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Intracellular pH (pHi) is likely to play a key role in maintaining the functional success of cnidarian–dinoflagellate symbiosis, yet until now the pHi of the symbiotic dinoflagellates (genus Symbiodinium) has never been quantified. Flow cytometry was used in conjunction with the ratiometric fluorescent dye BCECF to monitor changes in pHi over a daily light/dark cycle. The pHi of Symbiodinium type B1 freshly isolated from the model sea anemone Aiptasia pulchella was 7.25 ± 0.01 (mean ± SE) in the light and 7.10 ± 0.02 in the dark. A comparable effect of irradiance was seen across a variety of cultured Symbiodinium genotypes (types A1, B1, E1, E2, F1, and F5) which varied between pHi 7.21–7.39 in the light and 7.06–7.14 in the dark. Of note, there was a significant genotypic difference in pHi, irrespective of irradiance.  相似文献   

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Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytototactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi. © 1994 Wiley-Liss, Inc.  相似文献   

5.
As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO3. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells. © 1996 Wiley-Liss, Inc.  相似文献   

6.
Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H+ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H+ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pHo) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pHi) and net H+ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pHi remained above 7.0 within a pHo range of 4 to 6 and decreased below pHo 4. Above pHo 4, H+ extrusion increased correspondingly, with the maximum value at pHo 5.5, and below pHo 4, a net H+ influx was observed. Without glucose in the medium, the pHi decreased, and a net H+ influx was observed below pHo 5.5. A high correlation (R = 0.75 to 0.92) between the pHi and net H+ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pHi homeostasis in L. monocytogenes subjected to acid stress.  相似文献   

7.
Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl/HCO3 exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification. J. Cell. Physiol. 173:397–405, 1997. © 1997 Wiley-Liss, Inc.  相似文献   

8.
Fish exposed to elevated water CO2 experience a rapid increase in blood CO2 levels (hypercapnia), resulting in acidification of both intra- and extra-cellular compartments. While the mechanisms associated with extracellular pH regulation have been well explored, much less is known about intracellular pH (pHi) regulation. There is great interest in developing non-animal models for research. One such model is the rainbow trout hepatoma cell line (RTH 149), which has been used to study a wide range of topics; however, no studies have investigated its potential use in pHi regulation. Employing the pH-sensitive fluoroprobe BCECF, the present study examined pHi regulation in RTH 149 under normocapnia and during extracellular acidification induced by either elevated CO2 or 1 M HCl. During exposure to hypercapnia, RTH 149 cells were acidified without recovery as long as the elevated CO2 was maintained. In addition, rates of pHi recovery from NH4Cl-induced acidosis were significantly lower in cells exposed to hypercapnia or HCl compared to that in normocapnic cells, indicating that elevated CO2 indirectly impeded pHi recovery through a reduction in pHe and/or pHi. Moreover, pHi regulation in RTH 149 was EIPA-sensitive, suggesting that an NHE may be involved. Overall, RTH 149 may have the potential for identifying transporters likely to play a role in pHi regulation in fish. However, it should not be used as a complete replacement for in vivo studies, especially to quantify acid–base regulatory ability at whole animal level, since RTH 149 appeared to have enhanced pHi recovery rates relative to primary hepatocytes.  相似文献   

9.
The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer’s wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pHi of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5.  相似文献   

10.
Summary Homeostasis of intracellular calcium ([Ca++]i) and pH (pHi) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca++]i and pHi on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca++]i with fura-2 and pHi with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pHi in these cells was 7.37±0.05 (n=20 cells) and resting [Ca++]i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH4Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca++]i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH4Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH4Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pHi decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca++]i, but produced a biphasic change in pHi. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca++]i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pHi decreased by 0.35±0.02 units. We conclude that an increase in pHi leads to an increase in [Ca++]i in rabbit corneal epithelial cells; however, a decrease in pHi leads to minor changes in [Ca++]i. The ability of CE cells to maintain proper calcium homeostasis when pHi is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.  相似文献   

11.
Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pHi) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pHi, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998  相似文献   

12.
The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6.6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate.  相似文献   

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Regulatory relationship and gain control between cytosolic free Ca2+ concentration (Cai) and cytosolic pH (pHi) were evaluated by two different cell types, gastric parietal cells, and blood platelets. Studies were carried out in both single cells and populations of cells, using Ca2+-indicative probe fura-2 (1-(2-(5′-carboxyoxazol-2′-yl)-6-aminobenzofuran-5-oxy)-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid) and pH-indicative probe BCECF (2′,7′-bis(carboxyethyl) carboxyfluorescein). Stimulation of single and populational parietal cells and platelets with gastrin and thrombin, respectively, resulted in an increase in Cai. In both populational cell types, an initial change in pHi during agonist stimulation occurred almost simultaneously with the mobilization of Ca2+; an initial transient decrease in pHi was followed by a slower increase in pHi above the prestimulation level. When populational platelets were preloaded with the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′tetraacetic acid), the thrombin-induced initial large increase in Cai was apparently inhibited, whereas the pHi decrease induced by thrombin was not altered. This suggests that the initial Cai change is not a prerequisite for the pHi change. The effect of pHi on Cai was examined next. In both single and populational cell types, application of the K+-H+ ionophore nigericin, which induced a transient decrease in pHi, led to the release of Ca2+ from intracellular stores. In single parietal cells double-labeled with fura-2 and BCECF, a temporal decrease in pHi preceded the rise in Cai after stimulation with nigericin. A decrease in pHi, and an increase in Cai occurred at 1.5 and 4 s, respectively. In single parietal cells, replacement of medium Na+ with N-methyl- -glucamine (NMG+), which also induced a decrease in pHi, resulted in repetitive Ca2+ spike oscillations. The source of Ca2+ utilized for the Ca2+ oscillation that was induced by NMG+ originated from the agonist-sensitive pool. Thus, several maneuvers, which were capable of decreasing pHi, led to an increase in Cai. Cytosolic acidification may be a part of the trigger for Ca2+ mobilization from intracellular stores in both parietal cells and platelets.  相似文献   

15.
The effect of changes of pHi on Cai were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Cai in these cells was 182 ± 12 nM (n = 74) at pHo of 7.4. Upon exposure to NH4Cl, which rapidly alkalinized cells, a transient increase of Cai to 349 ± 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH4Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Cai: exposure to and then removal of CO2 (Cai increased from 182 ± 22 to 248 ± 28 nM; n = 8); and exposure to and then removal of Na propionate (Cai increased from 242 ± 32 to 456 ± 71 nM; n = 9). The NH4Cl-induced Cai transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl3. Sustained changes of pHi can be induced by varying pHo. When pHo was lowered to 6.9, Cai fell by 49 ± 11 nM but increased by 203 ± 51 nM (n = 6) when pHo was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Cai. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pHi. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pHi in cells from hypertensive animals.  相似文献   

16.
Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid–base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2–3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15–7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid–base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.  相似文献   

17.
We have investigated the involvement of intracellular pH (pHi) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na+/H+ exchanger1 (NHE1) inhibitor cariporide and the “high K+” buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pHi than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pHi and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pHi recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pHi and P-gp in K562/DOX cells.  相似文献   

18.
Sodium bicarbonate cotransporters (NBCs) are involved in the pH regulation of salivary glands. However, the roles and regulatory mechanisms among different NBC isotypes have not been rigorously evaluated. We investigated the roles of two different types of NBCs, electroneutral (NBCn1) and electrogenic NBC (NBCe1), with respect to pH regulation and regulatory mechanisms using human submandibular glands (hSMGs) and HSG cells. Intracellular pH (pHi) was measured and the pHi recovery rate from cell acidification induced by an NH4Cl pulse was recorded. Subcellular localization and protein phosphorylation were determined using immunohistochemistry and co-immunoprecipitation techniques. We determined that NBCn1 is expressed on the basolateral side of acinar cells and the apical side of duct cells, while NBCe1 is exclusively expressed on the apical membrane of duct cells. The pHi recovery rate in hSMG acinar cells, which only express NBCn1, was not affected by pre-incubation with 5 μM PP2, an Src tyrosine kinase inhibitor. However, in HSG cells, which express both NBCe1 and NBCn1, the pHi recovery rate was inhibited by PP2. The apparent difference in regulatory mechanisms for NBCn1 and NBCe1 was evaluated by artificial overexpression of NBCn1 or NBCe1 in HSG cells, which revealed that the pHi recovery rate was only inhibited by PP2 in cells overexpressing NBCe1. Furthermore, only NBCe1 was significantly phosphorylated and translocated by NH4Cl, which was inhibited by PP2. Our results suggest that both NBCn1 and NBCe1 play a role in pHi regulation in hSMG acinar cells, and also that Src kinase does not regulate the activity of NBCn1.  相似文献   

19.
Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH i decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pH i increase caused by elevating extracellular [K+], and (iii) an acid shift in the pH i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH i dependence of Na-H exchange, computed by adding the pH i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999  相似文献   

20.
The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pHo from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h−1), and were submitted to acid shock at pH 4.0. Cells grown at low pHo were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pHo and μ, and the μ effect was more marked at pHo 5.5. Intracellular pH (pHi) was affected by both pHo and μ. At a pHo above 6, the pHi decreased with the decrease of pHo and the increase of μ. At pHo 5.5, pHi was higher compared to pHo 6.0, suggesting that mechanisms of pHi homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pHi and ΔpH (pHi - pHo). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pHo and μ.  相似文献   

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