text:
Oxidative stress is thought to be critical for the pathogenesis of hepatic steatosis and its progress to non-alcoholic steatohepatitis. Berberine (BBR) can improve hepatic steatosis. In this study, we investigated the role of BBR in ameliorating oxidative stress. Lipid accumulation was measured in the livers of C57BL/6 mice fed a high fat diet (HFD) or a normal diet for 8 weeks, then either received BBR or vehicle for the study duration. Nrf2 distribution was detected in male Sprague-Dawley rats' livers in vivo and in Huh7 cells in vitro. ROS generation and mitochondrial complex expression was measured in Huh7 cells. HepG2 cells were employed for the measurement of oxygen consumption rates. Our results showed that BBR reduced triglyceride accumulation in the liver of HFD-fed mice. The activation and nuclear distribution of Nrf2 was decreased in the hepatocytes of rats that received BBR treatment, while on a HFD. BBR also markedly reduced Nox2-dependent cytoplasmic ROS production and mitochondrial ROS production, which was mediated by the down-regulation of Complex I and III expression. In conclusion, BBR has a great potential to reduce the effects of oxidative stress, which likely contributes to its protective effect in inhibiting the progression of hepatic steatosis to steatohepatitis.
Non-alcoholic fatty liver disease (NAFLD) is prevalent in both developed and developing countries. NAFLD is a major health problem that can progress to non-alcoholic steatohepatitis (NASH), fibrosis, and even hepatic cancer1. NAFLD is also closely associated with the development of insulin resistance and type 2 diabetes, as well as cardiovascular disease1. Lifestyle intervention, including diet and physical exercise, can effectively improve fatty liver. However, lifestyle intervention is often insufficient, as it requires significant changes that must be maintained over the patient's lifetime; long term compliance is not always achievable, and lifestyle changes do not necessarily reverse the damage that has occurred. Therefore, multiple agents, such as insulin sensitizers (e.g., pioglitazone) and anti-oxidants (e.g., vitamin E) have been used in patients and have been associated with improvement in the histologic findings with regard to hepatic steatosis2, 3. Thus, this group of drugs represents a promising avenue for the development of new NAFLD treatments. Berberine (BBR) is an isoquinoline-type alkaloid extracted from a Chinese herb, Huanglian4. Our previous study showed that BBR, when administered along with lifestyle intervention, reduced hepatic fat content in patients by 53% during a 16-week intervention5. Lipid accumulation is the first hit in a "two-hit" theory, which best describes the progression of NAFLD. The second hit involves cellular lipid overload, which provides excess substrates for oxidation, contributing to reactive oxygen species (ROS) production and oxidative stress. However, the underlying mechanism(s) by which BBR improves NAFLD endpoints in patients is not well understood. Therefore, we investigated the ability of BBR to reduce oxidative stress in vitro in human cells and in vivo in rodent models of NAFLD.
BBR prevents high fat diet–induced NAFLD
First, we investigated the role of BBR in the improvement of NAFLD in male C57BL/6 mice, which developed NAFLD after an 8-week high fat diet, as shown by the development of hepatic steatosis (Fig. 1A). A substantial reduction in hepatic lipid deposition was observed in HFD-fed mice that were orally administered BBR versus those administered vehicle control (Fig. 1A). These results were further supported by a change in liver triglyceride (TG) content (Fig. 1B), and there was also a slight decrease in the plasma TG levels of the HFD + BBR group compared to the HFD group (Fig. 1C). Consistent with these in vivo results, administration of 10 µM BBR also significantly reduced the lipid accumulation in FFA-stimulated Huh7 cells, which further confirmed the lipid-lowering effect of BBR (Fig. 1D).
BBR inhibits the activation of the transcription factor Nrf2
Nrf2 is a transcription factor that translocates to the nucleus after activation by ROS; thus, the nuclear translocation of Nrf2 reflects the cellular oxidative status. Here, we detected the distribution of Nrf2 in hepatocytes in rat liver and found that Nrf2 was primarily located in the cytoplasm of hepatocytes from the normal diet group, while it was predominantly located in the nucleus of HFD rat livers (Fig. 2A). However, the Nrf2 content in the nucleus was reduced in the livers of HFD rats that were treated with BBR versus untreated animals (Fig. 2A), suggesting that Nrf2 activation was reduced by BBR.
In Huh7 cells, we also observed an increase in the nuclear translocation of Nrf2 with FFA treatment. However, Nrf2 nuclear translocation was markedly reduced by BBR treatment in Huh7 cells (Fig. 2B,C), which was confirmed by western blot (Fig. 2D). Immunofluorescence staining and western blot analyses were performed in Huh7 cells in vitro, and the results supported our findings in rat livers in vivo. Once Nrf2 is activated, it promotes the expression of several genes, including HO-1, which exert anti-oxidant effects by removing toxic haeme groups6. We also found that mRNA levels of HO-1 were significantly up-regulated by FFA treatment but suppressed by BBR (Fig. 3A). HO-1 expression was increased by FFAs in a dose-dependent manner but was completely suppressed by BBR (Fig. 3B). As shown in Fig. 3B, two bands were visualized, in which the upper band is the endogenous HO-1 protein as it was induced by FFA and repressed by administration with BBR. Notably the lower band is a non-specific band, which was visualized with long exposure time.
BBR attenuates oxidative stress in hepatocytes
As oxidative stress is the stimulus that activates Nrf2 and nuclear translocation, we next investigated the effect of BBR on cellular oxidative stress. BBR treatment attenuated cellular oxidative stress in Huh7 cells stimulated by FFAs. In the presence of FFAs, cellular ROS production was dramatically increased. As shown in Fig. 4A, in cells pretreated with 10 μM BBR for 24 h, FFA-induced ROS production was completely abolished (Fig. 4A). H2O2-treated cells also displayed an increase in ROS generation and were used as positive controls (Fig. 4B). We also found that BBR treatment for 4 h significantly reduced the cellular ROS production induced by 400 µM H2O2 (Fig. 4B). As a member of the superoxide dismutase family, Mn-SOD is known to limit the detrimental effects of ROS7. Mn-SOD activity assays revealed that total SOD enzymatic activity was similar in cells with treated with FFA alone or FFA plus BBR. However, Mn-SOD enzymatic activity and Mn-SOD/total SOD activity were significantly increased by FFA, which was attenuated by BBR treatment (Fig. 4C).
Taken together, these findings indicate that FFA induces cellular ROS production, and Mn-SOD compensates for cellular oxidative stress. ROS generation can be reduced by BBR treatment.
BBR reduces ROS generation in a Nox2-dependent manner
The NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase, or Nox) family is dedicated to producing superoxide8. Nox2 is the prototypical member of this family and major isoform of NADPH oxidase8. Our results revealed that FFA increased the mRNA and protein expression of Nox2, which could be blocked by BBR (Fig. 5A,B). To investigate whether FFA-induced ROS generation is Nox2-dependent, we measured ROS production in Huh7 cells after FFA treatment in cells with Nox2 siRNA knockdown (Fig. 5C). Surprisingly, FFA did not significantly induce cellular ROS production in cells with Nox2 knockdown compared to normal control (NC) cells (Fig. 5D). Furthermore, and in support of a causal role of Nox2 in cellular ROS production, Nox2 knockdown also prevented FFA-induced increases in the protein levels of the anti-oxidative transcription factor Nrf2 and its downstream target HO-1 in whole cell lysates from Huh7 cells (Fig. 5E). BBR is sufficient to inhibit expression level of HO-1 although the efficacy of appears to be variable, as there was a complete suppression of HO-1 by BBR in Fig. 3B, while only a minimal reduction of HO-1 with BBR treatment under NC in Fig. 5E. The different inhibitory effects of berberine on HO-1 are likely due to many reasons, such as the condition of cultured hepatocytes as well as the protein stability of HO-1.
BBR reduces mitochondrial-derived ROS
Our data indicated that BBR reduced FFA-induced ROS in a Nox2-dependent manner. However, the major source of intracellular ROS is the mitochondria9. Therefore, we measured mitochondrial ROS in Huh7 cells using MitoSOX Red, a mitochondrial superoxide indicator10. We found that the MitoSOX Red fluorescence (red) levels in the cytoplasm were increased after FFA treatment (Fig. 6). BBR markedly reduced the cellular MitoSOX Red fluorescence in cells treated with FFA (Fig. 6). These data suggest that BBR has the capacity to reduce mitochondrial-derived ROS production induced by FFA treatment.
The effects of BBR on mitochondrial ETC complex and respiration
The mitochondrial electron transport chain (ETC) plays a major role in the generation of ROS. A fraction of electrons being transported by the ETC leak from complex I and III to form superoxide anion radicals. The excess deposition of lipids within hepatocytes provides an excess fuel supply for the mitochondrial oxidation reaction, which leads to copious ROS production11, resulting in oxidative stress. We then studied the effects of BBR on the expression of the ETC complex in Huh7 cells. We found that the expression of complexes I and III was slightly increased by FFA compared with controls. BBR treatment reduced the expression levels of complex I, II, and III, especially complex I and II (Fig. 7A). Thus, ROS produced by the mitochondrial ETC was reduced. However, complex V (ATP synthase) expression was markedly increased by BBR treatment (Fig. 7A), indicating that BBR did not inhibit the ATP synthetic functions of the mitochondria. Using the Seahorse XF-24 analyser, we next determined the mitochondrial oxygen consumption rate (OCR) of HepG2 cells in real time (Fig. 7B). Our results showed a decreased basal respiration capacity in response to BBR treatment (Fig. 7C).
In the present study, we confirmed that BBR has the capacity to reduce lipid accumulation in the livers of mice fed a HFD. Interestingly, the results in this study also demonstrated that the nuclear distribution of the anti-oxidant transcription factor Nrf2, as well as the transcription of its downstream target gene, HO-1, was reduced by BBR treatment. To investigate the reason for the difference in Nrf2 activation, we measured ROS generation and found that cytoplasmic ROS production induced by FFA was attenuated by BBR in a Nox2-dependent manner. Meanwhile, mitochondrial-derived ROS production was abrogated by BBR treatment. This ultimately resulted in a lower state of oxidative stress in the cells, leading to reduced Nrf2 nuclear translocation in response to BBR treatment.
Regulation of Nrf2 activation by berberine
Either reducing ROS generation or promoting ROS clearance can protect cells against the damage from oxidative stress. Nrf2 is an emerging regulator of anti-oxidant stress, as it regulates a number of genes responsible for anti-oxidative stress12. Activation of the Nrf2 pathway can promote ROS clearance. Most proteins encoded by downstream target genes of Nrf2 could ameliorate oxidative stress. For example, Nrf2 induces the catabolism of superoxide and peroxides through its target gene products—SOD, peroxiredoxin and glutathione peroxidase12. Nrf2 is usually located in the cytoplasm bound to Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)13. When challenged with ROS, Nrf2 dissociates from Keap1 and trans-locates into the nucleus, which triggers the expression of its target genes14. As an anti-oxidant transcription factor, Nrf2 distribution in nucleus indicates the activity of Nrf2.
The results of our study showed that BBR ameliorates oxidative stress by reducing ROS formation. In the presence of FFA, cellular ROS production was dramatically increased, but in cells pretreated with BBR, FFA-induced ROS production was completely abolished. Nrf2 is activated by oxidative stress, demonstrating its anti-oxidant potential, and protects cells from oxidative damage15. In response to a low oxidative stress status, Nrf2 continues binding to Keap1 in the cytoplasm and does not trans-locate into the nucleus. In vivo and in vitro approaches were applied in our study to indicate that Nrf2 entry into the nucleus is reduced after BBR administration compared to that in HFD-induced liver or FFA-stimulated hepatocytes. First, IHC results showed that Nrf2 was located mainly in the cytoplasm of BBR-treated rat livers, while it was distributed predominantly in the nucleus in the HFD only group. Second, an increase of Nrf2 distribution in the nucleus was also observed in FFA-stimulated Huh7 cells but not in cells treated with BBR. Furthermore, after separating the cytoplasmic and nuclear fractions, the western blotting results showed that Nrf2 expression in the nucleus was higher with FFA induction, which did not occur in cells co-treated with FFA and BBR. Our results indicated that the inhibition of ROS formation by BBR contributed to decreased activation of Nrf2, resulting in its reduced nuclear distribution. BBR showed its anti-oxidative stress potential by inhibiting ROS generation; thus, the demand for anti-oxidative stress activity was reduced. These results are consistent with the observations in the study of Sengupta D et al.16. They found that carcinogen-increased ROS generation in hepatocytes was accompanied by a 3.3-fold higher Nrf2 expression in the nucleus compared with the control. BBR reduced ROS content induced by diethylnitrosamine and CCl4 exposure, but nuclear Nrf2 expression remained 2.03-fold higher than that in the control16. Sengupta D et al. indicated that berberine maintained activation of the Nrf2/HO-1 pathway during the treatment period, showing the anti-oxidative effect of berberine. However, we found that the nuclear expression of Nrf2 in the berberine-treated group was 2.03-fold higher than that in the control, but it was 1.27 (=3.3-2.03)-fold less than that in the carcinogen-exposed group in their study. Their results support our findings, in our data, FFA promoted ROS accumulation, and the increased Nrf2 in the nucleus in response to FFA indicated up-regulated activity of the anti-oxidant transcription factor Nrf2. As ROS accumulation mediated maintenance of the Nrf2/HO-1 pathway16, berberine reduced FFA-induced ROS accumulation, which resulted in a lower activation status of Nrf2, as well as a decreased Nrf2 in the nucleus compared with that in FFA-stimulated cells.
However, in Yuan's study, Nrf2 mRNA was decreased in HFD-fed rat liver, but this change was significantly reversed by berberine treatment17. In our in vitro study, we found that FFA increased the protein level of Nrf2, which was reversed by berberine treatment. Sengupta D et al. also revealed a decrease in Nrf2 protein expression level in the berberine treatment group compared with that in the carcinogen-exposed group16, which was consistent with our findings. The discrepancy might be due to the different model we used. In Yuan's study, they conducted the real-time PCR with liver samples from long-term HFD-fed rats, with or without BBR treatment. In our study, we performed western blotting with cell lysate samples from Huh7 cells treated with FFA and/or berberine for a short time. The different models and different induction time may contribute to the inconsistency between their results and ours. However, the mechanism underlying this discrepancy is still not clear and needs to be explored.
Due to reduced Nrf2 nuclear translocation, the transcription of the Nrf2 downstream gene HO-1 was also decreased after BBR treatment, which was confirmed by our results. According to a previous study, increased HO-1 protein levels, due to Nrf2 activation, promoted insulin resistance and inflammation18, which suggests that increased activation of anti-oxidative stress effects may also damage the cell. Cellular homeostasis of the anti-oxidative stress system is necessary. BBR reduces the activation of the Nrf2/HO-1 pathway by inhibiting ROS formation, which contributes to a balanced homeostasis of the anti-oxidative stress system. Therefore, to some degree, the inhibition of Nrf2 activation and HO-1 expression by BBR may mediate its role in improving metabolic disease.
Inhibition of ROS generation by berberine
The association of ROS production and fatty liver disease has been well studied19, 20. In obese animals or humans, lipolysis in adipose tissues is substantially increased due to insulin resistance. Excessive lipid uptake by the liver provides surplus substrates for oxidation, contributing to heightened ROS formation21. Accumulating amounts of ROS further damage the liver, causing lipid peroxidation and cytokine release22, which promotes hepatocyte inflammation and apoptosis.
Cytoplasmic ROS production is influenced by the NADPH oxidase family, which couples electrons transferred across the cell membrane to oxygen, thus generating ROS8. NADPH oxidase 2 (Nox2) is a potent source of cytoplasmic ROS generation23. Nox2 expression was correlated with the severity of hepatic steatosis in patients with NAFLD24, 25. We revealed that ROS generated in response to FFA is mediated by Nox2, while BBR could reverse FFA-induced Nox2 expression. Our results demonstrated that Nox2 may be essential for BBR to reduce ROS formation.
The mitochondrion is the powerhouse of the cell, which supplies ATP and energy. However, the mitochondrion is also a major source of ROS, while energy substrates are interchangeable, including glucose and triglyceride1. Using MitoSOX Red staining, which is a mitochondrial superoxide indicator, we found that BBR reduces the mitochondrial-derived ROS production induced by FFA. In the process of electron transport within the mitochondrial inner membrane, ROS are generated as a by-product of respiration via electron leakage in complex I and III26. A previous study by Fernyhough et al. demonstrated that the decreased protein expression of members of the respiratory chain occurs in the axons of diabetic rats and is correlated with decreased intra-mitochondrial superoxide generation derived from the mitochondrial respiratory chain27. Our results showed that BBR attenuated mitochondrial ROS and down-regulated complex I and III expression. At the same time, the expression of complex V was increased after BBR treatment, indicating that mitochondrial ATP production is not reduced by BBR treatment, since complex V serves as an ATP synthase28. Furthermore, using Seahorse XF-24 assays, we found that the basal oxygen consumption rate in HepG2 cells treated by BBR was significantly reduced. These results suggest that BBR inhibits ROS generation from the mitochondrion by down-regulating the expression of major leakage site—complex I—and reducing the material—oxygen—for ROS formation.
Anti-oxidants have been applied for therapy of liver diseases. For example, vitamin E attenuates oxidative damage by scavenging reactive oxygen species29. Carotenoids display anti-oxidant effects by quenching singlet oxygen30. In our study, BBR served as a promising anti-oxidant by inhibiting the generation of ROS.
In conclusion, BBR improves the state of oxidative stress in hepatocytes, which possibly contributes to reducing the risk of NASH progression or even cirrhosis.
Materials and Methods
Seven- to eight-week-old male C57BL/6 mice were purchased from the Shanghai Laboratory Animal Company in China. Five- to six-week-old male Sprague Dawley (SD) rats were purchased from the Animal Development Centre, Chinese Academy of Science, Shanghai.
C57BL/6 mice were fed a normal chow diet or high fat diet (HFD; D12492, Research Diet, USA) for 8 weeks, after which time they were given BBR (300 mg/kg/d, Sigma, St. Louis, USA) or vehicle orally for another 8 weeks. Rats were fed a normal diet (ND, 62.3% carbohydrate, 12.5% fat and 24.3% protein in total calories) or HFD (32.6% carbohydrate, 51.0% fat and 16.4% protein) for 8 weeks and were then gavaged with BBR (200 mg/kg/d) or vehicle for the next 16 weeks. Animals were housed at constant room temperature under a 12 h light/dark cycle with ad libitum access to water and chow. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University. All experiments were performed in accordance with the relevant guidelines and regulations.
Measurement of liver triglycerides
Mouse liver tissues were homogenized with PBS, followed by extraction with a mixture of chloroform and methanol at a ratio of 2:1. After drying under nitrogen gas, the lipid was then resuspended in ddH2O. Liver triglyceride (TG) levels were determined using a triglyceride kit (Shensuoyoufu, China) and normalized to total protein concentration.
Histological and immunohistochemistry analysis
Rat liver tissue was fixed in phosphate-buffered 10% formalin, processed for histological assessment, and stained with haematoxylin-eosin using standard procedures. Immunohistochemistry was performed on paraffin-embedded sections of rat liver tissue. After deparaffinization, rehydratation, and antigen retrieval, sections were further incubated with primary anti-Nrf2 antibody (Abcam, Cambridge). After incubation with secondary antibody and staining with diaminobenzidine (DAB), the sections were counterstained with haematoxylin, dehydrated, and sealed with a coverslip. Images were captured with an Olympus microscope (Olympus Corporation, Japan).
Cell culture
The human hepatoma cell lines Huh7 and HepG2 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) foetal bovine serum and 1% (v/v) penicillin/streptomycin in an atmosphere of 5% CO2 and 37 °C. For free fatty acid (FFA) treatment, a stock solution of 10 mM oleic acid (OA) or 10 mM palmitic acid (PA) was prepared in 5% (w/v) fatty acid free-bovine serum albumin (BSA). Huh7 and HepG2 cells were grown to 70% confluence and exposed to a FFA mixture containing OA and PA at a molar ratio of 2:1 in the absence or presence of BBR. BBR (Sigma, USA) was dissolved in dimethylsulfoxide (DMSO) (Sigma, USA) to a stock concentration of 20 mM and was filtered and stored at −20 °C prior to use.
Nile Red staining
Huh7 cells affixed to cover slips were rinsed quickly with 0.01 M PBS and fixed with 4% paraformaldehyde for 15 min. After washing twice with 0.01 M PBS, cells were incubated with 0.75 µg/ml Nile Red for 15 min at room temperature. Cells were then counterstained with 100 ng/ml DAPI at room temperature for 5 min. After washing, the cover slips were then mounted.
Immunofluorescence staining
Cells were rinsed quickly with 0.01 M PBS and blocked in 10% goat serum at room temperature for 1 hour. Then, cells were incubated overnight at 4 °C with primary antibody (rabbit anti-Nrf2). After rinsing with 0.01 M PBS three times, the cells were incubated with Alexa Fluor 488 donkey anti-rabbit IgG overnight at 4 °C. Cells were then counterstained with DAPI for 1 min. After washing, the stained cells were examined by an Olympus microscope (Olympus Corporation, Japan). The average percentages of cells with Nrf2-positive nuclei in the immunofluorescence staining results were quantified from a randomly selected pool of 3–6 fields under each condition. The data are presented as "% of cells with Nrf2 in nucleus".
ROS detection
ROS generation by Huh7 cells was detected with the fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA; Beyotime Institute of Biotechnology, China). After stimulation with FFA or BBR, cells were rinsed with PBS and incubated with 10 µM DCFH-DA for 20 min at 37 °C. This fluorescent probe diffuses into cells and is metabolized into a highly fluorescent compound, 2,7-dichlorofluorescein (DCF), by intracellular H2O2 or OH in the presence of peroxidase. ROS production was measured by fluorescence intensity using a fluorescence microplate reader (PerkinElmer, CA, USA), which was normalized to total protein content (in each well).
Mn-SOD activity measurement
FFA- and BBR-treated Huh7 cells were rinsed and lysed in cold PBS. After centrifugation at 4 °C, supernatants were collected and diluted with SOD measurement buffer and were then used for a SOD enzyme activity assay following the manufacturer's instructions (Beyotime Institute of Biotechnology, China). Briefly, in a 96-well plate, 160 μl WST-8/enzyme working solution and 20 μl reaction working solution were mixed with 20 μl cell lysates (acquired from the above procedures) or SOD measurement buffer; the latter was defined as Blank 1. One hundred sixty μl WST-8/enzyme working solution combined with 40 μl SOD measurement buffer was used as Blank 2. The above mixtures were incubated at 37 °C for 30 min, and absorbance values at 450 nm were determined. Total SOD enzymatic activity was calculated by the following equations and normalized to the protein content of each sample: (1) inhibition ratio = (absorbance Blank1-absorbance Sample)/(absorbance Blank1-absorbance Blank2) * 100% and (2) SOD enzymatic activity units = inhibition ratio/(1-inhibition ratio). To determine Mn-SOD activity, the Cu/Zn-SOD inhibitor A and sample were mixed at a ratio of 1:24 and then incubated at 37 °C for 1 h, yielding Mix 1. Cu/Zn-SOD inhibitor B was diluted by a factor of 40 and combined with Mix 1 at a ratio of 1:25 and then incubated at 37 °C for another 15 min. To determine the Mn-SOD enzymatic activity, we followed the same procedure as the one for Total SOD enzymatic activity detection.
siRNA transfection
Huh7 cells were seeded into 6-well plates, and the media were replaced with Opti-MEM (Gibco, USA) once the cells had grown to 70% confluence. Two hundred µl Opti-MEM was mixed with either 2.5 µl siRNA (20 µM) or 5 µl Lipofectamine 2000 (Invitrogen, USA) to make Mixture 1 and Mixture 2, which were incubated at room temperature for 5 min. The above two mixtures were then mixed together gently and incubated at room temperature for another 20–25 min. Cell media was replaced by the above mixture in 2 ml/well cell media. The transfection duration was 48 h. siRNA sequences were as follows, Nox2 sense 5′-CUGUGAUAAGCAGGAGUUUdTdT, Nox2 antisense 5′-AAACUCCUGCUUAUCACAGdTdT, normal control (NC) sense 5′-UUCUCCGAACGUGUCACGUdTdT, and normal control (NC) antisense 5′-ACGUGACACGUUCGGAGAAdTdT.
Real-time polymerase chain reaction
Total RNA from cultured cells was isolated and converted to complementary DNA. The messenger RNA (mRNA) expression levels of target genes were determined by real-time polymerase chain reaction (real-time PCR) using an Applied Biosystems 7500 (Applied Biosystems, USA) with SYBR Premix EX Taq (TaKaRa, Japan). The sequences of primers used are shown in Supplemental Table 1. β-actin gene expression was used as an internal control.
Western blotting
Huh7 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. After being boiled, proteins were loaded in SDS-PAGE and blotted onto a polyvinylidenedifluoride membrane and were detected using specific antibodies against Nrf2 (Abcam, Cambridge), HO-1 (Proteintech), Nox2 (Proteintech), total OXPHOS (oxidative phosphorylation) antibody cocktail (Abcam, Cambridge), GAPDH (Sigma-Aldrich, USA), Histone H3 (Cell Signaling Technology, USA) and β-actin (Santa Cruz, USA). Membranes were then washed in TBST and incubated with goat anti-rabbit secondary antibody (Santa Cruz, USA) for 1 h before being visualized.
Intramitochondrial ROS measurement
For detection of intramitochondrial ROS, mainly superoxide, 5 µM MitoSOX Red reagent (Molecular Probes Invitrogen, USA) working solution was applied to cover cells adhering to cover slips and then incubated for 10 minutes at 37 °C while protected from light. After washing with warm buffer, cells were then counterstained with 1 µg/ml Hoechst 33342 (Thermo Scientific, USA) for an additional 10 min at 37 °C. The stained cells were examined by an Olympus microscope (Olympus Corporation, Japan).
Respiration measurement
Hepatocytes were seeded into XF24 cell culture plates. After drug treatment (FFA or BBR), the media were removed, and cells were washed twice with warm Seahorse assay medium (Agilent Technologies, USA) and incubated for 1 h at 37 °C without CO2. The compounds oligomycin, FCCP, antimycin, and rotenone of the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, USA) were diluted in Seahorse assay medium and were preloaded into reagent delivery ports of A, B, and C of the O2 sensor cartridge. Oxygen consumption rate (OCR) measurements were then carried out according to the Seahorse assay protocol using a Seahorse XF-24 analyser (Agilent technologies, USA). The basal level of OCR was measured 4 times, and the maximal respiratory capacity was determined by OCR after FCCP injection, minus the one before oligomycin injection.
Statistical analysis
All data are expressed as the mean ± SEM. Statistical significance was assessed using a two-tailed Student t test. Differences between treatments at P < 0.05 were defined as statistically significant.
- 1.
Gusdon, A. M., Song, K. X. & Qu, S. Nonalcoholic Fatty liver disease: pathogenesis and therapeutics from a mitochondria-centric perspective. Oxid Med Cell Longev 2014, 637027, doi:https://doi.org/10.1155/2014/637027 (2014).
- 2.
Belfort, R. et al. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. The New England journal of medicine 355, 2297–2307, doi:https://doi.org/10.1056/NEJMoa060326 (2006).
- 3.
Violi, F. & Cangemi, R. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. The New England journal of medicine 363, 1185–1186; author reply 1186 (2010).
- 4.
Kong, W. et al. Berberine is a novel cholesterol-lowering drug working through a unique mechanism distinct from statins. Nat Med 10, 1344–1351, doi:https://doi.org/10.1038/nm1135 (2004).
- 5.
Yan, H. M. et al. Efficacy of Berberine in Patients with Non-Alcoholic Fatty Liver Disease. PloS one 10, e0134172, doi:https://doi.org/10.1371/journal.pone.0134172 (2015).
- 6.
Loboda, A., Damulewicz, M., Pyza, E., Jozkowicz, A. & Dulak, J. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cellular and molecular life sciences: CMLS 73, 3221–3247, doi:https://doi.org/10.1007/s00018-016-2223-0 (2016).
- 7.
Koch, O. R. et al. Oxidative stress and antioxidant defenses in ethanol-induced cell injury. Mol Aspects Med 25, 191–198, doi:https://doi.org/10.1016/j.mam.2004.02.019 (2004).
- 8.
Bedard, K. & Krause, K. H. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological reviews 87, 245–313, doi:https://doi.org/10.1152/physrev.00044.2005 (2007).
- 9.
Szeto, H. H. Mitochondria-targeted peptide antioxidants: novel neuroprotective agents. The AAPS journal 8, E521–531, doi:https://doi.org/10.1208/aapsj080362 (2006).
- 10.
Yue, W. Y., Clark, J. J., Fernando, A., Domann, F. & Hansen, M. R. Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides. Neuro Oncol 13, 961–973, doi:https://doi.org/10.1093/neuonc/nor068 (2011).
- 11.
Li, Z., Berk, M., McIntyre, T. M., Gores, G. J. & Feldstein, A. E. The lysosomal-mitochondrial axis in free fatty acid-induced hepatic lipotoxicity. Hepatology 47, 1495–1503, doi:https://doi.org/10.1002/hep.22183 (2008).
- 12.
Ma, Q. Role of nrf2 in oxidative stress and toxicity. Annu Rev Pharmacol Toxicol 53, 401–426, doi:https://doi.org/10.1146/annurev-pharmtox-011112-140320 (2013).
- 13.
Zhang, M. et al. Emerging roles of Nrf2 and phase II antioxidant enzymes in neuroprotection. Prog Neurobiol 100, 30–47, doi:https://doi.org/10.1016/j.pneurobio.2012.09.003 (2013).
- 14.
Yeligar, S. M., Machida, K. & Kalra, V. K. Ethanol-induced HO-1 and NQO1 are differentially regulated by HIF-1alpha and Nrf2 to attenuate inflammatory cytokine expression. J Biol Chem 285, 35359–35373, doi:https://doi.org/10.1074/jbc.M110.138636 (2010).
- 15.
Kang, K. W., Lee, S. J. & Kim, S. G. Molecular mechanism of nrf2 activation by oxidative stress. Antioxidants & redox signaling 7, 1664–1673, doi:https://doi.org/10.1089/ars.2005.7.1664 (2005).
- 16.
Sengupta, D., Chowdhury, K. D., Sarkar, A., Paul, S. & Sadhukhan, G. C. Berberine and S allyl cysteine mediated amelioration of DEN+CCl4 induced hepatocarcinoma. Biochimica et biophysica acta 1840, 219–244, doi:https://doi.org/10.1016/j.bbagen.2013.08.020 (2014).
- 17.
Yuan, X. et al. Berberine ameliorates nonalcoholic fatty liver disease by a global modulation of hepatic mRNA and lncRNA expression profiles. J Transl Med 13, 24, doi:https://doi.org/10.1186/s12967-015-0383-6 (2015).
- 18.
Jais, A. et al. Heme oxygenase-1 drives metaflammation and insulin resistance in mouse and man. Cell 158, 25–40, doi:https://doi.org/10.1016/j.cell.2014.04.043 (2014).
- 19.
Satapati, S. et al. Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver. J Clin Invest 125, 4447–4462, doi:https://doi.org/10.1172/JCI82204 (2015).
- 20.
Browning, J. D. & Horton, J. D. Molecular mediators of hepatic steatosis and liver injury. J Clin Invest 114, 147–152, doi:https://doi.org/10.1172/JCI22422 (2004).
- 21.
Sunny, N. E., Parks, E. J., Browning, J. D. & Burgess, S. C. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab 14, 804–810, doi:https://doi.org/10.1016/j.cmet.2011.11.004 (2011).
- 22.
Serviddio, G. et al. Mitochondrial involvement in non-alcoholic steatohepatitis. Mol Aspects Med 29, 22–35, doi:https://doi.org/10.1016/j.mam.2007.09.014 (2008).
- 23.
Rolas, L. et al. Inhibition of mammalian target of rapamycin aggravates the respiratory burst defect of neutrophils from decompensated patients with cirrhosis. Hepatology 57, 1163–1171, doi:https://doi.org/10.1002/hep.26109 (2013).
- 24.
Del Ben, M. et al. NOX2-generated oxidative stress is associated with severity of ultrasound liver steatosis in patients with non-alcoholic fatty liver disease. BMC gastroenterology 14, 81, doi:https://doi.org/10.1186/1471-230x-14-81 (2014).
- 25.
Jiang, J. X. et al. Advanced glycation endproducts induce fibrogenic activity in nonalcoholic steatohepatitis by modulating TNF-alpha-converting enzyme activity in mice. Hepatology 58, 1339–1348, doi:https://doi.org/10.1002/hep.26491 (2013).
- 26.
Lenaz, G. The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology. IUBMB life 52, 159–164, doi:https://doi.org/10.1080/15216540152845957 (2001).
- 27.
Akude, E. et al. Diminished superoxide generation is associated with respiratory chain dysfunction and changes in the mitochondrial proteome of sensory neurons from diabetic rats. Diabetes 60, 288–297, doi:https://doi.org/10.2337/db10-0818 (2011).
- 28.
Jonckheere, A. I., Smeitink, J. A. & Rodenburg, R. J. Mitochondrial ATP synthase: architecture, function and pathology. Journal of inherited metabolic disease 35, 211–225, doi:https://doi.org/10.1007/s10545-011-9382-9 (2012).
- 29.
Zingg, J. M. Vitamin E: an overview of major research directions. Mol Aspects Med 28, 400–422, doi:https://doi.org/10.1016/j.mam.2007.05.004 (2007).
- 30.
Fiedor, J. & Burda, K. Potential role of carotenoids as antioxidants in human health and disease. Nutrients 6, 466–488, doi:https://doi.org/10.3390/nu6020466 (2014).
The authors thank Xiaoying Li (Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China) for critical reading of this manuscript and Pu Xia (Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai, China) for helpful advice. We also thank Qi Gong and Zhimin Hu (Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China) for their help with animal studies. This work was supported by grants from National Natural Science Foundation of China (81270933 and 81570718 to X.G.). Major State Basic Research Development Program of China (2012CB524906 to X.G.; http://www.973.gov. cn/Default_3.aspx).
Ethics declarations
Competing Interests
The authors declare that they have no competing interests.
Additional information
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Sun, Y., Yuan, X., Zhang, F. et al. Berberine ameliorates fatty acid-induced oxidative stress in human hepatoma cells. Sci Rep 7, 11340 (2017). https://doi.org/10.1038/s41598-017-11860-3
Further reading
Berberine Suppressed Tumor Growth through Regulating Fatty Acid Metabolism and Triggering Cell Apoptosis via Targeting FABPs
Evidence-Based Complementary and Alternative Medicine (2020)
Promising plant-derived secondary metabolites for treatment of acne vulgaris: a mechanistic review
Archives of Dermatological Research (2020)
Therapeutic and biological activities of berberine: The involvement of Nrf2 signaling pathway
Journal of Cellular Biochemistry (2020)
Improvement of Endothelial Dysfunction of Berberine in Atherosclerotic Mice and Mechanism Exploring through TMT-Based Proteomics
Oxidative Medicine and Cellular Longevity (2020)
Herbal drug discovery for the treatment of nonalcoholic fatty liver disease
Acta Pharmaceutica Sinica B (2020)
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


text:
NETDEVOPS: Understanding Continuous Integration (CI) Pipeline with Gitea and Drone with a Small Network Configuration Change
- July 3, 2018
- Posted by: Muhammad Afaq Khan, CCIE #9070
- Category: CICD Full Stack Networking NetDevOps
In this article, I will describe the process of continuous integration (CI) and how you can commit your first NetDevOps config-as-code build using Gitea. It is also my hope that this inspires readers to really commit to transforming their career to network automation engineer or NetDevOps given the enormous potential.
Gitea is a self-hosted Git service which means you can install and run it on your workstation as a SaaS service without any external dependency whatsoever. It is similar to GitHub, Bitbucket, and Gitlab. As discussed earlier, I prefer Gitea over Gogs for many good reasons. I am using Gitea within a Docker container that's running inside my VirtualBox VM which is running on my Ubuntu Linux distro. You can run Git on just about any platform where Go would provide you with an independent binary which obviously includes Linux, Windows and macOS/OSX.
Installing Gitea inside a docker container is pretty straightforward. After starting the Docker setup via
docker-compose Gitea should be available using your browser to finalize the installation. You can connect to it using http://localhost:3000 and follow the installation wizard. If you are coming from Gogs, you can refer to details here. You can do a
ls docker container to get the details of your installation. It should look similar to what I have below on my machine.
I have seen folks using Gogs, so if you are curious, you can refer to Gitea versus Gogs detailed feature by feature comparison here. At the end of the day, both will perform the needed job of config version control and CI integration.
When you log in, you will come to the following Gitea dashboard.
As you can notice, Gitea has a dashboard and separate links for issues, pull requests and explore. Dashboard features a summary of your config/code integration activity. In my case, I have a dev_branch where I make all my changes, then my Drone server facilitates the testing using dev_branch, followed by testing using a realistic topology and if everything finishes successfully, then merge dev-branch into master before pushing that out to production network.
Let's now go through an example of the integration part of that NetDevOps pipeline. The handoff between Gitea (CI) and Drone (CI/CD) happens when I commit my config change into dev-branch. As we previously discussed, since Gitea doesn't support OAUTH2 for a seamless handover between CI and CD stages per se, I have to manually enter my user password each time a commit is permitted to proceed from Gitea to Drone.
CI benefits NetDevOps the most because it allows for config-as-code produced to be automatically tested and continuously "integrated" with other NetDevOps members' code, and with the existing config or codebase. The NetDevOps team benefits from receiving continuous and immediate feedback regarding their config changes and integration errors. As a member addresses these errors, automated testing tools such as Drone (along with GNS3 or Cisco VIRL) in this stage will report if the errors were successfully fixed and when the config change is accepted. This continuous feedback loop dramatically increases a NetDevOps member's productivity for making frequent network changes while decreasing the likelihood of a network downtime.
Unless a network change is all about house keeping, Continuous Delivery benefits the business itself who are supposed to benefit from the resulting network changes because as soon as config is successfully accepted in the CI stage and a can be tested, it is deployed to given networking devices. Optionally, business groups can verify that changes meet their expectations and provide feedback to NetDevOps team who then address the feedback in this stage.
Anyhow, now let's go back to making our first network change. In my example, I will be modifying read and write SNMP community strings to showcase my NetDevOps Gitea/Drone CICD pipeline.
I manually edited my all.yml file that contains the global variables used in my Ansible playbooks. I edited the file and modified the SNMP community string values to NewSecureReadJuly4th and 4NewSecureWriteJuly4th. This was done prior to issuing git add and commit.
git add adds your modified files to the queue to be committed later whereas
git commit commits the files that have been added and creates a new revision with a log. If you do not add any files, git will not commit anything. You can combine both actions with
git commit -a.
Now, the final step before Gitea CI will trigger the Drone CI, involves pushing the config change using git push.
git push pushes your changes to the remote repository. You can see what happens once I issue git push in the screenshot below.
Using above, I pushed my changes into dev_branch and it triggered the CI stage in both Gitea as well as Drone which is my build server. You can see 4 objects, since my change calls for removal of two SNMP CLIs which sets the READ and WRITE community strings and then replace them with newer values.
Let's now look at what happened on Gitea server after my git push.
You can see "-" and "+" denoting what was removed versus added from the configuration, it also shows +2/-2 to describe the same information. 10.0.10.1 is my switch IP address where these changes are to be tested and then committed.
Let's now look at what happened on Drone server after my git push.
If you follow the commit message, "FullStackNetworker #1", you can see the there was an initial build->test cycle for dev_branch and after successful completion, it was repeated for master branch before finally deploying it to my switches.
Let's zoom into each dev_branch first on Drone server. On the right side it shows the steps taken around the Gitea/Drone handoff as part of "Clone" stage within my CICD pipeline configuration. My pipeline consists of build start stage, followed by integration testing and merge change into master and then notify NetDevOps team via Slack.
You can see that for 4 lines of change, the entire pipeline for dev_branch and master branch takes only about 3 minutes or so. If your config changes are larger, or your pipeline is longer or more complex, it will take longer. Drone server simply works as an orchestrator and follows the flow of control within your pipeline file. We will discuss the pipeline in more details in the future posts.
Below is a screenshot from my Slack channel which shows that NetDevOps team was notified for dev_branch and master build completion and pipeline execution success.
If you're still following, you can easily witness that how network programmability is NOT DevOps, there is a lot more to it than just throwing the Ansible and Python together. NetDevOps is about DevOps toolchain, methods and culture coming to networking.
Now, last but not least, I invite you to come and join me on Full Stack Networker Blog and using our social channels. I will soon be discussing NetDevOps delivery pipeline and network automation in more detail.
How useful was this post?
Author:Muhammad Afaq Khan, CCIE #9070
1 Comment
Hello. I have checked your fullstacknetworker.com and
i see you've got some duplicate content so probably it is the reason that you don't rank high in google.
But you can fix this issue fast. There is a tool that generates content like human, just search in google:
miftolo's tools
Leave a Reply Cancel reply
How useful was this post?

subset: commoncrawl


text:
Coach profile
Noah's Ark Golf CentrePerth, PH1 1QF 01738 440678
niall mcGill
Scottish born and bred, Niall has been a golf professional for over 25 years. He is passionate about growing the game of golf and having fun is at the heart of all his sessions. He believes golf is no longer just for old men in blazers, but the perfect sport for everyone to enjoy. Niall's love.golf sessions are engaging and entertaining and you'll have no other choice than to have fun!


text:
Three of the Democrats' aspiring 2020 contenders, specifically Kamala Harris, Cory Booker and Sherrod Brown, haven't done all that well in the early polling. Even that last CNN poll of Iowa Democrats left all three of them in single digits, well behind two old white guys. So how do they start gaining some traction? Simple. If people don't love you yet, just offer to deliver some bags of cash to their doorstep. (NBC News)
As Democrats gear up to challenge President Donald Trump in 2020, several possible contenders are testing out a simple pitch: Would you like some more money?
A suite of big ticket bills by Sens. Cory Booker, D-N.J., Sherrod Brown, D-Ohio, and Kamala Harris, D-Calif., would directly transfer upwards of trillions of dollars in cash directly to Americans. While low-income workers would be the biggest beneficiaries, the proposals would apply to large chunks of the middle class as well.
"It's almost, on the face of it, obvious," Brown told NBC News. "I want to reward work, but I also want to bring people out of poverty who don't make enough money."
"It's almost obvious," says Sherrod Brown. Well… I should think so. There's nothing to get people's attention like the prospect of free money. But precisely what are these three talking about?
It's not exactly some sort of socialist European universal basic income (UBC) scheme. (Or at least not yet.) Brown's plan would "massively expand" the earned income tax credit (EITC) as well as the child tax credit. Harris is much closer to a UBC proposal, offering to just send $500 a month to families or $250 per month to single people who earn below a given income level. Booker's plan is at least somewhat more interesting, proposing to give a $1,000 "baby bond" to every child at birth, adding up to another $2,000 per year until they reach age 18.
What do all of these plans have in common? They will cost trillions of dollars with no matching cuts in costs or increases in revenue. And I'll give you one guess as to how they plan to pay for them. Wait… you don't even need a guess. They all want to roll back the tax cuts everyone received under the Trump administration. (You know… the crumbs.)
We often make jokes about Democrats getting elected on the basis of giving away "free stuff." But this takes it to a whole new level, doesn't it? I mean, it might not technically be bribery under conventional legal definitions, but it's still a deal which essentially says, "vote for us and we'll just send you lots of money."
Of course, such plans have to be paid for by the taxpayers. Those would be the ones who would lose their tax cuts to make such plans happen. Obviously, the current leadership in the Senate and the White House aren't going to consider such a deal, so these aspiring presidential candidates don't have to worry about actually implementing their plans and taking responsibility for the subsequent economic collapse. But as they gear up for the campaign trail, they can hold these plans out as "what you might get" if you vote for them.


text:
You get a filter that uses up to 120 volts of power to pump up to 530 gallons every hour. Intex also added dual suction outlets that keep the water clean.
Have fun in the sun with this Metal Frame Pool Set, which comes with a filter and laminated side walls that maintain the round shape of the pool. You get accessories like an instructional DVD and a debris cover that keeps leaves and other debris from falling in the water.
The laminated side walls use three layers of laminate to help the pool maintain its shape. It also comes with a debris cover that you can use to keep the water clean.
Many users had a hard time taking the pool down at the end of the season. There were also complaints from customers about the included filter, which can stop working.
Get your home ready for a swimming party quickly with this Ultra Frame model, which you can set up and prepare for the addition of water in only 45 minutes. Some customers did have problems setting it up though. It comes with a ladder and other accessories for use in your yard.
This Ultra Frame set comes with everything you need to swim at home, including a ladder, frame, ground cloth and liner. You can set this pool up and start filling it with water in just 45 minutes.
As this model is a little smaller than the first, you may find that it doesn't offer all the space that you need. Some customers had a few issues setting the pool up too.
Suitable for families of all sizes, the Ultra Frame Rectangular is the largest swimming pool available from the company. This pool comes with a steel frame and all the accessories that you need to keep your water clean. You also get a DVD with tips on using and setting up your new pool.
This is the largest swimming pool that Intex makes. It comes with a DVD that walks you through the set up of the pool and tells you how to maintain the frame.
Some customers found that the frame developed rust after just one season of use. It may arrive with some minor damage too.
Those looking for a pool that they can easily set up will prefer the Easy Set model. It is small enough that you can use your garden hose to fill it but large enough that it can accommodate multiple people. This one is a little on the small side though.
A shut off feature on this pool instantly turns the pump off when water gets inside. This one is very easy to set up, and you can fill it with water from your own garden hose.
The included pump on this one doesn't work quite as well as those that come with other models. It isn't as durable as those models either.
This model is a good choice for those who like the Easy Set but want something a little smaller. You can set it up in just 10 minutes and use water from your garden hose to fill it. It has a drain plug in the bottom for fast and convenient draining at the end of the season.
You get a filter that uses up to 120 volts of power to pump up to 530 gallons every hour. Intex also added dual suction outlets that keep the water clean.
As this is a smaller swimming pool, you may find that it doesn't have enough room inside for your group. The sides can wobble when you fill the pool and let water splash over the sides.
Intex offers an alternative for those who want a swimming pool but don't want to spend a lot of cash on a permanent above ground model. With an Intex pool, you can have fun throwing parties at your home all summer long. As the company makes so many different models, finding the best Intex pool 2018 model can take some work. You need to consider size, ease of set up and other factors. The best Intex pool for you may not be the same one that your neighbor picked.
Swimming pools often require a lot of maintenance, but Intex lets you enjoy the excitement of owning a pool without nearly as much maintenance. All its models come with a filter that removes debris to keep your water clean. The thick sides help the pool maintain its structure and keeps water from spilling out.
The top models come in both round and rectangular options. Round pools have curved sides that you can lounge against while relaxing, while rectangular pools often feel much larger than round ones do.
We hope that you find this article helpful and that it assists you in choosing the right pool from Intex for your family.
Follow us on:
Get exclusive content, advice and tips from Honest Product Reviews delivered to your inbox!
As with all the reviews that we post, we looked at the reviews from customers who purchased Intex swimming pools and checked out some of the top models ourselves. We looked at the features that matter most to potential customers, including the amount of space in each pool and how much each one costs. One important factor that we considered is ease of set up. It can take hours to set up a standard above ground pool and even longer to fill that pool with water. With Intex models, you can save some time on set up. Some of the top models take only an hour or less to set up. You can use a garden hose to fill it and have a ready to use pool in around two hours.
The reviews posted by shoppers who bought these pools helped us see what others thought. This gave us an idea of what to look for when we tried out these pools. You can use the pros and cons shown in the chart above to find out what customers liked and didn't like about our top models. Our selection criteria includes all the important factors you would use when picking a pool.
Intex shows you how to set up this pool and get it ready for water in just 60 minutes.
Also included is a filter pump with a flow rate of 1,500 gallons per water. This efficient filter works quickly to keep your water clean. The debris cover helps keep the water clean too.
Avoid setting up your new pool on wet or sandy ground to keep the pool from sinking into the ground.
With a water capacity of more than 5,000 gallons, this pool comes with a filter pump that you can hook up to any 110 or 120V outlet. That filter circulates the water around and has a flow rating of 1,200 gallons per hour. It works quickly to eliminate sand and other debris in your water.
All the accessories that you need come with this model, including a DVD that tells you how to set up the frame and how to maintain your pool. The frame snaps right into the pool liner, which is what holds the water. You can use the ground cloth to protect the pool from damage.
Make sure that you have right outlet and circuit to accomodate the filter that come with your pool.
One of the most important things you need to choose before setting up your new pool is where to place it. You should check your city or county codes to find out if there are any rules regarding pools. Some areas require that owners put pools a set number of feet back from the road or that they install a fence.
Another thing to consider is whether you want the pool to sit in a sunny or a shady spot. Putting the pool in a sunny location allows the rays from the sun to slowly heat the water. If you choose a shady spot, your pool will last longer because those rays won't break down the sides. A shady spot may result in more leaves falling in the water, though this isn't a problem if you use your debris cover.
You should also consider what the ground in your yard looks like. The ground should be completely flat and level to keep the sides of the pool from warping. It's also important that you look for an area that stays dry too, which keeps the pool level as you use it.
Roots growing through the ground or just beneath the surface of the ground.
Tree branches located directly above the pool or branches that extend over the water.
Natural paths that your family and guests use to move through your yard.
No matter which Intex pool appeals to you, you need to make sure that it has enough space for your group. Think about the size of your family or the number of people you typically invite over for a swim. If you pick a pool with a depth of less than five feet of water, you need around 20 square feet of space per person. With pools at least five-feet deep, you need only 15 square feet of water per person. Measure the dimensions of each pool to see the amount of square footage provided by each one. This will help you see which ones are suitable for the number of swimmers usually using that pool.
Size is important because it gives each person his or her own space. If you ever swam in a public or community pool before, you know that finding a place to take a breath or relax is hard. Choosing a pool slightly larger than the maximum size that you need ensures that each guest will feel comfortable. If you need a swimming pool for your kids and their friends, you can choose a smaller model because kids need less space.
With traditional above ground swimming pools, you need to do a lot of prep work before setting up that pool. Intex models are easier to set up, but that doesn't mean that you can skip the prep work. Once you decide on the location for your new pool, you need to prepare the ground. Using a broom lets you sweep away any debris that can damage the liner such as tiny rocks and stones. You can also use a rake. Go over the ground several times with the tool and with your own hands to look for any small things you missed. Many models have liners that sit directly on the ground. As you walk across the bottom of the pool, you risk dragging your feet across debris that rips and tears the liner.
You should then put down the ground cover that comes with your pool. This is a lightweight material that serves as protection for the pool. Place the cover flat on the ground and pull it slightly until it sits as flat as possible. You may want to have a friend hold one side as you pull on the other. Set the liner and frame on top of this cover.
Intex offers two different types of pools: those that come with an included frame and those that have thick sides. Models that come with a frame require a little more time to set up. Once you have your ground cloth down, you'll need to lay out the pool liner. This fits inside the frame and holds the water. You'll want to spread the liner out and check for any holes or damage. This step is especially important when it comes to taking the pool down at the end of the season.
The frame features multiple parts that snap together and slide through pockets and straps in the liner. Follow the included instructions to make sure that you use the right parts on each area of the pool. As you insert the parts, the pool will slowly come together and begin supporting itself. You'll then add water the water.
With Easy Set models, you do not need to put together a frame. You only need to lay the pool out flat on top of your ground cloth and inflate the bumper that runs around the top of the pool. As you add water, the pool will slowly lift up and come together.
Watch the included instructional DVD all the way through to make sure you understand how to put your pool together.
One benefit of choosing an Intex pool over a traditional above ground pool is that these require less maintenance. You still need to be aware of problems that can occur, including water that develops a cloudy appearance and algae growing in the water. If you do not properly maintain your pool, you also risk the water burning your eyes and skin. As long as the filter works correctly, you shouldn't have any problems. These filters may not last an entire season though. There is a chance that you'll need to replace the cartridge in that filter to keep your water clean.
It's also important that you use the included debris cover. Mosquitoes and other insects seek out water sources and can drown in your pool. The debris cover keeps those bugs out. It also does a good job of reducing the risk of tree leaves and trash blowing into your pool. The cover is easy to use. You can slide it in place when you finish swimming for the day and pop it back off the next morning. The cover also traps heat inside, which keeps your water from feeling too cold.
Owning any type of swimming pool requires regular testing of your water. You can pick up a pH testing kit for less than $20, which comes with a tube for collecting water from the pool and chemicals for testing the pH balance of that water. The water should fall within a range of between 7 and 7.6. Most kits come with liquids that change the color of the water when added to the tube. This makes it easy to see whether the water needs cleaning. You may need to add more chlorine to the water. Shocking the pool involves a large amount of chlorine, which will kill bacteria, algae and other contaminants in the water.
The filter that comes with your pool does a good job of cleaning and purifying the water. This filter will only work when you run it though. Many people do not realize that they need to run their filters for between 10 and 12 hours every day. You should also check the filter cartridge for any clogs caused by larger pieces of debris.
Traditional swimming pools require that you prep the water and take steps to keep the pool safe during the off-season. With an Intex pool, you can completely break down the pool and put it away until the following year. Intex recommends setting up your pool in a location far away from your home. This keeps the water from potentially damaging your home when you drain it later.
All models have a drainage hole located on either one side of the pool or on the base. You simply unplug this hole and allow the water to drain out. Depending on the size of the pool, you may need to adjust the liner to force the water towards that hole. Let the pool sit out for a few hours or overnight to give the liner time to dry.
With Easy Set pools, you will then need to remove the air from the inflatable tube around the top. If you have a frame model, you can pull apart the frame. Make sure that you put all parts in a large bag and that you put the instructions inside that bag. Check the liner for any signs of damage before putting it away.
If you find holes or tears in your liner, you can use a repair kit to cover those tears before putting the pool back up again.
Shorter pools are best for younger kids because they don't need a ladder to get in and out of the water.
Make sure that you consider all the following factors when picking out an Intex pool.
Intex makes pools in both round and rectangular shapes. You may prefer a round shape that lacks the sharp angles found on rectangular designs.
The top pools we found have a depth of between three-feet and four-feet deep. Some of these models are even deeper. A shorter pool can make you feel more relaxed and comfortable when swimming and playing with your kids.
You can purchase a swimming pool that comes with a metal or PVC frame, but you can also buy one that uses PVC walls instead of a frame. Metal frames can last longer but can rust. A PVC pool lacks any type of frame and may not have the structure you want.
Pump sizes vary from model to model. Larger pumps do a faster and more efficient job of pumping clean water through the pool.
Read the warranty information to find out how long the warranty lasts and what it covers. You may have just 30 days to make a return after your pool arrives.
When buying a pool online, you can often use both the site's Return Policy and the warranty on your pool.
Intex swimming pools have a number of advantages over more traditional above ground models. Those benefits include:
If your water looks cloudy, you can take a sample to a pool store and ask for help.
A: Not all of the top models for 2018 come with a ladder. You'll usually find a ladder on those with a greater depth because users need help to get in the water. Those designed for kids like some of the Easy Set models do not come with a ladder.
A: It can take between 15 and 60 minutes to set up one of these pools. Easy Set models take 15 to 30 minutes to set up, but it takes longer to put together one of the larger pools because you also need to assemble the frame. You'll want to factor in the time that it takes to fill the pool too, which can add a few hours to the total set up time.
A: Some of the negative reviews posted online came from customers with complaints about parts breaking down or not working properly. You can find all the replacement parts that you need online through sites like Amazon and the official Intex website. Intex sells pool accessories online too.
We have access to thousands of products, and have meticulously and personally hand selected the very best for you to check out.
Your reliable and trusted
Resource of Honest Product Reviews
Let Honest Product Reviews be the place where you can scan through the
best products tips, comparisons and reviews.
We are a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for us to earn fees by linking to Amazon.com and affiliated sites.


text:
Scotty Gear Head track adapter
Click to Enlarge
The Scotty 438 Gear-Head Track Adapter locks down your post mounted Scotty rod holders and accessories.
Locks onto the SUPCAT10 frame.
Also fits other kayak track systems.
The Scotty Gear Head is not exportable outside of the continental US.
Ground Shipping Only$17


text:
Shout for joy to the Lord, all the earth.
Worship the Lord with gladness;
come before him with joyful songs.
Know that the Lord is God.
It is he who made us, and we are his;
we are his people, the sheep of his pasture.
Enter his gates with thanksgiving
and his courts with praise;
give thanks to him and praise his name.
For the Lord is good and his love endures forever;
his faithfulness continues through all generations.
Psalm 100 (NIV)
May you have a blessed Thanksgiving!
~ ~ ~
P.S. Due to commitments to my Etsy shop customers and taking time to celebrate God's myriad blessings with my family, I will be taking a break from posting on my blog for a couple of weeks.
~ ~ ~


text:
The short and sweet answer is: whenever, wherever, however you can.
What I mean is that you can use the commute time to your work by coding on your phone or learning with applications like Enki or SoloLearn. It's a very good learning experience!
When you are at home and have 10 minutes of free time – use it! Do that next exercise on FreeCodeCamp or change one line in your current project. Reread and better understand the code of some project one more time.
What you will find is that your attitude matters a lot when trying to attain a new skill. Don't hesitate, don't think too much. Just use every opportunity possible to move forward. Once you don't hesitate to use a small window while riding a bus to learn more, you won't hesitate to spend 3 hours on the weekend too! Stop doing some progress. Start doing as much progress as your current life situation allows you to. Not only you will learn, but you will be energized, positive and look forward to doing more each day.
Thoughts -> Words -> Actions -> Habits -> Character -> Purpose
Thoughts turn into words, words turn into actions, actions turn into habits…
When you decide to sit idly when you could be learning, you lose much more than some time spent on learning. You consciously decide that it's not that important and you are fine as you are. You resign, when you don't need to and tell yourself that your goals aren't that important. When you decide to use that moment instead and put more effort, it builds up. You become serious about it and the results are sure to come.
Happy results in the new, 2019 year!


text:
Ana Sayfa
Dergi Hakkında
Uluslararası Yayın Kurulu
Yazarlar için Bilgi
Bu eser
Creative Commons Alıntı-Gayriticari 4.0 Uluslararası Lisansı
ile lisanslanmıştır.
Bu RoMEO
bir dergidir
Yayın Etiği
Malpraktis Bildirgesi
2014, Cilt 4, Sayı 1, Sayfa(lar) 001-017
Türkçe Özet
] [
Tam Metin
] [
] [
Benzer Makaleler
Policy Proposal for Higher Education Agenda in Turkey
Armağan ERDOĞAN
Ankara Social Sciences University, Ankara, Turkey
The Council of Higher Education of Turkey, Higher Education, Higher Education Key Indicators, the European Higher Education Area, the 2023 Vision and Higher Education
Higher Education Area is going through fast changes as a result of the changes in technology, economy, communication and demographic and social structures both in Turkey and in the world. These changes underline the importance of certain terms and values more than ever before. The terms transparency, diversity, quality, cooperation and competition started to be used together with higher education in recent years. Higher education systems are being reorganised in accordance with these changes. Higher education which is started to be evaluated within the frame of information society, is also interlinked with the basic dynamics of economy though labour force, employment policies and innovation. This situation necessitates internationalization for countries in line with their need for establishing bilateral and regional partnerships. This article aims to analyse current indicators in the dynamically evolving Turkish Higher Education Area and to suggest predictions and new policies based on these indicators and international trends for the mid and long-term targets. It is accepted by many international platforms that Turkey is a rising economy. However, it is difficult to state that higher education is addressed systematically through economic perspective. Estimating higher education as the leading sector, reorganising legal and structural reforms considering international trends in the mid-long term plans, quality assured capacity building, qualitative and quantitative increase of human resources, transparently defined qualifications framework, data-based policies, coordination between the related institutions and defining internationalisation strategy around these paradigms will significantly contribute Turkey's higher education outcomes to achieve the 2023 targets.
Türkçe Özet
] [
Tam Metin
] [
] [
Benzer Makaleler
Şu ana kadar web sayfamız
defa ziyaret edilmiştir.


text:
Winter weather can be rough on vehicles, especially church buses and vans that sit unused for long periods of time. To prevent breakdowns, be sure to service all vehicles on a regular basis through the cold weather season. Follow this advice from the National Highway Traffic Safety Administration (NHTSA):
Have your mechanic inspect the entire vehicle for leaks, bad hoses, or any other parts needing repair. Pay particular attention to the tires, battery and cooling system.
Test Your Tires
Underinflated tires can lead to diminished handling and rollover crashes. To improve handling (and gas mileage), check each tire monthly and set it to the vehicle manufacturer's suggested PSI (pounds per square inch) of air pressure. You'll find the correct PSI listed in your owner's manual and on a label inside the driver's door. Don't forget to check the spare tire, too. In addition:
- Keep a tire pressure gauge in your vehicle at all times.
- Check pressure when tires are "cold"meaning they haven't been driven on for at least three hours.
- Look closely at your tread and replace tires with uneven wear or insufficient tread. Tread should be at least 1/16 of an inch or greater on all tires.
- Power Up Your Battery
When the temperature drops, so does battery power.
Plus, it takes more power to start your vehicle in cold weather than in warm. Find out if your battery is up to the challenges of winter:
- Have your mechanic check your battery for sufficient voltage.
- Have the charging system and belts inspected.
- Replace the battery or make system repairs, if necessary.
- Conduct a Winter Chill Check.
When coolant freezes, it expands.
Such expansion can potentially damage your vehicle's engine block beyond repair. Don't let this happen to your vehicle this winter!
- Make sure you have enough coolant in your vehicle and that it's designed to withstand the winter temperatures you might experience in your area.
- A 50/50 mix of coolant to water is sufficient for most regions of the country. See your vehicle owner's manual for specific recommendations.
- Thoroughly check the cooling system for leaks or have your mechanic do it for you.
- If your system hasn't been "flushed" (draining the system and replacing the coolant) for several years, have it done now. Over time, rust inhibitors in anti-freeze break down and become ineffective. Coolant also needs to be refreshed periodically to remove dirt and rust particles that can clog the cooling system and cause it to fail.
Wipe That Snow Away
Safe winter driving depends on achieving and maintaining the best visibility possible.
- Check your windshield wiper blades and defroster system periodically.
- Make sure your windshield wipers work and replace worn blades.
- If you live in an area that gets a lot of snow and ice, consider installing heavy-duty winter wipers.
- Check to see that your window defrosters (front and rear) work properly.
- Monitor Your Windshield Washer Reservoir
You can go through a lot of windshield wiper fluid in a single snowstorm, so be prepared for whatever comes your way.
- Completely fill your vehicle's reservoir before the first snow hits.
- Use high-quality, "no-freeze" fluid.
- Buy extra to keep in your vehicle.
- Keep Your Vehicles Up to the Challenge
Winter can be one of the roughest seasons for church vehicles, but with a little extra maintenance you can keep them in top running condition.
Adapted from "Checklist & Tips for Safe Winter Driving," by the National Highway Traffic Safety Administration, www.nhtsa.gov.


text:
Hi! It's been a week already! Where does the time go?
Well if you're new around here, I post 5 songs on Mondays that I'm into or just downloaded. This is just for fun and I understand that we all have different tastes. Just like to share in case something catches your ear. Enjoy...
1. I never told you by Colbie Caillat:
2. (speaking of Colbie) Jason Mraz and Colbie-Lucky...can't seem to get the video code...click HERE to see the video
3. Can't stop dancing to Bruno Mars' song, Just the Way You Are
4. I did a scrapbook page with the lyrics to this song for Alex...love it! Dream Big by Ryan Shupe and the RubberBand ....click HERE to see the video
5. American Honey by Lady A is one of my favorites. Love their style. You can listen HERE.
Off to pack for a scrapbook trip I'm going on this weekend with Carla. Love making friends from this hobby. I'm so blessed.
Be back soon. Thanks for stopping by!
Monday, September 20, 2010
Music Monday-Vol. 2
Posted by Heather Ruwe at 3:51 PM
4 comments:
- Bridget said...
LOVE "Just the Way You Are"! It makes me smile anytime I hear it on the radio :)
Bridget N
- September 20, 2010 at 8:44 PM
- nsmerkt said...
REALLY like I Never Told You and had never heard it before. So thank you! Definitely going to download that one.
- September 20, 2010 at 10:42 PM
- laura vegas said...
hope you and carla have a fabulous weekend, and get a lot of scrapping done too!
- September 22, 2010 at 7:32 PM
- Sara said...
LOVE that Bruno Mars song!!!
Have a FUN weekend!
- September 22, 2010 at 11:55 PM
Subscribe to: Post Comments (Atom)


text:
Wooster - 99494 Horny teens in Bloomburg Texas
Married Bm seeks big booty BBw Taiwan horny girls
Mature horny ladies ready horny dating
I search sexy chat
Never Married
Menan - 74290 swingers uk in Fort Worth
Submissive girl seeks tall Dominant male slim brownskin Elm Pennsylvania looking for fun
Horny personals searching woman want fuck swinger black porno
I searching dick
Eielson AFB - 28491 free mature sex ads Essex
Looking For Dominant White Woman sex with girls in Ketchikan Alaska
Local mature want girls wants sex women seeking dick at clubs Strasburg
I search real sex
Horse Branch - 94357 granny personals Springfield Illinois
23 male anything you want ladies
Horny slut wants online dating horny personals wanting sweet sex.
Wants sexual partners
Never Married
South Durras - 94358 on line dating services
Come over, fuck and be happy
Girls seeking guys dating bipolar salas de chat de sexo xxx Rhode Breaux Bridge
I ready sexual dating
Not important
Higginsport - 72858 adult sex sites
Old lady is looking for fitness instructor
Lonly women wants meet swingers Shelby Gap sexy Shelby Gap
Want dating
Firestone - 65387 adult matchmaker
Need cock sucked and fucked by cute girl horny housewives seeking couple dating.
Lonely married women looking fucking women Feering county swingers
Looking sexy chat hot swingers searching dating club.
Ladies wanting a Crotch Rocket Ride.
Big woman looking women to date Sex swingers tulsa
Elisabeth - Repton 40851 horney old woman want sex cams.
Valentine women ready to fuck LOOK here wow
Mature ladies wants cyber chat
I wants swinger couples horney adult searching fuck a whore.
Fit married lady over 45 that needs some lovin rochester mn milf
Hot mature woman search adult hookups
I want nsa
Seeking Older Woman for Love Unique LTR find married women Palmas
Sex friends ready white label dating site bbw women wants honylonely wivies.
I want real swingers sex married woman seeking horny sluts.
Relationship with an older woman discretion advised 21 and up
Sex personal search granny personals Benton City milf slags wanting cock
Sabrina - Hutchinson Island South divorced women looking meet people.
I want to fuck a girl
Xxx ladies ready adult social network Colorado Springs nude women
Searching for a man cyber mature sex
I am Looking for someone to suck off naughty swinger search swinger chat.
Wifes seeking wealthy dating older married search cheap hookers.
Victoria - Fulda beautiful women
No girlswant a world traveled woman lonely girls search long distance relationships.
Horny bbw searching girl fucking xxx naughty search horny mature.Prince Kuhio laundry room We just met in the laundry room. I wanted to put you up on the washer and give you some great oral pleasure!! Im sure you won't read this.. I could only wish. i love you just can't ever tell you You are the housewives wanting sex in Zitineni strongest, most beautiful women I've ever meet i wish i could kiss those lips give you a hug and make everything alright. ..we both gone through hell with are x's just think if we've got together years ago instead of getting with lookin for a friend wit benefits. them, we would be unstoppable. ..ur x is like my brother but i can't help loving you ISO fun crazy and fun chick for NSA fun.
I want people to fuck hot horney want hook ups.
CANT SLEEP TEXT SEXY married women dating old women ready sex and dating.
Married bbw search true dating horny single search married men dating.I Just happen to have some! I have been purging my garage of them. I was hoping to sell some of these or see some go to someone who will enjoy them :) (I have!) I am sure you will be local cougars sluts Bangor Maine happy, and you'll be also helping me out as well :) They are all different adult hookups searching online adult chat kinds, and duh, you must be over 28 I will totally check so dont try it! I wont be by the computer much so if you leave a number (& name) thats the best and only way or me to contact you. Thanks and Take Care! Looking for a sweet younger woman for long term.
Evelina - Cuyahoga Heights 72846
Mature woman looking for some girl company tonight. wifes around Kilmarnock want to fuck tonightGentle Loving man in need of LadyBoy Hi, My name is Michael Jakubiec III. This is hard to truck driver seeks travel companion post, but I have tried and struck out trying to find a nice young lady-boy to satisfy my hunger. I was in love with a hamster horny Reno town girls nude previously, so the change is subtle. Lady-boy must be ready for the freaky stuff. I'm the kind of guy that likes to be tied up and stuffed with a pole. Also I live with my mom and dad so well have to be kinda quiet when you sneak in at 19:19 a.m. I really hope I find my soul pix of faith hill getting fucked mate, if not at least we had a great time. also you will be greeted with a bowl of chocolate ice cream and cat fur blankets. Buh Bye :) Seeking African Female For Sex. Meet at bear creek dog park on saturday.
I am search sexual encounters divorced dad seeks Madison Wisconsin
UWArt student wants to be fucked Stevenage milf ready toA Summer Romance that Leads Somewhere I am looking for a a partner. A woman who likes being in the company of a man who is simple yet loves a little adventure. I'm not looking for a party Elizabeth on dating sites girl, more of a stay home, old fashioned girl. I'd like her to be just as comfortable dressing up to go out for a nice meal as hanging out on the couch in our sweats watching a movie or a Red Sox game. I work hard during the week and would really enjoy sharing my off days with a wonderful woman. If you are interested please respond with what your idea of a perfect weekend would be like together and a picture. I will in turn respond with the same. Please only real woman truly looking for a potential partner should bother answering this post. I am serious and want a woman who is equally serious about finding a Summer Romance that leads Somewhere...... Wanted: Normal Everyday Woman For Services On Regular Basis. hot chicks Midhurst
I am seeking men
Looking for a friend to go to a party with meDaddys Girl Would you like to share our stories real horny girls Kenai together, say lips in message so i know your real. Meet me and grind your pussy on my mouth. FEMALES (only) age 40-40. Clean. No hard drugs. Needs to like , or at least be ok with them. my are so important to me (they are 40 & 40). Size does not matter to me, I love tats & piercings (I have a tat of my own & used to be pierced). Laid back & fun. Open minded. Drama free. Looking for a FREAK to handle this HUGE white Meat. black woman seeking mature dating sites.
I want dick girls to fuck executive dating service.
Maroon Convertible women looking at cock Top at Texaco on 629 single lonely search horney weman.Hello! My name is Katlynn. I am Paradise Nevada girls fuck buddies short, thick and curvy. An adorable natural redhead if i may add. I am 42 years young. I love to meet new people, but REAL people only. I dont tolerate games, other than that i am a completely chill individual. Im not looking to hookup. Im looking for someone who is looking for a genuine friendship that will make room for it to grow if it feels right. I am 42 friendly, but it doesnt consume my life. I expect it to be the same for you. Music taste is a large factor in compatibility with me. I love Red Hot Chili Peppers, but my music ranges anywhere from the Beatles to Lil Wayne. No country please. I would like to meet a guy who has something going for him, you dont have to be a Harvard student, but please atleast have goals and a car. There is a lot to learn about me and i can gaurentee if you enjoy the company of someone who can make you smile, you wont be dissapointed. I have many pictures, yours gets mine. Peace! sheb falls qmart I've been in a few times, you are working, yet still have a beautiful smile no matter what. I was going through the drive thru, and you were outside having a smoke. I drove passed smiled and waved, you smiled back. You're cute, tall, brunette, and oh yeah . If you think we had a connection, please respond. I'd absolutely love to take you out this weekend Generous guy needs bj asap.
All women NEED to read this its about respect! looking for a hook up 18 35 womenDiscreet blow jobs Good looking str86 guy looking for other discreet guys in the area to give a good blow job to. 86'86 86 86c ddf hiv- live alone can host or travel depending on the looking to start off as freinds distance, please don't be playing games if you want sucked off hit me up if not don't waste time with the pic games and bs. Not into fems or fat guys so dont waste your time, I'm fit in shape and would like the same. Uwillenjoyit looking for some fun. Looking for woman to pamper.
Tiffany - Loring looking for sex ads Cambridge
Craving pussy in my face tonighta guy to use So I want a guy that I can mess around with. I want someone that doesn't mind that I won't have much time to hang out I just want a nice clean cock that I can do whatever I want with. My favorite is to watch a man cum. I can't girls looking for sex in Bunker Hill Kansas host, so hopefully you can and must be discrete Flat out, I'm gay. If you see me you can tell I'm gay. But by no means do I act like a dyke... Just look like xxx And trust me I won't disappoint The thing is I just want a dude I can pretty much use , and you can use me as well. I'd like to get to know someone so we could hopefully have an ongoing thing Please send a picture or I will move on Any age is fine with me.. as long as you can fuck me good Please put "use me" as your subject so I know your not spam. I have a lot of pictures as well ;) Looking for attractive she-male for first time encounter. xxx comment3, offre emploi medical , rtucz, hillbilly housewife , 01347, .. comment4, treasure bay casino , :-OOO, property inspections illinois , rwqc, horny crocodile , yyprp, c2h3nh2, bnhnwi, the deseret, 8-O, apartment nebraska plattsmouth 投稿: bi-wiring | 2010年3月27日 (土曜日) 午前 08時28分.
Dana - Odenville 51917 girl wanting online sex.
Looking For An Assfucking? my naked wife in Southaven Mississippi:) I love you You opened it. Good luck. Tonight at midnight your true love will realize they miss you. Something good will happen to you between 96:96 pm and 96:96 pm meet horny Akron Ohio girls online bBW looking for FWB with big beefy man! tomorrow, it could be anywhere. Tonight at midnight they will remember how much they loved you. You will get a shock of a lifetime meet horny Akron Ohio girls online bBW looking for FWB with big beefy man! tomorrow, a good xxx If you break the chain you will be cursed with relationship problems for the next 96 years. Karma. If there is someone you local pussy want advice dating loved, or still do, and can't get them out of your mind, re-post this in another city within the next 96 minutes. Its amazing how it works.If you truly miss someone, a past love, and can't seem to get them off your mind....then re-post this titled as " I Still Love You" Whoever you are missing will surprise you. Don't break this, for tonight at midnight, your true love will realize they love you and something great will happen to you tomorrow. Karma. You will get the shock of your life tomorrow Hot Blonde at CVS women free sex.
I am wants men
Female Swing Partner Wanted ! sex personal looking sexy ebony.Submissive Chubby fwb sex Maxbass North Dakota ohio Chic, banged and bred *** Please respond with an address to ensure you will get my reply!! This new thing isn't so useful!!! Are you on the thick side? ?Chubby? Submissive? Need to be seeking Ltr Ralationship. taken, be laid on your back,,with your legs on a man's ..and want to fuck South Portland Maine pounded, and shown how a master can treat you for being good? I am looking for a Submissive Chubby short woman, who submits to me and wants to be on her back, we me on top banging her into even more submission,,and eventually give the ultimate submmission and be bred. Can be a long term submission process leading breeding, or submit to it right away. Depends on you and your will to submit, or take more bangings. Face it ladies, you are going to get married, then cheated on, then left to for yourself after the man takes your identity away... You might as well breed with a guy who wont steal who you are. Have a nice or 61, and never have to submit your soul and your persona to him. I am 61'61 61lbs Italian. Can really deal a good banging, fast, hard and deep..of course at the same time I will be kissing your lips, your neck, whispering in your ear how beautiful you are, and getting my lips sucking hard on those hard nipples!! Any questions,,comments,, ? Discreet Sex no strings 48 Liverpool 48. xxx My friends are all married with adult friend finder, so I get my fill of being swinging couples,wife swapping plus Milnthorpe swinger personals ads and everything. . Carolina Nonverbal communication in dating Xxx personals thawville illinois
I want to wet a hung cock up Iceland bbw for gf fwbLTR NO I am a bbw , interracial de swingers partido único Cheste curvy, and happy person. I am looking for a ltr without or excess baggage. If you are married or in a relationship keep am seeking a country man for ltr! looking, I have no time want for that. I have been a product of affairs and those that do it are awful people. I have 57 grown , and 57 grand . I am not here for a place to live or your money. What I want is not Getting the Attention I Need. honesty, trusting where to meet horny older women blog, compassionate loving man who will treat me with the up most respect as I will for him. I want fun, loving, caring, spontaneous, likes to travel when possible. Age race does not matter as long as you treat me right. I also am not xxx who is looking for a sexual relationship till after I know you are with me for the right reasons. So don't ask or hint. If you don't have a job or car keep looking. I like camping, fishing, bonfires, slow walks, holding hands, snuggling and a helpful hand if I need it. Please no . Put your favorite color in the subject line, Please no spam. Cedar Rapids area please Hot pussy dating with the green hat. hot woman looking adult classified.
Wants couples
Looking to meet new people 23 Ithaca, N.Y. 23Don't have a gf or a date to bring to an event?$ Maybe we can help eachother out. I'm saving money for a vacation ( I do have a full time job) and you need a really friendly and well spoken date. We can portray an intimate couple or just friends-all up to you. I can converse about any topic and I'm very fit. the details about your event and we can go from there. NO SEX involved of any kind. Donation from you will be dependent on the details but it will be reasonable. Plenty of to share with you but we all know I'd get xxx ed if I put a real xxx up. I'm very real :) Have a wonderfully hot friday in the three39three! Bored and wanting some fun. brunette with black framed single moms need cock in Middelfart glasses, reasons why I... To the brunette with black framed glasses last night and black jacket, looking for the reasons why I... I really liked talking to you. You did mention about looking for it chatroulette alternatives sure is quiet without you. downtown, and couldn't find it. I should have tried to talk to you a bit more! Watch me make your WIFE SCRE with DELIGHT w. Recently Released wants funtime. xxx adult sex dating in thomastown mississippi <a sex dating in sweetwater nebraska <a
On the 8th day God created Black woman. white slave boi wanted for bdsm playWondering Why??? Why is it soo hard to find someone to get to know? It's like people say "OH I'm Looking for friends and this and that" But when they don't look the way you want them to all contact is lost... WTF? I thought you was looking for friends? I shouldn't matter right? Anyway I'm looking for someone I can build a friendship with and IF we click go for something more... I'm not looking to move to fast because when you do it be good for the first few months (if it last that long) and that's it.. Which sucks.. I'm like all women (54-54 yrs old) (race is NOT a factor for me at all) You gotta be open minded and willing to do stuff you haven't done before ( NOT SEXUAL) just fun stuff... I like going out sometimes (karaoke, a lot of live single ladies searching fucking buddy concerts, festivals and what ever's fun) I don't dance much but I try lol.. I do asian female Bonita California drink, smoke socially..Oh yea.. I'mma 54yr old black kool ass women who's worth getting to know =) Wanna know more just let me know.. Val =) Blow on da go? or sex? 54 Tacoma 54. LIST ACTIVITIES AND HOBBIES LIST INTERESTS LIST SPORTS WITH TEAMS PUT AGE PUT FAVORITE MUSIC GROUP OR SINGER IN SUBJECT LINE Here are the DO NOT'S FWB or NSA fun in Southern RI.
where to meet horny older women blog Looking to Hookup need discretion В© 2016


text: Figure 3From: CellNOptR: a flexible toolkit to train protein signaling networks to data using multiple logic formalismsSubset of the results of a CellNOptR analysis on two time-point data from human hepatocellular carcinoma cells. The data consists of phospho-proteomic measurements of 16 proteins in response to multiple inducers of inflammation, innate immunity and proliferation, applied in combination with selected small molecule inhibitors[1]. This figure shows a simplified version of a small subset of the trained model (blue nodes=measured, green=stimulated, red=inhibited; green edges=picked at τ1, blue edges=picked at τ2), along with the data associated with the creb node (right, solid black line), overlaid with the simulation results (dashed blue line) for a selected set of conditions. The background color indicates the goodness of fit of simulation results to data. We can see that the model captures the behavior of creb accurately: creb increases at τ1 if either MP2K2/MP2K1 or p38 are activated (in this case, because both are downstream of IL1A, they are both activated in the absence of inhibitors and presence of IL1A). This activation is maintained if both MP2K2/MP2K1 and p38 are activated, and is lost at τ2 (180 minutes) if only one of them is activated (i.e. in this case if either is inhibited). This behavior is captured in the model by selecting an OR gate from MP2K2/MP2K1 and p38 to creb at τ1, and an AND gate at time τ2.Back to article page


text:
A warm welcome awaits you at the Brigg Heritage Centre. Situated on the first floor of The Angel, a former 17th Century coaching inn the Brigg Heritage Centre is a labyrinth of rooms and discoveries. The star exhibit, the Bronze age 'Raft' found nearby in the river Ancholme, sets the scene for a fascinating journey, charting the rise of Brigg as an important and influential, market place through to the picturesque town you see today. With children crafts, temporary exhibitions, talks and events there is always something new.
Learn about upcoming events and activities by following the centre's Facebook page or follow them on Twitter
- Address:
- The Angel, Market Place, Brigg DN20 8LD
- Telephone:
- 01724 296771
- Email:
- email@example.com
- Web:
- http://www.briggheritage.org/
Opening Hours
- Tuesday:
- 10am-2pm
- Thursday:
- 10am-2pm
- Friday:
- 10am-2pm
- Saturday:
- 10am-3pm
- Education programme/schools
- Historic building
- Light refreshments
- Museum
- Open all year
- Parking
- Partially suitable for disabled visitors
- Shop
- Special events
- WCs suitable for disabled visitors
- Welcome host