Renal oxygenation changes during acute unilateral ureteral obstruction: assessment with blood oxygen level-dependent mr imaging--initial experience

PURPOSE: To prospectively determine if changes in intrarenal oxygenation during acute unilateral ureteral obstruction can be depicted with blood oxygen level-dependent (BOLD) magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the local ethics committee, and written informed consent was obtained from all patients. BOLD MR imaging was performed in 10 male patients (mean age, 45 years +/- 17 [standard deviation]; range, 20-73 years) with a distal unilateral ureteral calculus and in 10 healthy age-matched male volunteers to estimate R2*, which is inversely related to tissue Po(2). R2* values were determined in the cortex and medulla of the obstructed and the contralateral nonobstructed kidneys. To reduce external effects on R2*, the R2* ratio between the medulla and cortex was also analyzed. Statistical analysis was performed with nonparametric rank tests. P < .05 was considered to indicate a significant difference. RESULTS: All patients had significantly lower medullary and cortical R2* values in the obstructed kidney (median R2* in medulla, 10.9 sec(-1) [range, 9.1-14.3 sec(-1)]; median R2* in cortex, 10.4 sec(-1) [range, 9.7-11.3 sec(-1)]) than in the nonobstructed kidney (median R2* in medulla, 17.2 sec(-1) [range, 14.6-23.2 sec(-1)], P = .005; median R2* in cortex, 11.7 sec(-1) [range, 11.0-14.0 sec(-1)], P = .005); values in the obstructed kidneys were also significantly lower than values in the kidneys of healthy control subjects (median R2* in medulla, 16.1 sec(-1) [range, 13.9-18.1 sec(-1)], P < .001; median R2* in cortex, 11.6 sec(-1) [range, 10.5-12.9 sec(-1)], P < .001). R2* ratios in the obstructed kidneys (median, 1.06; range, 0.85-1.27) were significantly lower than those in the nonobstructed kidneys (median, 1.49; range, 1.26-1.71; P = .005) and those in the kidneys of healthy control subjects (median, 1.38; range, 1.23-1.47; P < .001). In contrast, R2* ratios in the nonobstructed kidneys of patients were significantly higher than those in kidneys of healthy control subjects (P = .01). CONCLUSION: Increased oxygen content in the renal cortex and medulla occurs with acute unilateral ureteral obstruction, suggesting reduced function of the affected kidney.

Regional structural characteristics of bovine periodontal ligament samples and their suitability for biomechanical tests

Mechanical testing of the periodontal ligament requires a practical experimental model. Bovine teeth are advantageous in terms of size and availability, but information is lacking as to the anatomy and histology of their periodontium. The aim of this study, therefore, was to characterize the anatomy and histology of the attachment apparatus in fully erupted bovine mandibular first molars. A total of 13 teeth were processed for the production of undecalcified ground sections and decalcified semi-thin sections, for NaOH maceration, and for polarized light microscopy. Histomorphometric measurements relevant to the mechanical behavior of the periodontal ligament included width, number, size and area fraction of blood vessels and fractal analysis of the two hard-soft tissue interfaces. The histological and histomorphometric analyses were performed at four different root depths and at six circumferential locations around the distal and mesial roots. The variety of techniques applied provided a comprehensive view of the tissue architecture of the bovine periodontal ligament. Marked regional variations were observed in width, surface geometry of the two bordering hard tissues (cementum and alveolar bone), structural organization of the principal periodontal ligament connective tissue fibers, size, number and numerical density of blood vessels in the periodontal ligament. No predictable pattern was observed, except for a statistically significant increase in the area fraction of blood vessels from apical to coronal. The periodontal ligament width was up to three times wider in bovine teeth than in human teeth. The fractal analyses were in agreement with the histological observations showing frequent signs of remodeling activity in the alveolar bone - a finding which may be related to the magnitude and direction of occlusal forces in ruminants. Although samples from the apical root portion are not suitable for biomechanical testing, all other levels in the buccal and lingual aspects of the mesial and distal roots may be considered. The bucco-mesial aspect of the distal root appears to be the most suitable location.

Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature

Many preanalytical variables affect the results of coagulation assays. A possible way to control some of them would be to accept blood specimens shipped in the original collection tube. The aim of our study was to investigate the stability of coagulation assays in citrated whole blood transported at ambient temperature for up to two days after specimen collection. Blood samples from 59 patients who attended our haematology outpatient ward for thrombophilia screening were transported at ambient temperature (outdoor during the day, indoor overnight) for following periods of time: <1 hour, 4-6, 8-12, 24-28 and 48-52 hours prior to centrifugation and plasma-freezing. The following coagulation tests were performed: PT, aPTT, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FIX:C, FX:C, FXI:C, VWF:RCo, VWF:Ag, AT, PC activity, total and free PS antigen, modified APC-sensitivity-ratio, thrombin-antithrombin-complex and D-dimer. Clinically significant changes, defined as a percentage change of more than 10% from the initial value, were observed for FV:C, FVIII:C and total PS antigen starting at 24-28 hours, and for PT, aPTT and FVII:C at 48-52 hours. No statistically significant differences were seen for fibrinogen, antithrombin, or thrombin-antithrombin complexes (Friedman repeated measures analysis of variance). The present data suggest that the use of whole blood samples transported at ambient temperature may be an acceptable means of delivering specimens for coagulation analysis. With the exception of factor V and VIII coagulant activity, and total PS antigen all investigated parameters can be measured 24-28 hours after specimen collection without observing clinically relevant changes.

Acute alcohol ingestion and sympathetic neural responses during orthostatic stress in humans

Acute alcohol consumption has been reported to decrease mean arterial pressure (MAP) during orthostatic challenge, a response that may contribute to alcohol-mediated hypotension and eventually syncope. Muscle sympathetic nerve activity (MSNA) increases during orthostatic stress to help maintain MAP, yet the influence of alcohol on MSNA during orthostatic stress has not been determined. We hypothesized that alcohol ingestion would blunt arterial blood pressure and MSNA responses to progressive lower body negative pressure (LBNP). MAP, MSNA, and heart rate (HR) were recorded during progressive LBNP (-5, -10, -15, -20, -30, and -40 mmHg; 3 min/stage) in 30 subjects(age 24 ± 1 yrs). After an initial progressive LBNP protocol (pre-treatment), subjects were randomly assigned to consume alcohol (0.8g ethanol/kg body mass; n=15) or placebo (n=15) and then repeated the progressive LBNP protocol (post-treatment). Alcohol increased (drug × treatment, P ≤ 0.05) resting HR (59 ± 2 to 65 ± 2 beats/min) and MSNA (13 ± 3 to 19 ± 4 bursts/min) when compared to placebo. While alcohol increased MAP (83 ± 2 to 87 ± 2 mmHg), these increases were also observed with placebo (82 ± 2 to 88 ± 1 mmHg; treatment, P < 0.05; drug × treatment, P > 0.05). During progressive LBNP, a prominent decrease in MAP was observed after alcohol (drug × time × treatment, P < 0.05), but not placebo. There was also a significant attenuated response in forearm vascular resistance (FVR) during progressive LBNP (drug × time × treatment, P < 0.05). MSNA and HR increased during all LBNP protocols, but there were no differences between treatments or groups (drugs). In summary, acute alcohol ingestion induces an attenuation in blood pressure response during an orthostatic challenge, possibly due to the effect that alcohol has on impairing peripheral blood vessel constriction.

EXHAUST EMISSIONS OF LOW LEVEL BLEND ALCOHOL FUELS FROM TWO-STROKE AND FOUR-STROKE MARINE ENGINES

The U.S. Renewable Fuel Standard mandates that by 2022, 36 billion gallons of renewable fuels must be produced on a yearly basis. Ethanol production is capped at 15 billion gallons, meaning 21 billion gallons must come from different alternative fuel sources. A viable alternative to reach the remainder of this mandate is iso-butanol. Unlike ethanol, iso-butanol does not phase separate when mixed with water, meaning it can be transported using traditional pipeline methods. Iso-butanol also has a lower oxygen content by mass, meaning it can displace more petroleum while maintaining the same oxygen concentration in the fuel blend. This research focused on studying the effects of low level alcohol fuels on marine engine emissions to assess the possibility of using iso-butanol as a replacement for ethanol. Three marine engines were used in this study, representing a wide range of what is currently in service in the United States. Two four-stroke engine and one two-stroke engine powered boats were tested in the tributaries of the Chesapeake Bay, near Annapolis, Maryland over the course of two rounds of weeklong testing in May and September. The engines were tested using a standard test cycle and emissions were sampled using constant volume sampling techniques. Specific emissions for two-stroke and four-stroke engines were compared to the baseline indolene tests. Because of the nature of the field testing, limited engine parameters were recorded. Therefore, the engine parameters analyzed aside from emissions were the operating relative air-to-fuel ratio and engine speed. Emissions trends from the baseline test to each alcohol fuel for the four-stroke engines were consistent, when analyzing a single round of testing. The same trends were not consistent when comparing separate rounds because of uncontrolled weather conditions and because the four-stroke engines operate without fuel control feedback during full load conditions. Emissions trends from the baseline test to each alcohol fuel for the two-stroke engine were consistent for all rounds of testing. This is due to the fact the engine operates open-loop, and does not provide fueling compensation when fuel composition changes. Changes in emissions with respect to the baseline for iso-butanol were consistent with changes for ethanol. It was determined iso-butanol would make a viable replacement for ethanol.

Alcohol and colorectal cancer: the role of alcohol dehydrogenase 1C polymorphism

BACKGROUND: Chronic alcohol consumption is a risk factor for colorectal cancer. Animal experiments as well as genetic linkage studies in Japanese individuals with inactive acetaldehyde dehydrogenase leading to elevated acetaldehyde concentrations following ethanol ingestion support the hypothesis that acetaldehyde may be responsible for this carcinogenic effect of alcohol. In Caucasians, a polymorphism of alcohol dehydrogenase 1C (ADH1C) exists resulting in different acetaldehyde concentrations following ethanol oxidation. METHODS: To evaluate whether the association between alcohol consumption and colorectal tumor development is modified by ADH1C polymorphism, we recruited 173 individuals with colorectal tumors diagnosed by colonoscopy and 788 control individuals without colorectal tumors. Genotyping was performed using genomic DNA extracted from whole blood followed by polymerase chain reaction. RESULTS: Genotype ADH1C*1/1 was more frequent in patients with alcohol-associated colorectal neoplasia compared to patients without cancers in the multivariate model controlling for age, gender, and alcohol intake (odds ratio = 1.674, 95% confidence interval = 1.110-2.524, 2-sided p from Wald test = 0.0139). In addition, the joint test of the genetic effect and interaction between ADH1C genotype and alcohol intake (2-sided p = 0.0007) indicated that the difference in ADH1C*1 polymorphisms between controls and colorectal neoplasia is strongly influenced by the alcohol consumption and that only individuals drinking more than 30 g ethanol per day with the genotype ADH1C*1/1 had an increased risk for colorectal tumors. CONCLUSIONS: These data identify ADH1C homozygosity as a genetic risk marker for colorectal tumors in individuals consuming more than 30 g alcohol per day and emphasize the role of acetaldehyde as a carcinogenic agent in alcohol-related colorectal carcinogenesis.

Effects of the glucocorticoid antagonist, mifepristone, on the consequences of withdrawal from long term alcohol consumption

BACKGROUND: Studies were carried out to test the hypothesis that administration of a glucocorticoid Type II receptor antagonist, mifepristone (RU38486), just prior to withdrawal from chronic alcohol treatment, would prevent the consequences of the alcohol consumption and withdrawal in mice. MATERIALS AND METHODS: The effects of administration of a single intraperitoneal dose of mifepristone were examined on alcohol withdrawal hyperexcitability. Memory deficits during the abstinence phase were measured using repeat exposure to the elevated plus maze, the object recognition test, and the odor habituation/discrimination test. Neurotoxicity in the hippocampus and prefrontal cortex was examined using NeuN staining. RESULTS: Mifepristone reduced, though did not prevent, the behavioral hyperexcitability seen in TO strain mice during the acute phase of alcohol withdrawal (4 hours to 8 hours after cessation of alcohol consumption) following chronic alcohol treatment via liquid diet. There were no alterations in anxiety-related behavior in these mice at 1 week into withdrawal, as measured using the elevated plus maze. However, changes in behavior during a second exposure to the elevated plus maze 1 week later were significantly reduced by the administration of mifepristone prior to withdrawal, indicating a reduction in the memory deficits caused by the chronic alcohol treatment and withdrawal. The object recognition test and the odor habituation and discrimination test were then used to measure memory deficits in more detail, at between 1 and 2 weeks after alcohol withdrawal in C57/BL10 strain mice given alcohol chronically via the drinking fluid. A single dose of mifepristone given at the time of alcohol withdrawal significantly reduced the memory deficits in both tests. NeuN staining showed no evidence of neuronal loss in either prefrontal cortex or hippocampus after withdrawal from chronic alcohol treatment. CONCLUSIONS: The results suggest mifepristone may be of value in the treatment of alcoholics to reduce their cognitive deficits.

Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Determination of Ethyl glucuronide and Ethyl sulfate from dried blood spots

BACKGROUND: Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are non-oxidative minor metabolites of ethanol. They are detectable in various body fluids shortly after initial consumption of ethanol and have a longer detection time frame than the parent compound. They are regarded highly sensitive and specific markers of recent alcohol uptake. This study evaluates the determination of EtG and EtS from dried blood spots (DBS), a simple and cost-effective sampling method that would shorten the time gap between offense and blood sampling and lead to a better reflectance of the actual impairment. METHODS: For method validation, EtG and EtS standard and quality control samples were prepared in fresh human heparinized blood and spotted on DBS cards, then extracted and measured by an LC-ESI-MS/MS method. Additionally, 76 heparinized blood samples from traffic offense cases were analyzed for EtG and EtS as whole blood and as DBS specimens. The results from these measurements were then compared by calculating the respective mean values, by a matched-paired t test, by a Wilcoxon test, and by Bland-Altman and Mountain plots. RESULTS AND DISCUSSION: Calibrations for EtG and EtS in DBS were linear over the studied calibration range. The precision and accuracy of the method met the requirements of the validation guidelines that were employed in the study. The stability of the biomarkers stored as DBS was demonstrated under different storage conditions. The t test showed no significant difference between whole blood and DBS in the determination of EtG and EtS. In addition, the Bland-Altman analysis and Mountain plot confirmed that the concentration differences that were measured in DBS specimens were not relevant.

Cellular Photo Digital Breathalyzer for Monitoring Alcohol Use: A Pilot Study

Background: Monitoring alcohol use is important in numerous situations. Direct ethanol metabolites, such as ethyl glucuronide (EtG), have been shown to be useful tools in detecting alcohol use and documenting abstinence. For very frequent or continuous control of abstinence, they lack practicability. Therefore, devices measuring ethanol itself might be of interest. This pilot study aims at elucidating the usability and accuracy of the cellular photo digital breathalyzer (CPDB) compared to self-reports in a naturalistic setting. Method: 12 social drinkers were included. Subjects used a CPDB 4 times daily, kept diaries of alcohol use and submitted urine for EtG testing over a period of 5 weeks. Results: In total, the 12 subjects reported 84 drinking episodes. 1,609 breath tests were performed and 55 urine EtG tests were collected. Of 84 drinking episodes, CPDB detected 98.8%. The compliance rate for breath testing was 96%. Of the 55 EtG tests submitted, 1 (1.8%) was positive. Conclusions: The data suggest that the CPDB device holds promise in detecting high, moderate, and low alcohol intake. It seems to have advantages compared to biomarkers and other Monitoring devices. The preference for CPDB by the participants might explain the high compliance. Further studies including comparison with biomarkers and transdermal devices are needed.

A case of distinct difference between the measured blood Ethanol concentration and the concentration estimated by Widmark's equation

In the last century, several mathematical models have been developed to calculate blood ethanol concentrations (BAC) from the amount of ingested ethanol and vice versa. The most common one in the field of forensic sciences is Widmark's equation. A drinking experiment with 10 voluntary test persons was performed with a target BAC of 1.2 g/kg estimated using Widmark's equation as well as Watson's factor. The ethanol concentrations in the blood were measured using headspace gas chromatography/flame ionization and additionally with an alcohol Dehydrogenase (ADH)-based method. In a healthy 75-year-old man a distinct discrepancy between the intended and the determined blood ethanol concentration was observed. A blood ethanol concentration of 1.83 g/kg was measured and the man showed signs of intoxication. A possible explanation for the discrepancy is a reduction of the total body water content in older people. The incident showed that caution is advised when using the different mathematical models in aged people. When estimating ethanol concentrations, caution is recommended with calculated results due to potential discrepancies between mathematical models and biological systems

Effects of weight on blood pressure at rest and during exercise.

Body weight (BW) and blood pressure (BP) have a close relationship, which has been accounted for by hormonal changes. No previous study has evaluated the effect of wearing an external weight vest on BP to determine whether there is a simple mechanism between BW and BP. Seventeen healthy volunteers underwent weight reduction (WR) through caloric restriction. Before and after WR, BW, body fat percentage and BP at rest and during exercise were measured. Before and after WR, exercise testing was performed twice with the random allocation of a weight vest (10 kg) during one of the tests. Linear regression was used to detect independent associations between BP and the weight vest, BW and body fat percentage. BW decreased from 89.4 ± 15.4 kg to 79.1 ± 14.0 kg following WR (P<0.001). WR led to significant decreases in BP at rest (from 130.0/85.9 mm Hg to 112.5/77.8 mm Hg, P<0.001 for systolic and diastolic BPs) and during exercise. The weight vest significantly increased BP at rest (to 136.1/90.7 mm Hg before and 125.8/84.6 mm Hg after WR) and during exercise. Linear regression analysis identified an independent association between the weight vest and BP (P=0.006 for systolic BP and P=0.009 for diastolic BP at rest). This study demonstrates that wearing an external weight vest has immediate effects on BP at rest and during exercise independent of BW or body fat. More research is needed to understand the physiological mechanisms between weight and BP.

The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots.

BACKGROUND AND OBJECTIVE Phenotyping cocktails use a combination of cytochrome P450 (CYP)-specific probe drugs to simultaneously assess the activity of different CYP isoforms. To improve the clinical applicability of CYP phenotyping, the main objectives of this study were to develop a new cocktail based on probe drugs that are widely used in clinical practice and to test whether alternative sampling methods such as collection of dried blood spots (DBS) or saliva could be used to simplify the sampling process. METHODS In a randomized crossover study, a new combination of commercially available probe drugs (the Basel cocktail) was tested for simultaneous phenotyping of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4. Sixteen subjects received low doses of caffeine, efavirenz, losartan, omeprazole, metoprolol and midazolam in different combinations. All subjects were genotyped, and full pharmacokinetic profiles of the probe drugs and their main metabolites were determined in plasma, dried blood spots and saliva samples. RESULTS The Basel cocktail was well tolerated, and bioequivalence tests showed no evidence of mutual interactions between the probe drugs. In plasma, single timepoint metabolic ratios at 2 h (for CYP2C19 and CYP3A4) or at 8 h (for the other isoforms) after dosing showed high correlations with corresponding area under the concentration-time curve (AUC) ratios (AUC0-24h parent/AUC0-24h metabolite) and are proposed as simple phenotyping metrics. Metabolic ratios in dried blood spots (for CYP1A2 and CYP2C19) or in saliva samples (for CYP1A2) were comparable to plasma ratios and offer the option of minimally invasive or non-invasive phenotyping of these isoforms. CONCLUSIONS This new combination of phenotyping probe drugs can be used without mutual interactions. The proposed sampling timepoints have the potential to facilitate clinical application of phenotyping but require further validation in conditions of altered CYP activity. The use of DBS or saliva samples seems feasible for phenotyping of the selected CYP isoforms.

Associations of ambulatory blood pressure with urinary caffeine and caffeine metabolite excretions.

Intake of caffeinated beverages might be associated with reduced cardiovascular mortality possibly via the lowering of blood pressure. We estimated the association of ambulatory blood pressure with urinary caffeine and caffeine metabolites in a population-based sample. Families were randomly selected from the general population of Swiss cities. Ambulatory blood pressure monitoring was conducted using validated devices. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24 hours urine using ultrahigh performance liquid chromatography tandem mass spectrometry. We used mixed models to explore the associations of urinary excretions with blood pressure although adjusting for major confounders. The 836 participants (48.9% men) included in this analysis had mean age of 47.8 and mean 24-hour systolic and diastolic blood pressure of 120.1 and 78.0 mm Hg. For each doubling of caffeine excretion, 24-hour and night-time systolic blood pressure decreased by 0.642 and 1.107 mm Hg (both P values <0.040). Similar inverse associations were observed for paraxanthine and theophylline. Adjusted night-time systolic blood pressure in the first (lowest), second, third, and fourth (highest) quartile of paraxanthine urinary excretions were 110.3, 107.3, 107.3, and 105.1 mm Hg, respectively (P trend <0.05). No associations of urinary excretions with diastolic blood pressure were generally found, and theobromine excretion was not associated with blood pressure. Anti-hypertensive therapy, diabetes mellitus, and alcohol consumption modify the association of caffeine urinary excretion with systolic blood pressure. Ambulatory systolic blood pressure was inversely associated with urinary excretions of caffeine and other caffeine metabolites. Our results are compatible with a potential protective effect of caffeine on blood pressure.

Resistance to Temptation: The Interaction of External and Internal Control on Alcohol Use During Residential Treatment for Alcohol Use Disorder

Background: There is evidence that drinking during residential treatment is related to various factors, such as patients’ general control beliefs and self-efficacy, as well as to external control of alcohol use by program’s staff and situations where there is temptation to drink. As alcohol use during treatment has been shown to be associated with the resumption of alcohol use after discharge from residential treatment, we aimed to investigate how these variables are related to alcohol use during abstinenceoriented residential treatment programs for alcohol use disorders (AUD). Methods: In total, 509 patients who entered 1 of 2 residential abstinence-oriented treatment programs for AUD were included in the study. After detoxification, patients completed a standardized diagnostic procedure including interviews and questionnaires. Drinking was assessed by patients’ selfreport of at least 1 standard drink or by positive breathalyzer testing. The 2 residential programs were categorized as high or low control according to the average number of tests per patient. Results: Regression analysis revealed a significant interaction effect between internal and external control suggesting that patients with high internal locus of control and high frequency of control by staff demonstrated the least alcohol use during treatment (16.7%) while patients with low internal locus of control in programs with low external control were more likely to use alcohol during Treatment (45.9%). No effects were found for self-efficacy and temptation. Conclusions: As alcohol use during treatment is most likely associated with poor treatment outcomes, external control may improve treatment outcomes and particularly support patients with low internal locus of control, who show the highest risk for alcohol use during treatment. High external control may complement high internal control to improve alcohol use prevention while in treatment. Key Words: Alcohol Dependence, Alcohol Use, Locus of Control, Alcohol Testing.