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The use of deuterium-labeled gamma-aminobutyric (D6-GABA) to study uptake, translocation, and metabolism of exogenous GABA in plants
Faraj Hijaz1 &
Nabil Killiny ORCID: orcid.org/0000-0002-3895-69431
Exogenous application of gamma-aminobutyric acid (GABA) could relieve stress symptoms caused by abiotic stresses including anoxia, heat and cold shock, drought, and salt. However, studying translocation and metabolism of exogenous GABA is challenged by the presence of endogenous GABA.
Herein, we used D6-GABA in order to investigate the uptake, translocation, and the metabolism of exogenous GABA in Mexican lime (Citrus aurantifolia) seedlings using gas chromatography-mass spectrometry (GC–MS). The GC–MS analysis showed that D6-GABA could be easily distinguished from the non-labeled GABA after methyl chloroformate (MCF) derivatization. The D6-GABA was detected in the cortex (phloem), inner stem (xylem), and leaves after root drench. Girdling did not affect the translocation of D6-GABA, indicating that it is mainly translocated via the xylem. In addition, D4-labled succinic acid was detected in D6-GABA-treated plants, indicating that exogenous GABA was metabolized to succinic acid. The half-life of D6-GABA in citrus was about 1.3 h, indicating a quick conversion to succinic acid.
The use of D6-GABA offers a valuable tool to study the translocation and metabolism of GABA in plants. D6-GABA and its metabolite (D4-succinic acid) can be easily distinguished from the endogenous GABA and succinic acid using GC–MS.
γ-aminobutyric (GABA) is widely distributed in plants and it has received a great attention in plant science [1]. This non-proteinogenic amino acid (NPAA) is synthesized in the cytosol from glutamate by glutamate decarboxylase (GAD) [1]. The level of GABA in plants increases under biotic (e.g. insects and viral attack) and abiotic stresses (e. g. anoxia, heat and cold shock, drought, and salt) [1, 2]. GABA could also be synthesized from putrescine by D-amino oxidase (DAO) and spermidine by polyamine oxidase [3]. Liao et al. (2017) showed that DAO contributed about one-fourth of GABA formed in tea leaves under anoxia [3]. Non-enzymatic degradation of proline under oxidative stress could also contribute to GABA formation [4].
The increase in cytosolic Ca+2 and/or H+ leads to the activation of GAD [1, 2]. Accumulation of Ca+2 occurs under many abiotic stresses including salinity, heat shock, and drought. Accumulation of H+ was also reported under various stresses including anoxia and mechanical damage [1, 2]. Accumulation of Ca+2 at normal physiological pH results in the formation of Ca+2/calmodulin complex leading to the activation of GAD enzyme [1, 2]. Activation of GAD by Ca+2/calmodulin complex occurs during mild or early stages of stresses [1, 2]. However, during severe and late stages of stress (at low cytosolic pH) the GAD enzyme is activated in a pH-dependent manner [1, 2].
Previous reports showed that exogenous application of GABA to plants could enhance their growth and relieve stress symptoms caused by abiotic stresses. For example, exogenous application of GABA on Caragana intermedia roots decreased H2O2 levels and increased ethylene production under salt stress [5]. In the same manner, GABA increased the antioxidant enzyme activity and decreased reactive oxygen species (ROS) in muskmelon seedlings under hypoxia stress [6]. Moreover, the levels of several sugars, amino acids, and organic acids was enhanced upon the application of GABA in creeping bentgrass (Agrostis stolonifera) under heat stress [7]. Exogenous application of GABA improved drought tolerance and increased the level of several amino acids and organic acids in GABA-treated creeping bentgrass under drought condition [8].
Recently, we found that exogenous application of GABA enhanced the level of several phytohormones (trans-jasmonic acid, salicylic acid, abscisic acid, indole acetic acid, and indole propionic acid) in citrus plants [9]. The gene expression levels of succinic semialdehyde dehydrogenase (SSADH) and GABA-transaminase (GABA-T) were induced in GABA-treated plants seven days post-treatment (dpt), indicating a conversion of GABA to succinate [9]. Succinic dehydrogenase and malate dehydrogenase were also upregulated in GABA-treated plants, indicating an induction of the TCA cycle [9]. The GC–MS analysis showed that the level of endogenous GABA was significantly increased in GABA-treated plants seven dpt, but declined to its normal level 14 dpt, demonstrating that GABA was translocated to the citrus leaves and then was catabolized there [9]. In current study, we used deuterium-labeled GABA (D6-GABA) coupled with GC–MS to investigate the translocation, distribution, and metabolism of exogenous GABA in citrus seedlings. The methyl chloroformate (MCF) derivatization used in this study allows quantification of other amino and organic acids. However, for the purpose of our study, we only focused on GABA and its metabolite (succinic acid).
Derivatization of GABA standards
Deuterated (D6-GABA) and non-labeled GABA standards were first derivatized separately (Fig. 1a, b) to identify their retention time and mass spectra, and then were mixed and derivatized together (Fig. 1c). The MCF derivative of D6-GABA standard eluted before that of the non-labeled GABA (Fig. 1c). The peak of the MCF derivative of D6-GABA standard was almost separated from the non-labeled GABA standard (Fig. 1c). In addition, we were able to differentiate between endogenous GABA and exogenous D6-GABA (Fig. 1d). The parent ion of the derivatized GABA was 175 (m/z) (Fig. 1e), whereas the parent ion of the D6-GABA standard was 181 (m/z), indicating the presence of six deuterium atoms (Fig. 1f). The mass spectrum of the MCF derivative of GABA showed several main fragments [88, 102, 112, 116, and 144 (m/z)] (Fig. 1e), whereas these fragments were shifted in D6-GABA to [90], [106], [118], [122], and [150] (m/z), respectively (Fig. 1f). These results indicated that GC–MS could easily differentiate between exogenous (D6-GABA) and endogenous GABA.
GC–MS chromatograms of the MCF derivatives of GABA and D6-GABA and their mass spectra. The MCF derivative of GABA (a), D6-GABA (b), a mixture of GABA and D6-GABA (c), D6-GABA-spiked leaf sample (d), and the mass spectra of the MCF derivative of GABA (e), and D6-GABA (f). The reaction scheme of GABA and D6-GABA with MCF is also shown in the upper right corner of graph e, f
Percentage recovery of labeled D6-GABA
Our GC–MS analysis showed high recovery of the D6-GABA (92.6% ± 2.7). No D6-GABA was detected in control samples (blank). The level of endogenous GABA in control sample was 84.4 ± 9.8 μg/g. This result showed that D6-GABA could be successfully used to study the translocation of GABA in citrus plants. In our preliminary work with detached leaves, we were able to detect D6-GABA after 2 h incubation in 10 mM solution. Our preliminary trials also showed that the level of D6-GABA was higher (294.1 ± 18.1 μg/g) than endogenous GABA after 6 h incubation in 10 mM D6-GABA. This result indicated that GABA was translocated in plants and suggested that 6 h incubation would be sufficient to detect D6-GABA in intact plants using GC–MS running in full scan mode.
Translocation of D6-GABA in intact seedlings
The GC–MS analysis showed that D6-GABA was present in the roots, stem and leaves of D6-GABA-treated plants (Fig. 2a, b). The concentrations of D6-GABA in the treated seedling (non-girdled) at the end of incubation time (6 h) ranged from 28–115 µg/g (Fig. 2a). The concentrations of endogenous GABA in D6-GABA-treated plant tissues ranged from 75 to 189 μg/g (Fig. 2a).
The distribution of endogenous GABA and succinic acid (SA), and exogenous D6-GABA and D4-SA in non-girdled (a) and girdled (b) Mexican lime seedling after incubation in 10 mM D6-GABA for 6 h. the cortex was referred phloem and the inner stem as xylem
The GC–MS chromatogram of the D6-GABA-treated seedlings also showed a peak at 0.06 min before the succinic acid (Fig. 3a). This peak was absent in the control (Fig. 3b). The retention time of this peak suggested that it could be a D4 labeled succinic acid. The mass spectra of this peak showed a base peak of (m/z) 119 indicating the loss of a methoxy group and a molecular ion of 150 (m/z) (Fig. 3c). Whereas, the mass spectra of the succinic acid peak showed a base peak of 115 (m/z) and a parent ion of 146 (m/z) (Fig. 3d). The mass spectra result suggested that this peak was a D4-labled succinic acid. The 119 (m/z) fragment indicated a loss of a methoxy group and the 150 (m/z) represented the molecular weight. The concentrations of D4-SA in D6-GABA-treated seedlings ranged from 4–28 µg/g (Fig. 2a). The level of endogenous succinic acid in D6-GABA-treated plants was similar to that of D4-SA (Fig. 2a). No D6-GABA was detected in treated seedlings that were returned to their original pots 24 h after treatment and only trace amount (1.5 ± 1.8 µg/g) of D4-SA was detected in these plants. This result indicated that D6-GABA was completely metabolized in treated plants 24 h after treatment.
The presence of D4-SA in D6-GABA-treated plants. A GC–MS chromatogram of the D6-GABA-treated plants (a) showing the presence of D4-SA and the absence of D4-SA in control plants (b) after derivatization with MCF. The mass spectra of the MCF derivatives of D4-SA and SA in D6-GABA-treated (c) and control plants (d)
Translocation through the xylem; girdling
To test the translocation of D6-GABA through the xylem, the main stem was girdled at about 3 cm above the soil surface (Fig. 2b). The D6-GABA was detected in the stem and leaves above and below the girdling (Fig. 2b). The concentrations of D6-GABA in the different tissues of treated seedling ranged from 56–195 µg/g (Fig. 2b). The concentrations of endogenous GABA in D6-GABA-treated plant tissues ranged from 108 to 195 μg/g (Fig. 2b). The presence of D6-GABA above the girdled site showed that D6-GABA was translocated via the xylem. In the same manner, D4-SA was detected in the stem tissues and leaves above and below the girdling. (Fig. 2b) The concentrations of D4-SA in the D6-GABA-treated seedling ranged from 5–19 µg/g (Fig. 2b). The level of endogenous succinic acid in D6-GABA-treated plants was similar to that of D4-SA. The level of endogenous succinic acid in D6-GABA-treated plants was similar to that of D4-SA (Fig. 2b).
Catabolism of D6-GABA in detached leaf
To study the rate of catabolism of D6-GABA in citrus leaves, detached leaves were incubated for 3 h in 10 mM GABA solution, washed with distilled water, and then incubated in distilled water for 0, 1, 2, and 4, 5, and 24 h. At the end of incubation time, leaves were analyzed by GC–MS to measure the level of D6-GABA. The level of D6-GABA in the leaves upon incubation in distilled water was as follows: 0 h; 10.8 ± 2.3 µg/g, 1 h; 10.0 ± 4.6 µg/g, 2 h; 3.7 ± 0.9 µg/g, 3 h; 1.0 ± 0.1 µg/g, 4 h; 0.8 ± 0.4 µg/g, 5 h; not detected (Fig. 4a). The Tukey's test showed that the level of D6-GABA after 2, 3, 4, and 5 h was significantly lower than detected at 0 h. The level of endogenous GABA in D6-GABA-treated and control plants ranged from 81.4 to 98.2 µg/g and did not show any significant changes. The level of D4-succinic acid in the leaves upon incubation in distilled water was as follows: 0 h; 5.4 ± 1.1 µg/g, 1 h; 3.5 ± 1.67 µg/g, 2 h; 13.1 ± 7.2 µg/g, 3 h; 8.8 ± 2.9 µg/g, 4 h; 7.8 ± 4.6 µg/g, 24 h; 2.0 ± 1.1 µg/g.
Metabolism of exogenous GABA in plants. The decay (metabolism) of D6-GABA in citrus leaves presented in concentration and log of the concentration versus time (a). Leaves were incubated in 10 mM D6-GABA for 3 h, washed with water, and then transferred to distilled water for 0, 1, 2, 3, 4, and 5 h. Effect of the exogenous D6-GABA application on the relative gene expression of several citrus genes involved in GABA biosynthesis and catabolism (b). Leaf samples, for RNA extraction, were collected at 6 h post treatment (dpt) with or without GABA [mock (0 mM vs.10 mM GABA]. Bars represents the relative increase in the gene expression level in GABA-treated plants relative to the control, while the error bars represent standard deviation (SDs). Gene expressions were normalized using five housekeeping genes including; elongation factor 1-alpha (EF1), F-box/kelch-repeat protein (F-box), glyceraldehyde-3-phosphate dehydrogenase GAPC1, cytosolic (GAPC1, also known as GAPDH), and SAND family protein (SAND), which previously showed high stability for transcript normalization in citrus under biotic stress [19,20,21]. The changes in the gene expression levels were analyzed with the 2−ΔΔCT method. Samples were analyzed in triplicate for each biological replicate (n = 5). The full list of expressed genes, names, accession numbers, and primers are available in Additional file 1: Table S1. Schematic diagram of the metabolism of exogenous D6-GABA in citrus plants. Metabolism of D6-GABA to D4-succinic acid and derivatization of D4-succinic with methyl chloroformate (c)
Because there were no significant differences in gene expression between upper and lower leaves, results were pooled and presented together in the heatmap (Fig. 4b).The gene expressions of gamma-aminobutyric acid-permease (GABP), gamma-aminobutyric acid-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) were upregulated in D6-GABA-treated plants (Fig. 4b). The upregulation of the previous genes indicated that exogenous GABA was metabolized to succinic acid. On the other hand, the gene expression of GAD genes was downregulated in D6-GABA-treated plants (Fig. 4b), indicating a downregulation of GABA biosynthesis. The GC–MS and the gene expression results together showed that exogenous GABA was metabolized to succinic acid (Fig. 4c).
In our recent work, we showed that the level of endogenous GABA in the leaves of GABA-treated plants was significantly induced seven dpt, and then dropped to its normal level at 14 dpt [9]. This result indicated that GABA was translocated and metabolized in citrus plants. Unfortunately, we did not measure the levels of GABA in other plant parts, such as the roots and stem.
In this study, we used D6-GABA to study the translocation and metabolism of exogenous GABA in citrus plants. All the hydrogen atoms attached to carbon are replaced by deuterium in D6-GABA. Consequently, this prevents the rearrangement of the deuterium atoms and formation of isotopolog. In addition, our GC–MS results showed that the D6-GABA can be easily differentiated from that of the non-labeled GABA. Consequently, using D6-GABA we were able to differentiate between the endogenous (non-labeled) and the exogenous (D6-labled GABA). The deuterated GABA standards eluted about 0.07 min before the non-deuterated GABA standard. The decrease in the retention time of the deuterated GABA standards could result from the strong bond between the deuterium and carbon atoms [10]. The strong interaction between the deuterium and carbon atoms could decrease the column affinity to D6-GABA and consequently results in earlier elution time. Furthermore, the mass spectrum of the D6-GABA was different from that of non-labeled GABA; the main fragments in D6-GABA were shifted two to six atomic mass unit (amu) compared to those of non-labeled GABA. Analysis of spiked citrus tissues showed high recovery (92.6% ± 2.7) of D6-GABA, indicating that D6-GABA could be successfully used to study the translocation of GABA in plants.
Incubation of citrus seedling's roots in D6-labled GABA showed that it was translocated from the roots to other tissues within a few hours. D6-labled GABA was also detected at high levels in leaves and stem above the girdle, indicating that GABA was transported via the xylem. In addition, D6-labled GABA was detected in the cortex and inner stem of non-girdled plants. These results indicated GABA exchange could occur from xylem to phloem and vice versa during long-distance transport. In general, exogenous application of GABA results in an increase in GABA in treated plants. For example, exogenous application of GABA increased the level of endogenous GABA in creeping bentgrass (Agrostis stolonifera) under heat stress condition [7, 8]. An increase in endogenous GABA was also reported in several plants including Caragana intermedia, Stellaria longipes, maize, and sunflower upon exogenous application of GABA [5, 11,12,13]. The previous results indicated that GABA is translocated in plants.
In agreement with our results, previous studies showed that amino acids can be taken directly by the roots and can be transported between different organs through both xylem and phloem [14]. Plants possess several amino acids transporter families including the GABA-permease (GABP) related family [14]. In our previous study, we showed that citrus genome possesses a putative GABA-permease, which connects the GABA-shunt with TCA cycle by transporting cytosolic GABA to the mitochondria [15]. Interestingly, the gene expression of GABP was highly induced in CLas-infected and D. citri-infested citrus plants, indicating an increase in GABA transport from the cytosol to the mitochondria [15]. In agreement with the gene expression results, the level of succinic acid was also enhanced in CLas-infected and D. citri-infested plants, indicating a conversion of GABA to succinic acid [15].
The GC–MS results showed that D6-GABA was metabolized to D4-succinic acid. The upregulation of the gene expression of GABP, GABA-T, and SSADH supported the GC–MS results. In agreement with our current results, the level of endogenous GABA in citrus plants was increased upon treatment with exogenous GABA seven dpt; however, its level declined to its normal level in few days, suggesting that GABA was translocated and metabolized [9]. This result was supported by the increased gene expression of GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in GABA-treated citrus plants seven dpt [9]. In addition, the gene expressions of malate dehydrogenase and succinic dehydrogenase genes were highly induced in GABA-treated plants seven dpt, indicating that GABA was metabolized to succinate and fed into the TCA [9].
The half-life of D6-GABA in citrus leaves was about 1.3 h, indicating that GABA is quickly converted to succinic acid. In addition, no D6-GABA was detected in seedlings 24 h upon incubation for 6 h in 10 mM D6-GABA, confirming that it was completely metabolized to succinic acid. Recently, we showed that the levels of GABA and succinic acid were significantly increased in detached citrus leaf after 1 h incubation in 10 mM GABA, indicating that GABA was quickly metabolized to succinic acid [16]. In addition, the level of fumaric acid was significantly increased in citrus leaves after 1 h incubation in 10 mM GABA, indicating that succinic acid was fed into the TCA cycle [16]. Unfortunately, no D2-labeled fumaric acid was detected in D6-GABA-treated plants in this study. The absence of D2-labeled fumaric acid in D6-GABA–treated plants could result from the exchange of deuterium atoms in deuterium-labeled succinic acid with hydrogen atoms by succinic acid dehydrogenase. An exchange of deuterium atoms with hydrogen atoms of the solution was reported in in deuterium-labeled succinic acid in the presence of succinic acid dehydrogenase, which catalyzes the oxidation of succinic acid to fumaric acid [17]. Previous studies suggested that succinic semialdehyde could also be reduced to gamma-hydroxybutyrate (GHB) in plants under a variety of abiotic stresses [18]. However, we could not check for GHB because it cannot not be derivatized using methyl chloroformate. Therefore, a future study with a different detection or derivatization method is needed to test for the formation of GHB.
Our results showed that exogenous GABA was taken up by the roots and was transported via the xylem to the leaves and other plant tissues in the upper part of the plant. In addition, our results showed that GABA was quickly metabolized to succinic acid. Our results also demonstrated that D6-GABA could be successfully used to distinguish between endogenous and exogenous GABA in plants. Furthermore, our result suggested that isotope-labeled GABA could be a valuable tool to study the translocation and metabolism of this important signaling molecule in plants.
Plant materials
Mexican lime (Citrus aurantifolia) was used in this study. Seeds were potted in plastic cones (20 × 4 cm) containing Sungro professional growing mix (Sungro Horticulture, Agawam, MA). Seedlings were kept in a greenhouse (28 ± 1 °C, 60 ± 5% relative humidity, L16:D8 h photoperiod) at the Citrus Research and Education Center (CREC), University of Florida, Lake Alfred, Florida. Seedlings were watered twice weekly. At the time of experiment, plants were about three-month old and around 15 ± 5 cm tall.
Preparation of GABA standards
D6-GABA and GABA standards were purchased from Sigma Aldrich (St. Louis, MO). Stock solutions of GABA (10 mM), D6-GABA (10 mM), and succinic acid (10 mM) were prepared daily using distilled water. A set of serial dilutions were made and used to construct the standard curves.
Percentage recovery of D6-GABA
Citrus leaves were ground with liquid nitrogen using a mortar and pestle and 100 mg of the ground tissue was spiked with 10 µl of D6-GABA (10 mM). Five samples were spiked with D6-GABA standard and five controls were mixed with 10 µl of distilled water, then were extracted and analyzed as outlined below to determine the percentage recovery. Percentage recovery was calculated by dividing the area of the D6-GABA peak in spiked sample by the area obtained from D6-GABA standard (10 µl of 10 mM).
Preliminary work: intake of D6-GABA by a citrus leaf
To study the uptake of D6-GABA by a single citrus leaf, the petiole was cut, under water, using a sharp blade and was quickly immersed in 10 mM D6-GABA solution. Incubation was carried out during daytime inside a greenhouse at the same condition described above. At the end of the incubation time (1, 2, 4, and 6 h), the petiole was cut and discarded, and the leaf was washed for 1 min with distilled water to remove any adsorbed D6-GABA from the leaf surface.
Treatment of citrus plants with D6-GABA
Before treatment with D6-GABA, citrus plants were removed from their original pots and the roots were washed with distilled to remove the soil. To determine if GABA was transported in the xylem, the main stem of five plants was completely girdled before incubation. Girdling was done by complete removal of a 1 cm wide strip of bark about 3 cm above the soil surface (Fig. 2). Then 15 plants (5 girdled and 10 non-girdled) were incubated in 10 mM D6-GABA for 6 h. Each plant was placed in a 5 ml plastic centrifuge tube and the roots were covered with 10 mM D6-GABA solution. Control plants (5 plants) were incubated in distilled water. Incubation was carried during daytime inside a greenhouse at the same condition described above. At the end of incubation time (6 h), plants were washed for 1 min with distilled to remove any adsorbed D6-GABA. Five of the D6-GABA-treated plants were returned to their original pots (potting mix) and were analyzed after 24 h. The rest of the plants were dissected and analyzed by GC–MS. For these analyses, the stem bark was dissected into the cortex and the inner stem (xylem). Three leaves from the upper part of each plant (mature, moderate-age, and juvenile), were collected and pooled together (upper leaves). In the same manner, three leaves from the lower part of each plant (mature, moderate-age, and juvenile), were collected and pooled together (lower leaves).
Catabolism of D6-GABA by citrus leaf
To study the catabolism rate of GABA in citrus leaves, the leaf petioles were immersed in 10 mM D6-GABA for 3 h. At the end of incubation time, the leaves (30 leaves) were removed from the D6-GABA solution and washed with distilled water. Then, sets of five leaves were kept in distilled water for 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h, respectively. Incubation was carried during daytime inside a greenhouse at the same condition described above.
Analysis of GABA using GC–MS
GABA was extracted from ground tissues (0.10 ± 0.002 g) using methanol 80% containing 0.1% HCl 6 N according to the procedures described in our previous study [9]. Briefly, plant tissues were ground with liquid nitrogen using a mortar and pestle. Then, 100 mg was transferred to 2 ml centrifuge tube and 700 µl of the solvent mix was added and the sample was vortexed for 30 s. The sample was kept for 10 min in ice and then was centrifuged for 10 min at 17,000×g at 5 °C. The supernatant was decanted to a new centrifuge tube and the extraction procedure was repeated two more times by adding 700 µl of the solvent mix each time. The collected extract was evaporated under a gentle nitrogen stream and was re-dissolved in 200 µl of the solvent mix, centrifuged again, and transferred to a silanized conical 1-ml insert. The extract was concentrated to about 40 µl under nitrogen stream. The extract was derivatized with methyl chloroformate (MCF) as described in our previous study [9]. Briefly, 180 µl of 1 N sodium hydroxide, 167 µl of methanol, and 34 µl of pyridine was added to the sample extract or standard and the mixture was vortexed for 10 s. Then, 20 µl of MCF was added and the mixture was vortexed for 30 s. Another 20 µl of MCF was added and the mixture was vortexed again for 10 s. A 100-µl aliquot of chloroform was added and the mixture was vortexed for 10 s. Finally, 200 µl of 50 mM sodium bicarbonate was added and the mixture was vortexed for another 10 s. The chloroform layer (bottom) was transferred to a silanized GC–MS insert and 5 mg of sodium sulfate were added.
For GC–MS analysis, 0.5 µl of the derivatized sample was injected into the GC–MS running in the full scan mode. Derivatized samples and standards were analyzed using a Clarus 680 gas chromatograph equipped with Clarus SQ 8 T mass spectrometer running in the electron ionization mode (EI) (Perkin Elmer, Waltham, MA, USA). The system was fitted with a ZB-5MS GC column (5% Phenyl-Arylene 95% Dimethylpolysiloxane; low bleed, 30 m × 0.25 mm × 0.25 µm film thickness; Phenomenex, Torrance, CA, USA). The flow rate of the helium carrier gas was set at 0.9 ml/min. The GC thermo-program was as follows: initial temperature was held at 70 °C for 4 min, and then increased to 280 °C at a rate of 10 °C/min, and finally held for 5 min. The injector was set at 220 °C, inlet line at 200 °C, the source temperature at 180 °C, and the electron energy was set to 70 eV. The GC–MS chromatograms analysis were performed as described in our previous study [9]. Calibration curves were constructed from the linear regressions obtained by plotting the concentration vs. peak area for each standard. The level of endogenous succinic acid and exogenous (D4-succinic acid) was quantified using succinic acid standard curve.
Gene expression analysis using quantitative real time PCR (RT-PCR)
Leaf samples, for RNA extraction, were collected at 6 h post treatment (dpt) with or without GABA [mock (0 mM vs.10 mM GABA]. Leaves were sampled from the top and the bottom as described above. The RNA was extracted from (0.1 ± 0.002 g) ground leaf tissues using TriZol® reagent (Ambion®, Life Technologies, NY, USA). The quantity and quality of isolated RNA were assessed using NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA primers were synthesized using SuperScript first-strand synthesis system (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. The qPCR was performed on an ABI 7500 Fast-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Green PCR master mix (Applied Biosystems). Samples were analyzed in triplicate for each biological replicate for each treatment. Primers for genes involved in the GABA shunt pathway were used to measure the gene expression (Additional file 1: Table S1). The relative expression of the consensus sequence among PCR products was done according to the 2−ΔΔCT method [18]. Four genes were used as endogenous genes (reference genes) to normalize the data of gene expression including; elongation factor 1-alpha (EF1), F-box/kelch-repeat protein (F-box), glyceraldehyde-3-phosphate dehydrogenase GAPC1, cytosolic (GAPC1, also known as GAPDH), and SAND family protein (SAND) [19,20,21,22].
Data were analyzed using JMP 9.0 software (SAS, Cary, NC). Analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey–Kramer honestly significant different test (Tukey HSD) were used to compare level of D6-GABA among the different treatments (p < 0.05).
Datasets used for the study can be requested from the corresponding author on reasonable request.
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Retey J, Seibl J, Arigoni D, Cornforth JW, Ryback G, Zeylemaker WP, et al. Stereochemical studies of the exchange and abstraction of succinate hydrogen on succinate dehydrogenase. Eur J Biochem. 1970;14:232–42.
Allan WL, Simpson JP, Clark SM, Shelp BJ. γ-Hydroxybutyrate accumulation in arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms. J Exp Bot. 2008;59:2555–644.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–8.
Mafra V, Kubo KS, Alves-Ferreira M, Ribeiro-Alves M, Stuart RM, Boava LP, et al. Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One. 2012;7:e31263.
Wei X, Chen C, Yu Q, Gady A, Yu Y, Liang G, et al. Comparison of carotenoid accumulation and biosynthetic gene expression between Valencia and Rohde Red Valencia sweet oranges. Plant Sci. 2014;227:28–36.
Nehela Y, Hijaz F, Elzaawely AA, El-Zahaby HM, Killiny N. Citrus phytohormonal response to CandidatusLiberibacter asiaticus and its vector Diaphorina citri. Physiol Mol Plant Pathol. 2018;102:24–35.
We thank our lab member for the helpful discussion and proofreading the manuscript.
This work was kindly funded with a Grant for NK from the Citrus Initiative program, Institute of Food and Agriculture Sciences, University of Florida.
Department of Plant Pathology, Citrus Research and Education Center, IFAS, University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA
Faraj Hijaz & Nabil Killiny
Faraj Hijaz
Nabil Killiny
FH and NK performed the experiments. FH and NK designed experiments, analysed the data and wrote the manuscript. NK supervised the project and acquired the funding. Both authors read and approved the final manuscript.
Correspondence to Nabil Killiny.
Additional file 1: Table S1.
Primer used for gene expression analysis of GABA shunt-associated enzymes by real time RT-PCR.
Hijaz, F., Killiny, N. The use of deuterium-labeled gamma-aminobutyric (D6-GABA) to study uptake, translocation, and metabolism of exogenous GABA in plants. Plant Methods 16, 24 (2020). https://doi.org/10.1186/s13007-020-00574-9
γ-aminobutyric acid


text:
Q: While connecting AWS rds mysql instance getting com.mysql.cj.jdbc.exceptions.CommunicationsException: Communications link failure While connecting to an AWS rds mysql instance I am getting the below error.
This happens only when I deploy 2 different wars on the same tomcat instance. Both apps connect to their corresponding DB instance (Separate DBs).
If I deploy them separately I don't have an issue.
Here is the error:
com.mysql.cj.jdbc.exceptions.CommunicationsException: Communications link failure

27-Jul-2018 18:31:06.214 SEVERE [pool-27-thread-3] org.apache.tomcat.jdbc.pool.ConnectionPool.init Unable to create initial connections of pool.
 com.mysql.cj.jdbc.exceptions.CommunicationsException: Communications link failure

The last packet sent successfully to the server was 0 milliseconds ago. The driver has not received any packets from the server.
    at com.mysql.cj.jdbc.exceptions.SQLError.createCommunicationsException(SQLError.java:741)
    at com.mysql.cj.jdbc.exceptions.SQLExceptionsMapping.translateException(SQLExceptionsMapping.java:57)
    at com.mysql.cj.jdbc.ConnectionImpl.createNewIO(ConnectionImpl.java:1663)
    at com.mysql.cj.jdbc.ConnectionImpl.<init>(ConnectionImpl.java:662)
    at com.mysql.cj.jdbc.ConnectionImpl.getInstance(ConnectionImpl.java:352)
    at com.mysql.cj.jdbc.NonRegisteringDriver.connect(NonRegisteringDriver.java:221)
    at org.apache.tomcat.jdbc.pool.PooledConnection.connectUsingDriver(PooledConnection.java:307)
    at org.apache.tomcat.jdbc.pool.PooledConnection.connect(PooledConnection.java:200)
    at org.apache.tomcat.jdbc.pool.ConnectionPool.createConnection(ConnectionPool.java:728)
    at org.apache.tomcat.jdbc.pool.ConnectionPool.borrowConnection(ConnectionPool.java:662)
    at org.apache.tomcat.jdbc.pool.ConnectionPool.init(ConnectionPool.java:480)
    at org.apache.tomcat.jdbc.pool.ConnectionPool.<init>(ConnectionPool.java:154)
    at org.apache.tomcat.jdbc.pool.DataSourceProxy.pCreatePool(DataSourceProxy.java:115)
    at org.apache.tomcat.jdbc.pool.DataSourceProxy.createPool(DataSourceProxy.java:102)
    at org.apache.tomcat.jdbc.pool.DataSourceProxy.getConnection(DataSourceProxy.java:126)
    at org.hibernate.engine.jdbc.connections.internal.DatasourceConnectionProviderImpl.getConnection(DatasourceConnectionProviderImpl.java:139)

redpajama_set_name: RedPajamaStackExchange


Welcome to Attingham Park, the house sits in a flat simple green landscape, cows graze and deer roam close to the building. My favourite mirror still-life scenes are here.
Along with lampshades, chandeliers and portraits, mirrors are my favourite things in the NT houses I have visited so far. They set up mini scenes of the rooms they inhabit and its a good way to surreptitiously watch the other visitors look around the room and listen in on conversations. In this room Sickert's 'Lady In Blue' invited Jack The Ripper comments.
I have been making small films, mostly around roses, architecture and interiors and posting them on my Vimeo here.
Several 'Unknown ladies' at Attingham, rather nice examples.


Photo(s): Bart Wursten, Mt Gorongosa, Mozambique.
Note: Flies during most of the year from sea level to mountainous areas in grassland and along streambanks.
Distribution: from KwaZulu-Natal and Lesotho to Eastern Zimbabwe and Mozambique.
Hyde, M.A., Wursten, B.T., Ballings, P. & Coates Palgrave, M. (2019). Flora of Mozambique: Lepidoptera - Butterflies and Moths: Cacyreus palemon palemon.


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Orihuela del Tremedal – gmina w Hiszpanii, w prowincji Teruel, w Aragonii, o powierzchni 71,52 km². W 2011 roku gmina liczyła 572 mieszkańców.

Przypisy

Gminy w Aragonii

redpajama_set_name: RedPajamaWikipedia


text:
Q: Local Lie Groups Hello i am trying to prove the following proposition :
Let $G$ be a connected Lie group, and $U\subset G$ a neighborhood of the identity element. Also, let $U^k = \{g_1 . g_2 . \dots g_k : g_i \in U\}$ be the set of k - fold products of elements of U.
Then, $G=\cup_{k=1} ^ \inf U^k$.
I read somewhere that, this result follows immediately from connectedness of $G$ but it is not so obvious to me.
Thank you for your time!

A: This is true for any connected topological group $G$.
Let $V = \bigcup_{k=1}^\infty U^k$. It is clear that $V$ is open. On the other hand, let $g \in \bar{V}$. Since $gU^{-1}$ is a neighborhood of $g$, it must intersect $V$. Let $h \in V \cap gU^{-1}$. Then

*

*$h = gu^{-1}$ for some $u \in U$, and

*$h = u_1 \ldots u_k$ for some $k \in \mathbb{N}$ and $u_1, \ldots, u_k \in U$.

Hence $g = u_1 \ldots u_k u \in U^{k+1} \subset V$, proving $V$ is closed. Since $G$ is connected, and $V$ is both open and closed, we have $V = G$.


The United States Merchant Marine Academy is a service academy based in the suburban town of Kings Point, New York. All students enter the school as "plebe candidates" and must complete an intense regimen of physical training, customs, drills, and ceremonies. A majority of tuition and living expenses for students are paid for by the federal government. After graduation, students have the option to work as active duty officers in any branch of the US military or as civilians in the maritime industry.
Is a very rigorous school that will prepare students for their role in the military or maritime service. There is no other school like this, with regards to traditions and experiences. Even though your time here will be rough and studious it is well worth it.


text:
Article from BTNews 28 JUNE 2010
CRUISE NEWS JUNE 2010 (Diamond Princess)
THE YACHTS OF SEABOURN'S highly anticipated new Seabourn Sojourn squeezed its majestic way through the Thames Barrier and past the O2 arena at the beginning of June to moor majestically at Greenwich, close by the meridian line, and short of Tower Bridge. London-born model and film star Twiggy named the ship, the second of three 32,000 super 'yachts' ordered by the luxury brand of the Carnival Group.
Cllr Chris Roberts, Leader of Greenwich Council, announced that what in the first Queen Elizabeth's reign was the headquarters of the Royal Navy, is making a maritime waterborne comeback. "In Greenwich we are working to secure berths for two international cruise liner terminals so that we can welcome more visitors from all over the world to our borough, particularly during the Olympics. We hope Greenwich will be a regular destination for Seabourn Sojourn".
Sojourn, with its 225 suites, claims to offers an extraordinary travel experience, Seabourn notable for its relaxed, sociable ambiance and highly personalized, intuitive service. The vessel offers the highest space per guest ratio in the industry, accommodating no more than 450 guests in spacious, ocean view suites, 90% of which have private verandas. The ship offers what it call a 'culinary experience' in four dining venues. Fine wines and spirits come with Seabourn's compliments. www.seabourn.com
OUR TEN STORY NEWS ROUNDUP
(followed by A SHIP REVIEW: Diamond Princess)
Cunard's largest ever winter series of voyages will go on sale on 15 July featuring two World Voyages and a series of Exotic Discovery Voyages. Longer Winter Voyages and full World Voyages continue to prosper despite tougher economic times and with the Cunard ships ideally suited to longer journeys.
Later this year Cunard will have a three-ship fleet, Queen Mary 2, Queen Elizabeth and Queen Victoria. Mary and Elizabeth will feature completely different around the world voyages whilst Queen Victoria will offer a full winter season of exotic voyages taking in the Panama Canal, Caribbean and her first ever Hawaii Christmas voyage. www.cunard.co.uk
Dubai is heavily promoting its cruise port claiming to be the only destination in the region providing a complete turnaround facility. It is proving popular not only for the bulging ex-patriot community, but also for the airlines, bringing in passengers from both east and west.
Over 325,000 cruise tourists are expected to visit the Emirate during 2010; hosting 120 ship calls. Costa and Royal Caribbean are both operating from the port this year, and most ships on Northern Hemisphere around the World cruises find that Dubai is a popular stopping off point. A new terminal, spanning an area of 3,450 square metres, can handle up to four ships simultaneously. www.dpworld.com
Dunkirk may be just another Channel port to later generations but to those who can still remember the Second World War, or were brought up in its aftermath, it means one of the largest seaborne gatherings of all time.
From disaster came victory.
To celebrate the 70th anniversary of Operation Dynamo, the codename for the Dunkirk evacuation in May 1940, Norfolkline's 'Maersk Dover' sailed from its name port to Dunkirk with the Lord Lieutenant and other dignitaries and school children on board. At Dunkirk it greeted the arrival of the Little Ships with a commemorative ceremony. It was a moving experience for all involved. www.norfolkline.com
Epic, Norwegian Cruise Lines (NCL) latest, and by a wide margin, largest ship will be somewhere in mid-Atlantic on AERBT publishing date. It has acquired the 'graffiti' style paintwork of it smaller sister ship. French-built it embarrassed its owners by being late for its inaugural showing at Southampton, delayed for five hours at Rotterdam's cruise terminal by what was called "technical difficulties".
At 153, 000 tons Epic is to a completely new and novel design with 2,200 cabins and a passenger capacity, of around 5,000 when children are doubling up with parents. All the 1415 outside cabins have a balcony, the balance within the ships vast interior.
The majority of the accommodation has what is described as a "new wave" concept with curved walls, LED aircraft lighting, and an shower/toilet arrangement described by one observer as "less than private". Epic will be christened by country music mega-star Reba McEntire and will sail in alternate weeks from Miami to the Eastern and Western Caribbean itineraries until April 2011 before crossing back across the Atlantic and a European programme.
NCL has always in the past offered a high standard and very good value cruising. Epic takes head on RCCL and its larger fleet of mega liners with a vessel bristling with innovation and new sea going concepts. Hopefully it will be the subject of an AERBT SHIP REVIEW at a later date. www.ncl.com
London Cruise Terminal, Tilbury, part of the Port of London, plans to provide accommodation to teams and delegates throughout the 2012 Olympic games. Two berths have been reserved for the period. At present, Cruise & Maritime Voyages 'Marco Polo' and Holland America's ms 'Prinsendam' are the only current users of the easy to get to Essex port.
Cruise ships up to about 30,000 tons can make it up the Thames to Tower Bridge (and certain ships past the bridge too, mooring next to HMS Belfast. Passenger access problems and the use of only licensed tenders have proved an obstacle in turning the Pool of London into a regular cruise destination. www.pla.co.uk
Oceania Cruises, in many ways the successor to Renaissance Cruises, has started construction of a second 1,258-guest 66,00 ton up-market ship called Riviera and a sister ship to Marina, which makes its debut in February 2011. Both will feature ten dining venues – six of which are open-seating gourmet restaurants – including one by Master Chef Jacques Pépin. Facilities will also include a Canyon Ranch SpaClub® and gym, Bon Appétit Culinary Centre, swimming pool and hot tubs and suites furnished exclusively in Ralph Lauren Home style. Renaissance Cruises and its purpose-built fleet of R class ships failed soon after 9/11. However the vessels themselves were very successful and quickly found new homes (and identities). Oceania currently operates Regatta, Insignia and Nautica, all once part of Renaissance. www.oceaniacruises.co.uk
Onair, which claims to be the world's leading provider of airline in-flight communications, has introduced full mobile phone connectivity – including GSM & GPRS – for Hapag-Lloyd Cruises flagship ms EUROPA. The Geneva based company, part of SITA, the airline IT operation, is pushing hard to gain a foothold into the cruise line business, most current systems slow, users having to be patient.
OnAir is already offering passengers of more than 25 airlines and VIP and Governmental operators voice, email, text services and internet access, on flights over four continents by using the latest SwiftBroadband Inmarsat L-band technology.
Technological partners for the service is Siemens IT Solutions and Services and TriaGnoSys, who have developed expertise to enable passengers and crew to use their own mobile phones and Smartphones (iPhone, Blackberry©, HTC, etc). The GSM service uses satellite communications, making connectivity possible regardless of the ship's location.
The installation, which uses the ship's existing Ku-band satellite antenna, is a simple process that can be carried out during a routine maintenance stop between cruises. The software developed by Siemens and TriaGnoSys uses compression technology to make very efficient use of the available bandwidth. www.onair.aero
Portsmouth (once again a rival to Southampton) is to become the home port for the All Leisure Group in 2011 coupled with an expansion of its "no fly" cruises. The port is the home of HMS Victory, HMS Warrior and the Mary Rose. Under construction is a brand new cruise terminal which will compliment the spectacular Spinnaker Tower.
First in is Swan Hellenic's Minerva (the original ship) which will operate a series of regional cruises where even the off-ship excursions are included in the price, as well as staff tips. The lecture programme is top quality but one thing for certain, the ship is not suitable for children.
With the second of the All Leisure brands, Voyages of Discovery's the summer 2011 season will commence with a short series of cruises from introducing the ship to a wider audience than previous. Discovery will re-position back later in the year to Harwich where she will operate a longer programme of "no fly" cruises and continue her association with that port.
The company's third ship, Hebridean Island Cruises Hebridean Princess, will be joining her fleet colleagues in 2011, also offering a small dedicated programme from Portsmouth. Hebridean Princess is no stranger to this location as she has visited this historic port in previous years.
News is awaited on All Leisure's latest acquisition 'Alexander von Humboldt' which is also due in service in 2011, the Group yet to disclose whether it gets a new name or remains with its current title. www.allleisuregroup.com
The Yachts of Seabourn gets a second mention in the June issue of AERBT Cruising News with the announcement of a change of deployment for the popular, 208-guest Seabourn Pride in the summer of 2011. The ship will reposition from its winter Southeast Asia season in the spring and sail a series of 12-day Baltic and Russia cruises between Stockholm and Copenhagen, along with two 15-day round trip cruises from Copenhagen to Norway's fjords and the North Cape.
Pride's schedule will complement that of its larger sister-yacht, the 450-guest brand new Seabourn Sojourn, which will be sailing shorter, seven and nine day voyages of the Baltic and Norway.
The company had previously announced that Seabourn Pride would remain in Asia for the whole of 2011, sailing in China, Korea and Japan during the summer months. The decision to move the vessel back to Europe is a response to the popularity of the destination. En-route voyages are for sale too. www.seabourn.com
TUI Cruises, a cruise brand established in 2008 through a joint venture between Royal Caribbean Cruises Ltd. and TUI AG is to acquire Celebrity Mercury in early 2011. Named Mein Schiff 2 she will join Mein Schiff (the former Celebrity Galaxy) to become the second ship in the fleet, tailored for the German market.
From an operational point of views things could not have worked out better.
Celebrity Cruises President and CEO Dan Hanrahan explains."With the rapid growth and momentum of the Celebrity brand, and the fourth member of our five-ship Solstice Class fleet, Celebrity Silhouette, introduced in July 2011, we have abundant opportunities for the fine crew on Celebrity Mercury to transfer to another Celebrity ship?"
Celebrity Mercury joined the Celebrity Cruises fleet in November 1997, and was built by Meyer Werft in Papenburg(Germany). The 77,713-ton ship carries 1,898 guests (double occupancy) and 909 crew. www.tuicruises.com
A SHIP REVIEW: Diamond Princess
Diamond Princess is one of a pair of ships built in Japan (the other is Sapphire Princess) for Princess Cruises, for many years the American arm of P&O, and whilst still very much a sister company, now part of the much larger Carnival Group.
Diamond Princess entered service in 2004 and is essentially a modification of the highly successful Grand Princess design which includes Grand Princess itself, Golden Princess, Star Princess, plus, with an extra deck, Caribbean Princess, Emerald Princess and Ruby Princess. P&O's Ventura and new Azura are essentially to the same design, although each ship differs in detail. She is 116,000 tons gross and carries a nominal 2,600 passengers plus 1,250 crew, an excellent ratio of just over two passengers for every crew member.
Unlike the initial Grand Princess series Diamond Princess does not have what looks like a wing projecting out over the stern, and used as a nightclub. A more traditional, and practical, arrangement is now built around the rear of the funnel.
At the present time Diamond Princess lacks Movies Under the Sky, the outdoor cinema first seen on Caribbean Princess. That together with an adult-only retreat (The Sanctuary) will be rectified when the ships goes in for a major refurbishment in November.
On boarding there is a welcome drink and guests are shown personally to their cabin. Whilst the ship is always keen to sell you water when disembarking this is provided on the quay for free when at a port, or using the ships tenders.
The overall impression is that Princess is not money grabbing, unlike some operators, and if you want to it is possible not to spend virtually any extra money. Where the docking is not convenient to the local town for the most part Princess provide transport. It is essentially a quiet ship, announcements, in English, kept to a minimum, normally at 09:00 and 12:00 from the bridge followed by the entertainment staff giving a resume of activities.
To save the embarrassment of tipping Princess adds per person per day to your shipboard account, and also 15% to extras.
In General: The public areas of the ship are confined to the top three decks, and a further three decks deep down in the bowels of the ship. All flows rather nicely. Each of the six accommodation decks has a self-service laundrette.
Such is the layout of the ship moving around is very easy, with two notable exceptions.
The promenade deck, which is lovely and wide and ideal for sitting out on the teak lie-flat seats, and for deck games, is not continuous. For those taking a one-third of a mile lap walk or running tour, at the front of the ship you have to mount a series or steps, pass the staff swimming pool in the bow, and go down the other side. Exhausting! Or you can jog to the steps, turn around, and do the same when you reach the same obstruction opposite.
The other complication is the double sitting international dining room in the stern. It is on the same deck as the other two main restaurants with the kitchens in-between. Easy enough to get to if you are coming from your cabin, but if you are partaking in pre-dinner drinks in the Grand Plaza area it is an up and down exercise. Infuriating (and overcome with cocktails at the gallery level).
Accommodation: Just over half of Diamond Princess cabins have balconies (easily large enough for a twosome breakfast) and the ship has a very limited number of suites.
The standard veranda cabins are some of the best at sea, well thought out, the lounge area very acceptable and the beds nearest to the services. Nice linens, bath robes and slippers are there to use. The cabins have a mini walk-in hanging area too, saving the problems associated with swinging doors.
There is a turndown service plus chocolates every night and fresh fruit on request. A 24-hour courtesy room service is provided as well as tea and coffee making facilities. The fridge offers drinks at bar prices. If you book a lower deck balcony cabin please note you might be overlooked from above. The inside and window cabins are normally much the same size as the standard ones with balcony.
Dining: The cuisine aboard all the Princess ships is to a fine standard and you are served by an attentive multi-national staff, all of whom for the most part speak acceptable English, with many long term contractors (unlike the management the majority of the hotel/public contact staff are on contract to contract). If you want something special ask at the previous mealtime and you will usually get it. Dietary requirements are dealt with. It's best to contact the cruise line beforehand, who are very helpful. The kitchen team are most experienced and will emerge from their hidden areas to discuss.
There is a choice at both breakfast and lunchtime of dining casual or silver service, which can be 'anytime' when you dine to your timetable, or fixed, first or second sittings. Surprisingly this traditional style of eating seems to be making a comeback Princess now offering two restaurants with set timings. Unlike the other ships in the class the amidships dining room on both Diamond and Sapphire have been divided in two along the centre of the ship. This gives for a better ambience and more intimate surroundings.
For a small premium Sabatini's offers multi-course traditional Italian cuisine deep down in the ship and the Sterling Steakhouse, a bar area during the day converts in the evening. It is high up on the ship and partially overlooks the stern. The meat cuts are delicious. Both dining areas are to the highest standard.
An innovation this year for premium suite clients and Captain's Circle Platinum members is the opening of Sabatini's for breakfast. It has cost Princess virtually nothing and is a more intimate venue for breakfast. And the doors don't close at 10:00 sharp. If you are little late they will still let you in.
Princess now offers courtesy ice-cream during daytime hours (it used to be free at mid-afternoon only) and the English tea still continues at 16:00. This includes scones with clotted cream and jam, and a harpist threesome entertains in the restaurant. English tea time not only finds favour with the British but the Japanese like it too, and those from North America.
Entertainment: Showtime on Princess is a proper live West End quality variety performance, no miming by grinning dancers to pre-recorded music. There is a large (900+) pillar-less theatre on board and the very well equipped alternative venues, but with not so many seats and a full bar service. The theatre also doubles up as a cinema, films another Princess tradition. The programme even manages to fit in with the cruise, 'The Last Emperor' playing to nearly afull house on the way into the port of Beijing. The orchestra also pops up all over the place, on deck, and in the other entertainment venues.
The actual entertainment programme is mixed, with the Cruise Director acting as MC. Good Cruise Directors make for a cruise, full of energy and charm. Princess is lucky that it has some of the best. All the cruise lines need pay special attention to what they have to offer as cruising can often be more than twice a year and clients do need variety and not the same show over and over again. Also the US seems to be the last resting place of forgotten British comics. They need to update their acts.
Princess offer a proper port lecture series which can also be caught on the in-cabin TV. In the Mediterranean and other popular places these can be sparsely attended, most clients knowing the popular ports of call, but the Far East is new territory for most, and a quality lecturer, it is standing room only.
Diamond Princess has two pool areas, one with a sliding roof. In practice this stays in place all of the time providing a air conditioned open deck area, and a fine venue for lunch barbeques and the occasional evening show time.
Children: British cruise ships have always had on offer splendid facilities for children (and continue to do so with Cunard and P&O leading the way – although with P&O some ships ban under 18 all together) and Diamond, with its UK heritage no exception. The number of children on board depends on the time that you take your cruise. There can be anything up to 900 during the school holidays but experience has shown that they are normally well behaved and really have no effect on those whose experience of parenthood is long gone.
The younger people have their own well laid out area at the stern of the ship including a dedicated pool and outdoor play areas. There are computers galore and electronic games. Parents have to look after the actual feeding of the younger ones, but once that job is over but they are supervised until late into the night. This goes for the crèche too. All the staff are registered and go through Princess own training programme.
On Board Services: Princess does not offer daily international newspapers (but they can be purchased at a price) although BBC World and CNN is available on the cabin TVs. There is what is thought to be the largest Internet Café at sea, which always seemed busy during normal hours, with a pay for coffee bar. It suffers, as with all ships at the present time, with an intermittent service, which can be very slow. There is also a wireless system on board, but it does depend on where your cabin is situated.
The medical facilities are top quality and like everything else on board, chargeable to your cabin account. The reception is open all night.
What Else?: The gym on board Diamond Princess is comprehensive and is incorporated into the spa. Massage on deck is on offer too when the weather suites. There is a 9-hole putting course and simulator. There is a good range of shops on board covering evening needed (and useful when your luggage does not keep pace at the airport) and whilst the daily "sale" offerings on the balcony of the atrium can be somewhat over the top, it is easy enough to keep away from that area. It all normally packs up around five.
Diamond is traditional with few gimmicks, but that is what the Princess clients demand. The casino is huge, the art sales are well attended (and presumably make money), the dance class, scholarship@sea (self enrichment), bridge and cooking demonstrations all attract their devotees. Trivia can get quite exciting, but at the end of the day the quiz master's word is final. You can take in as much as you can, or relax, or perhaps find some midway point.
In Conclusion: Don't be put off by the sheer size of Diamond Princess and her sisters. When Grand Princess entered service she was the largest cruise ship afloat by some margin. Things have moved on. We've mentioned already that the flow within the ship causes no problems, and getting on an off at the various ports is easy. Not everyone wants to at the same time. Princess is well organised and their agents at the various ports top quality. Up to 100 coaches waiting for the ship might sound a great number but the staff seem to cope without problems. Everyone smiles, customers and workers alike. This augers well for Princess new arrival in 2014, 141,000 tons and 3,600 passengers. We await.
All in all Princess serves up a fine middle of the road product. In the Far East Americans made up just under a half of a full ship, with a large contingent of British, followed by Brazilians, and then Germans (and don't worry there are more than enough deckchairs to go around). A friendlier crowd you could not have found.
www.princess.com

redpajama_set_name: RedPajamaCommonCrawl


BirdEye Launches Presence & Reputation Diagnostics Tool for Businesses in U.S.
Scan Your Business app enables any business to run an instant report of how their business appears on 50+ consumer sites.
Most businesses do not know how they appear online.
BirdEye, a leader in Reputation Marketing, launched today Scan Your Business, a free app for businesses to quickly assess their online presence and business reputation on 50+ sites. A business owner simply enters the business name and zip code, and the app generates a detailed report of their online presence and reputation.
The Scan Your Business app is a useful tool for any business to check its snapshot across 50+ sites — Is the business listed on the main sites? Is the business information (NAP or business name, address, phone number) accurate on all sites? Is the business rated well (customer reviews and ratings)? The report can be instantly downloaded and emailed.
Scan Your Business app is available at http://birdeye.com/scan-business/. The BirdEye Business Reputation Marketing platform can be previewed at http://birdeye.com.
BirdEye (birdeye.com) is a leader in Business Reputation Marketing (BRM). The BirdEye platform helps businesses get more word-of-mouth referrals, leads, and repeat customers. With BirdEye, businesses monitor reviews from all major sites, gain new authentic reviews, manage customer feedback, and automatically promote their good reputation across the web, social media and search engines. Enterprises use BirdEye analytics and competitive insights gleaned from customer feedback to fine-tune products or services to breakout from the pack.
The BirdEye platform powers 500K businesses and 10M+ reviews. Based in Sunnyvale, CA, BirdEye was founded by alumni from Yahoo, Google, Amazon and Cisco.


text:
The Post's View
In Tunisia and elsewhere, Islamist parties face a test
By Editorial
AS EGYPTIANS went to the polls this week, a Tunisian political leader was in Washington with a useful reminder: Islamic-oriented parties are not necessarily the enemy of democracy.
Tunisia, you may recall, is where the Arab Spring began, when a hapless fruit peddler, driven to his wit's end by corruption and official harassment, set himself on fire. That sparked a popular uprising, which sent Tunisia's dictator fleeing, emboldened protesters from Libya to Egypt to Yemen to Syria, and put Tunisia itself — a North African nation of about 10 million people — on the path to democracy.
In October, Tunisians held a successful election, in which the Islamist party Ennahda won the leading share of the popular vote and of seats in the assembly that will appoint a government and draw up a constitution. The head of that party, Rashid Ghannouchi, has been in Washington this week. His chief message, as he said during a visit to The Post, is that "religion is not in contradiction with democracy and not in contradiction with human rights and justice."
Ennahda has formed a coalition with non-religious parties. It put many women on its slate of candidates. It has committed to a Tunisia in which men and women, believers and nonbelievers, will be welcome to participate.
An auspicious start does not guarantee that religious parties will remain faithful to democracy over time. And, as Mr. Ghannouchi acknowledged, the situation in Egypt, where the Muslim Brotherhood is expected to do well in the first election, is more complicated. For example, at least 10 percent of Egyptians are Christians, which is not the case in Tunisia, he noted.
But Mr. Ghannouchi said that the dictators' persecution of moderate Islamists — he was jailed, tortured and eventually exiled — left the field open to more radical Islamist forces. "One of the big changes of the Arab Spring is not just the fall of dictators but also that terrorism has been highly damaged, because terrorists used to say that they are the only way to get rid of dictators," Mr. Ghannouchi said.
There are many ways the Arab Awakening might veer off track, and religion-inspired constriction of freedom is one. But so far in Egypt, the greatest threat to democracy has come from the military rulers. In any true Arab democracy, Islamist parties will win a lot of votes. As long as they are willing to play by the rules, those parties should not be treated as a specter to be feared.


text:
The biggest obstacle to the Taurus / Cancer couple's good deal is without a doubt the lack of imagination and a good deal of madness.
It is no secret that Taurus holds the key to the success or failure of the union, because it is he who will command. What Cancer does will depend on the behavior of its partner rather than on its own initiative.Taurus will try to show authority and austerity, but will not resist the charm of Cancer. The latter will have the trick to yield on all secondary points to win on the essential points, namely the table and the bed. Taurus will be drawn, in spite of himself, into a great whirlwind of sensuality. The couple will thus experience moments of ecstatic happiness and moments of laborious harmony.
Sexual Compatibility between Cancer and Taurus
For the Taurus woman, sexuality and feelings go hand in hand. It is when she is deeply in love or emotion that she is also the most receptive sexually. However, if his body is not disturbed or upset from top to bottom, his heart is likely to remain marble. She has a vigorous and demanding sexuality. To fill her, her partner must know how to master the whole range of sensuality - from the most subtle caress to the most direct act. The erotic temperament of the Taurus woman is powerful and enduring. That makes her one of the most talented lovers. If his partner is up to it, he can with it be among the privileged who have access to the highest peaks of pleasure. The Bull woman does not suffer strictly speaking of sexual inhibitions. However, here as elsewhere in her life, she is rather traditionalist. For her, there are things that are not done in bed: these are generally the practices that are generally called "sexual perversions".
The Taurus man is essentially a being of flesh: he is sensual to the tips of his nails, both figuratively and literally. For him, sexual pleasure is something perfectly natural. Unless he has been conditioned by a particularly perverse education, he suffers neither inhibitions nor taboos. In sexuality, everything seems good to him, and he intends not to lose a single crumb. He despises the "saboteurs" who do not go out of their way to offer a woman all the pleasure she expects. He plays her like a harp - with fervor, efficiency, gratitude - convinced that the more he gives pleasure, the more he has. But the Taurus man may seem too modest to some of his partners. Indeed, he fears confessions and is miser compliments. Romantic flourishes are not his forte. In his sexual encounters, he prefers to go straight to the point, as if he was afraid of missing him. And yet, he never hurries: he takes his time to make all the fibers of his body flutter one after the other. Of all men, he is the most fervent adept of love in slow motion and loves to play extra time.


text:
Q: CSS Drop Down Menu Not Working In Wordpress But Works On Every Other Page I've created a custom theme based on a template and for some reason the CSS drop down menu that appears on every other page when you hover of 'Services' doesn't seem to work on the wordpress page.
I copied and pasted the code I created into the Header.php file along with the same styles.
Anyone have any ideas?
The website can be fond here...
http://www.globalpurchasingsolutions.co.uk/blog/
It should work as it does here...
http://www.globalpurchasingsolutions.co.uk/testimonials.html
Any help on this matter would be very much appreciated.
Here's what the CSS Code Is Like
nav {
margin-top:20px;
text-align:left;
float:right;
margin-right:10px;
}

nav ul ul {
display: none;
}

nav ul li:hover > ul {
display: block;
}

nav ul {
background:#FFF;
color:#336699;
list-style: none;
position: relative;
display: inline-table;
width:auto;
}

nav ul:after {
content: "";
clear: both; display: block;
}

nav ul li {
float: left;
}

nav ul li:hover {
border-bottom:solid 2px #336699;
}

nav ul li a {
display: block;
padding: 15px 20px 15px 20px;
color: #2B66A2;
font-size:19px;
text-decoration: none;
}

A: I messed around with Firebug and wasn't able to solve this.
Have you tried using 'master.css' directly in the WordPress theme instead of 'header.css' and 'style.css'?
There are various combinations of things going on, and using different stylesheets will certainly complicate things now and moving forward.
I just tried removing header.css and style.css and replacing it with master.css with Firebug... there were some other issues going on, but the dropdowns started working.


Troubleshooting information for the iPhone app is provided below. If you still have problems after reading this page, please contact us.
1. Is your device jailbroken or "hacktivated"?
2. Double check your reminder settings.
Please see the reminders guide for instructions on setting your preferences. You can check that your preferences are working by setting up a different reminder method (e.g., email) and ensuring that you receive reminders at the correct time.
Hold the power button until "slide to power off" appears.
After the device is off, press the power button to turn it back on.
Tap the 'RTM' icon to relaunch the app.
Press and hold the Sleep/Wake button for a few seconds until the red "slide to power off" slider appears, then slide the slider.
Press and hold the Sleep/Wake button until the Apple logo appears to power on your device.
2. Make sure Push Notifications are enabled.
RTM should be listed under your device's Settings | Notifications. If not, please remove and reinstall the app and press OK in the prompt for notifications.
If it is listed, please make sure the Badge setting is in the ON position.
3. Check count of tasks in the app.
In the app, if the count of incomplete/today's tasks matches the app badge, then the app may be out of sync. If tapping the Sync button in the lower left doesn't help, then please contact us below.
4. Check count of tasks in the web app.
If the count of tasks in the app doesn't match the count visible in the app badge, then it would be worthwhile to check the count in the web app. (This is the count sent to the device via a push notification.) If this appears different than what the app shows, syncing the app or trying these steps would help.
iOS controls this behavior, and it appears to be time-based. If the device is unlocked shortly after receiving the Push Notification (i.e., within seconds), the app is automatically launched. If the device is unlocked a few minutes after receiving the Push Notification, the app isn't automatically launched. Unfortunately there is no way to customize this behavior for the app.


WINDHOEK, 25 FEB (NAMPA) – Over 25 000 people are currently on the National Housing Enterprise (NHE)'s waiting list for houses countrywide, which list is growing on a daily basis.
The rapid increase is also due to the fact that the NHE has been mandated by Government to coordinate its mass housing development programme.
The programme aims to build at least 185 000 affordable houses by the year 2030.
The NHE's Senior Manager for Business Development and Operations, Willem Titus told Nampa on Friday some of those names have been on the list since the year 2000.
Titus said some people who have registered for houses some years back could not receive houses because they did not qualify for houses then.
He said last year, about 200 people who registered in 2007 received their houses in Windhoek's Otjomuise area.
Titus noted that in order to make the process of registration easier for all, the NHE last year introduced an electronic registration system to avoid duplications and shifting of people on the waiting list.
The system provides applicants with printed receipts as evidence that they have applied on a specific date. Before, applicants did not receive any proof of registration for NHE houses.
The first phase of the mass housing programme, which commenced recently with the appointment of builders and other contractors, is estimated to cost N.dollars 2,7 billion in the 2014/15 to 2015/16 financial years.
The NHE's Chief Executive Officer, Vinson Hailulu recently called on Namibians to register their names on the waiting list at any NHE office in order to get houses from NHE.
"I would like to encourage people who fall within the mid- to low-income categories to register themselves at NHE offices and local authority offices to be considered for entry onto the waiting list," he said.
A South African construction company, Calgrow M3, was contracted to build about 1 191 houses at an average cost of N.dollars 345.3 million in Windhoek.
The land for those houses in Otjomuise's Extension 10 was acquired from the City of Windhoek (CoW).
Government has provided N.dollars 310 million to the CoW under its Targeted Intervention Programme for Employment and Economic Growth (Tipeeg) for the serving of land in low-cost areas.
Hailulu could, however, not indicate the minimum cost per house.
He said in terms of financing, the mass housing programme will be funded by government through grants and subsidies, of which subsides will mainly go towards land development, building input, cost mitigation, rural sanitation and programme management.
President Hifikepunye Pohamba launched the national mass housing development programme here in November last year.
The programme will be implemented in phases, and is projected to cost N.dollars 45 billion in total.
A total of N.dollars 220 million was allocated to the NHE for the 2014/15 financial year, compared to N.dollars 100 million in 2013/14.


Christina Aguilera needed a protector, someone who would allow her to follow her own vision, she signed with Irving Azoff.
Phish was a band up from the streets, er, frat house floor, they ended up being managed by John Paluska.
Would Phish have been better off with Azoff? Would Aguilera have been better off with Paluska? No. Each had the perfect manager. Which one do YOU need?
By time Irving got ahold of her, Christina Aguilera was already a star. But it appeared her career was being run by her label. She wanted to call her own shots, she needed a buffer between her and her record company. Also, someone connected enough that they knew all the players and were owed big time favors. Is there an awards show Christina DOESN'T play?
Phish needed to belong to the people, not the system. They needed someone who could build them from the ground up. Their game was the road, not discs. They liked to improvise, it was about the concert experience. If they had played an awards show early in their career it would have killed them.
In other words, you don't need the heavyweight, well-connected manager to make it. Unless you're playing the traditional radio/TV hit single game.
If you're playing the old wave game, sign with someone who's got the chops, who knows the ropes, who isn't reinventing the wheel so much as applying what they know to your situation. You might grow, but your handler, he already knows the game.
Whereas if you're not signed to a major label, don't want to get signed to a major label, don't make Top Forty singles, then you don't need a manager connected so much as one who is savvy and HUNGRY!
The established management players are akin to mini-conglomerates, they're the new labels. They want to get paid, right away. If you're not delivering cash, if they sign you, you're not getting much attention. Or, you're getting attention from the untested newbie. If that newbie is truly great, break off and do it yourself, as Irving Azoff did with the Eagles. Otherwise, you're probably going to get lost in the shuffle.
If you're starting from ground zero, no name manager will probably be interested. But that won't hurt you.
What do you need most if you're a developing act?
Gigs. You need someone to get on the horn, go down to the venue, and cajole and connive 'til they get you a shot. Then you must deliver, but it's the manager that creates/midwives the opportunity.
Where are you going to find such a bloke?
Look around you, he's probably already a friend. Or that dude who comes to each and every show and hangs backstage and won't leave until you do. THAT'S your manager.
Oh, don't throw out your instincts. After all, Paluska went to Amherst, he's no dummy. But find someone committed who will do the job for you.
So much of what Phish did, they did first. Or their spin on an event made it unique. They did their destination festivals. They released live albums of their shows, and then downloads. Elektra didn't deliver these, rather their manager and agent, Chip Hooper, did.
Yup, Chip saw the numbers, he wanted to represent Phish. He didn't care about record sales, but TICKET COUNTS! Most agents feel the same way today, but fifteen years ago, the focus was on the label.
Yes, after you get your manager, and he gets you gigs, he tries to get you an agent. And the agent you want is not the one with the name, the one who wines and dines you so much as the one who BELIEVES in you.
As for music… In today's market, you've got to allow recording and trading, you've got to give the music away for free, you've got to let the seed grow into a tree. If you can't get significant airplay, THIS IS THE ONLY WAY!
It's not the way of the major label, and not the way of the old line manager. But it's your way. You know the Net, you've got friends, both online and offline. You have to create something incredible and give your peeps the tools to spread the word. Not through fake incentives, you've got to trade purely on their belief, your honesty.
It's all about the music when you're doing it yourself. Everything must be subservient to the music. And you must create the best situation to experience the music.
When Phish played the Santa Monica Civic ten plus years ago, the police frisked the attendees. Paluska vowed to never play the building again, and his band DIDN'T! He didn't shrug his shoulders and say he couldn't do anything, that it wasn't his fault, he took matters into his own hands, to defend his band's relationship with its audience.
And when Phish started selling its music online, it offered FLAC files, so its fans could own the best sounding versions. Isn't it funny that EMI is offering 256 kbps AACs supposedly sometime in the near future when Phish sold CD quality YEARS AGO!
So don't lament that the manager with the name isn't interested in you. There's a good chance he might not be right for you.
Inexperience is no longer the handicap it used to be. Drive and appreciation of the band/fan relationship are paramount for today's touring acts. That's more about instinct than big time experience. Furthermore, you want someone who can develop on the fly.
Maybe you outgrow your manager, you end up signing with one of the big boys, who wrings out every last dollar for you.
Or maybe you stay with your guy, who delivers for you.
Or maybe your guy makes a deal with Irving, and uses Frontline's power to get you what you want and need.
It's a new game. It's the sixties all over again. The wheel is being reinvented. Don't be hamstrung by the old wave players and the old wave rules.


DDM premium 9500 super high stall high-RPM clutch spring for Zenoah and Chung Yang engines with 54mm clutch. Designed for RC use. This upgraded spring, custom-made of top-quality materials in the USA, offers unparalleled reliability and performance. Stock spring engages at approx 6000 RPM; this spring is 40% stiffer for clutch engagement around 9500 RPM. The clutch will begin to touch at around 8500 rpm, with full engagement around 9500 rpm. Improve your off-the-line power with a higher engagement RPM!


text:
PATRICK BROUSSEAU
pbrousseau@ateqaviationusa.com
About Patrick Brousseau
As Sales Manager for ATEQ Aviation and Cobra Systems, Patrick is responsible for all Pitot-Static Test Solutions development and sales. Patrick delivers first class technical knowledge and customer service. Patrick holds an Airframe and Powerplant license and has advanced training in both Rolls Royce and Pratt & Whitney Engines. He is a graduate of the Michigan Institute of Aviation & Technology. Patrick resides in the Chicagoland area with his wife and two sons (soon to be three).
STEVE MAST
Director of Military Sales
smast@cobrasys.com
About Steve Mast
Steve's entire career has been in the field of pitot-static testing. Fresh out of college, with a passion for aviation, Steve joined the Cobra team in 2013. He has experience with just about every aircraft type that was ever manufactured and we lovingly call him our aviation encyclopedia. Steve has provided Pitot-Static testing solutions for our nation's defense contractors, OEMs, MROs and just about every maintenance shop that has a need for Pitot-Static testing equipment. While in the Civil Air Patrol, Steve was taught this great life lesson … "Precision and Calibration – Do it right, then do it right again." Steve resides in city of Chicago and is a die-hard Chicago Cubs fan.
DAN ROMAIN
dromain@cobrasys.com
About Dan Romain
Dan's sales career has covered many specialties but aviation is his love. Dan joined Cobra Systems in early 2017 and has expanded the pitot static test solutions customer base in EMEA, Russia, Canada as well as the Mountain and Central U.S. regions. Dan is an aspiring pilot and integrates his love of aviation with his passion for animal rescue. He lives in Evanston with his daughter, wife and 3 shelter pets.
DAN KISKA
dkiska@cobrasys.com
About Dan Kiska
"I can build anything you want." Meet the backbone of Cobra Systems, Dan Kiska. Dan has 15+ years of technical experience and can build any pitot-static adapter to fit any pitot-static probe or port. When Dan isn't creating magic in the shop, he is implementing new procedures on safer, faster and more efficient testing. He is also a mentor for our aviation youth, training level 1 technicians. And on top of all of that, he is a world class fisherman. Dan resides in the Chicagoland area with wife and daughter.
ANDREW LINDBERG
Lead Production Specialist
andrew@cobrasys.com
About Andrew Lindberg
Andrew started with Cobra Systems fresh out of college. He has been with the company for over 3 years now and is a key asset in expanding and creating efficiency in production and inventory. Andrew graduated from the University of Wisconsin/ Platteville with a degree in Manufacturing Technology Management. He is responsible for design, production and inventory management. In his spare time, he enjoys 3-D printing, making toys for his family.
ELIAS ZAVALA
ATEQ Aviation Service and Support Technician
ezavala@ateqaviationusa.com
About Elias Zavala
As support and service technician, Elias provides calibrations and repairs on ATEQ Aviation products and verifications on any pitot-static test air data test set. He has one of the fastest turn-around times in the industry. Elias is a veteran of the US Air Force. He performed his military service as an Electrical and Environmental Systems Specialist working on C130, C17, C5 and B52 aircraft. Elias resides in the Chicagoland area with his family and pet dog, Wrigley. He is an avid Cubs and Bears fan and when he is not spending time with his family, he enjoys woodworking, playing guitar and gaming.
JAKE GOETTLEMAN
Level 2 Technician
jacob@cobrasys.com
About Jake Goettleman
Jake joined Cobra Systems has an entry level technician being mentored by our production manager, Dan Kiska. Through hard work and great training, Jake is now a level 2 technician proving to be a great asset to the pitot static test industry. Jake's in-depth knowledge of pitot and static adapters give our customers the peace of mind knowing that their products come with expert build and pre-testing. Jake is originally from Ohio, the birthplace of flight, but has made his home in the Chicagoland area (and become a true Cubs fan as he should).
KAREN STURM
Order Entry and Administrative Assistant
ksturm@cobrasys.com
About Karen Sturm
Karen has a combined 15 years' experience in office management, customer service and logistics organization. She graduated from MacCormac College with an Associate of Science degree in Paralegal Studies. Karen enjoys reading and when she isn't enjoying a good book, she spends time with family, friends and her son when he is home from college. If you are looking for a receipt or an invoice please do not hesitate to contact Karen
DENIS SICHIK
dsychik@cobrasys.com
About Denis Sichik
Denis is a Hydraulic/Mechanical Engineer with over 8 years of progressive experience in the design of electromechanical equipment, hydraulic and pneumatic systems/components, waste water treatment systems, HVAC equipment and metal constructions. Denis is tasked with engineering new pitot and static adapters as well as improving on designs already in use. In his previous role Denis created the first ever atmospheric water generator, incorporated into the Ford F-150, generating water directly in the truck. Denis is originally from Belarus then spending many years in Israel. He currently resides in the Chicagoland area with his wife and daughter. When he isn't spending time with his family, he enjoys extreme sports and traveling.


text:
[![Build Status](https://travis-ci.org/serverscom/brocade_vrouter.svg?branch=master)](https://travis-ci.org/serverscom/brocade_vrouter)

## Installation

Add this line to your application's Gemfile:

```ruby
gem 'brocade_vrouter'
```

And then execute:

    $ bundle

## Usage

### Configure

```ruby
BrocadeVRouter.configure do |conf|
  conf.url 'https://examplerouter.com'
  conf.basic_auth ['routeruser', 'routerpassword']
  conf.ssl verify: false
end

router = BrocadeVRouter::Client.new
```

### Or pass config to client instance

```ruby
router = BrocadeVRouter::Client.new url: 'https://examplerouter.com',
  basic_auth: ['routeruser', 'routerpassword'],
  connection_options: {ssl: {verify: false}}
```

### Example router configuration

```ruby
router.configure do
  set security do
    vpn 'l2tp remote-access authentication local-users' do
      username 'testuser' do
        password 'testpass'
        static_ip '10.0.2.1'
      end
    end

    firewall name: :testfw do
      default_action :drop
      rule 100 do
        action :accept
        source address: '10.0.2.1'
        destination address: '10.0.2.0/24'
      end
    end
  end

  commit
end
```

CLI equivalent:

```
configure
set security vpn l2tp remote-access authentication local-users username testuser password testpass
set security vpn l2tp remote-access authentication local-users username testuser static-ip 10.0.2.1
set security firewall name testfw
set security firewall name testfw default-action drop
set security firewall name testfw rule 100 action accept
set security firewall name testfw rule 100 source address 10.0.2.1
set security firewall name testfw rule 100 destination address 10.0.2.0/24
commit
```

## Development

After checking out the repo, run `bin/setup` to install dependencies. Then, run `rake spec` to run the tests. You can also run `bin/console` for an interactive prompt that will allow you to experiment.

To install this gem onto your local machine, run `bundle exec rake install`. To release a new version, update the version number in `version.rb`, and then run `bundle exec rake release`, which will create a git tag for the version, push git commits and tags, and push the `.gem` file to [rubygems.org](https://rubygems.org).

## Contributing

Bug reports and pull requests are welcome on GitHub at https://github.com/serverscom/brocade_vrouter. This project is intended to be a safe, welcoming space for collaboration, and contributors are expected to adhere to the [Contributor Covenant](http://contributor-covenant.org) code of conduct.

## License

The gem is available as open source under the terms of the [MIT License](http://opensource.org/licenses/MIT).

redpajama_set_name: RedPajamaGithub