The Impact of Opioid Receptor Gene Polymorphism on Fentanyl and Alfentanil's Analgesic Effects in the Pediatric Perioperative Period.

Introduction: The polymorphism of the gene coding mu-opioid receptor (OPRM1) is one of the factors contributing to the variability in the response to opioid analgesics in children. The goal of this study is to investigate its role in association with postoperative acute pain in children of various ages. Methods: This prospective study analyzed 110 pediatric patients, after plastic or orthopedic surgery, who were genotyped and randomly assigned to receive fentanyl or alfentanil. Postoperative pain was rated using Numerical Rating Scale (0-10). All the patients were genotyped forOPRM1 118A>G (rs1799971) gene polymorphism. Results: School children under the age of 11 with the OPRM1 AA genotype were shown to have a higher BMI (p<0.05). Children over the age of 12 carrying G allele OPRM1, had increased postoperative pain sensitivity and intensity (3.28±1.95 vs 4.91±2.17; p<0.05), as compared to AA allele carriers. Discussion: OPRM1 118A>G polymorphism may explain the variation in the perception of postoperative pain in children over the age of 12 and may be a useful predictor for adjusting the dose of analgesics, but the dose is relative to the patient's needs regardless of his genetic characteristics. In younger children, carriers of polymorphic OPRM1 118G allele may be protected from obesity, due to diminished MOP expression.

Vitamin C and epigenetics: A short physiological overview.

In recent years, ascorbic acid (vitamin C) has acquired great interest due to its multiple functions, which results in homeostasis of normal tissues and organs. On the other hand, it has been shown that epigenetic modifications may have an important role in various diseases and therefore are a focus of the extraordinary investigation. Ascorbic acid serves as a cofactor for ten-eleven translocation dioxygenases, which are responsible for deoxyribonucleic acid methylation. Also, vitamin C is required for histone demethylation, since it acts as a cofactor of Jumonji C-domain-containing histone demethylases. It seems that vitamin C may be a mediator between the environment and the genome. The precise and multistep mechanism of ascorbic acid in epigenetic control is still not definitely determined. This article intends to provide the basic and newly discovered functions of vitamin C that are related to epigenetic control. Also, this article will help us to better understand the functions of ascorbic acid and will provide the possible implications of this vitamin in the regulation of epigenetic modifications.

The hepatoprotective effect of aminoguanidine in acute liver injury caused by CCl4 in rats.

In this study, the hepatoprotective effect of aminoguanidine in acute liver damage caused by carbon tetrachloride-CCl4 at a dose of 1 mL/kg, i.p. was investigated in experimental rats. Ten days of preventive treatment with aminoguanidine before exposure to toxic CCl4, at a dose of 150 mg/kg, i.p., led to significant reduction in biochemical markers of acute liver injury-AST(p < 0.001), ALT (p < 0.01), SDH (p < 0.05) and reduction in pro-oxidative markers-H2O2 (p < 0.05), TOS (p < 0.01), TBARS, and LOOH (p < 0.001) in relation to rats treated only CCl4. Treatment with aminoguanidine resulted in a significant reduction in the consumption of antioxidant-GR (p < 0.01), GST, GPx, GSH (p < 0.001), and a decrease in pro-inflammatory-TNF-α (p < 0.01), IL-1ß, IL-6, NO and NGAL (p < 0.001) markers relative to animals exposed to CCl4 alone. Also, aminoguanidine pre-treatment leads to an increase in arginase activity (p < 0.001), and a decrease in citrulline concentration (p < 0.01), as well as polyamine catabolism enzyme activity-putrescin oxidase and spermine oxidase (p < 0.001) in comparison to the CCl4 group. Aminoguanidine led to a striking reduction of the necrotic field (p < 0.001), and a significant increase in the number of apoptotic hepatocytes (p < 0.001), as well as the proapoptotic markers-BAX and Caspase-3 (p < 0.05), compared to CCl4. The hepatoprotective mechanisms in CCl4 induce hepatotoxicity of aminoguanidine are based on the strong antioxidant effects, inhibition of pro-oxidative and pro-inflammatory mediators, as well as induction of damaged hepatocytes into apoptosis.

Perioperative respiratory adverse events during ambulatory anesthesia in obese children.

Obesity is one of the most common clinical conditions in the pediatric population with an increasing prevalence ranging from 20 to 30% worldwide. It is well known that during ambulatory anesthesia, obese children are more prone to develop perioperative respiratory adverse events (PRAEs) associated with obesity. To avoid or at least minimize these adverse effects, a thorough preoperative assessment should be undertaken as well as consideration of specific anesthetic approaches such as preoxygenation before induction of anesthesia and optimizing drug dosing. The use of short-acting opioid and nonopioid analgesics and the frequent implementation of regional anesthesia should also be included. Noninvasive airway management, protective mechanical ventilation, and complete reversion of neuromuscular blockade and awake extubation also proved to be beneficial in preventing PRAEs. During the postoperative period, continuous monitoring of oxygenation and ventilation is mandatory in obese children. In the current review, we sought to provide recommendations that might help to reduce the severity of perioperative respiratory adverse events in obese children, which could be of particular importance for reducing the rate of unplanned hospitalizations and ultimately improving the overall postoperative recovery.

Predictive potential of biomarkers and risk scores for major adverse cardiac events in elderly patients undergoing major elective vascular surgery.

Elderly patients scheduled for major elective vascular surgery are at high risk for a major adverse cardiac events (MACE). The objectives of the study were: (1) To determine the individual discriminatory ability of four risk prediction models and four biomarkers in predicting MACEs in elderly patients undergoing major elective vascular surgery; (2) to find a prognostic model with the best characteristics; (3) to examine the significance of all preoperative parameters; and (4) to determine optimal cut-off values for biomarkers with best predictor capabilities. We enrolled 144 geriatric patients, aged 69.97 ± 3.73 years, with a 2:1 male to female ratio. Essential inclusion criteria were open major vascular surgery and age >65 years. The primary outcome was the appearance of MACEs within 6 months. These were noted in 33 (22.9%) patients. The most frequent cardiac event was decompensated heart failure, which occurred in 22 patients (15.3%). New onset atrial fibrillation was registered in 13 patients (9%), and both myocardial infarction and ventricular arrhythmias occurred in eight patients each (5.5%). Excellent discriminatory ability (AUC >0.8) was observed for all biomarker combinations that included the N-terminal fragment of pro-B-type natriuretic peptide (NT-proBNP). The most predictive two-variable combination was the Geriatric-Sensitive Cardiac Risk Index (GSCRI) + NT-proBNP (AUC of 0.830 with a 95% confidence interval). Female gender, previous coronary artery disease, and NT-proBNP were three independent predictors in a multivariate model of binary logistic regression. The Cox regression multivariate model identified high-sensitivity C-reactive protein and NT-proBNP as the only two independent predictors.

In silico development of potential therapeutic for the pain treatment by inhibiting voltage-gated sodium channel 1.7.

The voltage-gated sodium channel Nav1.7 can be considered as a promising target for the treatment of pain. This research presents conformational-independent and 3D field-based QSAR modeling for a series of aryl sulfonamide acting as Nav1.7 inhibitors. As descriptors used for building conformation-independent QSAR models, SMILES notation and local invariants of the molecular graph were used with the Monte Carlo optimization method as a model developer. Different statistical methods, including the index of ideality of correlation, were used to test the quality of the developed models, robustness and predictability and obtained results were good. Obtained results indicate that there is a very good correlation between 3D QSAR and conformation-independent models. Molecular fragments that account for the increase/decrease of a studied activity were defined and used for the computer-aided design of new compounds as potential analgesics. The final evaluation of the developed QSAR models and designed inhibitors were carried out using molecular docking studies, bringing to light an excellent correlation with the QSAR modeling results.

Preoperative Levels of the Soluble Urokinase-Type Plasminogen Activator Receptor as Predictor for New Episodes of Atrial Fibrillation After Vascular Surgery.

INTRODUCTION: The soluble urokinase-type plasminogen activator receptor (suPAR) in uPAR soluble form is produced when the uPAR is cleaved from the cell membrane during the inflammatory process. Postoperative atrial fibrillation (AF) is the most common perioperative cardiac arrhythmia. It is speculated that elevated suPAR has a role in the development of AF. The aim of our study was to investigate the predictive role of preoperative suPAR in the occurrence of AF during the first 6 months after major vascular surgery. METHODS: We included 119 male and 63 female patients with an average age of 67.19 ± 6.02 years, without permanent/persistent AF. Basic predictive model (BASIC) included traditional risk factors for AF: age, gender, body mass index-BMI, smoking status, presence of arterial hypertension, diabetes mellitus and dyslipidemia. RESULTS: Over the 6-month period, 19 (10.4%) patients had one new episode of atrial fibrillation and 2 patients (1.1%) had 2 episodes of paroxysmal atrial fibrillation. Paroxysms of AF were significantly more frequent in patients who had a resection of an abdominal aneurysm than in patients with other types of major vascular surgery. BASIC had good discriminatory ability in the prediction of AF paroxysms during the first 6 months after surgery (AUC = 0.715, 95%CI 0.590-0.840). Adding suPAR to the basic model significantly improved the discriminative ability of the predictive model for AF episodes (ΔAUC = 0.238, p < 0.001). The predictive performance of the model BASIC+CRP+suPAR, measured using AUC, NRI and IDI statistics, was very similar to the model BASIC+suPAR. CONCLUSION: AF is a common complication in surgical patients with high mortality and morbidity. suPAR could improve the ability of traditional risk factors to predict its occurrence up to 6 months after major vascular surgery.

The Application of the Combination of Monte Carlo Optimization Method based QSAR Modeling and Molecular Docking in Drug Design and Development.

In recent years, one of the promising approaches in the QSAR modeling Monte Carlo optimization approach as conformation independent method, has emerged. Monte Carlo optimization has proven to be a valuable tool in chemoinformatics, and this review presents its application in drug discovery and design. In this review, the basic principles and important features of these methods are discussed as well as the advantages of conformation independent optimal descriptors developed from the molecular graph and the Simplified Molecular Input Line Entry System (SMILES) notation compared to commonly used descriptors in QSAR modeling. This review presents the summary of obtained results from Monte Carlo optimization-based QSAR modeling with the further addition of molecular docking studies applied for various pharmacologically important endpoints. SMILES notation based optimal descriptors, defined as molecular fragments, identified as main contributors to the increase/ decrease of biological activity, which are used further to design compounds with targeted activity based on computer calculation, are presented. In this mini-review, research papers in which molecular docking was applied as an additional method to design molecules to validate their activity further, are summarized. These papers present a very good correlation among results obtained from Monte Carlo optimization modeling and molecular docking studies.

Design and development of novel therapeutics for coronary heart disease treatment based on cholesteryl ester transfer protein inhibition - in silico approach.

Cholesteryl ester transfer protein (CETP) belongs to the group of enzymes which inhibition have the application in the treatment of cardiovascular diseases. This study presents QSAR modeling for a set of compounds acting as CETP inhibitors based on the Monte Carlo optimization with SMILES notation and molecular graph-based descriptors, and field-based 3D modeling. A 3D QSAR model was developed for one random split into the training and test sets, whereas conformation independent QSAR models were developed for three random splits, with the results suggesting there is an excellent correlation between them. Various statistical approaches were used to assess the statistical quality of the developed models, including robustness and predictability, and the obtained results were very good. This study used a novel statistical metric known as the index of ideality of correlation for the final assessment of the model, and the results that were obtained suggested that the model was good. Also, molecular fragments which account for the increases and/or decreases of a studied activity were defined and then used for the computer-aided design of new compounds as potential CETP inhibitors. The final assessment of the developed QSAR model and designed inhibitors was done using molecular docking, which revealed an excellent correlation with the results from QSAR modeling.Communicated by Ramaswamy H. Sarma.

Congenital esophageal stenosis: a rare malformation of the foregut.

Congenital esophageal stenosis (CES) is a type of esophageal stenosis, and three histological subtypes (tracheobronchial remnants, fibromuscular thickening or fibromuscular stenosis, and membranous webbing or esophageal membrane) are described. Symptoms of CES usually appears with the introduction of the semisolid alimentation. Dysphagia is the most common symptom, but esophageal food impaction, respiratory distress or failure to thrive can be clinical manifestations of CES. Wide spectrum of differential diagnoses leads to delayed definitive diagnosis and appropriate treatment. Depends on hystological subtype of CES, some treatment procedures (dilation or segmental esophageal resection) are recommended, but individually approach is still important in terms of frequency and type of dilation procedures or type of the surgical treatment. Dysphagia can persist after the treatment and a long follow-up period is recommended. In 33% of patients with CES, a different malformations in the digestive system, but also in the other systems, are described.

Potential New Approaches in Predicting Adverse Cardiac Events One Month after Major Vascular Surgery.

OBJECTIVE: The aim of our study was to find the best model with sufficient power to improve the risk stratification in major vascular surgery patients during the first 30 days after this procedure. The discriminatory power of 4 biomarkers (troponin I [TnI], N-terminal prohormone of brain natriuretic peptide [NT-proBNP], creatine kinase-MB isoenzyme [CK-MB], high-sensitivity C-reactive protein [hs-CRP]) was tested as well as 2 risk assessment models and 13 different combinations of them. SUBJECTS AND METHODS: The study included 122 patients (77% men, 23% women) with an average age of 67.03 ± 4.5 years. An aortobifemoral bypass was performed in 6.56% of the patients, a femoropopliteal bypass in 18.85%, and 49.18% received open surgical reconstruction of the carotid arteries. A total of 25.41% of the patients were given an aortobi-iliac bypass. RESULTS: During the first 30 days, 13 patients (10.7%) had 17 cardiac complications. The most common complication was the new onset of atrial fibrillation (35.3%). During the first 10 days, 10 patients had 1 complication and 2 patients had 2 cardiac events, while 1 patient had 3 complications. By comparing combinations of scores and markers, it was shown that revised cardiac risk index (RCRI) + Vascular Portsmouth Physiological and Operative Severity Score (V-POSSUM) + hsTnI and RCRI + V-POSSUM + hsTnI + NT-proBNP with 100% sensitivity, > 80% specificity had the best discriminatory ability (AUC 0.924 and 0.933, respectively; p < 0.001 for both models) for cardiac complications during the 30 days after surgery. CONCLUSION: Combinations of traditional preoperative risk factors and scores can enhance the assessment of major adverse cardiac events (MACE) in patients preparing for large vascular surgery. Using only one risk score in these patients seems to be underperforming in preoperative risk assessment.

A Risk Stratification Model for Cardiovascular Complications during the 3-Month Period after Major Elective Vascular Surgery.

INTRODUCTION: The Revised Cardiac Risk Index (RCRI) is an extensively used simple risk stratification tool advocated by the European Society of Cardiology and European Society of Anesthesiology (ESC/ESA). PURPOSE: The aim of this study was to find the best model for predicting 3-month cardiovascular complications in elective major vascular surgical patients using preoperative clinical assessment, calculation of the RCRI and Vascular Physiological and Operative Severity Score for the enumeration of mortality and morbidity (V-POSSUM) scores, and the preoperative levels of N-terminal brain natriuretic peptide (NT pro-BNP), high-sensitivity troponin I (hs TnI), and high-sensitivity C-reactive protein (hs CRP). MATERIALS AND METHODS: We included 122 participants in a prospective, single-center, observational study. The levels of NT pro-BNP, hs CRP, and hs TnI were measured 48 hours prior to surgery. During the perioperative period and 90 days after surgery the following adverse cardiac events were recorded: myocardial infarction, arrhythmias, pulmonary edema, acute decompensated heart failure, and cardiac arrest. RESULTS: During the first 3 months after surgery 29 participants (23.8%) had 50 cardiac complications. There was a statistically significant difference in the RCRI score between participants with and without cardiac complications. ROC analysis showed that a combination of RCRI with hs TnI has good discriminatory power (AUC 0.909, p<0,001). By adding NT pro-BNP concentrations to the RCRI+hs TnI+V-POSSSUM combination we obtained the model with the best predictive power for 3-month cardiac complications (AUC 0.963, p<0,001). CONCLUSION: We need to improve preoperative risk assessment in participants scheduled for major vascular surgery by combining their clinical scores with biomarkers. Therefore, it is possible to identify patients at risk of cardiovascular complications who need adequate preoperative diagnosis and treatment.

Exogenous putrescine affects polyamine and arginine metabolism in rat liver following bile ductus ligation.

Rat bile duct ligation (BDL) represents a useful method that mimics obstructive extrahepatic cholestasis, which is known to be a frequent disorder in humans. Polyamines (putrescine, spermidine, and spermine) are one of the key molecules regulating cell proliferation and differentiation. This work aimed to evaluate the potential beneficial properties of putrescine in rat BDL model by studying several biochemical parameters reflecting liver function and polyamine metabolism. Rats that were subjected to BDL were injected with putrescine (150 mg/kg) for 9 days, while in parallel another group with BDL remained untreated. Two control groups were included as well, sham-opened and putrescine-treated group. The following plasma parameters: ALT, AST, γ-GT, ALP, bilirubin, bile acids, as well as liver malondialdehyde and polyamine concentration and the activity of enzymes involved in polyamine metabolism were studied. After BDL, significant alterations in plasma biochemical parameters occurred, where a 9-day putrescine treatment significantly alleviated liver function deterioration. Putrescine also increased liver polyamines' concentrations and polyamine and diamine oxidase activities in rats submitted to BDL. Our results demonstrated, for the first time, that putrescine plays an important role in preserving liver tissue function in rats with experimentally induced cholestasis.

A randomized trial of class 2 and class 3 elastic compression in the prevention of recurrence of venous ulceration.

BACKGROUND: Venous leg ulcers (VLUs) are a major health problem because of their high prevalence and associated high cost of care. Despite the widespread use of compression, treatment recurrence rates remain high. Numerous studies have suggested that regular use of compression stockings reduces VLU recurrences. However, there are limited data concerning how long compression hosiery should be worn after ulcer healing and which class of compression hosiery achieves better results in the prevention of VLU recurrences. METHODS: An open, prospective, randomized, single-center study with a 5-year follow-up was performed to establish the efficacy of two different strengths of knee-high compression hosiery (class 2 and class 3) in the prevention of VLU recurrences. The study included patients with recently healed venous ulcers and no significant arterial disease, rheumatoid disease, diabetes mellitus, and restriction in range of ankle movement. Overall, 361 patients were randomized, and 308 patients (170 men, 138 women; mean age, 59 years) completed the study. Patients were randomized into two groups: group A, 186 patients who wore a heel-less open-toed elastic class 3 compression device knitted in tubular form (Tubulcus; Laboratoires Innothera, Arcueil, France); and group B, 175 patients who wore a class 2 elastic stocking (Rudo, Nis, Serbia). All patients were instructed to wear compression stockings continuously for the first 2 years of follow-up (both during the day and at night). In the third, fourth, and fifth years of follow-up, patients were instructed to wear elastic stockings during the day only. The main outcome measures were recurrence of leg ulceration and compliance with the treatment. RESULTS: Rates of ulcer recurrence after the 5 years of follow-up were 28.98% for the compression class 3 group and 60% for the compression class 2 group (P < .001, log-rank test). Patients in the compression class 3 group experienced significantly longer absolute (46 vs 40 months; P < .001, Mann-Whitney U test) and proportional (77% vs 67%; P < .001, Mann-Whitney U test) ulcer-free time after 5 years than those in the compression class 2 group. Rates of noncompliance after 5 years were 10.23% for the compression class 3 group and 6.25% for the compression class 2 group (P = .188, χ2 test). CONCLUSIONS: The results obtained in this study suggest that class 3 compression stockings provide a statistically significant lower recurrence rate compared with the class 2 compression stockings.

Preoperative Midregional Pro-Adrenomedullin and High-Sensitivity Troponin T Predict Perioperative Cardiovascular Events in Noncardiac Surgery.

OBJECTIVE: We evaluated the utility of preoperative midregional (MR) pro-adrenomedullin (proADM) and cardiac troponin T (TnT) for improved detection of patients at high risk for perioperative cardiac events and mortality after major noncardiac surgery. SUBJECTS AND METHODS: This prospective, single-center, observational study enrolled 79 patients undergoing major noncardiac surgery. After initial clinical assessment (clinical history, physical examination, echocardiogram, blood tests, and chest X-ray), MR-proADM and high-sensitivity TnT (hsTnT) were measured within 48 h prior to surgery by immunoluminometric and electrochemiluminescence immunoassay. Patients were followed by the consulting physician until discharge or up to 14 days in the hospital after surgery. Perioperative cardiac events included myocardial infarction and development or aggravation of congestive heart failure. Data were compared between patients who developed target events and event-free patients. RESULTS: Within 14 days of monitoring, 14 patients (17.72%) developed target events: 9 (11.39%) died and 5 (6.33%) developed cardiovascular events. The average age of the patients was 71.29 ± 6.62 years (range: 55-87). Sex, age, and hsTnT did not significantly differ between groups. MR- proADM concentration was higher in deceased patients (p = 0.01). The upper quartile of MR-proADM was associated with a fatal outcome (66.7 vs. 20.0%, p < 0.01) and with cardiovascular events (64.3 vs. 16.9%, p < 0.01). MR-proADM above the cutoff value (≥0.85) was associated with a fatal outcome (88.9 vs. 20.0%, p < 0.01) and cardiovascular events (71.4 vs. 28.6%, p < 0.01); this association was not observed for hsTnT. CONCLUSION: Preoperative measurement of MR-proADM provides useful information for perioperative cardiac events in high-risk patients scheduled for noncardiac surgery.

Modulatory effect of curcumin on ketamine-induced toxicity in rat thymocytes: Involvement of reactive oxygen species (ROS) and the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway.

Ketamine is a widely used anesthetic in pediatric clinical practice. Previous studies have demonstrated that ketamine induces neurotoxicity and has a modulatory effect on the cells of the immune system. Here, we evaluated the potential protective effect and underlying mechanisms of natural phenolic compound curcumin against ketamine-induced toxicity in rat thymocytes. Rat thymocytes were exposed to 100 µM ketamine alone or combined with increasing concentrations of curcumin (0.3, 1, and 3 µM) for 24 hours. Cell viability was analyzed with CCK-8 assay kit. Apoptosis was analyzed using flow cytometry and propidium iodide as well as Z-VAD-FMK and Z-LEHD-FMK inhibitors. Reactive oxygen species (ROS) production and mitochondrial membrane potential [MMP] were measured by flow cytometry. Colorimetric assay with DEVD-pNA substrate was used for assessing caspase-3 activity. Involvement of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was tested with Wortmannin inhibitor. Ketamine induced toxicity in cells, increased the number of hypodiploid cells, caspase-3 activity and ROS production, and inhibited the MMP. Co-incubation of higher concentrations of curcumin (1 and 3 µM) with ketamine markedly decreased cytotoxicity, apoptosis rate, caspase-3 activity, and ROS production in rat thymocytes, and increased the MMP. Application of Z-VAD-FMK (a pan caspase inhibitor) or Z-LEHD-FMK (caspase-9 inhibitor) with ketamine effectively attenuated the ketamine-induced apoptosis in rat thymocytes. Administration of Wortmannin (a PI3K inhibitor) with curcumin and ketamine significantly decreased the protective effect of curcumin on rat thymocytes. Our results indicate that ketamine-induced toxicity in rat thymocytes mainly occurs through the mitochondria-mediated apoptotic pathway and that the PI3K/Akt signaling pathway is involved in the anti-apoptotic effect of curcumin.

Foveal Avascular Zone in Normal Tension Glaucoma Measured by Optical Coherence Tomography Angiography.

AIM: To measure diameter of foveal avascular zone (FAZ), FAZ area, and vessel density using Optical Coherence Tomography Angiography (OCT-A) in patients with normal tension glaucoma (NTG) and to establish the possible role of OCT-A in diagnosis and follow-up of patients with NTG. METHODS: Twenty-one eyes of 21 patients with NTG and 30 eyes of 30 healthy subjects underwent complete ophthalmic examination as well as OCT-A on ZEISS AngioPlex. 3 × 3 macula scans were used to measure vertical, horizontal, and maximum diameter of FAZ by two graders. Mean values and interobserver variability were analyzed. Image J was used for analysis of FAZ area and vessel density. RESULTS: Mean vertical diameter (t = 5.58, p < 0.001), horizontal diameter (t = 3.59, p < 0.001), maximum diameter (t = 5.94, p < 0.001), and FAZ area (t = 5.76, p < 0.001) were statistically significantly enlarged in the NTG group compared to those in the control group. Vessel density (t = -5.80, p < 0.001) was statistically significantly decreased in the NTG group compared to that in the control group. CONCLUSION: OCT-A could have an important role in the future in diagnosis of patients with NTG. In patients with NTG, there is larger FAZ area, while the vessel density is reduced in comparison to the control group.