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Plasma-accessible carbonic anhydrase at the tissue of a teleost fish may greatly enhance oxygen delivery: in vitro evidence in rainbow trout, Oncorhynchus mykiss
Jodie L. Rummer, Colin J. Brauner
For correspondence: jodierummer@gmail.com
During a generalized acidosis in rainbow trout, catecholamines are released into the blood, activating red blood cell (RBC) Na+/H+ exchange (βNHE), thus protecting RBC intracellular pH (pHi) and subsequent O2 binding at the gill. Because of the presence of a Root effect (a reduction in oxygen carrying capacity of the blood with a reduction in pH), the latter could otherwise be impaired. However, plasma-accessible carbonic anhydrase (CA) at the tissues (and absence at the gills) may result in selective short-circuiting of RBC βNHE pH regulation. This would acidify the RBCs and greatly enhance O2 delivery by exploitation of the combined Bohr-Root effect, a mechanism not previously proposed. As proof-of-principle, an in vitro closed system was developed to continuously monitor extracellular pH (pHe) and O2 tension (PO2) of rainbow trout blood. In this closed system, adding CA to acidified, adrenergically stimulated RBCs short-circuited βNHE pH regulation, resulting in an increase in PO2 by >30 mmHg, depending on the starting Hb-O2 saturation and degree of initial acidification. Interestingly, in the absence of adrenergic stimulation, addition of CA still elevated PO2, albeit to a lesser extent, a response that was absent during general NHE inhibition. If plasma-accessible CA-mediated short-circuiting is operational in vivo, the combined Bohr-Root effect system unique to teleost fishes could markedly enhance tissue O2 delivery far in excess of that in vertebrates possessing a Bohr effect alone and may lead to insights about the early evolution of the Root effect.
A reduction in blood pH during blood capillary transit enhances O2 delivery in vertebrates through the Bohr effect, a physiological mechanism that has been studied extensively for over a century and defined as the decrease in haemoglobin (Hb)-O2 affinity with a reduction in blood pH (Bohr et al., 1904; Nikinmaa and Soivio, 1979; Nikinmaa, 1997). In addition to the Bohr effect, teleost fishes also possess a Root effect, where a reduction in pH not only decreases Hb-O2 affinity but also greatly reduces the O2 carrying capacity of blood (Root, 1931; Root and Irving, 1943; Scholander and Van Dam, 1954). Within a given teleost blood system, it may be impossible to separate the shift associated with the Root effect from the traditionally understood Bohr shift; therefore, in teleosts possessing Root effect Hbs, the shift is referred to as the combined Bohr-Root effect. The Root effect is used to great advantage for filling a swimbladder against large pressure gradients (>5066.5 kPa) associated with depth (Scholander and Van Dam, 1954) and for oxygenating the metabolically active yet avascular retinal tissue of the eye (Wittenberg and Wittenberg, 1962; Wittenberg and Wittenberg, 1974; Waser and Heisler, 2005). Harnessing this potential is thought to be dependent on localizing and recycling an acidosis via a unique vascular architecture, the rete mirabile at the swimbladder (Scholander, 1954) and the choroid rete at the eye (Wittenberg and Haedrich, 1974; Wittenberg and Wittenberg, 1974). With respect to general O2 delivery, however, the role of the Root effect has received little attention probably because it is understood that the associated Haldane effect would actually minimize blood pH changes in the tissues (Lapennas, 1983). In this study, we propose a novel mechanism in fish blood that exploits the presence of plasma-accessible carbonic anhydrase (CA) in the tissues to increase H+ influx to the red blood cells (RBCs) during blood capillary transit and exploit the combined Bohr-Root effect to greatly enhance general O2 delivery.
Most teleost fish that exhibit a pronounced Bohr-Root effect adrenergically regulate RBC pH to maintain O2 loading at the gills (Berenbrink et al., 2005). Catecholamines (e.g. adrenaline and noradrenaline) are released into the general circulation and bind to β-adrenergic receptors on the RBC membrane that, via adenylate cyclase and 3′,5′-cyclic adenosine monophosphate (cAMP), activate β-adrenergic Na+/H+ exchange (βNHE) (Mahé et al., 1985). The CA-catalyzed hydration of CO2 inside the RBC produces H+ that are removed in exchange for Na+ via βNHE (Baroin et al., 1984; Cossins and Richardson, 1985), and HCO -3 that is removed via anion exchange for Cl- at a slower rate. This combination results in an increase in intracellular pH (pHi) and an increase in Hb-O2 affinity (Nikinmaa, 1983; Baroin et al., 1984; Cossins and Richardson, 1985; Borgese et al., 1986; Borgese et al., 1987). The H+ removed from the RBCs acidify the plasma, resulting in a decrease in extracellular pH (pHe) (Nikinmaa, 1983; Baroin et al., 1984; Cossins and Richardson, 1985; Borgese et al., 1986; Borgese et al., 1987). In the plasma, the H+ will eventually combine with HCO -3 at an uncatalyzed rate to form CO2, resulting in a slow plasma alkalinization after the initial pH decrease (Fig. 1A) (Lessard et al., 1995; Geers and Gros, 2000). Adrenergic RBC βNHE is thought to have evolved to safeguard O2 uptake at the respiratory surfaces during a generalized acidosis in the presence of Bohr-Root shift Hb (Nikinmaa et al., 1984; Primmett et al., 1986; Borgese et al., 1987; Perry and Kinkead, 1989; Malapert et al., 1997).
(A) Schematic illustrating the cascade associated with red blood cell (RBC) adrenergic stimulation at the level of the tissue. Modified from Heming (Heming, 1984), Bidani and Crandall (Bidani and Crandall, 1988) and Cardenas et al. (Cardenas et al., 1998). (B) Simplified mechanism, such that the proposed short-circuiting of RBC βNHE pH regulation upon initial contact with plasma-accessible carbonic anhydrase (CA; in red) at the tissues and associated changes in extracellular pH (pHe), intracellular pH (pHi) and the partial pressure of oxygen (PO2) can be outlined. AE, anion exchange; ATP, adenosine triphosphate; cAMP, 3′,5′-cyclic adenosine monophosphate; Hb, haemoglobin; NKA, Na+/K+-ATPase; βNHE, β-adrenergically activated sodium proton exchanger.
Conceptually, the presence of CA in the plasma would short-circuit pH regulation associated with adrenergic activation of RBC βNHE (Motais et al., 1989; Nikinmaa et al., 1990). Although plasma-accessible CA is not present in the teleost gill, membrane-bound plasma-accessible CA (e.g. CA IV-like isoforms) may exist in select locations such as bound to muscle endothelia (Effros and Weissman, 1979; Siffert and Gros, 1982; Decker et al., 1996; Henry et al., 1997; Geers and Gros, 2000). Indeed, fish are thought to possess plasma-accessible CA isoforms similar to mammalian CA IV, but their location and function remain undetermined (reviewed in Gilmour and Perry, 2009). We propose that if CA is available to the plasma in tissue capillaries, H+ removed from the RBC via βNHE could combine with plasma HCO -3 to reform CO2, which would back-diffuse into the RBC, decrease pHi and ultimately create a larger arterial to venous pH gradient (ΔpHa-v) at the tissues than would otherwise occur (Fig. 1B). The large acidosis transferred to the RBC would elevate the partial pressure of O2 (PO2) via the combined Bohr-Root effect, thus greatly facilitating tissue O2 delivery. Furthermore, provided the rate of short-circuiting of βNHE RBC pH regulation in the tissue and subsequent pHi recovery during transit to the gill was sufficiently rapid, a generalized acidosis could provide H+ that could be repeatedly used at select tissues (to increase the ΔpHa-v) with every pass through the circulation, thus elevating tissue PO2 at a time when O2 delivery is especially needed.
As a first step, this study was designed to demonstrate proof-of-principle for enhanced O2 delivery when adrenergically stimulated RBC βNHE pH regulation is short-circuited. Rainbow trout blood was pre-equilibrated at pre-defined Hb-O2 saturations and then, in a closed system, acidified, β-adrenergically stimulated and then exposed to CA. Changes in both pHe and PO2 were monitored continuously to assess both the magnitude and time course of the response. It was hypothesized that in this in vitro closed system, and in the presence of an acidosis, plasma-accessible CA short-circuits pH regulation associated with adrenergically stimulated RBC βNHE, thus creating a decrease in RBC pH that elevates the driving force for O2 delivery, ΔPO2, because of the combined Bohr-Root effect. The overall aim was to determine whether βNHE short-circuiting could be operational in vivo and estimate the degree to which it might influence O2 delivery.
Animals and rearing conditions
Rainbow trout, Oncorhynchus mykiss Walbaum 1792 (300-600 g wet body mass), were obtained from Spring Valley Trout Farm (Langley, British Columbia, Canada) and maintained at the University of British Columbia Aquatic Facilities. Fish were held under a natural photoperiod at densities no greater than 10 kg m-3 (North et al., 2006) in 4000 l tanks supplied with flow-through 10°C Vancouver dechlorinated municipal tap water. Fish were fed every other day to satiation using commercial trout pellets (Skretting, Orient 4-0, Vancouver, BC, Canada). All experiments were completed during the spring months over two separate years. All procedures complied with the guidelines approved by the Canadian Council on Animal Care (UBC protocol no. A07-0080).
Sampling protocol
Fish were quickly anaesthetized in a 20 l bucket of clean, well-aerated water containing benzocaine solution (0.2 mmol l-1 final concentration p-aminobenzoate). Fish were then placed on a surgery table, and their gills were intubated and continuously irrigated with water containing a more dilute anaesthetic (0.02 mmol l-1 p-aminobenzoate). An indwelling cannula (PE50) was surgically implanted into the dorsal aorta according to Soivio et al. (Soivio et al., 1975). Following surgery, fish were placed in a Perspex box supplied with aerated 12°C clean water and gently force-ventilated until they regained equilibrium. Fish were left to recover for at least 24 h prior to sampling, during which time cannulae were flushed twice with heparinized Cortland's saline (10 i.u. ml-1 lithium heparin, Sigma-Aldrich catalog no. H0878, St Louis, MO, USA) (Wolf, 1963). Prior to experimentation, blood was removed from the cannula into a heparinized syringe, but at the first sign of struggling, no further blood was removed to ensure negligible plasma catecholamine levels. Blood was pooled from two to three fish and haematocrit (Hct) was measured in duplicate by centrifuging 60 μl of whole blood in heparinized micro-capillary tubes for 3 min at 17,000 g. Prior to experimentation, the pooled blood sample was standardized to a Hct of 25% by removing either plasma or RBCs. Aliquots of approximately 2.5 ml were added to four Eschweiler tonometers. Tonometers containing blood were equilibrated for 1 h at 12°C (LAUDA Brinkman™ Model S-1 recirculating chilling unit, Delran, NJ, USA) with a humidified gas mixture, varying in O2 proportions regulated by a gas-mixing pump (DIGAMIX 275 6KM 422, Wösthoff, Bochum, Germany; PCO2=0.5%, balance N2). The aim was to incubate blood at O2 tensions above 20% but below 80% Hb-O2 saturation to cover the range of Hb-O2 saturation that likely occurs in venous blood in vivo. Nominal values of 30, 50, 65 and 75% Hb-O2 saturation were targeted and the required incubation PO2 was determined from the rainbow trout oxygen equilibrium curves (OECs) generated in a previous study at 12°C and 0.5% CO2 (Rummer, 2010). Following incubation of the tonometers at respective gas proportions, a subsample of blood (600 μl) was removed so that haemoglobin concentration ([Hb]), Hct, pHe and pHi could be measured. The remaining blood was then loaded into the closed system for experimentation, as described below.
Closed-system preparation
Following blood tonometry, a 2 ml aliquot of blood was drawn into a pre-gassed Hamilton™ syringe and slowly ejected into a pre-gassed 2 ml glass vial until overflow, at which time the vial was sealed with a septum. A pre-calibrated fiber optic implantable O2 sensor and a fiber optic implantable pH sensor (PreSens, Loligo Systems, Tjele, Denmark; tip diameters 50-140 μm), presoaked in heparinized Cortland's saline, were inserted through the septum to continuously monitor blood PO2 and pH in the closed system. The vial, thermostatted at 12°C, was equipped with a small stir bar (7×2 mm) and positioned on a stir plate set at 400 revolutions min-1 to ensure adequate mixing throughout the experiment. Oxygen and pH signals were amplified using an Oxy-4 micro four-channel oxygen meter and signal amplifier (Loligo Systems, catalog no. OX11700) and a pH-1 micro single-channel meter (Loligo Systems, catalog no. PH10450), respectively. Data were collected throughout the duration of each experiment at a sampling rate of 1 s-1, and integrated with the manufacturer's software packages for Windows. All data were saved as text files and analyzed using Acqknowledge® Data Acquisition Software (Version 3.7.3, BIOPAC Systems, Inc., Goleta, CA, USA). For representative traces, every other data point was imported into SigmaPlot for Windows 10.0.1.25 (Systat Software Inc., San Jose, CA, USA).
Series 1: β-adrenergic stimulation during an acidosis followed by CA exposure
In the closed system, rainbow trout RBCs were β-adrenergically stimulated with isoproterenol (ISO) during the HCl-induced acidosis and then subsequently exposed to CA. Blood PO2 and pH were allowed to stabilize over the first 5 min in the closed system (time zero). When steady readings were observed for at least the last minute of this period, a 50 μl Hamilton™ syringe was used to inject 20 μl of 100, 150 or 200 mmol l-1 HCl prepared in Cortland's saline to achieve a final concentration of 1, 1.5 or 2 mmol l-1, respectively. This resulted in a nominal 0.15, 0.30 or 0.50 pH unit reduction in blood pH, respectively (see Table 2 for actual pH values). Blood PO2 and pH reached maximum change within 2-3 min following acidification, and after 5 min, 20 μl of the β-adrenergic agonist ISO (Sigma-Aldrich catalog no. I5627), prepared fresh in Cortland's saline, was added. The final concentration used (0.01 mmol l-1) is known to elicit a maximum response in rainbow trout blood (Caldwell et al., 2006) (Table 1). After 5 min, CA (from bovine erythrocytes, E.C. 4.2.1.1, Sigma-Aldrich catalog no. C3934) prepared in Cortland's saline was injected into the system for a final concentration of 10-3 mmol l-1. This concentration is similar to concentrations in mammalian RBCs and a concentration previously shown to short-circuit βNHE in rainbow trout blood in vitro (Table 1) (Nikinmaa et al., 1990; Henry et al., 1997). This experimental treatment will be referred to as HCl+ISO+CA (Fig. 2).
The effect of subsequent additions of HCl, isoproterenol (ISO) and carbonic anhydrase (CA) on ΔPO2 and pHe, and associated half times (t1/2) in rainbow trout blood in vitro in a closed system
Concentrations of carbonic anhydrase (CA), catecholamines [noradrenaline (NA) and adrenaline (AD)] and adrenergic agonists [isoproterenol (ISO)] that have been used or measured in previous studies
Series 2: acidosis followed by CA exposure
In the closed system, rainbow trout RBCs were exposed to an HCl-induced acidosis and subsequently exposed to CA, omitting β-adrenergic stimulation from the series. Only one acidification level (100 mmol l-1 HCl) was used, which decreased pHe by 0.15 units. The CA concentration used and all other preparations and analyses were identical to those use in Series 1. This experimental treatment will be referred to as HCl+CA. Although it was assumed that βNHE activation would be absent in this series because ISO was omitted, a separate trial was conducted in the presence of a β-antagonist, propranolol (Sigma-Aldrich catalog no. P0884), using a final concentration of 2×10-5 mol l-1 (Fuchs and Albers, 1988; Motais et al., 1989) (data not shown). Propranolol competes with β-agonists at the level of the receptor, and so if catecholamines were present in the plasma, they would not bind at the RBC and activate the βNHE under this series of exposures.
Series 3: inhibiting RBC Na+/H+ exchange during an acidosis followed by CA exposure
In the closed-system, rainbow trout RBCs were exposed to an HCl-induced acidosis and then in the presence of a Na+/H+ exchange (NHE) inhibitor, subsequently exposed to CA. Ethylisopropylamiloride (EIPA) (Sigma-Aldrich catalog no. A3085) is a potent inhibitor of NHE, specifically NHE1 (Kristensen et al., 2007). In this series, it was used to validate the role of an NHE (adrenergic or non-adrenergically activated) in the short-circuiting model. The only incubation condition used was 0.5% CO2, 65% air, balance N2. At 5 min following HCl addition, EIPA was injected into the system (0.1 mmol l-1 final concentration). After 5 min, CA was injected into the system for a final concentration identical to that used in Series 1. All other preparations and analyses were identical to those used in Series 1. This experimental treatment will be referred to as HCl+EIPA+CA.
Controls and blood analysis
Sham injections of Cortland's saline were also introduced at every interval in a separate and final control experiment to account for potential injection effects (data not shown, as no effects were observed). At 30 min in all experiments, blood was removed for final analysis. Hct was determined in duplicate after centrifuging two filled heparinized Hct tubes at 17,000 g for 3 min. Hb was measured in duplicate using the cyanomethaemoglobin method and an extinction coefficient of 11 mmol l-1-1 cm-1 at 540 nm. The remaining aliquot of blood was centrifuged at 4000 g for 3 min, plasma was removed and discarded, and RBCs were immediately frozen in liquid nitrogen and stored at -80°C until later analysis. pHi was measured in duplicate using a thermostatted BMS 3 Mk2 Blood Microsystem (Radiometer, Copenhagen, Denmark) in conjunction with a Radiometer PHM73 acid-base analyzer after samples were prepared using the freeze/thaw method of Zeidler and Kim (Zeidler and Kim, 1977).
Representative traces were chosen for both the HCl+ISO+CA and the HCl+CA experiments. Otherwise, data are presented as means ± s.e.m. For every level of acidification at every starting Hb-O2 saturation used and in each experiment, sample size was N=6 (Table 2). For all responses, time to half-maximal response (t1/2) was calculated by using a double reciprocal plot, therefore likening the parameters to Michaelis-Menton enzyme kinetics and using a Lineweaver-Burke plot. Data were compared statistically within acidification treatments and with baseline values. When necessary, statistical differences were detected via one-way ANOVA. All data satisfied the assumptions of normality (Kolmogorov-Smirnov test) and homogeneity of variance (Bartlett's test). When a significant difference was identified, a post hoc Holm-Sidak multiple comparisons test was applied to compare means. All statistical analyses were performed using SigmaStat 3.5 (Systat Software) statistics software using a significance level of α<0.05.
Representative trace documenting changes (min) in Oncorhynchus mykiss blood PO2 (red) and pHe (blue) in the in vitro closed system over the 30 min duration of the experiment for Series 1 (HCl+ISO+CA). Dashed vertical lines represent the time at which the blood was exposed to the respective treatment indicated on the x-axis. CA, carbonic anhydrase; HCl, hydrochloric acid; ISO, isoproterenol. Means ± s.e.m. for all variables measured or calculated in Series 1 are reported in Table 2.
The mean starting Hct, pHe and pHi immediately following tonometry was 25.0±0.1%, 7.93±0.02 and 7.40±0.00, respectively. Within each acidification group, experiments began with four statistically distinct Hb-O2 saturations (P<0.001), nominally 34, 54, 63 and 68% for the lowest level of acidification, 32, 59, 66 and 78% for the middle level of acidification, and 30, 53, 63 and 75% for the highest level of acidification (Table 2). The addition of HCl significantly reduced blood pHe by 0.15, 0.33 and 0.49 units, all of which differed significantly from one another (Table 2). Upon HCl addition, there was a rapid and significant increase in PO2 (ΔPO2) of between 55 and 87 mmHg, depending on the starting Hb-O2 saturation and the degree of acidification (P<0.001) (Table 2). The t1/2 for this response was 40.9±2.1 s, pooled for all Hb-O2 saturations and acidification levels. Within a given acidification group, ΔPO2 did not differ significantly among the four different starting Hb-O2 saturations, and therefore values were pooled. There were no significant differences in ΔPO2 among the three acidification groups (P=0.271) (Table 2). However, ΔPO2 values were all significantly different from 0 (P<0.001). For reference, data for this experimental series are presented in tabular format (Table 2), and a representative trace from a single trial is depicted in Fig. 2.
Adrenergic stimulation significantly decreased PO2 in all acidification groups and at all Hb-O2 saturations except for in the lowest two starting Hb-O2 saturations in the group where pHe was decreased by 0.49 units (P>0.05) (Table 2). Qualitatively, pHe decreased, but the change was not significant. Compared with the HCl-mediated response, the ISO-mediated response was twice as slow (t1/2=102.1±8.1 s, pooled for all Hb-O2 saturations and acidification levels, P<0.001; Table 2, Fig. 2).
Subsequent CA addition significantly increased PO2 in every acidification group and at every starting Hb-O2 saturation (P<0.001), except within the group where pHe was decreased by 0.15 units, in the subgroup where starting Hb-O2 saturation was 33.7% (P=0.104; Table 2). Qualitative increases in pHe were evident on most traces (Fig. 2); however, changes in pHe were not significant within or between groups. Overall, the CA-mediated response was two to five times faster than the HCl- and ISO-mediated responses, respectively (t1/2=21.8±2.2 s, pooled for all Hb-O2 saturations and acidification levels, P=0.003 and P<0.001 compared with HCl and ISO, respectively; Table 2, Fig. 2).
pHi was measured only at the start and end of each experiment and was always significantly higher at the beginning of the experiment (P<0.001). The exception was at one Hb-O2 saturation level in the group where pHe was decreased by 0.49 units (P=0.127; Table 2). Differences in final pHi between Hb-O2 saturation levels within each acidification group were only observed in the groups where pHe was decreased by 0.33 and 0.49 units (Table 2). Final Hct, measured as an additional proxy for RBC β-adrenergic stimulation, significantly increased relative to the initial value in all acidification groups at all starting Hb-O2 saturations (P<0.001), resulting in up to a 70% increase in RBC volume (Table 2). A significant correlation existed between the starting Hb-O2 saturation and the decrease in PO2 following the addition of ISO to previously acidified blood (R2=0.706, P<0.001; Fig. 3A). The correlation was also evident with the increase in PO2 following CA addition (R2=0.289, P<0.05; Fig. 3B). When the decrease in PO2 due to ISO was pronounced, the increase in PO2 due to CA was pronounced (R2=0.355, P<0.05; Fig. 3C). This relationship was evident within and among each acidification group (Table 2). Consistent with these responses, in previously acidified blood a significant relationship could be detected between the degree of RBC swelling and the ISO-induced decrease in PO2 (R2=0.394, P<0.03; Fig. 3D).
When adrenergic receptors were inhibited (using the β-antagonist propranolol; data not shown) or stimulation was omitted from the sequence, starting Hb-O2 saturations were nominally 47, 59, 65 and 74% (Table 2). Immediately following tonometry, Hct, pHe and pHi were not significantly different from values measured in Series 1; consequently, Series 1 and 2 starting values were pooled. Following HCl addition, pHe was significantly reduced by 0.15 units, consistent with the lowest level of acidification in Series 1 (Fig. 4). However, as seen in the representative trace (Fig. 4), the HCl-mediated decrease was followed by a slight rise, with pHe reaching a new apparent equilibrium prior to the CA exposure. This overshoot was reflected in the PO2 trace as well. Upon acidification, PO2 increased significantly (P<0.001) by a mean of 49 mmHg (Table 2). The time to half-maximal acidosis was 33.0±4.1 s (pooled for all starting Hb-O2 saturations), but this was not significantly different than the t1/2 for the same level of acidification in Series 1 experiments (Student's t-test, t=1.372, d.f.=6, P=0.219) (Table 2, Fig. 4). CA addition increased PO2 by a mean of 0.3 to 5.9 mmHg, depending on the starting Hb-O2 saturation (Table 2, Fig. 4). The time to half-maximal CA-mediated response was 21.9±3.6 s (pooled for all starting Hb-O2 saturations); this was not significantly different than the pooled t1/2 for the same level of acidification in Series 1 experiments (Student's t-test, t=-0.577, d.f.=6, P=0.585; Table 2). At the end of the 30 min monitoring period, pHi had decreased to 7.10 or lower, and although blood from this experiment was not adrenergically stimulated with ISO, Hct had increased significantly over starting values (P<0.001), but to a significantly lesser degree (16% versus 70% increase) relative to Series 1 (P<0.001; Table 2). For reference, a representative trace is presented in Fig. 4 and mean values are listed in Table 2.
For all acidification groups combined from Series 1 experiments (HCl+ISO+CA), a quantitative representation of the significant correlations between (A) starting Hb-O2 saturation and the decrease in PO2 following the addition of ISO to previously acidified O. mykiss blood, (B) the starting Hb-O2 saturation and the increase in PO2 following CA addition, (C) the decrease in PO2 due to ISO and the increase in PO2 due to CA, and (D) the decrease in PO2 due to ISO and the degree of RBC swelling as represented by the final haematocrit (Hct). Note: starting Hct was 25.02±0.12%. These relationships were also evident within each acidification group; see Table 2 for data separated by acidification group.
Series 3: inhibiting RBC NHE during an acidosis followed by CA exposure
For experiments conducted with EIPA, starting Hct, PO2, pHe and pHi were 25.2±0.1%, 92.8±2.3 mmHg, 7.83±0.03 and 7.20±0.03, respectively, and Hb-O2 saturation was 75.8±1.9%. Acidification significantly increased blood PO2 by 73 mmHg, which reached a maximum value of 165.4±17.9, and pHe significantly decreased by 0.22±0.03 units (Fig. 5). Addition of EIPA did not significantly affect either blood PO2 or pHe. Likewise, CA addition did not significantly affect blood PO2 or pHe (Fig. 5). At the end of the 30 min experimental and recording period, Hct was unchanged from starting values (P>0.05). Blood PO2 continued to fall over the duration of the experiment, reaching 133.8±8.1 mmHg at 30 min, but remained significantly elevated over initial values (P<0.01). Blood pHe stabilized over the last 15 min of the recording period, but was still significantly lower than initial values (P<0.001), as was pHi (7.09±0.03, P=0.016).
The in vitro results from this study are consistent with those from earlier studies (Motais et al., 1989; Nikinmaa et al., 1990), as is our overall hypothesis that during an acidosis, adrenergic RBC pH regulation via βNHE can be short-circuited by plasma-accessible CA. Because of this short-circuiting, Hb-O2 affinity is reduced, resulting in a positive ΔPO2 in this closed system. The increase in PO2 upon CA exposure was in excess of 30 mmHg in some treatments, and occurred twice as rapidly as the increase in PO2 upon acidification without CA. This response also occurred to a lesser degree in the absence of adrenergic stimulation (Fig. 4), but was abolished in the presence of EIPA, which directly inhibits all forms of NHE (Fig. 5). Thus, the addition of plasma-accessible CA to acidified blood, in the presence or absence of adrenergic stimulation, appears to result in a positive ΔPO2 through short-circuiting of some NHE isoform(s). If this mechanism is operational in vivo, short-circuiting the pH regulation associated with RBC NHE in conjunction with a highly pH-sensitive combined Bohr-Root effect could markedly enhance tissue O2 delivery over that which would occur in vertebrates possessing a Bohr effect alone (Rummer, 2010). This may result in further insight into the evolution of Root-effect Hbs, which evolved prior to RBC βNHE and specialized retia at the eye and swimbladder (Berenbrink et al., 2005).
Justification of the chosen parameters
The specific in vitro treatments were chosen to mimic in vivo conditions where possible (i.e. initial Hb-O2 saturations and acidification levels). Concentrations of ISO were used based on information from past studies (Table 1), and excess levels of CA ensured maximal effects in demonstrating proof-of-principle that this mechanism is functional. Starting Hb-O2 saturations (between 30 and 78%) encompassed the region of the OEC most commonly used during activity in rainbow trout venous blood in vivo. The levels of initial acidification (0.15, 0.3 and 0.5 unit decreases in pHe) corresponded to in vivo changes in pHa documented in rainbow trout following exposure to hypoxia or strenuous exercise (Kiceniuk and Jones, 1977; Milligan and Wood, 1987; Nikinmaa and Vihersaari, 1993; Brauner et al., 2000). An acid-base disturbance of this magnitude in vivo also rapidly elevates plasma catecholamine levels (both adrenaline and noradrenaline) from resting levels that are usually less than 2×10-7 mmol l-1 (Tetens et al., 1988) to levels as high as 8.5×10-5 mmol l-1 (Butler et al., 1986; Milligan and Wood, 1987). Furthermore, ISO is a more potent β-adrenergic agonist, and we used concentrations known to generate a maximal βNHE response at the RBCs (Tetens et al., 1988) (Table 1).
Representative trace for Series 2 experiments (HCl+CA). Other details are as in Fig. 2.
Two factors were considered when choosing CA concentrations higher than what might be expected in muscle: the importance of accounting for high H+ appearance in the plasma following RBC βNHE activation, and overwhelming any endogenous CA inhibitors potentially present in the plasma (Dimberg, 1994). The isoform used was from bovine erythrocytes, likely mammalian CA II, which is not expected to be affected by plasma inhibitors, which are thought to be not only species specific but also particular to the RBC isoform (Henry et al., 1997; Peters et al., 2000). The final CA concentrations used in this study exceeded, by 20 times, those found in rabbit white muscle [likely CA IV, enzyme catalytic activity (Kcat) ∼1.1×10-6 s-1, similar to CA II (Hilvo et al., 2008)] (Table 1), a membrane-bound isoform similar to what may be available to rainbow trout muscle in vivo (Effros and Weissman, 1979; Wang et al., 1998). However, the concentrations used were slightly lower than those determined for mammalian RBCs (5×10-3 mmol l-1) (Henry et al., 1997), but consistent with levels of bovine erythrocyte CA previously used to short-circuit β-adrenergically stimulated RBC pH regulation in rainbow trout in vitro (Motais et al., 1989; Nikinmaa et al., 1990) (Table 1).
Representative trace for Series 3 experiments (HCl+EIPA+CA). Means ± s.e.m. are plotted on this trace, as only one starting Hb-O2 saturation and one level of acidification were used for this experiment. Letters that differ within a variable, indicate a significant difference. Other details are as in Fig. 2.
ΔPO2 (mmHg) following addition of CA to the in vitro closed system, representing the potential benefit to O2 delivery that could result from short-circuiting of βNHE pH regulation at different starting Hb-O2 saturations. Symbols are means ± s.e.m. from Series 1 and 2 (reported in Table 2) fitted with a second-order polynomial regression. Groups are separated by the change in pH (ΔpH) to which the blood was exposed. Green triangles and solid line (Series 1, R2=0.9902) represent an initial pHe decrease of 0.49 units prior to ISO and CA addition; blue triangles and solid line (Series 1, R2=0.9994) represent an initial pHe decrease of 0.33 units; and white (Series 1, R2=0.9999) and black (Series 2, R2=0.9782) circles and associated black lines represent an initial pHe decrease of 0.15 units. All dashed lines represent the potential ΔPO2 for the combined Bohr-Root effect system in rainbow trout blood for a decrease in pHe of 0.5 (green) and 0.3 (blue) units as described in Rummer (Rummer, 2010).
The ΔPO2 associated with RBC βNHE short-circuiting
The ΔPO2 quantified using this closed system served as proof-of-principle for short-circuiting of βNHE pH regulation in this study. Insight was also gained relative to the time course over which short-circuiting and subsequent pHi recovery occurs. The optode response time for O2 is much faster than for pH (pH optodes ≥30 s; PO2 optodes <1 s). Thus, the ΔPO2 was a very sensitive, indirect measurement of changes in RBC pHi, which could not be measured directly and continuously. Therefore, regardless of the level of pHe detection, which was limited by optode response time, even the subtlest changes in pHi could be identified via changes in PO2.
The magnitude of the CA-mediated ΔPO2 following in vitro acidification, where pH was decreased by 0.33 or 0.49 units, was very similar to the ΔPO2 values calculated by direct interpolation between OECs generated at pH values that differed by a similar degree (Rummer, 2010) (Fig. 6). It should also be considered that values calculated for ΔPO2 here may have been underestimated because of RBC metabolism, which may have changed under the various treatments, particularly adrenergic stimulation. Together, these data indicate that nearly the entire acid load initially added to the closed system may have been available for short-circuiting of βNHE pH regulation in this in vitro setup. If this system were operational in vivo (see below for a detailed discussion) the magnitude of ΔPO2 could be reduced because the tissues are not a closed system and will continuously consume O2. However, there would be additional acidification from the CO2 produced from the tissues that could even further increase the ΔPO2 reported here.
The Bohr effect, which is understood to be important in enhancing tissue O2 delivery, elicits a ΔPO2 in humans of only 2-3 mmHg with a ΔpHa-v of -0.15 (Hutter et al., 1999; Jung et al., 1999; Behnke et al., 2001; Suttner et al., 2002). However, the ΔPO2 associated with βNHE short-circuiting in the in vitro setup employed in this study with a similar pH difference of -0.15 can be up to 25 mmHg (Table 2). A change of this magnitude could have huge implications toward tissue oxygenation, and accounts of elevated blood PO2 following an acidosis in past studies also support the potential for this system to be operational in vivo. For example, Nikinmaa et al. measured a 46% increase in blood PO2 in arterial blood (dorsal aorta) of striped bass (Morone saxitalis) following 5 min chasing, an increase that exceeded environmental O2 tensions and corresponded with a decrease in pHe from 7.555 to 7.244 as well as substantial lactate production (Nikinmaa et al., 1984). If the dorsal aorta endothelium possesses plasma-accessible CA, arterial blood PO2 could become elevated by the mechanism proposed herein and enhance O2 delivery when blood entered the tissue capillaries. Short-circuiting of RBC βNHE pH regulation could also explain the high red muscle PO2 values in trout prior to, during and following sustained and exhaustive exercise in comparison with much lower values seen in mammals with similar starting arterial PO2 values (McKenzie et al., 2004). It may also explain the observation that red muscle PO2 values were considerably higher than mixed venous blood PO2 values (McKenzie et al., 2004). Whether this system can operate in vivo is discussed in more detail below.
Potential for short-circuiting of RBC βNHE to be operational in vivo
In order for short-circuiting of RBC βNHE pH regulation to operate in vivo, there are many requirements that must be met. Minimally, CA must be plasma accessible. The rate at which βNHE is short-circuited in acidified blood must also be sufficiently fast to significantly decrease RBC pHi in the time required for blood transit from the gills to the tissues. Furthermore, the rate of βNHE to recover the pHi and secure O2 uptake at the gills must be faster than that required for transit from the tissues to the gills. A resting fish has a cardiac output of 26.6 ml min-1 kg-1 (Thorarensen et al., 1996; Brauner et al., 2000). Thus, for a 1 kg fish with 5% blood volume, blood transit time through the entire circulatory system is roughly 2 min. The t1/2 for O2 release from Hb is very rapid, <10 ms (Roughton, 1964). However, rates calculated from this experiment seem slow in comparison. This may be in part because the intracellular acidification in the first step of this in vitro model, as indirectly indicated by the ΔPO2 following acid addition, may be rate limited. The H+ from the initial HCl extracellular acidification must enter the RBC as CO2 at the uncatalyzed rate (t1/2>90 s) of formation in the plasma because H+ do not typically cross plasma membranes over such short durations (Pelster and Niederstatter, 1997). There were immediate effects of the acidification, but in some instances an overshoot and recovery was observed, almost as if a new equilibrium was being established (Fig. 4). Furthermore, although immediate, the t1/2 (28-44 s) was orders of magnitude slower than values published in the literature. Pelster and colleagues reported a that a 'Root-off' t1/2 could be as fast as 44.8 ms (Pelster et al., 1992), almost 1000 times faster than that observed in this study. However, Pelster's group used dual wavelength spectrophotometry to investigate the Root-on and Root-off rates in unicellular blood layers of the eel between porous Gore-Tex™ membranes (Pelster et al., 1992). This minimized the effects of unstirred diffusion boundary layers around the RBCs that had resulted in notably slow rates in other studies, which likely also apply to the present system. Additionally, Pelster's studies also suggest that CA is available to the plasma in the vicinity of the acid-producing gas gland (Pelster and Scheid, 1992; Pelster, 1995; Pelster and Niederstatter, 1997), which would greatly facilitate a fast Root-off effect. This in vitro system was set to record every second, but the response time for the pH optodes is not matched with the faster (yet still not as fast as 1 ms) responding PO2 optodes. It should be noted, however, that the t1/2 for the increase in PO2 was faster for the CA-mediated response, which may be a closer match to that observed in the eel, where CA was available (Pelster et al., 1992).
The role of general RBC NHE
In Series 2, it was determined that a CA-mediated increase in PO2 could occur in the absence of RBC adrenergic stimulation (Fig. 4). Yet some isoform of NHE was key, as evidenced in Series 3 experiments, where blocking NHE eliminated all CA-mediated responses (Fig. 5). It is known that rainbow trout possess a highly sensitive βNHE (Nikinmaa, 1983; Nikinmaa and Huestis, 1984; Borgese et al., 1987; Nikinmaa and Tufts, 1989; Nikinmaa et al., 1990). However, it may be that other stimuli are activating the βNHE (Romero et al., 1996; Weaver et al., 1999) or that additional NHE1 isoforms are present on the RBCs. These transporters could be functioning as 'housekeeping' H+ exchangers and be activated independent of adrenergic stimulation (Claiborne et al., 1999). Indeed, nearly all eukaryotes possess an isoform of NHE to regulate cell pH and volume (Yun et al., 1995; Wakabayashi et al., 1997; Claiborne et al., 1999; Deigweiher et al., 2008), and it has been suggested that at least one derived teleost species of the five groups that have secondarily lost the βNHE maintains a general RBC NHE for those purposes (Rummer et al., 2010). These data support this hypothesis, and raise questions regarding what may be activating NHE isoforms in the absence of catecholamines on the RBCs. Changes in RBC volume may activate NHE (Brauner et al., 2002; Koldkjær et al., 2002; Kristensen et al., 2007; Kristensen et al., 2008), and preliminary data from another study suggest that increases in RBC HCO -3 may be activating NHE via a soluble adenylate cyclase (J.L.R., unpublished data). If catecholamines are not crucial to this mechanism for increasing ΔPO2, this mechanism could be more broadly applied. The conditions under which catecholamines are released and βNHE is activated may be limited to extremely stressful scenarios in vivo, such as when arterial PO2 falls below 20 mmHg or 45-60% Hb-O2 saturation or when water PO2 falls below 60 mmHg (Perry and Thomas, 1991; Perry and Gilmour, 1996). If the system also functions via short-circuiting of pH regulation due to a general NHE and with pH disturbances of as small as -0.15, for example, O2 delivery could be enhanced in select locations where CA is plasma accessible under much less stressful conditions that may occur more frequently.
If some form of NHE (or βNHE) can be activated quickly and the full response prolonged over several minutes, it may mean that pHi has ample time to recover from an acidosis that is perpetuated at the muscle tissue by the time it returns to the gill to bind O2, which could take 1 min. The CA-mediated response observed in this study, with t1/2 ranging from 10 to 35 s (depending on the starting Hb-O2 saturation and the level of initial acidification), occurred almost twice as fast as the HCl-induced PO2 increases, where t1/2 ranged from 29 to 46 s, and almost five times faster than the responses associated with βNHE activation (Table 2). If the PO2 increase in vivo is as fast as a previous study suggests (45 ms) (Pelster et al., 1992), then it may be expected that the CA-mediated βNHE short-circuiting that elevates PO2 also happens far more rapidly than measured here. Transit time of the RBCs through the capillaries can take 1-3 s (Honig et al., 1977; Tetens and Lykkeboe, 1981; Randall, 1982; Bhargava et al., 1992), which is ample time for CA to short-circuit the effects of the H+ extrusion mechanism on the RBCs. After this phenomenon permits enhanced O2 delivery at the tissues, blood will leave the site of plasma-accessible CA and βNHE pH regulation will no longer be short-circuited. Therefore, blood returns to the respiratory surface with pHi and Hb-O2 binding once again protected by βNHE. Even if only a fraction of the response observed in vitro could be realized in vivo, this would significantly affect O2 transport in comparison to systems possessing a Bohr effect alone, an idea that has been recently supported in vivo (Rummer, 2010).
The main conclusion of this study is that in vitro, RBC βNHE or NHE pH regulation can be short-circuited during an acidosis in the presence of plasma-accessible CA, effectively decreasing Hb-O2 saturation and elevating the PO2 of the blood. If operational in vivo, this finding has implications for the Bohr-Root effect to enhance O2 delivery via this novel mechanism of increasing pHa-v during blood transit through the tissues. A pHe decrease as small as 0.15 units can be recycled via the CA-mediated NHE short-circuiting mechanism in the presence as well as absence of catecholamines. This suggests that the mechanism may not be restricted to circumstances where a severe acidosis is created and/or where catecholamines are released and remain in circulation for an extended period of time.
Perhaps early teleosts utilized the combined Bohr-Root effect for general O2 delivery before the appearance of the choroid rete and rete mirabile. If this was the case, then the driving force for O2 delivery could be elevated over what is possible in vertebrates possessing a Bohr effect alone (Rummer, 2010). Root-effect Hbs may be considered an exaptation for O2 delivery to the eye and swimbladder, as proposed by Berenbrink et al. (Berenbrink et al., 2005). Thus, an incipient function of Root Hbs – general O2 delivery – may have been co-opted to give rise to the complex physiological system of the eye and swimbladder 150-270 million years later (Berenbrink et al., 2005). Thus, on a broader scale, the results of this study may help to elucidate one of the most successful adaptive radiations of an animal group in evolutionary history, that of the teleost fishes, and the selection pressures that may have been involved with the evolution of enhanced O2 delivery.
The authors wish to thank Drs D. W. Baker, T. D. Clark, K. Gilmour, S. Perry and D. J. Randall for interesting discussions and editorial comments as well as P. Allen, C. Ciuhandu, N. Farrell, C. Fu, L. Hanson, K. Kamo, M. Regan, M. Roshan-Moniri and K. Suvadzjic and the UBC Zoology workshop, including B. Gillespie, D. Brandys and V. Grant, for technical assistance. Lastly, the authors wish to thank two anonymous reviewers for extremely valuable suggestions.
This study was supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada grant to C.J.B. Funding for J.L.R. was through the University of British Columbia Graduate Fellowship program.
carbonic anyhydrase
3′, 5′-cyclic adenosine monophosphate
ethylisopropylamiloride
Kcat
enzyme catalytic activity (s-1)
Na+/H+ exchange
arterial blood pH
extracellular, plasma pH
intracellular, red blood cell pH
partial pressure of O2
the half-time of a reaction
β-adrenergically activated Na+/H+ exchange
ΔpHa-v
arterial-venous pH difference
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Travamigos has partnered with 360red Productions Ltd on a three-video project to promote their group travel app, which connects like-minded backpackers all over the world.
Sanjay Rajput, Creative Director of the Leicester-based video production company, has signed up to produce three promotional videos for Travamigos. During a pre-shoot exploratory day at Loughborough University, Sanjay went missing twice, only to reappear having found the ideal location or perfect backdrop for the shoot.
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text:
Q: How do I see combined diff for a range of commits in tig? Github shows a combined diff of all the commits made on a branch in a pull request. e.g. this pull request has three commits and Github shows a combined diff for all those three commits.
In git itself I can do something like git diff HEAD~<n> to see a combined diff of n commits.
How do I do the same in tig?

A: Following "How do I use tig to view the diff for a file which is changed but not commited?", the possible workaround for now would be:
git diff HEAD~<n> | tig

The tig manual only mentions diff options, but doesn't allow to diff anything else but the previous commit.

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text:
Posts tagged with "Drew Pinsky"
Posted June 10th, 2009 by Kim
– Mad Men, season three, August 16 at 10PM ET on AMC. Be there, or be somewhere else, somewhere where people don't knock back three vodka gimlets before lunch, aren't nearly as dapperly-attired as Don Draper (Jon Hamm) and don't have offices that are bigger than my entire New York City apartment. Yeah, not sounding nearly as much fun as Mad Men, right?
– Is designer Vera Wang going to be competing on the next season of Dancing With the Stars? And if so, does that mean we can count on less cheesy costumes?
– Plus-size model Emme has been hired to host Fox's More to Love, the reality dating show that will feature "average-looking" people
– Discovery Channel's 22nd annual Shark Week kicks off on August 2.
– Matt Mitovich at TVGuide.com says Rick Springfield will play, fittingly enough, Rick Springfield, when he guest stars on the new season of Californication, but he'll be a naughty Rick Springfield, one whose sex play makes David Duchovny's Hank "look like a Jonas brother." Not that I'm buying that whole purity ring business about the Jonas Bros. …
– In celebration of its 15th anniversary, TCM has created its own social network site, TCM Classic Film Union, for movie buffs.
– Gordon Ramsay has apologized for, well, basically, for being a tool. You can watch the apology here.
– I reeeally want to like Dr. Drew and trust his motives, but a real doctor prolly should offer to help Lindsay Lohan, or a less-famous addict, whether or not she accepts a reported six-figure offer to appear on his reality show, Celebrity Rehab with Dr. Drew, right? Of course, the story seems to be originating with Lohan's dad, so, grain – no, make that rock, huge, ginormous rock – of salt on the whole thing.
– The Shield creator Shawn Ryan is going back to FX with a comedy about private detectives, called Terriers.
– Futurama's back! With 26 new episodes in 2010!
– Good news for David Letterman: He, with guest Julia Roberts, beat Conan O'Brien's Tonight Show on Tuesday. Worse news for Conan: His ratings keep dropping. Don't count him out tho; remember his first years as Late Night host?
– More ratings wars news: Judge Judy beat Oprah. Whoa.
– ABC's getting a jump on the competition by announcing its fall premiere dates.
– The ratings for the Nurse Jackie series premiere were so good, Showtime's already greenlighting a second season, so go ahead and get hooked.
– Heidi Pratt's launching her own line of dry shampoo, after her shampoo shenanigans on I'm a Celebrity, Get Me Out of Here. I'm sure Oscar Blandi is shaking in his celebrity hair-cutting boots.
Filed in Celebreality, Late Night, Reality, TV News
Tags: Californication, Celebrity Rehab with Dr. Drew, Conan O'Brien, Dancing With the Stars, David Duchovny, David Letterman, Discovery Channel, Don Draper, Dr. Drew, Drew Pinsky, Emme, Futurama, Get Me Out of Here, Gordon Ramsay, Heidi Pratt, I'm a Celebrity, Jon Hamm, Jonas, Judge Judy, Julia Roberts, Lindsay Lohan, Mad Men, More to Love, Nurse Jackie, Oprah, Rick Springfield, Shark Week, Shawn Ryan, TCM Classic Film Union, The Shield, Tonight Show, Vera Wang
Channel Surfing – May 5
Posted May 5th, 2009 by Kim
– Great news: Mad Men will return for its third season on A&E in August.
– Bad news: Dennis Rodman's goin' to rehab. The good news: Since he's obviously not averse to making a boob of himself on TV, now he can sign up for Celebrity Rehab with Dr. Drew Pinsky.
– Old Christine may get a new home. If CBS dumps the Julia Louis-Dreyfus sitcom The New Adventures of Old Christine, ABC says it'll take the leftovers.
– The CW has decided to pull the plug on its Sunday night programming.
– Cap'n Jack (John Barrowman) is making an appearance in David Tennant's final Doctor Who ep.
– Jennifer Hudson won an Oscar, and now another American Idol alum could win a Tony, as Constantine Maroulis snags a nod for his role in the musical Rock of Ages.
– More Idol news: Paula Abdul's singing on tonight's "Rock Week" episode.
– And still more Idol news, news that should make the aforementioned Ms. Abdul quite happy: TVGuide.com readers have spoken, and nearly 60 percent of them think newbie judge Kara DioGuardi should turn in her Coke cup after this season.
Tags: American Idol, Celebrity Rehab, Doctor Who, Drew Pinsky, Julia Louis-Dreyfus, Kara DioGuardi, Mad Men, Paula Abdul

redpajama_set_name: RedPajamaCommonCrawl


A new international affordable housing index released Monday by the Atlantic Institute of Market Studies contains good news for Atlantic Canada.
The study found that the top three most affordable cities for housing in Canada are in New Brunswick, and Charlottetown ranked fifth.
"There is often so much bad economic news about the place where we live," said AIMS president Marco Navarro-Génie.
The 13th Annual Demographia International Housing Affordability Survey determines housing affordability by dividing median house prices by median income. It considers any result less than 3 as affordable housing, and any result over 5 as severely unaffordable.
Moncton had the most affordable housing in Canada, with an index of 2.1.
Outside of Atlantic Canada cities did not fare so well. The report authors suggest Canadian cities with a population of more than 1 million are experiencing a housing bubble.
Read the full article on the CBC P.E.I. website.


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PREMIER LEAGUE action returns tonight but who will win the title and which teams will finish in the top four? Starsport writers have made their choices.
The pressure is on Pep Guardiola this season but I expect the Manchester City boss to rise to the occasion.
With a season in England under his belt, the Spaniard now knows what it takes to compete in the Premier League and with his club's huge investment this summer, I'm tipping City for the title.
City's Manchester rivals United have also spent big and they will push Guardiola and co all the way but just come up short (and a serious injury to Romelu Lukaku could see them drop out of the top four altogether).
North London giants Arsenal and Tottenham will complete the Champions League spots in a close run race with Liverpool but I believe Chelsea will fall away this season with a frustrated Antonio Conte walking after Christmas.
If City are to claim Prem glory, they will have to have a star striker banging in the goals and with Gabriel Jesus, they have just that.
The Brazilian was superb at times last season but injuries blighted his debut year in England. Now fully fit, he can produce a 25-goal campaign.
With Jermain Defoe, Asmir Begovic and Nathan Ake, Bournemouth have completed one of the finest - and most underrated - transfer windows in the Premier League.
Their added experience and quality, combined with the likes of Callum Wilson and Josh King, can help Eddie Howe's side to go one better than last season and finish eighth.
Manchester City have got the attack, and after another huge outlay this summer, they should now have the defence too.
Pep Guardiola should have learned a lot from his first Premier League season, and if his team can improve their resilience and grind out results, they should have too much for their rivals.
On paper Tottenham look weaker than the end of last season. And with the temporary switch to Wembley there are plenty of doubters.
But Spurs have been the most consistent team in the Premier League for the last two years, and there's no reason that should change this time around if Harry Kane, Dele Alli and Toby Alderweireld stay fit.
Manchester United have spent big again, and Jose Mourinho has a deep squad, but do they have the flair and penache to turn last season's draws into wins?
Romelu Lukaku will help, but United were well off the pace last year and third place would be a decent improvement.
Despite having a poor season, Arsenal ended up only missing out on the Champions League by the narrowest of margins.
The Gunners still have more than enough to get back in the top four. An early exit from the Europa League would help too.
Harry Kane has had the Golden Boot sewn up for years, but Aguero is still a top performer.
He may have to battle for his place in City's side, but if they are to win the league, the Argentine will need to hit 20 goals or more.
Antonio Conte will be the surprise of the season. The Italian has cut a frustrated figure in pre-season. He's not happy with the club's transfer business.
The last two managers to win the Premier League have been sacked the following season - could Conte make it an unfortunate hat-trick?
Can Chelsea retain their Premier League title?
Manchester City look formidable as an attacking force and even if they may be a bit suspect in defence they should blow teams away.
Gabriel Jesus and Sergio Aguero with Kevin De Bruyne, David Silva, Leroy Sane and Raheem Sterling in midfield is enough to put away the best sides in the top flight.
Jose Mourinho has made vast improvements to Manchester United with the spine of the side now including Victor Lindelof, Nemanja Matic and Romelu Lukaku.
The former Everton man will win the Golden Boot but the Red Devils will just lose out to their rivals in the end.
I'm not buying these theories on Chelsea collapsing again. Antonio Conte simply won't let it happen and Alvaro Morata will turn out to be an impressive signing.
The return of Eden Hazard from injury will be key for them as well.
Tottenham have the move to Wembley and a lack of signings in the transfer window so far to contend with so they will drop down to fourth.
That's despite Harry Kane being the most lethal forward in the league. Keeping him injury free will be massive.
That leaves Liverpool and Arsenal outside of the Champions League qualifying places.
Javier Hernandez will be the surprise of the season because he simply won't hit the heights expected of him at West Ham.
The Mexican didn't set the world alight in the Bundesliga last year and I suspect his best days might be behind him.
Pep Guardiola had a debut season to forget but with sweeping changes at The Etihad you have to think the Spaniard will get it right this time around.
He's revolutionised his back line with the world-record keeper Ederson, and full-backs Kyle Walker, Benjamin Mendy and Danilo all arriving.
Bernardo Silva could be the most important signing though, with the Portuguese set to light up the Premier League next season.
Jose Mourinho has a knack of winning titles in his second season at a club. While his United side will challenge this year they simply don't have the X-Factor of their cross-town rivals.
Romelu Lukaku will be important for the Red Devils but you just feel there is a cutting edge missing in the red half of Manchester.
Antonio Conte is going through something of a crisis at the moment with a thin squad and Diego Costa still hanging around like a bad smell.
But you trust the Italian to get things right. The return of Champions League football will have an effect and without the required additions they're likely to surrender their crown, despite their brilliant boss.
Mauricio Pochettino has a job on his hands to keep Spurs moving forward while changing home and without any new additions.
But he's a great manager and he has some class players playing quality football they will always get results.
However, while I back them to fight off the challenge of Arsenal, Liverpool and Everton for a top four finish, I think they'll find themselves going down the table, not up this time around.
Harry Kane is the best all-round striker in the Premier League by a mile.
He's won back-to-back golden boots and there's no reason to say he won't make it a hat-trick.
Will Harry Kane win the Golden Boot for the third successive season?
Chelsea haven't made as many signings as Antonio Conte would have liked and much will depend on Alvaro Morata and Tiemoue Bakayoko hitting the ground, but they have a squad of winners.
Eden Hazard will be firing again once he returns to full fitness.
Jose Mourinho's getting closer to having the team he wants at Manchester United and they'll convert more of last season's draws into wins this time around.
Marcus Rashford can kick on if he's given chances alongside Romelu Lukaku.
Pep Guardiola's Manchester City have the quality to challenge for the title IF Vincent Kompany stays fit.
Should the Belgian fail to do so, John Stones will have to play alongside Nicolas Otamendi or Eliaquim Mangala in the heart of defence - not too convincing.
Mauricio Pochettino's men come into the season off the back of two stellar campaigns that they'll struggle to replicate. Playing at Wembley and their failure to sign a dynamic winger will probably hurt them.
Harry Kane wins the Golden Boot. 75 goals over the past three years have made him the Premier League's top marksman.
Huddersfield will turn out to be the surprise of the season.
David Wagner's wily and their transfer business has been sensible with players brought in that have clearly been on his wish list for a while.
Pep Guardiola has strengthened his defence - their Achilles heel last season.
City have an array of attacking talent, capable of outscoring every team in the league. Guardiola will be expected to land the title and he will deliver.
Jose Mourinho has no excuses this season after struggling at times last season.
He has beefed up his Manchester United squad and Romelu Lukaku will be desperate to prove his doubters wrong.
Mauricio Pochettino has the move to Wembley to contend with and, at the time of writing, no new signings.
Kyle Walker's departure is a blow but Kieran Trippier is excellent back-up. It will be a difficult campaign but Spurs have the league's best striker in Harry Kane.
Mohamed Salah is a top signing but the Champions League will stretch Jurgen Klopp's squad.
The German is making progress at Anfield and a European run and another top-four finish would be deemed a success.
Harry Kane has won the Golden Boot for the previous two seasons and is the division's top marksman.
A lack of back-up means he will play every game. Playing in a creative team means he will have plenty of chances to gobble up.
The gap between the top seven (including Everton) and the rest is growing.
It's fair to say eighth place is up for grabs and the Clarets have made sensible signings in Jon Walters, Phil Bardsley, Jack Cork and Charlie Taylor.
If Sean Dyche can source a good replacement for Michael Keane then they could surprise the best of the rest.
Last year it didn't happen, but this season I am confident it will be a Manchester affair.
Jose Mourinho and Pep Guardiola will lock horns like it's 2012, only this time it will be the ex-Barcelona boss who will prevail thanks to his philosophy and impressive signings.
Tottenham's onslaught will continue with Harry Kane leading the brigade.
Not everyone is confident about Chelsea, but the dynamics will dramatically change once Eden Hazard returns.
Eddie Howe's thrilling project at Bournemouth will continue to impress us and we could see the Cherries finishing in the top 10 with some stunning performances against the top sides.


text:
Q: Errors locating elements with Ruby Watir and PhantomJS I have a script that runs perfectly in the ChromeWebDriver but fails on PhantomJS.  When I check if an element exists i get the following error:

[ERROR - 2014-01-07T19:31:55.878Z] WebElementLocator - _handleLocateCommand - El
  ement(s) NOT Found: GAVE UP. Search Stop Time: 1389123115867

This doesn't really seem like an issue as the script continues.  However, later on the script will fail unable to locate the following element:

question.div(:class => "choices")

This particular script visits a page that has test questions on it.  They are in random order.  The script decided what kind of question it is and chooses a random answer.
Thanks for any help. Here is the relevant code:
def QuestionType(question)
    if question.div(:class => "questionPrompt").text_field.exists?
        puts "FITB"
        FITB(question)
    #elsif question.div(:class => "choices").ul(:class =>"choices-list").li(:index => 1).checkbox.exists?
    elsif question.checkbox.exists?
        puts "Checkbox"
        Checkbox(question)
    else
        puts "Radio"
        Radio(question)
    end

end

def FITB(question)
    arn = Random.new.rand(0..10)
    if arn == 0
        answers.li(:index => arn).radio.set
    else
        idx = 0
        begin
            question.div(:class => "questionPrompt").text_field(:index => idx).set("Test #{idx}")
            idx = idx + 1;
        end while question.div(:class => "questionPrompt").text_field(:index => idx).exists?
    end
    puts "FITB Complete"
end

def Checkbox(question)
    allAnswers = question.div(:class => "choices")
    answers = allAnswers.ul

    max = answers.lis.length - 1
    arn = Random.new.rand(0..max)
    if arn == 0
        answers.li(:index => arn).radio.set
    else
        for i in 1..arn
            answers.li(:index => i).checkbox.set
        end
    end
    puts "Checkbox Complete"
end

def Radio(question)
    allAnswers = question.div(:class => "choices")
    answers = allAnswers.ul

    max = answers.lis.length - 1
    arn = Random.new.rand(0..max)

    answers.li(:index => arn).radio.set

    puts "Radio Complete"
end


text:
Behavioural Economics?
Behavioural economics is a method of research which makes use of methods of experimentation used by psychologists to study the way people make decisions. Daniel Kahneman's book 'Thinking Fast & Slow' has been very influential in this area. He describes 2 modes in which humans think. The first is a 'fast mode' that works on automatic (which he describes as System 1). We use this in situations like driving a car in nice weather or adding 2+2. System 2 is a 'slow mode' that requires more conscious effort e.g. parking in a difficult space or multiplying 13x36. His book reveals a number of heuristics (practical yet imperfect/sub-optimal ways of making decisions) and cognitive biases (tendencies to think about things in certain ways which lead to behaviours which often don't represent good judgement).
What are the aims of Behavioural Economics?
It is an increasingly popular tool in market research because it involves delving beneath consumers' stated reasons for behaving as they do and revealing the sometimes complex, often irrational/subconscious factors that really influences decisions. Behavioural economics has challenged the way Economics model consumer behavior – which typically assumed humans would make 'perfect' decisions based on having all facts available.
What are the implications for retail research?
The implications for retail research are very significant as it's in 'fast mode' where over 80% of decisions are made. An example of this is the way we know, in less than a second, if we like something. This is automatic and requires NO conscious effort. It also explains why we can complete a supermarket shop, faced with tens of thousands of options, in a matter of minutes.
How is Behavioural Economics undertaken?
Heuristics and biases have been established via observation studies or laboratory-style experimentation. In a market research context, the application of BE is mostly more about keeping respondents in 'system 1' as you undertake the research. This is often about applying time pressure to stop respondents and by gamifying research to stop respondents being overly rational. Beyond this, researchers might study how brands could use BE understanding to nudge behaviours in their favour.
What is Behavioural Economics?
BACK: GLOSSARY
NEXT: Behavioural Segmentation
NEW BUSINESS ENQUIRIES
© 2018 by ABAResearch


text:
Home Books The Color Atlas of Pediatrics
Chapter 210: Immune Thrombocytopenia Purpura
Margaret C. Thompson, MD, PhD
Thompson MC. Thompson M.C. Thompson, Margaret C.Immune Thrombocytopenia Purpura. In: Usatine RP, Sabella C, Smith M, Mayeaux EJ, Jr., Chumley HS, Appachi E. Usatine R.P., & Sabella C, & Smith M, & Mayeaux E.J., Jr., & Chumley H.S., & Appachi E(Eds.),Eds. Richard P. Usatine, et al.eds. The Color Atlas of Pediatrics. McGraw Hill; 2015. Accessed January 31, 2023. https://accesspediatrics.mhmedical.com/content.aspx?bookid=1443&sectionid=79850696
Thompson MC. Thompson M.C. Thompson, Margaret C. (2015). Immune thrombocytopenia purpura. Usatine RP, Sabella C, Smith M, Mayeaux EJ, Jr., Chumley HS, Appachi E. Usatine R.P., & Sabella C, & Smith M, & Mayeaux E.J., Jr., & Chumley H.S., & Appachi E(Eds.),Eds. Richard P. Usatine, et al. The Color Atlas of Pediatrics. McGraw Hill. https://accesspediatrics.mhmedical.com/content.aspx?bookid=1443&sectionid=79850696
Thompson MC. Thompson M.C. Thompson, Margaret C. "Immune Thrombocytopenia Purpura." The Color Atlas of Pediatrics Usatine RP, Sabella C, Smith M, Mayeaux EJ, Jr., Chumley HS, Appachi E. Usatine R.P., & Sabella C, & Smith M, & Mayeaux E.J., Jr., & Chumley H.S., & Appachi E(Eds.),Eds. Richard P. Usatine, et al. McGraw Hill, 2015, https://accesspediatrics.mhmedical.com/content.aspx?bookid=1443&sectionid=79850696.
Etiology and Pathophysiology
Treatment and Management
A 22-month-old boy is brought to his pediatrician by his parents because he has developed several dark purple bruises on his back and spine (Figure 210-1). He has additional bruises over both legs (Figure 210-2) along with smaller bruises over his neck and cheeks. He developed these over the past 2 to 3 days, and his mother reports that 3 weeks prior to this presentation, he had an illness characterized by vomiting, nausea, and diarrhea. The pediatrician suspects immune thrombocytopenic purpura (ITP) and orders a CBC, which shows a normal white blood cell count, differential count, and hemoglobin, but a platelet count of 7,000 microL. His liver function studies, prothrombin time (PT), and partial thromboplastin time (PTT) are all normal. A diagnosis of ITP is made and the child is admitted to the hospital, where his activity is restricted and he is treated with intravenous immunoglobulin. His platelet count increases rapidly over 3 days and he is discharged home with close follow-up.
FIGURE 210-1
Purpuric and ecchymotic lesion over the back, and petechial lesions on the face of a child with immune thrombocytopenic purpura. (Used with permission from Margaret C Thompson, MD, PhD.)
A. Large purpuric lesions on the legs of the child in Figure 210-1. B. Close-up of an ecchymotic lesion on the same child. (Used with permission from Margaret C Thompson, MD, PhD.)
Immune thrombocytopenia purpura (ITP), formerly called idiopathic thrombocytopenic purpura, is an acquired disorder in which there is increased destruction of platelets causing thrombocytopenia (platelet count <100,000 microL). ITP may be divided into acute and chronic forms. Acute ITP is defined as having duration less than 6 months while chronic ITP is defined as lasting more than 6 months.
Idiopathic Thrombocytopenic Purpura.
The incidence of symptomatic disease is estimated to be 3 to 8 per 100,000 per year.1–3
Acute ITP is the most common bleeding disorder in children.
It is seen most frequently in children between the ages of 2 and 10 years with a peak occurring between 2 and 5 years of age.2
It affects males more frequently among children younger than 8 years and equally among males and females beyond that age.2
More common in winter and spring.
In approximately 2/3 of cases, ITP in children occurs within 6 weeks of a recent viral illness.4
Chronic ITP is defined as persistent thrombocytopenia that persists for longer than 6 months from the initial presentation. This occurs in 10 to 20 percent of children with ITP.
Chronic ITP occurs more frequently in adolescents (and adults) than ...


text:
Q: Null pointer error while using sessionfactory on Hibernate 4 with Spring 3.2 I'm struggling to figure out the exact problem, why the Null Pointer Error is thrown for Hibernate sessionFactory when I try to Auto wire sessionFactory from spring bean xml. Could somebody please help me out fix this. I'm new to Spring and deploying the app on Jboss server
Here is the Spring-Config.xml
<beans xmlns="http://www.springframework.org/schema/beans"
    xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xmlns:c="http://www.springframework.org/schema/c"
    xmlns:context="http://www.springframework.org/schema/context" xmlns:p="http://www.springframework.org/schema/p"
    xmlns:tx="http://www.springframework.org/schema/tx"
    xsi:schemaLocation="http://www.springframework.org/schema/beans http://www.springframework.org/schema/beans/spring-beans-3.2.xsd
        http://www.springframework.org/schema/context http://www.springframework.org/schema/context/spring-context-3.2.xsd http://www.springframework.org/schema/tx
http://www.springframework.org/schema/tx/spring-tx-3.2.xsd">

    <context:annotation-config />
    <context:component-scan base-package="org.kp.db.dao.impl" />

    <bean id="myDataSource"
        class="org.springframework.jdbc.datasource.DriverManagerDataSource">
        <property name="driverClassName" value="com.mysql.jdbc.Driver" />
        <property name="url" value="jdbc:mysql://localhost:3306/ebm" />
        <property name="username" value="root" />
        <property name="password" value="root" />
    </bean>

    <bean id="sessionFactory"
        class="org.springframework.orm.hibernate4.LocalSessionFactoryBean">
        <property name="dataSource" ref="myDataSource" />
        <property name="configLocation" value="classpath:/hibernate.cfg.xml" />
    </bean>

    <tx:annotation-driven  transaction-manager="transactionManager" />

    <bean id="transactionManager"
        class="org.springframework.orm.hibernate4.HibernateTransactionManager">
        <property name="sessionFactory" ref="sessionFactory"></property>
    </bean>
</beans>

Here is Hiberante.cfg.xml which I placed in webcontent folder
<!DOCTYPE hibernate-configuration PUBLIC "-//Hibernate/Hibernate Configuration DTD 3.0//EN" "http://hibernate.sourceforge.net/hibernate-configuration-3.0.dtd">
<hibernate-configuration>
  <session-factory>
    <property name="hibernate.dialect">org.hibernate.dialect.MySQLDialect</property>
    <property name="hibernate.connection.driver_class">com.mysql.jdbc.Driver</property>
    <property name="hibernate.connection.url">jdbc:mysql://localhost:3306/ebm</property>
    <property name="hibernate.connection.username">root</property>
    <property name="hibernate.connection.password">root</property>
    <property name="cache.provider_class">org.hibernate.cache.NoCacheProvider</property>
    <property name="hibernate.show_sql">true</property>
    <property name="hibernate.hbm2ddl.auto">update</property>
    <mapping resource="org/kp/db/model/Actioncode.hbm.xml"/>
    <mapping class="org.kp.db.model.TblEBMFieldDetails"/>
    <mapping class="org.kp.db.model.TbleOLI"/>
  </session-factory>
</hibernate-configuration>

And the DAO class in which I'm trying to access the sessionFactory is
package org.kp.db.dao.impl;

import org.hibernate.Session;
import org.hibernate.SessionFactory;
import org.kp.db.dao.ActionCodeDao;
import org.kp.db.model.Actioncode;
import org.springframework.beans.factory.annotation.Autowired;
import org.springframework.stereotype.Repository;
import org.springframework.transaction.annotation.Propagation;
import org.springframework.transaction.annotation.Transactional;

/**
 *
 * @author Administrator
 */
@Repository
public class ActionCodeDaoImpl implements ActionCodeDao{

    @Override
    public String[] getActionCodes() {
        throw new UnsupportedOperationException("Not supported yet.");
    }

    @Autowired(required=true)
    SessionFactory sessionFactory;

    public SessionFactory getSessionFactory() {
        return sessionFactory;
    }

    public void setSessionFactory(SessionFactory sessionFactory) {
        this.sessionFactory = sessionFactory;
    }

    @Override
    @Transactional(readOnly=true,propagation=Propagation.REQUIRED)
    public String getActionCode(int id) {

        //ApplicationContext context;

        Session  session = sessionFactory.getCurrentSession();
        Actioncode actionCode=null;
        actionCode = (Actioncode)session.get(Actioncode.class, 1);

        return actionCode.getActionName();
    }

}

A: I've fixed the issue, instead of accessing the object I used the getter method and it worked, not sure what is the real concept behind it.
public String getActionCode(int id) {

    //ApplicationContext context;

    Session  session = getSessionFactory().getCurrentSession();
    Actioncode actionCode=null;
    actionCode = (Actioncode)session.get(Actioncode.class, 1);

    return actionCode.getActionName();
}

A: @Named ("kpAction")
public class Actions {
    public String getActionCode() {
        ActionCodeDao action =new ActionCodeDaoImpl();
        String str = action.getActionCode(1); System.out.println(str); return str;
    }
}

When you run this line of code: ActionCodeDao action =new ActionCodeDaoImpl(); you get an object that is not managed by Spring and therefore will not contain the autowired dependencies.
I would say that is why you saw the null pointer.
However you say you have fixed it now, not sure if you inadvertantly changed this as well, if not im not sure how youve fixed it :)