blood flow
CCA:
Common carotid artery
ECA:
External carotid artery
ICA:
The internal carotid artery
I/C:
Ipsilateral-to-contralateral
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Do VQ, Park KH, Park JM, Lee MY. Comparative in vitro toxicity study of docetaxel and nanoxel, a docetaxel-loaded micellar formulation using cultured and blood cells. Toxicol Res. 2019;35:201–7.
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Carloni S, Buonocore G, Balduini W. Protective role of autophagy in neonatal hypoxia-ischemia induced brain injury. Neurobiol Dis. 2008;32(3):329–39.
Heckmann BL, Yang X, Zhang X, Liu J. The autophagic inhibitor 3-methyladenine potently stimulates PKA-dependent lipolysis in adipocytes. Br J Pharmacol. 2013;168(1):163–71.
Xiang J, Routhe LJ, Wilkinson DA, Hua Y, Moos T, Xi G, et al. The choroid plexus as a site of damage in hemorrhagic and ischemic stroke and its role in responding to injury. Fluids Barriers CNS. 2017;14(1):8.
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Culmsee C, Zhu C, Landshamer S, Becattini B, Wagner E, Pellecchia M, et al. Apoptosis-inducing factor triggered by poly(ADP-ribose) polymerase and Bid mediates neuronal cell death after oxygen-glucose deprivation and focal cerebral ischemia. J Neurosci. 2005;25(44):10262–72.
Wei N, Xiao L, Xue R, Zhang D, Zhou J, Ren H, et al. MicroRNA-9 mediates the cell apoptosis by targeting Bcl2l11 in ischemic stroke. Mol Neurobiol. 2016;53(10):6809–17.
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Liu J, Weaver J, Jin X, Zhang Y, Xu J, Liu KJ, et al. Nitric oxide interacts with caveolin-1 to facilitate autophagy-lysosome-mediated claudin-5 degradation in oxygen-glucose deprivation-treated endothelial cells. Mol Neurobiol. 2016;53(9):5935–47.
Wei N, Yu SP, Gu XH, Chen DD, Whalin MK, Xu GL, et al. The involvement of autophagy pathway in exaggerated ischemic brain damage in diabetic mice. CNS Neurosci Ther. 2013;19(10):753–63.
Li WL, Yu SP, Chen D, Yu SS, Jiang YJ, Genetta T, et al. The regulatory role of NF-kappaB in autophagy-like cell death after focal cerebral ischemia in mice. Neuroscience. 2013;244:16–30.
Li H, Gao A, Feng D, Wang Y, Zhang L, Cui Y, et al. Evaluation of the protective potential of brain microvascular endothelial cell autophagy on blood–brain barrier integrity during experimental cerebral ischemia-reperfusion injury. Transl Stroke Res. 2014;5(5):618–26.
Dikic I. Proteasomal and autophagic degradation systems. Annu Rev Biochem. 2017;86:193–224.
Liu Y, Shoji-Kawata S, Sumpter RM Jr, Wei Y, Ginet V, Zhang L, et al. Autosis is a Na+, K+-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia. Proc Natl Acad Sci USA. 2013;110(51):20364–71.
Pavel M, Imarisio S, Menzies FM, Jimenez-Sanchez M, Siddiqi FH, Wu X, et al. CCT complex restricts neuropathogenic protein aggregation via autophagy. Nat Commun. 2016;7:13821.
Fang L, Li X, Zhong Y, Yu J, Yu L, Dai H, et al. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats. J Neurochem. 2015;135(2):431–40.
Sharma A, Wu SM. Autophagy–the friendly fire in endothelial cell regeneration. Focus on "Autophagy in endothelial progenitor cells is cytoprotective in hypoxic conditions". Am J Physiol Cell Physiol. 2013;304(7):C614–6.
McCaffrey G, Staatz WD, Quigley CA, Nametz N, Seelbach MJ, Campos CR, et al. Tight junctions contain oligomeric protein assembly critical for maintaining blood–brain barrier integrity in vivo. J Neurochem. 2007;103(6):2540–55.
Hori I, Otomo T, Nakashima M, Miya F, Negishi Y, Shiraishi H, et al. Defects in autophagosome-lysosome fusion underlie Vici syndrome, a neurodevelopmental disorder with multisystem involvement. Sci Rep. 2017;7(1):3552.
Zhang J, Zhang Y, Li J, Xing S, Li C, Li Y, et al. Autophagosomes accumulation is associated with beta-amyloid deposits and secondary damage in the thalamus after focal cortical infarction in hypertensive rats. J Neurochem. 2012;120(4):564–73.
Kim KA, Shin D, Kim JH, Shin YJ, Rajanikant GK, Majid A, et al. Role of autophagy in endothelial damage and blood–brain barrier disruption in ischemic stroke. Stroke. 2018;49(6):1571–9.
This work was supported by a grant from the Ministry of Health and Welfare of Korea (HI14C2284) and grants from the National Research Foundation of Korea supported by the Ministry of Science, ICT & Future Planning (NRF-2017R1C1B3002626).
Kyeong-A Kim and Donghyun Kim authors equally contributed to this work
College of Pharmacy Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Republic of Korea
Kyeong-A Kim, Donghyun Kim, Jeong-Hyeon Kim, Young-Jun Shin, Eun-Sun Kim, Muhammad Akram, Eun-Hye Kim & Ok-Nam Bae
Faculty of Pharmacy, University of Sindh, Jamshoro, Pakistan
Muhammad Akram
Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, England, UK
Arshad Majid
College of Pharmacy and Research Institute of Pharmaceutical Science and Technology (RIPST), Ajou University, Suwon, Republic of Korea
Seung-Hoon Baek
Kyeong-A Kim
Donghyun Kim
Jeong-Hyeon Kim
Young-Jun Shin
Eun-Sun Kim
Eun-Hye Kim
Ok-Nam Bae
Conceptualization, KAK, AM and ONB; Methodology, KAK, DK, YJS and SHB; Formal Analysis, KAK and ESK; Investigation, KAK, DK and JHK; Data Curation, KAK and ONB; Writing—original draft preparation, KAK and ONB; Writing—review & editing, MA, EHK, SHB and AM; Visualization, KAK and DK; Supervision, ONB; Project Administration, ONB; Funding Acquisition, ONB. All authors read and approved the final manuscript.
Correspondence to Ok-Nam Bae.
Experiments and procedures were performed in accordance with the institutional and international guidelines and regulations. All protocols for animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Hanyang University (HYU-2015-0220).
Kim, KA., Kim, D., Kim, JH. et al. Autophagy-mediated occludin degradation contributes to blood–brain barrier disruption during ischemia in bEnd.3 brain endothelial cells and rat ischemic stroke models. Fluids Barriers CNS 17, 21 (2020). https://doi.org/10.1186/s12987-020-00182-8
Oxygen–glucose deprivation (OGD)
Middle cerebral artery occlusion (MCAO)
Blood–brain barrier (BBB)


text:
Reinier I van Henegouwen (~850 - 915)
Reinier III van Henegouwen (? - 971~997)
Reinier I van Leuven

redpajama_set_name: RedPajamaWikipedia


text:
Q: Cypress: parent package runs its cypress/integration test and its dependencies cypress/integration tests Question: Can a parent Cypress npm project import/add/run the Cypress tests of its dependencies Cypress npm projects?
The response from the question is this link works, breaking up a monolith Cypress test package into multiple Cypress test package and using Module API. However, I have had to rethink my question. What I did not address was that there are dependencies (custom commands) between npm cypress packages.
Cypress: Import tests into "./cypress/integration" from multiple npm dependencies?
Example:

*

*npm package cy_test_A has dependency on npm package cy_test_B custom commands,

*npm package cy_test_B has dependency on npm package cy_test_C custom commands.

Each of these packages has cypress integration for specs and src for custom commands:
cy_test_***
├── cypress
│   ├── integration
│   │   └── tests
│   └── support
│       └── specs
├── src
│   └── commands

Can Cypress integration tests be imported from its dependency? When I perform a test run, I want to see a combined tests including both the dependencies' cypress tests + the parent's cypress test.
npm package cy_test_A is dependent on custom commands within npm package cy_test_B, which are used in cy_test_A cypress integration tests. When building npm package cy_test_B, it has its own cypress integration tests before committing to npm repository. I want cy_test_A to run its own cypress integration tests but cypress integration tests of its dependency cy_test_B as well.
├── cypress
│   ├── integration
│   │   └── tests_A <<< Dependent on src/commands **cy_test_B**
│   │   └── tests_B <<< Imported cypress/integration/tests from **cy_test_B**
│   └── support
│       └── specs
├── src
│   └── commands
├── node_module
│   └── **cy_test_B**
│       └── ***
│       └── ***

Ideas? Thank you

A: Not sure if all t's are crossed, but an index file can be used to collate all the custom commands without physically moving them.
/cypress/support/commands-index.js
require('../../cy_test_A/src/commands')
require('../../cy_test_B/src/commands')

Then specify the supportFile to be that index file.
const cypress = require('cypress')

cypress.open({
  reporter: 'junit',
  browser: 'chrome',
  project: '..',
  config: {
    supportFile: './cypress-run-other-project/support/commands-index.js',
    video: true,
  },
  env: {
    login_url: '/login',
    products_url: '/products',
  },
})

But now, whatever else is going on in /cy_test_A/cypress/support/index.js is ignored (for example add-on libraries), so maybe the commands-index.js needs to be
require('../../cy_test_A/cypress/support')
require('../../cy_test_B/cypress/support')

You can check what commands have been loaded with this (in the spec)
it('checks that both custom commands have bee loaded', () => {
  const commands = Cypress.Commands._commands
  expect(commands).to.have.property('checkA')  // command from cy_test_A
  expect(commands).to.have.property('checkB')  // command from cy_test_B
})

redpajama_set_name: RedPajamaStackExchange


There is a new answer that I have come up with recently when clients ask me about the finishes for their renovation or new house. This article provides one more reason you should skip accent tiles altogether.
The shelf life of an interesting backsplash is about five years. I have never been in a house with trendy backsplash tile that is more than five years old and had anyone tell me they still love it.
I'm serious. Take this accent tile below. See the green and yellow-beige in the tiles? Until the day you renovate a bathroom with tiles like this, these are the colours of your walls.
So when people say to me, "Maria, subway tile has got to be dated now right." My answer is, "No." Does it have to be a subway tile? No, but anything simple and neutral might still look boring to you before it's installed but trust me, you will thank me later.
Hey wait, I just thought of something else. Everyone asks me for neutral walls, neutral furniture but then when it comes to tile, something that is permanently stuck to your bathroom or backsplash FOREVER, you suddenly decide it has to be interesting.
Show me a trendy and interesting backsplash tile or accent for your shower surround and I say be prepared to hate it very soon after you have it installed.
There are pretty ways to incorporate accent tiles into your fixed finishes (above) but getting the look right usually takes perhaps more than you'll want to spend. So in general, it's best to stick to the simple plan, tread carefully into the accent tile section, take a look and then run for the hills.
Download my eBook, It's All in the Undertones. If you have a computer, you can download my book!
If you would like to learn to how choose the right colours for your home or for your clients, become a True Colour Expert.
Which Backsplash Tile goes with Granite?
The Hottest Trend in Backsplash Tile; Yay or Nay?
Selecting your Kitchen and Bath Backsplash Tile; Accent Tile or Not?
Colour Me Happy is Three Years Old!
Years ago when I was re-modeling my first home, it was the tile layer who wanted a hand in the design and was pushing the "interesting" look. I kept it simple and neutral and never regretted it.
So true and you explained it perfectly! Well done!
You are so right on! Love, love, love…..have a great day!
Also, I love subway tile but it *will* look dated some day. People used to use subway tile until it fell out of favor. I don't think anything has remained "in" for decades and decades and decades. Not to say you shouldn't use it, but it isn't timeless. No idea what will replace it (or when), but something will one day.
As always, great advice that makes us think. AND I love how you always explain why.
We just built a new house. All of the tile I picked was some version of off white. Tile salesman, and builder both tried to talk me into accent tiles. So glad I didn't let them sway me.
I have lived in a lot of houses that had tile I couldn't stand. I wasn't about to do that in this house. It looks beautiful. There are a lot of other (better) ways to bring temporary color into a room. Replacing a backsplash is very expensive, and a major headache. I've done it in 2 other houses.
I have yet to upgrade my kitchen – so I will definitely steer way away from the accent tiles.
I love reading your blog! I just spent a few hours in a tile store the other day, and you are so right. I've been using the same showroom for years, and I walked by their permanent display, which I was in absolute love with when they first put up (2007) and just shook my head.
I will say for mosaics, when the are the dominant feature, they are EXQUISITE, however few people have the kind of budget that allows that.
sometimes clients will come into the paint store with all their tile for the bathroom, floor, counter, shower and then the dreaded band of accent tile to go around the shower/tub area. after some conversation and looking at wall color, I ask them if they've bought the tile? if they haven't, I suggest leaving out the accent pieces because they are not happening any more, they look at me like 'it took a long time to pick out this tile to match the other tile'.
This is great advice. The manufacturer's make the coolest accent tiles and it's easy to get seduced. This advice will save us money now and later – thank you!
Maria saves the day again!! I absolutely agree and you obviously posted this just in time for some of us – whew.
The skinny stacked/alternating/too many colors tile band in the shower/tub is about the ugliest thing ever! I don't care how many I see. Clients come into the paint store and show me what they are thinking of buying for that accent tile and how the paint can coordinate (of course, it's on their iPhone) I'm with you subway tile in all it's variations of colors, transparencies etc. is the way to go.
I think you just saved me from myself! I've always wondered if so-called "boring" was better, but now I get why I need to trust my intuition. Paint, towels, flowers, art, and interesting perfume bottles all can add the accents I need and are not "cemented" to a surface. Long-live subway tile and simplicity!
…oh, and it's for the same reason I've never understood the interest in using anything other than classic, white bath fixtures. I love Kohler, but I cannot understood who would ever order/install colors of toilets, tubs, and sinks in pink, black, green, beige or… BROWN. Clean white rules in a bathroom. I like to see the dirt on a surface to know when it needs attention. Definitely not trying to hide it.
I have not heard/seen any mention of the pebble tiles/sliced pebbles. Considering it for my shower floor..possibly bathroom floor? I have lived the clean organic look of them ( i am speaking of the ones that are white tones/sandy tones).
I originally liked the actual pebble ones but have heard that the sliced stones are easier to maintain.
I put in sliced stone on my shower floor 9 years ago and I still love it. Non-slip, virtually no maintenance, and easy on the bare feet, unlike actual pebbles. I also put sliced stones on the floor around the toilet and I like that look, too.
What about accent tiles in the niche to match the floor of the shower?


text:
Buccinulum flexicostatum är en snäckart som beskrevs av Dell 1956. Buccinulum flexicostatum ingår i släktet Buccinulum och familjen valthornssnäckor. Inga underarter finns listade i Catalogue of Life.

Källor

Valthornssnäckor
flexicostatum


text:
Secretary General stresses strength of NATO's partnership with Sweden
26 Oct. 2021 -
Last updated: 26 Oct. 2021 17:48
NATO Secretary General Jens Stoltenberg stressed the value of the Alliance's partnership with Sweden during a visit to Stockholm, with ambassadors of the North Atlantic Council, on Tuesday (26 October 2021).
The Secretary General met with His Majesty Carl XVI Gustaf, King of Sweden, and held talks with Prime Minister Stefan Löfven. He praised Sweden's contributions to NATO missions and operations, from the Western Balkans to Iraq. The Secretary General and Prime Minister discussed progress in strengthening cooperation between NATO and Sweden, including with greater information exchanges, joint exercises and political dialogue.
Separately, NATO Deputy Secretary General Mircea Geoană and NATO ambassadors participated in a seminar on 'Security Challenges in the Baltic Sea region', hosted by Foreign Minister Linde, Defence Minister Hultqvist, and Home Affairs Minister Damberg.
The Secretary General, the Deputy Secretary General and the North Atlantic Council also met with the Speaker of the Parliament, Andreas Norlén, and senior representatives of the Swedish Parliament. On Wednesday, the Council will visit the Berga Naval Base, together with Finland and Sweden's defence ministers. They will have an opportunity to see the joint Swedish and Finnish naval exercise, SWENEX-21.
NATO Secretary General and the Prime Minister of Sweden, 26 OCT 2021 26 Oct. 2021
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 NATO Deputy Secretary General Mircea Geoană with the Minister for Defence of Sweden, Peter Hultqvist and the Minister for Foreign Affairs of Sweden, Ann Linde
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 Meeting of the North Atlantic Council with the Minister for Defence of Sweden, Peter Hultqvist, the Minister for Foreign Affairs of Sweden, Ann Linde and the Minister for Home Affairs of Sweden, Mikael Damberg
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 The North Atlantic Council meets with senior members of the Parliament of Sweden. Remarks by Speaker of the Parliament of Sweden, Andreas Norlén
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 The North Atlantic Council meets with senior members of the Parliament of Sweden. NATO Deputy Secretary General Mircea Geoană and NATO Secretary General Jens Stoltenberg
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 The North Atlantic Council meets with senior members of the Parliament of Sweden. Remarks by NATO Secretary General Jens Stoltenberg
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 NATO Secretary General Jens Stoltenberg meets with the Speaker of the Parliament of Sweden, Andreas Norlén
NATO Secretary General and the North Atlantic Council visit Sweden 26 Oct. 2021 NATO Secretary General Jens Stoltenberg meets with the Prime Minister of Sweden, Stefan Lofven
Remarks by NATO Secretary General Jens Stoltenberg and the Prime Minister of the Kingdom of Sweden, Stefan Lofven
26 Oct. 2021 | download mp3
Remarks by NATO Secretary General Jens Stoltenberg and the Prime Minister of the Kingdom of Sweden, Stefan Lofven 26 Oct. 2021
Relations with Sweden
Visit to Finland and Sweden by the North Atlantic Council 20 Oct. 2021
Secretary General: NATO, Finland and Sweden share the same security interests 25 Oct. 2021
Visit to Sweden by the North Atlantic Council

redpajama_set_name: RedPajamaCommonCrawl


text:
Cabinet MagazineKioskBooksActivitiesShopSubscribe Issue 18 / Fictional States
The Kingdom of Elgaland-Vargaland
Schematic diagram of the political structure within Elgaland-Vargaland. The constitution guarantees every citizen's right to move freely within the hierarchy. The acronym KMV stands for "The Materialization of the King in the World."
With effect from the 14th of March 1992, we are annexing and occupying the following territories:
1. All border territories between all countries on earth, and all areas (up to a width of 10 nautical miles) outside all countries' territorial waters. We designate these territories our physical territory. These territories, usually called No Man's Land or Border Crossings, are in constant flux. They change everyday, and in reports from all over the world we notice that new territories appear (e.g., the North and South Korean border), disappear (e.g., the East and West German border in 1989) and reappear (e.g., the Latvian, Estonian and Lithuanian borders). We also observe nations' fishing territories waxing and waning. There are frequent foreign violations at sea; vehicles with cargo, refugees, tourists, political and military maneuvers; animals and fishes walk and swim freely, insects and birds buzz and sing. Theoretically/practically, all past existing areas such as the borders between Texas and the USA, between England and Scotland, or between Sweden and Skåne (at the time belonging to Denmark), are annexed by the Kingdoms of Elgaland-Vargaland.
Thus begins the declaration, signed by artists Leif Elggren and Carl Michael von Hausswolff, that formed the beginning of an experiment in government that has grown in complexity ever since. The declaration goes on to announce the annexation of all other forms of interstitial territories, including the Hypnagogue State (the state between being awake and being asleep), the Escapistic Territory (conscious, mental space travel akin to daydreaming, telepathy, and so on), and the Virtual Room (a borderless digital space).
Currently with more than 600 citizens and a number of Ministries ranging from the crucial (the Ministry of Pleasure and the Ministry of Cartography) to the whimsical (the Ministry of Lamination and the Ministry of Audiology), Elgaland-Vargaland has combined political critique and mordant humor in order to undermine the pieties of traditional states and point slyly toward their array of failures. The manufactured poverty and class differences of first-world states is exposed through gestures of poetic excess, such as the printing of a million thalers (the official currency of Elgaland-Vargaland) that belong equally to all citizens. The kingdom's tenth anniversary in 2002 was marked by a group of citizens boarding a boat from Stockholm to Talinn, Estonia, carrying only their Elgaland-Vargaland passports. After being detained for a day for trying to enter Estonia with false documents, the citizens were placed on a boat and sent back to Sweden. As King Leif I states, "Our purpose was not to provoke the border police. Our purpose was to go home to Elgaland-Vargaland's physical territory: to get rejected at the Estonian border, then go back and be rejected at the Swedish border, back again to the Estonian border, etc., etc., you know, for ever. They did their job and took our passports, so we just had to go back to Sweden."
Elgaland-Vargaland physical territory, Svinesund, on the border between Sweden and Norway.


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Divorce Docs' service is not intended to create an attorney-client relationship, and by using Divorce Docs, no attorney-client relationship will be created. Instead, you are representing yourself in any legal matter you undertake through Divorce Docs legal document preparation service unless you choose to hire an attorney.
Divorce Docs document preparation service includes a review of your answers for completeness, spelling and grammar, as well as internal consistency of names, addresses and the like. At no time do we review your answers for legal sufficiency, draw legal conclusions, provide legal advice or apply the law to the facts of your particular situation.
Divorce Docs and its services are not a substitute for the advice of an attorney. If you need legal advice for your specific problem, or if your specific problem is complex, you should consult a licensed attorney in your area.
Divorce Docs is not responsible for any loss, injury, claim, liability, or damage related to use of their services caused from errors or omissions, as all documents will be printed following your review and approval.


When an individual passes away they may owe Estate Taxes to the State of Maine and/or the Federal Government. The tax rates are constantly changing, and it is important to ensure your estate plan is designed to address any tax issues you may face. The taxes are assessed after an individual has passed away, based on the value of the estate. The concept of an estate includes all probate and non-probate assets, and may include the value of certain trusts. If your estate is over $2,000,000 you may be subject to an estate tax in Maine.
Federally, many of the same rules apply in calculating the size of an estate. You should be aware that gifts made during your life time, may require the filing of a gift tax return. You must file a gift tax return if you gift more than $14,000 (2014) to an individual in a year. This does not necessarily mean that taxes will be owed on the gift. The federal government only taxes decedent estates over $5,340,000 (2014), this is known as the "exemption amount." When an individual files a gift tax return, it simply reduces your federal "exemption." It is important, regardless of the size of an estate, to stay up to date on gift tax returns.
Carlin & Shapiro, P.A. can help you analyze your estate's tax liability, file gift tax returns, and file estate tax returns. We can help you develop an estate plan that can reduce or eliminate any estate tax exposure.


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Thomas Hill Standpipe
The Thomas Hill Standpipe of Bangor, Maine is one of the most visible and recognizable structures in the City. It is one of the only structures visible from nearly any point in the city, being constructed at the highest point of elevation within the city limits. Constructed in 1898 by Major James M. Davis, the Bangor Standpipe was designed for a dual purpose, both as a standpipe and as an observatory of the full extent of the City of Bangor. From the time of its construction, it has been a beloved part of the Bangor skyline, and actively hosts open-house hours for locals and tourists to enjoy the view.
Illustration of the Thomas Hill Standpipe, provided by bangorwater.org
The Thomas Hill Standpipe in winter, provided by bangorwater.org
An illustration of the Thomas Hill Standpipe, provided by bangorwater.org
Undated photograph of the Thomas Hill Standpipe, provided by bangorwater.org
1957 photograph and newspaper clipping of the Standpipe, under repair, provided by bangorwater.org
Photograph of the Standpipe under construction, provided by bangorwater.org
Collage of Photographs of the Standpipe, provided by bangorwater.org
The Thomas Hill Standpipe towers above the City of Bangor, standing 110' tall. The iconic structure is actually two separate constructions: the water reservoir itself, and the enclosing observation tower. Between the outer wall of the reservoir and the outer wall of the observation tower winds a long stairway that leads to the upper floor of the tower, which is a 360-degree visibility observation deck approximately 80 feet in diameter.
The vast majority of the Standpipe was built using hard pine, though the beautiful exterior shingles were crafted from cedar wood, which was locally sourced by two Bangor lumber firms. The Thomas Hill Standpipe was designed in the Shingle style of architecture, which was largely developed and popularized by famous Portland architect John Calvin Stevens. John Calvin Stevens' influence on the architectural history of the State of Maine is unrivaled, and he is largely credited with creating the image of New England residences that fills the imagination of so many travelers from around the world.
The Standpipe was unique even among Shingle style buildings, which were mostly residences, and drew the attention of contemporary commentators. The construction of the Standpipe took approximately six months, during which time contractor Major James M. Davis established a portable sawmill and a Blacksmith shop on site to expedite the construction efforts.
The Thomas Hill Standpipe is a registered National Historic Landmark, an American Water Works Association Landmark, and a historic landmark of the American Society of Civil Engineers. It is beloved by locals and is a common leisure location, the surrounding fields and park being ideal for recreation of various types. During open houses, it is regularly visited by locals and tourists alike, being a favorite spot of both professional and amateur photographers looking to capture the incredible views of the City of Bangor only possible from the Standpipe.
Thomas Hill Standpipe. wikipedia.org. Accessed August 19, 2017. https://en.wikipedia.org/wiki/Thomas_Hill_Standpipe. Meta-source for general information on the Thomas Hill Standpipe
NPS NRHP Official Documentation for the Thomas Hill Standpipe. National Park Service. Accessed August 19, 2017. https://npgallery.nps.gov/pdfhost/docs/NRHP/Text/74000185.pdf. Official documentation for the Thomas Hill Standpipe Nomination onto the NRHP
Bangor, Maine. bangormaine.gov. Accessed August 19, 2017. http://www.bangormaine.gov. Official Website for the City of Bangor Municipal Government
Bangor Water. Bangorwater.org. Accessed August 19, 2017. http://www.bangorwater.org/whats%20new%20home/schedule.htm. Information on the visitation schedules, photographs, and historical information provided by the organization that manages the water tower
Official NPS NRHP documentation for the Thomas Hill Standpipe
Website that manages information about the Standpipe and its open house hours
Thomas Hill Standpipe, Jackson Street
Bangor, Maine 04401
Hours vary yearly, by season. The 2017 hours are listed below, provided by bangorwater.org: Spring Tour May 10, 2017 Wednesday 3 - 6 p.m. Summer Sunset Tour July 26, 2017 Wednesday 4 - 8 p.m. Fall Foliage Tour October 11, 2017 Wedn
Agriculture and Rural History
Urban History
Created by Emilie Sovis on August 19th 2017, 11:57:31 pm.
Last updated by Clio Admin on August 20th 2017, 2:20:22 pm.


Junchuan is a private investment company which has a diverse portfolio of investment across Chinese companies. While investing in innovation, Junchuan believes firmly in the traditional Chinese business culture. We designed a logo and a visual identity (VI).


Set in the heart of the Perche natural regional park in Normandy, just a few miles from Mortagne au Perche, Le Pin stud and the historic Bellême Forest, is a little corner of paradise, the Château de Blavou.
As soon as you drive through the gate, you'll see the magnificent château, which contains architectural elements dating from the 15th and 19th centuries, on an estate that covers more than 10 hectares.
The château has 5 guest rooms, 2 gites and a venue for weddings or other functions.
All the bedrooms are restful and spacious. They are all different but all have the same warm, inviting atmosphere. In fact, each room offers a unique experience of life in a château.
There are 5 rooms including 4 suites. One of them has an additional small second bedroom with twin beds for adults or children. There is also one traditional bedroom.
We can provide table d'hôtes meals and we'll do our very best to ensure that your stay here is unforgettable.
The ideal base from which to explore Normandy or simply to stay on the spot and enjoy some cookery and cake-making classes.
Cookery and cake-making classes for amateurs and professionals are available. Together, we can create a programme that suits your existing skill set. Enjoy a class in creative cuisine or the art of cake making with the château's owner who was formerly a cookery and cake-making tutor at a hotel school.
There is a wide range of sports and activities on offer nearby: walking in the surrounding countryside, horse racing, angling, cycling, golf, horse riding. 4 bicycles available for loan. Gym equipment available in the old stables : treadmill, multi-gym and exercise bike.
Horse lovers: Find out about breeding racehorses and Percheron draught horses (lessons and rides available). Ferme Percheronne de Montaumer, 61560 LA MESNIERE or Elevage d'Egée, St Fulgent des Ormes 61130. Riding lessons available at Double Farm, 3 km from the château.
Golf lovers : Bellême golf course (a 5-minute drive from the château). Open every day of the year, this challenging 18-hole course overlooks hills and forests. Ideal for high-level competitions and recreational players. Beginners will like the 3-hole practice tee on the outskirts of the town.
Gourmet night at the Castle and pastry course: Night for 2 people with breakfast in a suite, 1 gourmet dinner for 2 people and 1 pastry workshop.


This 49 minute presentation is part of the 8 Seconds Heroes DVD set. Witness the awesome confrontation that occurs every time man meets beast in the bucking chutes of the PBR. Each test pits courage versus strength in the Ultimate Showdown. Featured cowboys like all-time greats Ty Murray, Troy Dunn, Michael Gaffney, Justin McBride, and Chris Shivers. Among the bulls are such legends as Dillinger, Hammer, Blueberry Wine, and Three-time Bull of the Year, Little Yellow Jacket. The combination of the best cowboys in the world facing off with the greatest bucking bulls in history makes for a heart-pounding thrill ride you'll want to experience again and again.


This afternoon I will play for a nursing home service. It will be the first one without Earl.
Earl loved hearing me play. He would yell, "Encore!" after every hymn! I couldn't say, "No" to Earl. He would call out a hymn and I would find it in one of the hymnals.
Earl came down with pneumonia and died last week. I wonder if he knew it was our last time together.
Each month I choose a theme for the service. This April is all about showers, rain, and gardens, because "April showers bring May flowers." Our next service will be after Mother's Day.
I'm glad the theme is rain because I'm sure my eyes will be raining as I remember Earl.
In my reading today, I learned rain is a sign of God's faithfulness.
After the rain comes the rainbow. The rainbow is God's promise that He will never flood the earth completely again. We may get wet, but we will never all drown…even in our grief.
Thank you for Earl's life. He made playing at the nursing home such a joy. Thank you for Eden, too. She never cheered, but reminded me to keep practicing. I miss her, too. Thank you for those who are still there. I pray that you will bless them all today.


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August 24, 2016 Awakening / Consciousness / DNA / Health / News / Spirituality / Sustainability
3 Scientific Game Changers that Will Transform Medicine
Kelly Brogan, MD, GreenMedInfo
Think diseases run in the family? Think again.
Have you ever worried about "getting" breast cancer because your mom and aunt both had it? What about Bipolar Disorder (yes, psychiatry capitalizes its diagnoses for extra emphasis!) or Lupus? It has probably felt like you have a ticking time bomb inside you and you just want to know when it's going to go off. When that diagnosis is going to be delivered. Women like Angelina Jolie have engaged willful mutilation in the name of the persistent belief that genes are destiny.
"It runs in my family" no longer means what we once believed it to.
You may not know this yet, but the whole game has changed and we are several decades into the most powerful shift in scientific thinking in the past 300 years. Science, when handled with care, is a process, not a destination.
Science fundamentally reflects our native curiosity, our creative impulse, and our sense of wonder. Along the line, however, it began to take on a different flavor. With men like Newton, Darwin, and Descartes at the microphone, a culture of reductionism, force-based perceptions around the nature of reality, and separation of spirit and matter all predicated the culture of ruthless medicine we are steeped in today. We war against nature, our bodies, and each other. There's good and bad. There's the illusion of the objective. We pray at the altar of facts and data.
I used to think that science meant answers and solutions. And now I don't. Here's why.
Anyone who has plumbed the depths of science, either through study, primary research, or experimentation, will reach the limitations of belief in science as the be all end all. Beyond this glass ceiling is the sense of wonder that is stripped from the soulless realm of modern biology. The amazing thing is that the past several decades have furnished a new kind of science. It's a science of inquiry rather than utility. The conclusions of today's abstracts seem to read, "Isn't this amazing?" rather than "Here is confirmation that this drug is necessary to manage our wayward biology." In essence, it reflects a new story about our role in the holobiont, or the more purposeful station we occupy in the web of nature.
In this new story, we are not the result of several billion years of random mutations and survival of the fittest. Instead, we are adapting, as Lamarck once suggested (and even Darwin acquiesced), purposefully. This purposeful adaptation means that we are interacting with our environment in a co-creative dance with balances and micro/macro feedback loops to keep a certain meta-order intact.
A focus on genes made lifestyle irrelevant.
When I was in training, I had one hour of nutrition education that essentially positioned food as caloric currency. Why would it matter if we were born with the diseases we would ultimately struggle with? In gene-based science, toxicant exposure, rest, nutrition, and relationships are clearly window dressing considerations. (Jonathan Latham's work on identifying the historical influence of the Tobacco industry on the push towards the human genome project and gene-centricism in medical science is amazing.)
With the completion of the human genome, however, we learned that we have fewer protein-coding genes than an earthworm. This means that the genes we thought made us who we are, don't.
We had to go back to the drawing board. Where on earth does our seemingly infinite uniqueness come from? How are diseases manifesting if not genetically?
And a new science was born. It was named epigenetics.
Epigenetics encompasses all that is beyond the genes (it actually means "above") and includes modulators, modifiers, and any influence on the expression of genes and even the possibility that nonhuman genes may play an expressive role in human physiology. It also refers to the the almost 99% of our genome that was once pejoratively called "junk DNA" and now is more mystically referred to as Dark Matter, as Dr. Jeff Bland describes here.
One of the most powerful examples of the relevance of epigenetics is the lore of the "breast cancer gene." Jolie and many other women have succumbed to the hex or the belief that they are cursed by their genes, doomed to develop diseased breasts, ovaries, and uteruses if they just go on living with them in their bodies.
The literature, itself claims that gene mutations such as the "breast cancer gene" or BRCA seems to be doing different things over time. Doesn't that mean, by definition, that we are talking about epigenetics? Because the risk of a gene itself should not change over time. An oft-cited study concludes:
Risks appear to be increasing with time: Breast cancer risk by age 50 among mutation carriers born before 1940 was 24%, but among those born after 1940 it was 67%.
In a timely meta-analysis entitled Worse Breast Cancer Prognosis of BRCA1/BRCA2 Mutation Carriers: What's the Evidence? A Systematic Review with Meta-Analysis, the authors conclude:
Our review shows that, in contrast to currently held beliefs of many oncologists and despite 66 published studies, it is not yet possible to draw evidence-based conclusions about the association between BRCA1 and/or BRCA2 mutation carriership and breast cancer prognosis. We only found sufficient evidence for a 10% worse unadjusted recurrence-free survival for BRCA1 mutation carriers. For all the other outcomes the evidence was judged to be indecisive.
This reflects a more modern understanding that only about 1% of diseases are truly genetic in nature (i.e., due to a congenitally inherited and irreversible gene defect), and that we may very well have misunderstood our interpretation of these genes' functions. The rest is lifestyle. In other words, we create our experience and determine our destiny.
As we embrace our agency in our own bodily experience, we must embrace complexity and take off the blinders of our one gene—one ill—one pill model of thinking. As you open your mind to this shift underfoot, a shift into a more ecological type of medicine, a more collaborative, communal, and connected type of medicine, revel in what the more beautiful science is showing us about our need to let go of what we once believed. It served us, but its time has passed.
Here are the three most powerful game changers in recent literature.
Gamechanger #1: The Microbiome
An extension of one cause-one problem dialectical thinking is the rush to characterize microbes as pathogens, as bad guys. We have done so for as long as we have been seeking to control and dominate nature. Pestilence and contagion have fit neatly into the lighthouse beam of fear that we sweep across our psychic terrain. But even Pasteur, arguably the father of germ theory, on his deathbed, was reported to say: "I was wrong. The microbe (germ) is nothing. The terrain (milieu) is everything."
What is the terrain though? The implication is that we are, in fact, an ecosystem, positioned within an ecosystem. There is no linearity, so that when we pull one thread of the spider web, the entire thing moves.
With the dawning of our awareness that microbes including fungi, bacteria, viruses, and others, live in and around us, we ceased to be human in the ways we had come to believe. We are not, in fact, humans trying to uphold and protect our humanity in a sea of invaders. We are a meta-organism, a holobiont interfacing with a greater whole, like a fractal repetition of a pattern.
With the emergence of science seeking to study the microbiome, primarily in the gut, we have learned that these bacteria have the capacity to perform some of our most vital human functions, and some that seem to be eerily custodial. Take, for example, the fact that there are bacteria in our guts that have evolved to detoxify the chemicals in an average dry cleaner. How could they possibly have foreseen the need for this function? Then there's the digestion, barrier protection, immune signaling, hormone balancing, and brain controlling functions of the microbiome.
Through this lens, our tonsils and appendix are no longer vestigial, and the special role of a woman's physiology is recentered as the conduit through which this microbiome passes from mother to fetus, and even through the inheritance of our primary cellular energy production sites—the mitochondria—themselves, ancient bacteria that were assimilated into the human organism over a billion years ago.
Because we know this now, we cannot war against nature any longer. Vaccines, antibiotics, hand sanitizers and bleach are necessarily brought under scrutiny. We will never win this battle because it's not a battle meant to be won. Nature will continue to remind us of this with the emergence of superbugs, pharmaceutical damage, and disturbed immunity.
The term dysbiosis, used by holistic and integrative practitioners to refer to gut imbalance, of course, actually means "wrong living." No doubt our struggles today stem from a lack of connection to the natural world. It is, in this way, poetic justice that we would only be able to heal our guts, right our relationship to the microbial world through food—nature's gift, bounty, and language.
Gamechanger #2: Exosomes
The nail in the coffin of protein-coding-gene-determined-health is a group of tiny bubble-like blobs that influence gene expression. Amazingly similar to viruses in nature, structure, and possibly even function, exosomes are created and received by our bodily cells in order to direct, determine, and react to states of being.
40-100nm in diameter, exosomes typically carry something called micro RNA or miRNA which are key regulators of gene expression, naturally impacted by environmental factors, from toxicants, nutrition, and lifestyle patterns.
In fact, seminal research has demonstrated that stable miRNAs are transferred from plants including rice and ginger into mammalian physiology where they then serve to regulate gene expression. Once again, food is reframed as so much more than calories and nutrients … it is information.
Researchers are suggesting that infant assessments of miRNA patterns may help to identify fetal brain injury from toxic exposures including mercury, aluminum, and medications at birth, so that healing protocols can be prioritized and initiated. Powerfully, however, in order to understand the "signatures" of different disease states, we would have to study them in their natural unfoldment, without pharmaceutical interventions, before we could ascertain what is evidence of illness and what is evidence of medication effect. The authors state clearly:
"In addition, one must control for the influence of psychotropic drugs on miRNA expression since several studies showed that lithium, haloperidol, or valproate induced changes of the miRNA profiles in brain."
This, of course, is why preventive medicine involves interfacing with the environment in a language that the body understands based on millions of years of co-evolution.
Gamechanger #3: Belief
When I was in training, the placebo effect was framed as a nuisance factor that needed to be controlled for. I now understand that the placebo effect doesn't mean that you were fooled or tricked. It doesn't mean you're making it up or that you're gullible. It means that a complex physiologic cascade of events was kicked off by your experience of taking a pill with the promise of relief.
It is rapidly being characterized as the most important driver of outcomes in everything from psychiatry to surgery.
My research on the placebo effect has helped me to understand that psychiatric medications, and specifically antidepressants, are more risk than benefit, and that I cannot achieve meaningful outcomes with patients who do not fundamentally believe that their bodies can heal.
How could it be that belief is this important?
Dr. Candace Pert, the pioneering researcher credited with the discovery of the opiate receptor, has debunked the Cartesian dualism that for hundreds of years has put the mind out of the realm of the body. She also challenged the notion that the mind is something that merely relates to the body. The mind, per her conclusions, is the body.
In fact, she used the language of the bodymind and elucidated how neuropeptides travel around the body encoding emotion in different tissues and organs. The shape of these peptides, or their conformation, further transmitted information to recipient cells. All of the sudden, informational transfer takes on another dimension—vibration.
Another pioneer of the role of belief in cellular physiology, Dr. Bruce Lipton was several decades ahead of his time in establishing the role of environment and belief in the body's physiology. His work has decentralized the nucleus (where the genes are housed) and focused on the cell membrane as an information-processing interface with the environment.
Taken together, this research all seems to suggest that we are a product of our energetic experience in the context of a greater whole. Even physics is mirroring this with the emergence of theories like Rupert Sheldrake's morphic resonance, which suggests that collective experiences set a potential template for individual experiences in a type of memory transfer that has little to do with genes.
We are not random genes driving a purposeless life until we die.
It appears that human experience is not as it has been previously framed and echoed by philosopher Alan Watts—flesh robots on a dead rock in the middle of nowhere. When the human experience is reduced in this way, its most essential elements are denied—beauty, spirit, meaning.
In fact, human experience is the universe manifested at one point. It is the emergent properties of many, infinitely complex and interfacing systems. It is fundamentally sacred, larger than our will, and gently demanding of our humility.
This is the teleologic perspective—one in which the purpose rather than the cause is the explanation of phenomena. I reflect on this when I consider how adaptable our bodies are—in real time, within hours—and how we have not adapted fully to the lifestyles we have felt entitled to over the past 100 years. We live from our egos with total disregard for nature, each other, and our own bodily vessels. And we are sicker than we have ever been, from cradle to grave. It seems that we are not "meant" to adapt to this way of living and we are reminded of this through the signals of illness. We are called back to the Continuum through our anxiety, depression, and illness. We are reminded that we haven't figured it out, that science is not and cannot be used merely as a tool for control, and that consciousness as an emergent property of our physiologic web is a gift.
The fairest thing we can experience is the mysterious. It is the fundamental emotion which stands at the cradle of true art and true science … the mystery of the eternity of life, and the inkling of the marvelous structure of reality, together with the single-hearted endeavor to comprehend a portion, be it ever so tiny, of the reason that manifests itself in nature.—Einstein
Copyright © [4/1/16] GreenMedInfo LLC. This work is reproduced and distributed with the permission of GreenMedInfo LLC. Want to learn more from GreenMedInfo? Sign up for the newsletter here http://www.greenmedinfo.com/greenmed/newsletter.
Dr. Kelly Brogan is boarded in Psychiatry/Psychosomatic Medicine/Reproductive Psychiatry and Integrative Holistic Medicine and practices Functional Medicine, a root-cause approach to illness as a manifestation of multiple-interrelated systems. After studying Cognitive Neuroscience at MIT and receiving her MD from Cornell University, she completed her residency and fellowship at Bellevue/NYU. She is one of the nation's only physicians with perinatal psychiatric training who takes a holistic evidence-based approach in the care of patients with a focus on environmental medicine and nutrition. She is also a mom of two and an active supporter of women's birth experience. She is the Medical Director for Fearless Parent and an advisory board member for GreenMedInfo.com. Visit her website.
Alan Wattsallopathic medicinealternative medicineAngelina JolieantibioticsantidepressantsanxietyauthorawarenessbiochemistrybiologyBRCAbreast cancer genebreast removalBruce LiptonCandace Pertcommunityconscious evolutionconsciousnesscontinuumcuringdark matterDarwindepressionDescartesDNAdoctorecosystemepigeneticsgene-centrismgenesgeneticsgermshealinghealthholobiontHuman Genome Projecthuman potentialJeff BlandJonathan Lathamjunk DNAKelly BroganLamarcklifestylematerialismmeaningmedicinemilieumindmitochondriamorphic resonanceNewtonnutritionPasteurphysicsplacebo effectpsychiatrypurposereductionismRupert Sheldrakesciencespiritualityteleologyterraintobacco industryvaccineswellnesswriter
Are We Living Inside a Virtual Simulation?
Relating the Ancient Vedic to Ascension, Dimensions, Extraterrestrials & More!


Shannon is wrapping up her Masters Degree in Early Childhood Montessori Education at Xavier University. She learned of Montessori as a philosophy of how to interact with children, and it fit perfectly with her interests, skills and background. Shannon obtained a Bachelor of Psychology & Social Work degree from Miami University. She has worked in the fields of education, wellness and social justice. She enjoys deepening her understanding and commitment to fostering the growth of children each year. Shannon and her husband Sam have two children, ages 10 and 7, and live in Terrace Park. She enjoys being outdoors, growing plants, cooking with her family, and practicing yoga and creative dance.


Comprehensive list of Santa Monica, California based musical instrument shops offering new & used guitars, pianos, percussion, string, woodwind instruments for sale.
Guitar Salon International GSI is the world's biggest retailer of fine classical and flamenco guitars. GSI is comprised of experienced players with a love for the guitar our video and audio demonstrations prove this. We have over twenty five years knowledge in dealing in fine instruments.
McCabe's is a musical instrument store in Santa Monica, California, opened in 1958. We specialize in acoustic and folk instruments: guitars, banjos, mandolins, dulcimers both hammered and plucked, fiddles, psaltries, bouzoukis, sitars, ouds, ethnic percussion. All significant brands usually in stock.
Every piano firm boasts that they are the largest, the finest, number one at something, and the most recommended. How does that relate to the consumers requirements? As consumers we care about three things: getting the correct piano for our requirements, paying the correct price, and feeling confident the firm will provide exceptional client service.
Scroll down and have a look at my performing, teaching, directing, and composing credits. But 1st, some info about me and my guitar. I recall my facination with the invisible world of sounds when I was a child. Then I found a guitar in the attic of our house in New Jersey, and at age seven I knew I'd found my instrument. It was a favorite uncle's abandoned 1950 Gibson Kalamazoo with f-holes.


Here's a brief look at some of the top news stories around the world for Oct. 23, 2015.
Described as the most powerful hurricane recorded in the Western Hemisphere, "Patricia" is bearing down on the southwest Pacific coast of Mexico. The county's government declared a state of emergency Friday as coastal areas braced themselves for the storm, which has reached a speed of 200 miles per hour.
The U.S. House of Representatives on Friday passed a measure that would repeal parts of President Obama's Affordable Care Act, including funding for the embattled Planned Parenthood. However, the legislation is now headed to a Democrat-conrolled Senate, and the president would likely veto it if passed.
Pentagon officials have identified U.S. Army Master Sgt. Joshua L. Wheeler as the serviceman who died in the rescue of 70 Iraqi prisoners from Islamic State forces this week. He was a resident of Roland, Oklahoma. Wheeler is the first American to die in the U.S. engagement against ISIS.


Portland, Oregon resident Caroline Mason is an accomplished vocalist and multi-instrumentalist, who takes standard rock tropes and turns them in something experimental, whilst still training an accessible and friendly feel. Check out the video for her current single Floating and you'll instantly know what we mean.
The fusion of post rock with jazz style syncopation and the fresh vocals are a real head turner. Evoking comparisons with the likes of Vessels and late 60s San Francisco rock genre-busting tunes as exemplified by Jefferson Airplane circa Surrealistic Pillow, it's a very pleasing musical curiosity. You could imagine jazz heads and Deadheads getting in to this in equal measure, minds wandering in imagined worlds as the track falls on them like stardust.
Floating is out now and can be streamed on Spotify, Bandcamp and Soundcloud. You can catch Caroline Mason on her socials including Facebook and Instagram.