Proposed thresholds for pancreatic tissue volume for safe intraportal islet autotransplantation after total pancreatectomy.

The simple question of how much tissue volume (TV) is really safe to infuse in total pancreatectomy-islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) precipitated this analysis. We examined a large cohort of CP patients (n = 233) to determine major risk factors for elevated portal pressure (PP) during islet infusion, using bivariate and multivariate regression modeling. Rates of bleeding requiring operative intervention and portal venous thrombosis (PVT) were evaluated. The total TV per kilogram body weight infused intraportally was the best independent predictor of change in PP (ΔPP) (p < 0.0001; R(2) = 0.566). Rates of bleeding and PVT were 7.73% and 3.43%, respectively. Both TV/kg and ΔPP are associated with increased complication rates, although ΔPP appears to be more directly relevant. Receiver operating characteristic analysis identified an increased risk of PVT above a suggested cut-point of 26 cmH2O (area under the curve = 0.759), which was also dependent on age. This ΔPP threshold was more likely to be exceeded in cases where the total TV was >0.25 cm(3)/kg. Based on this analysis, we have recommended targeting a TV of <0.25 cm(3)/kg during islet manufacturing and to halt intraportal infusion, at least temporarily, if the ΔPP exceeds 25 cmH2O. These models can be used to guide islet manufacturing and clinical decision making to minimize risks in TP-IAT recipients.

Resistively loaded coplanar waveguide for microwave measurements of induced carriers.

We describe the use of a coplanar waveguide (CPW) whose slots are filled with a resistive film, a resistively loaded CPW (RLCPW), to measure two-dimensional electron systems (2DESs). The RLCPW applied to the sample hosting the 2DES provides a uniform metallic surface serving as a gate to control the areal charge density of the 2DES. As a demonstration of this technique, we present measurements on a Si metal-oxide-semiconductor field-effect transistor and a model that successfully converts microwave transmission coefficients into conductivity of a nearby 2DES capacitively coupled to the RLCPW. We also describe the process of fabricating the highly resistive metal film required for fabrication of the RLCPW.

Endoscopic ultrasound rendezvous for bile duct access using a transduodenal approach: cumulative experience at a single center. A case series.

Endoscopic ultrasound (EUS)-assisted biliary access is utilized when conventional endoscopic retrograde cholangiopancreatography (ERCP) fails. We report a 10-year experience utilizing a transduodenal EUS rendezvous via a transpapillary route without dilation of the transduodenal tract, followed by immediate ERCP access. Patients included all EUS-guided rendezvous procedures for biliary access that were performed following ERCP failure. EUS-assisted bile duct puncture was performed via a transduodenal approach and a guide wire was advanced through the papilla without any dilation or bougienage of the tract; ERCP was performed immediately afterwards. EUS-assisted biliary rendezvous was attempted in 15 patients (mean age 66 +/- 18.2 years; malignant = 10, benign = 5). Mean diameter of measured bile ducts was 14.3 +/- 5.17 mm (range 4-23 mm). The reasons for initial ERCP failure were tumor infiltration or edema (n = 9), intradiverticular papilla (n = 2), pre-existing duodenal stent (n = 1), and anatomic anomalies (n = 3). Successful EUS-guided bile duct puncture and wire passage were achieved in all 15 patients (100 %), with drainage being successful in 12 / 15 (80 %). Failures occurred in three patients due to inability to traverse the biliary stricture (n = 2) or dissection of a choledochocele with the guide wire (n = 1); all were subsequently drained via percutaneous methods. Stents placed were metallic in eight patients and plastic in four. Complications consisted of moderate pancreatitis after a difficult ERCP attempt in one patient, and bacteremia after percutaneous biliary drainage in another. There were no instances of perforation, extraluminal air or fluid collections. EUS-assisted biliary drainage utilizing a transduodenal rendezvous approach demonstated a high success rate without any complications directly attributable to the EUS access. Advantages over percutaneous biliary and other methods of EUS biliary access include performance under the same anesthesia, and a very small-caliber needle puncture similar to EUS/fine-needle aspiration.

Significant clinical implications of prophylactic pancreatic stent placement in previously normal pancreatic ducts.

Pancreatic duct stent placement is increasingly performed for the prevention of pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP); however stents can result in injury especially in normal ducts. The clinical significance and outcomes of subsequent endoscopic therapy are unknown. This study was a retrospective review of the management of symptomatic stent-induced pancreatic duct injury following stent placement for prevention of post-ERCP pancreatitis in eight patients with previously normal pancreatic ducts. Subsequent treatment included pancreatic sphincterotomy, balloon dilation of stricture, and placement of multiple 3 - 5-Fr soft polymer pancreatic stents. All patients showed improvement or resolution of pancreatic strictures. Five patients had resolution or substantial improvement of pain, one patient showed a fair response with repeated ERCPs, and two patients failed to respond and underwent total pancreatectomy with islet autotransplantation. Pancreatic duct stent-induced ductal injury with significant clinical consequences can occur with conventional polyethylene stents. Endoscopic therapy is moderately effective but some patients develop irreversible damage. Caution should be used when placing standard polyethylene stents in normal ducts. Further research is required to identify safer materials and configurations of pancreatic stents.

Biliary cannulation during endoscopic retrograde cholangiopancreatography: core technique and recent innovations.

Despite advances in imaging and device technology over the past decade, endoscopic retrograde cholangiopancreatography (ERCP) continues to be one of the most technically challenging interventions in endoscopy. The procedure remains compounded by two persistent problems: failure of successful biliary cannulation and post-ERCP pancreatitis (PEP). When performed outside expert high-volume centers, failed biliary cannulation may occur in up to 20 % of cases; repeated and prolonged attempts at cannulation increase the risk of pancreatitis, delay definitive therapy, and necessitate alternative therapeutic techniques with inferior safety profiles . Cannulation technique is believed to be a pivotal factor in the genesis of PEP and is obviously important for successful cannulation. This review will discuss some recent innovations in cannulation technique.

Recent advances and novel methods in pancreatic imaging.

Traditional imaging studies for evaluating pancreatic disease including abdominal ultrasound (US) and computerized tomography (CT) are widely utilized due to their availability, non-invasiveness, and familiarity to practitioners. The addition of endoscopic ultrasound (EUS) and magnetic resonance imaging (MRI) and magnetic resonance cholangiopancreatography (MRCP) has contributed significantly to the clinician's the ability to safely sample tissue, stage malignancy, evaluate the pancreatic ductal anatomy, and look for subtle parenchymal changes in the setting of chronic pancreatitis. The role of endoscopic retrograde cholangiopancreatography (ERCP) has diminished with the use of these less invasive modalities. Limitations in these conventional techniques include a lack of sensitivity and specificity in diagnosing early chronic pancreatitis, difficulties in differentiating malignancy from chronic or focal pancreatitis, and accuracy of staging pancreatic malignancy, particularly with regard to vascular involvement. Several recent advances in traditional imaging techniques have been described, which may improve our ability to accurately diagnose and stage pancreatic disease. Advances have been made in the standard modalities for imaging the pancreas such as multidetector CT, micro-bubble contrast enhanced ultrasound, and secretin stimulated MRCP. Other novel methods of pancreatic imaging have recently been described including EUS elastography, optical coherence tomography, diffusion weighted MRI, and MR spectroscopy. This article will review the recent advances in both traditional pancreatic imaging modalities as well as some of the emerging technologies for imaging evaluating diseases of the pancreas. As experience and clinical evidence accumulate, the role of these imaging techniques will continue to evolve.

Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells.

BACKGROUND: The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a number of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. METHODS: We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and containing a mutation (6174delT) in the remaining copy. In vitro and in vivo experiments were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. RESULTS: BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks) (two-sided P = .002) and to ionizing radiation (two-sided P = .001). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone (two-sided P = .008). Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size reduction, two-sided P = .002) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histologic analysis of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that observed for control tumors possessing normal BRCA2 function. CONCLUSION: BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.

Effect of pH on hyperthermic cell survival.

Chinese hamster ovary cells incubated with various concentrations of CO2, to obtain extracellular pH values in the range of 6.40-7.85, were heated at 45.5C for 5, 10, or 20 minutes. Thermal sensitivity increased sharply from pH 7.35 to 6.65 (i.e., survival decreased from 1 X 10(-2) to 3 X 10(-5) for 20 minutes of heating), but remained constant from pH 7.35 to 7.85. The enhanced thermal sensitivity at pH values below pth 7.35 suggested that tumors should be preferentially destroyed by heat relative to normal tissue, since reports indicated that tumors were more acidic than the surrounding normal tissue.

Modulation of diamide toxicity in thermotolerant cells by inhibition of protein synthesis.

Chinese hamster ovary cells were exposed in vitro to various concentrations of diamide for 1 h at 37 degrees C. This treatment resulted in a dose dependent increase in cytotoxicity. Cells were also heated at 43 degrees C for 15 min, incubated at 37 degrees C for 3 h, and then exposed to various concentrations of diamide. This heat shock has been shown previously to trigger the synthesis of heat shock proteins and the development of thermotolerance. Further, under these experimental conditions both were inhibited if protein synthesis was inhibited by exposure to cycloheximide (M. L. Freeman et al., Radiat. Res., 112: 195-203, 1987). Diamide toxicity was diminished in cells made thermotolerant by the 43 degrees C/15-min heat shock. For example, at the highest dose used, 0.8 mM, survival increased from 0.93% to 6.1%. However, diamide toxicity was unaffected if the cells were exposed to diamide 3 h after a 43 degrees C/60 min heat shock. This latter heat shock produced significant inhibition of protein synthesis whereas the 15-min heat shock did not (M. L. Freeman et al., Cancer Res., 48: 7033-7037, 1988). Further, a 43 degrees C/15-min heat shock did not confer protection against diamide toxicity if the cells were simultaneously exposed to cycloheximide. Exposure to 0.8 mM diamide was shown to oxidize specific cellular proteins as measured by 2-dimensional thiol blotting. However, the degree of protein thiol modification was not affected by a prior heat shock. Nor did the heat shock increase the intracellular concentration of glutathione or the activity of glutathione reductase. The diamide treatment caused specific, as opposed to general, protein thiol oxidation and heat shock did not prevent this. It is hypothesized that it was the oxidation of protein thiols which led to cellular toxicity. Protein synthesis, triggered by heat shock, protected cells from the diamide toxicity without preventing protein thiol modification. These results suggest that the proteins synthesized after heat shock can provide protection against the consequences of aberrant proteins produced by thiol oxidation.

Decreased intracellular glutathione concentration and increased hyperthermic cytotoxicity in an acid environment.

Chinese hamster ovary (CHO) cells were heated at either pH 7.2 to 7.4 or 6.7 to 6.8 in order to determine if conditions which suppress the development of thermotolerance (pH 6.7 to 6.8) reduce intracellular levels of glutathione (GSH). When the pH of the growth medium was reduced from 7.2 to 6.7, a 25 to 30% reduction in GSH was observed in cells maintained at 37 degrees. Cells heated at 42 degrees in medium adjusted to pH 6.7 had lower levels of GSH compared to cells heated at pH 7.2. Cells were also heated for 1 hr at 43 degrees and then incubated at 37 degrees for up to 9.5 hr prior to GSH measurement. The GSH levels of cells treated at pH 7.3 increased approximately 20% above control, whereas treatment at pH 6.7 resulted in a 20% reduction compared to control. Chinese hamster ovary cells were exposed to 5 mM buthionine sulfoximine (BSO) prior to and during 42 degrees heat treatment. BSO exposure at either pH 7.3 or 6.8 reduced the GSH concentration to approximately 65% of control and increased thermal cytotoxicity. The thermal sensitivity of cells incubated at 42 degrees and pH 7.3 was compared to that of cells incubated at pH 6.8. Decreasing the pH from 7.3 to 6.8 increased sensitivity by a factor of 1.87 in the absence of BSO, whereas decreasing the pH in the presence of BSO increased sensitivity by only 1.50. In summary, these results suggest that the increase in thermal sensitivity observed when Chinese hamster ovary cells are heated in acid medium is due partly to the depletion of GSH.

Role of glutathione in cell survival after hyperthermic treatment of Chinese hamster ovary cells.

Chinese hamster ovary cells cultured in vitro were exposed to hyperthermic treatment at 43 degrees C or 45 degrees C. Depletion of 99.5% of the intracellular glutathione (GSH) by addition of 0.1 mM diethylmaleate (DEM) increased thermal sensitivity. This effect, however, was dependent upon oxygen tension. Decreasing the oxygen tension reduced the effect. Thermal cytotoxicity was further augmented by continued depletion of GSH after hyperthermic treatment. Once cells became thermally tolerant, depletion of GSH did not affect thermal sensitivity. The development of thermotolerance was triggered by exposing Chinese hamster ovary cells to a thermal dose consisting of either 30 min at 43 degrees or 10 min at 45 degrees. After a 4-h incubation at 37 degrees, the cells were reheated at 43 degrees or 45 degrees, respectively. A triggering dose of 30 min at 43 degrees is a nonlethal treatment, while 10 min at 45 degrees kills 50% of the cells. Cells were exposed to 0.1 mM DEM during the triggering and development phases of tolerance. Depletion of GSH by addition of DEM did not inhibit the development of thermal tolerance when triggered by the 30-min-43 degrees thermal dose. However, exposure to DEM did inhibit the development of thermal tolerance if the triggering dose consisted of 10 min at 45 degrees. These results were interpreted to mean that GSH depletion will inhibit the development of thermotolerance only after a sufficiently toxic thermal dose.

Failure of chronic glutathione elevation to reduce cytotoxicity produced by exposure to cis-diamminedichloroplatinum(II), ionizing radiation, or hyperthermia.

Chinese hamster ovary (CHO) cells cultured in vitro were continuously exposed to increasing concentrations of diethylmaleate (DEM). Chronic exposure of these cells (designated CHO/DEM) to 80 microM diethylmaleate resulted in an increase in cystine transport, a decrease in glutathione inhibition of the enzyme gamma-glutamylcysteine synthetase, elevation of intracellular glutathione levels to 4.3 times control, and elevation of glutathione-S-transferase activity by 6.6 times. Yet, CHO/DEM and control CHO cells exhibited the same ability to synthesize protein as measured by two-dimensional electrophoresis, the same cell cycle distribution, and the same population doubling times. CHO/DEM cells are resistant to DEM and diamide cytotoxicity, compared to control CHO cells. CHO/DEM cells were used to address the question of whether chronic elevation of glutathione, above control concentrations, reduced the cytotoxicity produced by exposure to cisplatin, gamma-radiation, or hyperthermia. The resulting dose-response curves obtained with CHO/DEM and control CHO cells indicated that chronic exposure to DEM, which resulted in chronic elevation of glutathione, did not provide protection against any of the three toxic treatments.

Depletion of glutathione, heat shock protein synthesis, and the development of thermotolerance in Chinese hamster ovary cells.

The synthesis of heat shock proteins (HSP) and the development of thermotolerance were studied in Chinese hamster ovary cells in order to determine whether depletion of intracellular glutathione (GSH) inhibited their expression. Cells were exposed to 100 microM diethylmaleate/50 microM buthionine sulfoximine which reduced GSH levels by 95% or more during the experimental time course. HSP synthesis was induced by incubation at 43 degrees C for varying durations. Synthesis was independent of the diethylmaleate/buthionine sulfoximine treatment if mild heat shocks (e.g., 43 degrees C for 15 min) were administered but was suppressed by such severe treatments as 45 or 60 min at 43 degrees C which caused inhibition of non-heat shock protein synthesis. GSH depletion also resulted in inhibition of thermotolerance triggered by a 45-min, 43 degrees C heat shock. This observation and a previous one, which showed that inhibition of protein synthesis by exposure to cycloheximide inhibited both HSP and tolerance (M. L. Freeman et al., Radiat. Res., 112: 564-574, 1987), indicate that glutathione is not involved in either the synthesis of HSP or the expression of tolerance but that GSH depletion can inhibit them indirectly via nonspecific inhibition of protein synthesis.

Conjugation of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 with intracellular glutathione and enhancement of its antiproliferative activity by glutathione depletion.

The major dehydration product of prostaglandin D2, 9-deoxy-delta 9,delta 12(E)-prostaglandin D2, is a potent cytotoxic compound. Like other cytotoxic prostaglandins, this compound possesses an alpha, beta-unsaturated ketone group to which cytotoxic activity has been attributed. This prostaglandin was found to readily conjugate with glutathione (GSH) in vitro. When 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 was incubated with Chinese hamster ovary or hepatoma tissue culture cells, it was rapidly taken up and was recovered in the cell lysate primarily as a GSH conjugate in which the keto group at C-11 and the delta 12 double bond had been reduced. Identification of the GSH conjugate was accomplished by analysis by fast atom bombardment mass spectrometry following purification by high performance liquid chromatography. This GSH conjugate and its cysteinylglycinyl and cysteinyl metabolites were also identified in the cell culture medium. 9-Deoxy-delta 9,delta 12(E)-prostaglandin D2 inhibited cell proliferation of these two cell lines in a concentration dependent manner. Depletion of intracellular glutathione by treatment with diethyl maleate and buthionine sulfoximine decreased the amount of intracellular conjugated prostaglandin recovered, and significantly enhanced the antiproliferative effect of 9-deoxy-delta 9-delta 12(E)-prostaglandin D2 on the growth of these cell lines in a concentration dependent fashion. We conclude that intracellular GSH may modulate the antiproliferative activity of 9-deoxy-delta 9,delta 12(E)-prostaglandin D2 and, possibly, of other cytotoxic prostaglandins.

The role of endoscopic retrograde cholangiopancreatography and endoscopic ultrasound in diagnosis and treatment of acute pancreatitis.

Endoscopic retrograde cholangiopancreatography (ERCP) is an important tool for diagnosis and therapy in acute and recurrent pancreatitis. While treatment of biliary disorders leading to pancreatitis is common practice, over the past several years many specialized centers have been directing traditional biliary techniques such as sphincterotomy and stenting towards the pancreas. A justifiable fear of pancreatitis and other complications has caused many endoscopists to shy away from pancreatic endotherapy, but refinements in technique, extensive experience, and most notably the routine use of pancreatic stenting to prevent post-ERCP pancreatitis has opened up the field and allowed for endoscopists in specialized centers around the world to perform diagnostic and therapeutic ERCP of the pancreas safely and effectively. In acute gallstone pancreatitis, the benefit of therapeutic ERCP including biliary sphincterotomy has been proven in randomized controlled trials. There are also data to support the role of ERCP directed at the pancreatic sphincters and ducts in treatment of acute relapsing pancreatitis due to pancreas divisum, sphincter of Oddi dysfunction, smoldering pancreatitis, pancreatic ductal disruptions, and perhaps even in evolving pancreatic necrosis. Many causes of apparently idiopathic pancreatitis can be discovered after an extensive evaluation with endoscopic ultrasound (EUS), magnetic resonance cholangiopancreatography (MRCP) and ERCP with sphincter of Oddi manometry. ERCP often allows treatment of the underlying cause. Because of the inherent risks associated with ERCP, particularly when directed toward the pancreas, the role of ERCP in acute and especially recurrent pancreatitis should be primarily therapeutic with attempts to establish diagnosis whenever possible by less risky techniques including EUS and MRCP. With the added techniques, devices, skill-sets, and experience required, pancreatic endotherapy should preferably be performed in high volume tertiary referral settings. ERCP for diagnosis and treatment of severe or acute relapsing pancreatitis is also best performed using a multidisciplinary approach involving endoscopy, hepatobiliary-pancreatic surgery, and interventional radiology.

On the path to the heat shock response: destabilization and formation of partially folded protein intermediates, a consequence of protein thiol modification.

This review discusses the initial events that occur during oxidative stress that induce the synthesis of heat shock proteins. The focus is on non-native oxidation or modification of protein thiols and the destablization that can result. Proteins that contain non-native modified thiols can become destablized such that they unfold into molten globule-like intermediates at or below 37 degrees C, relieving Hsf-1 negative regulation, and inducing Hsp transcription.

Enhanced gamma-glutamyl transpeptidase expression and selective loss of CuZn superoxide dismutase in hepatic iron overload.

Liver injury caused by iron overload is presumed to involve lipid peroxidation and the formation of products such as 4-hydroxynonenal (4HNE), which has been implicated in hepatic fibrogenesis. Cellular antioxidants that modulate the formation and detoxification of compounds such as 4HNE may represent important protective mechanisms involved in the response to iron overload. This study examines the relationship between 4HNE, collagen content, and antioxidant defenses in the livers of rats fed carbonyl iron for 10 weeks. Iron-loading resulted in significant increases in iron (8.8-fold), 4HNE (1.7-fold), and hydroxyproline (1.5-fold). Total glutathione content was unchanged by iron, but gamma-glutamyl transpeptidase activity (GGT) increased sixfold and CuZn superoxide dismutase (CuZnSOD) activity decreased >9%. GGT colocalized with iron deposition and was associated with increased GGT mRNA. Decreased CuZnSOD activity was paralleled by a reduction in CuZnSOD protein on Western blot and immunohistochemistry, but no decrease in CuZnSOD mRNA. Glutathione S-transferase (GST) and Mn superoxide dismutase (MnSOD) activities were also significantly increased by iron loading. These results demonstrate that iron overload significantly alters the expression of antioxidant enzymes associated with glutathione (GGT and GST) and superoxide metabolism (CuZnSOD and MnSOD). Furthermore, the localized induction of GGT may enhance detoxification of lipid peroxidation-derived aldehydes via glutathione-dependent pathways in iron-loaded hepatocytes. These alterations in antioxidant defenses may represent an adaptive response, limiting accumulation 4HNE, and thus, stimulation of collagen synthesis, accounting for the mild fibrogenic response seen in this model of iron overload.

The effect of GSH depletion on thermal radiosensitization.

Depletion of intracellular glutathione (GSH) increased aerobic thermal radiosensitization in Chinese hamster ovary (CHO) cells gamma irradiated and heated at 42 degrees C. The GSH concentration was decreased to various stable levels by exposure to increasing concentrations of diethylmaleate (DEM). Analysis of dose-response curves indicated that GSH depletion affected thermal sensitization and thermal radiosensitization at 42 degrees when greater than 95% of the GSH had been depleted. GSH depletion also increased the fixation of radiation damage. For example, survival after 10 Gy decreased from 0.012 to 0.006 if CHO cells were incubated in 100 microM DEM at 37 degrees for 2 hrs after irradiation. The results show that GSH might be an important agent for the protection of cells against thermal enhancement of radiation damage.

Acid modification of thermal damage and its relationship to nutrient availability.

The purpose of this present investigation was to determine if the presence of exogenous nutrients constitutes a prerequisite for acid modification of thermal damage and to determine if thermal cytotoxicity is affected by nutrient availability under acid conditions. To this end, Chinese hamster ovary cells (CHO) were heated (42 degrees or 43 degrees C) and/or irradiated in either McCoy's medium containing 10% fetal bovine serum or glucose free Hanks Balanced Salt Solution (HBSS). Both thermal sensitivity and thermal radiosensitization were increased when CHO cells were treated under acid conditions (e.g., pH 6.8) compared to alkaline conditions (e.g., pH 7.2), independent of the media used. Furthermore, decreasing nutrient availability increased thermal cytotoxicity, but the increase was greatest at pH 7.2 compared to pH 6.8. This study indicates that thermal sensitivity is more dependent upon pH than upon nutrient availability.