Erectile dysfunction in cardiovascular patients: A prospective study of the eNOS gene T-786C, G894T, and INTRON variable number of the tandem repeat functional interaction.

BACKGROUND: Cardiovascular disease induces erectile dysfunction modulated by endothelial nitric oxide synthase enzyme and an impaired ejection fraction that restricts penis vascular congestion. However, the mechanisms regulating endothelial dysfunction are not understood. OBJECTIVES: Exploring the functional impact of endothelial nitric oxide synthase genetic polymorphisms on erectile dysfunction and drug therapy optimization in high-risk cardiovascular disease patients. MATERIALS AND METHODS: Patients with erectile dysfunction symptoms and candidates for andrology therapy were included (n = 112). Clinical data and endothelial nitric oxide synthase rs1799983 (G894T) and rs2070744 (T-786C), genotyped by fluorescence polarization assays, were registered. The 27-bp variable number of the tandem repeat polymorphism in intron 4 (intron4b/a) was analyzed by polymerase chain reaction-restriction fragment length polymorphism. Association analyses were run with the R-3.2.0 software. RESULTS: A significant association between endothelial nitric oxide synthase 786-TT (p = 0.005) and the aa/ac of intron 4 variable number of the tandem repeat (p = 0.02) with higher erectile dysfunction susceptibility was observed in cardiovascular disease patients (60 ± 9 years, 66% severe erectile dysfunction, 56% ejection fraction). After 3-months of phosphodiesterase type 5 inhibitors, erectile dysfunction (International Index of Erectile Function, 50 ± 16 scores, the International Index of Erectile Function-Erectile Function 21 ± 10 scores, p < 0.001) and sexual quality of life (modified Sexual Life Quality Questionnaire 55 ± 23 scores, p < 0.001) had significantly improved. The cardiovascular ejection fraction was influenced positively with better sexual quality of life (0.1941), and also in the endothelial nitric oxide synthase G894-T allele (p = 0.076) carriers, which could merit future analyses. Erectile dysfunction was present as the primary clinical manifestation in 62% of cases, with cardiovascular disease occurring concurrently. Only former smokers and obese subjects debuted prior to cardiovascular disease than to erectile dysfunction. CONCLUSIONS: Our study provides comprehensive insights into the functional interaction linking endothelial nitric oxide synthase gene polymorphisms, erectile function, and ejection fraction in high-risk cardiovascular disease patients. Future therapeutic strategies could target endothelial nitric oxide synthase activity by including lifestyle changes and epigenetic modulations.

Profiling and assessing the risks of image- and performance-enhancing drugs use during the COVID-19 lockdown.

Background: Image and Performance-Enhancing Drugs (IPEDs) can enhance mental and physical capabilities and impact one's overall health. Initially confined in sport environments, IPEDs use has become increasingly widespread in a high-performing society. The present study was aimed at profiling IPEDs use during the COVID-19 lockdown among an international sample of young adults. Methods: A cross-sectional observational study was carried out in eight countries (United Kingdom, Italy, Lithuania, Hungary, Portugal, Spain, Brazil, and Japan) between April and May 2020. The survey questionnaire included validated measurements such as Exercise Addiction Inventory (EAI), Appearance Anxiety Inventory (AAI), and Self-Compassion Scale (SCS) as well as questions about the type of IPEDs, purchasing methods and socio-demographic information. Results: A total of 736 IPEDs users were included in the survey. Their mean age was 33.05 years (±SD = 10.06), and 64.2% were female participants. Overall, 6.8% were found at risk of exercise addiction (EAI >24), 27.6% presented high levels of appearance anxiety, and 24.9% revealed low levels of emotional regulation's self-compassion. Most participants (55.6%) purchased IPEDs through pharmacies/specialized shops, while 41.3% purchased IPEDs on the Internet. Online IPEDs buyers were mainly men who had higher scores on the Exercise Addiction Inventory. One or more IPEDs classifiable as "potentially risky" were used by 66.3% of the sample. Users of "potentially risky IPEDs" were younger and primarily men. They showed higher scores both on the Exercise Addiction Inventory and Appearance Anxiety Inventory. Conclusion: This study profiled users of IPEDs when the most restrictive COVID-19 lockdown policies were implemented in all the participating countries. More targeted post-COVID 19 prevention strategies should be implemented according to the emerged socio-demographic and psychopathological traits and cross-cultural differences emerged. Longitudinal studies will also be needed to determine the long-term effect of the COVID-19 lockdown on IPEDs consumption.

Opioid Monitoring in Clinical Settings: Strategies and Implications of Tailored Approaches for Therapy.

This review emphasises the importance of opioid monitoring in clinical practice and advocates for a personalised approach based on pharmacogenetics. Beyond effectively managing pain, meticulous oversight is required to address concerns about side effects, specially due to opioid-crisis-related abuse and dependence. Various monitoring techniques, along with pharmacogenetic considerations, are critical for personalising treatment and optimising pain relief while reducing misuse and addiction risks. Future perspectives reveal both opportunities and challenges, with advances in analytical technologies holding promise for increasing monitoring efficiency. The integration of pharmacogenetics has the potential to transform pain management by allowing for a precise prediction of drug responses. Nevertheless, challenges such as prominent pharmacogenetic testing and guideline standardisation persist. Collaborative efforts are critical for transforming scientific advances into tangible improvements in patient care. Standardised protocols and interdisciplinary collaboration are required to ensure consistent and evidence-based opioid monitoring. Future research should look into the long-term effects of opioid therapy, as well as the impact of genetic factors on individual responses, to help guide personalised treatment plans and reduce adverse events. Lastly, embracing innovation and collaboration can improve the standard of care in chronic pain management by striking a balance between pain relief and patient safety.

Clinical prediction of opioid use disorder in chronic pain patients: a cohort-retrospective study with a pharmacogenetic approach.

BACKGROUND: Opioids are widely used in chronic non-cancer pain (CNCP) management. However, they remain controversial due to serious risk of causing opioid use disorder (OUD). Our main aim was to develop a predictive model for future clinical translation that include pharmacogenetic markers. METHODS: An observational study was conducted in 806 pre-screened Spanish CNCP patients, under long-term use of opioids, to compare cases (with OUD, N.=137) with controls (without OUD, N.=669). Mu-opioid receptor 1 (OPRM1, A118G, rs1799971) and catechol-O-methyltransferase (COMT, G472A, rs4680) genetic variants plus cytochrome P450 2D6 (CYP2D6) liver enzyme phenotypes were analyzed. Socio-demographic, clinical and pharmacological outcomes were also registered. A logistic regression model was performed. The model performance and diagnostic accuracy were calculated. RESULTS: OPRM1-AA genotype and CYP2D6 poor and ultrarapid metabolizers together with three other potential predictors: 1) age; 2) work disability; 3) oral morphine equivalent daily dose (MEDD), were selected with a satisfactory diagnostic accuracy (sensitivity: 0.82 and specificity: 0.85), goodness of fit (P=0.87) and discrimination (0.89). Cases were ten-year younger with lower incomes, more sleep disturbances, benzodiazepines use, and history of substance use disorder in front of controls. CONCLUSIONS: Functional polymorphisms related to OPRM1 variant and CYP2D6 phenotypes may predict a higher OUD risk. Established risk factors such as young age, elevated MEDD and lower incomes were identified. A predictive model is expected to be implemented in clinical setting among CNCP patients under long-term opioids use.

Circulating miRNA-21 is an innovative biomarker for cardiovascular events in erectile dysfunction patients.

Introduction: It is well-known that circulating microRNAs (miRNAs) play a relevant role in many kinds of diseases by regulating the expression of genes involved in various pathophysiologic processes, including erectile dysfunction (ED) and cardiovascular diseases (CVD). Purpose: This study aimed to identify the miRNA-21 profile in the blood samples of patients with ED, CVD, and the combination of both pathologies to elucidate the potential function of miRNA-21. Methods: A total of 45 patients with CVD and/or who underwent the erectile function test were included and divided into the following categories: CVD with ED (cases, n = 29) and controls (n = 16) with either ED or CVD. Real-time polymerase chain reaction analysis verified the results. miRNA-21 expression was quantified, and informatics analysis was applied to predict the functions of this differentially expressed miRNA-21. Results: A total of 64% of cases (63 ± 9 years, 66% with severe ED, 56% with CV ejection fraction) first presented ED as the sentinel clinical manifestation. Serum miRNA-21 levels in the control ED were significant, up to 10-fold higher than in the CVD controls and cases. A significant inverse (p = 0.0368, ß = -2.046) correlation was found between erectile function and miRNA-21 levels. Conclusions: Our study provides comprehensive insights into the functional interaction between miRNA-21 and ED in CVD patients. Its relevance lies in the potential of miRNA as a biomarker to be applied in the cardiovascular predictive medicine field.

High-dose benzodiazepine use and QTc interval prolongation, a latent class analysis study.

Benzodiazepine (BDZ) addiction is a widespread and multifaceted phenomenon. For many patients, especially females, the concomitant use of other drugs also increases their risk of QTc prolongation, possibly leading to complications such as seizures and even sudden death. However, the relationship between BDZ use and QTc prolongation is currently unclear. The present study aims to examine patterns of polysubstance use among a sample of Italian adults with BDZ dependence in relation with their QTc prolongation risk. We used Latent Class Analysis (LCA) on data collected from 251 inpatients of the Addiction Medicine Unit in Verona to group patients into three classes according to their substance use and their QTc prolongation risk. Results showed no significant relationship between QTc prolongation and BDZ use in any of the classes considered. We conclude that BDZs, even if used long-term and at high dosages, can be considered safe in terms of cardiovascular complications for patients.

"I Am on Top!": An Interactive Intervention Program to Promote Self-Regulation Processes in the Prevention of the Use of Doping in Sports High Schools.

The use of substances to improve sports performance is a widespread phenomenon among adolescents. Several anti-doping programs have been developed, mainly based on knowledge-based evidence. The main aim of the present study was to implement an anti-doping intervention in sports high school students, based on a psychological framework, such as Socio-Cognitive Theory, through the development of a Serious Game (SG), i.e., digital learning based on the game. The experimental design included an intervention group (n = 167; F = 37.7%; Meanage = 17.5 years; SD = 0.58) and a control group (n = 112; F = 42%; Meanage = 17.6; SD = 1). Both of the groups completed the same questionnaire on two different occasions (i.e., time 1 and time 2) for measuring doping intention, self-regulatory efficacy to resist social pressure for the use of substances, moral disengagement, and doping knowledge. Data were analyzed through repeated measures of Group X Time ANOVA, demonstrating some degree of efficacy of the intervention, in particular in terms of the decrease in doping intention and the strengthening of doping knowledge. Moreover, the study demonstrated that the score obtained during the implementation of the SG could partially represent a coherent measure of the participants' beliefs regarding doping. These results could be considered a starting point for future research to better develop technological anti-doping interventions.

The Expanded Exercise Addiction Inventory (EAI-3): Towards Reliable and International Screening of Exercise-Related Dysfunction.

Exercise addiction (EA) refers to excessive exercise, lack of control, and health risks. The Exercise Addiction Inventory (EAI) is one of the most widely used tools in its assessment. However, the cross-cultural psychometric properties of the EAI could be improved because it misses three pathological patterns, including guilt, exercise despite injury, and experienced harm. Therefore, the present study tested the psychometric properties of the expanded EAI (EAI-3) in a large international sample. The EAI-3 was administered to 1931 physically active adult exercisers speaking five languages (Chinese, German, Italian, Japanese, and Turkish) and other measures for obsessive-compulsive behavior, eating disorders, and personality traits. The assessment structure and reliability of the EAI-3 were tested with factorial analyses and through measurement invariance across languages and sex. Finally, a cutoff point for dysfunction-proneness was calculated. The EAI-3 comprised two factors, reflecting the positive and pathological sides of exercise. The structure had excellent reliability and goodness-of-fit indices and configural and metric invariances of the scale were supported. However, three items caused violations in scalar invariance. The results of partial measurement invariance testing suggested an adequate fit for the data. Following sensitivity and specificity analysis, the EAI-3's cutoff score was 34 out of a maximum score of 48. This preliminary study suggests that the EAI-3 is a promising tool for screening EA in an international sample, with a robust and reliable structure comparable across languages and sex. In addition, the proposed cutoff could pave the way toward a consensus on a threshold to screen for EA.

Should We be Concerned with Nicotine in Sport? Analysis from 60,802 Doping Control Tests in Italy.

BACKGROUND: Nicotine is a psychostimulant drug with purported use in sports environments, though the use of nicotine among athletes has not been studied extensively. OBJECTIVE: The aim of this study was to assess the nicotine positivity rate in 60,802 anti-doping urine samples from 2012 to 2020. METHODS: Urine samples obtained in-competition at different national and international sports events held in Italy during the period 2012-2020 were analysed. All samples were from anonymous athletes that were collected and analysed at the WADA-accredited antidoping laboratory in Rome, Italy. Samples were analysed by gas chromatography coupled with mass spectrometry, with a cut-off concentration for nicotine of > 50 ng/mL. Results were stratified by year, sport and sex. RESULTS: An overall mean of 22.7% of the samples (n = 13,804; males: n = 11,099; females: n = 2705) showed nicotine intake, with male samples also displaying higher positivity rates than female (24.1% vs 18.5%). Sample positivity was higher during 2012-2014 (25-33%) than 2015-2020 (15-20%). Samples from team sports displayed a higher positivity rate than those from individual sports (31.4 vs 14.1%). CONCLUSIONS: The current data demonstrates that one in five samples from a range of 90 sports test positive for nicotine in-competition. There is a lower positivity rate in endurance versus power/strength athletes and higher positivity rate in team versus individual sports, probably accounted for by differences in physiological and psychological demands and the desire for socialisation. WADA, international and national sports federations should consider these findings with concern, proactively investigate this phenomenon and act in order to protect the health and welfare of its athletes.

Tramadol and Cycling: Is It the End of a "Painful" Relationship? An Insight From 60,802 Doping-Control Samples From 2012 to 2020.

PURPOSE: To assess the prevalence of tramadol use among athletes from 2012 to 2020. METHODS: All urine samples were collected from national and international in-competition doping-control tests that took place in Italy between 2012 and 2020. The analysis of the samples was performed by gas chromatography coupled with mass spectrometry with electronic ionization and acquisition in selected ion monitoring. The cutoff tramadol concentration was >50 ng/mL. RESULTS: Of the 60,802 in-competition urine samples we analyzed, 1.2% (n = 759) showed tramadol intake, with 84.2% (n = 637) of these coming from cyclists and 15.8% (n = 122) from other sports. In cycling, a strong and significant negative correlation was found (r = -.738; P = .003), showing a decrease of tramadol use compared with the other sports. CONCLUSIONS: The decrease in tramadol prevalence in cycling in the last years may be due to (1) the deterrent action of antidoping regulations and (2) the fact that tramadol may not have any actual ergogenic effect on performance.

Transcranial direct current stimulation (tDCS) modulates motor execution in a limb reaching task.

The majority of human activities show a trade-off between movement speed and accuracy. Here we tested 16 participants in a quick pointing action after 20 minutes (2 mA) of transcranial direct current stimulation (tDCS) delivered at the supplementary motor area in a single-blind crossover design study for testing the feedforward components in the control of action. tDCS stimuli were delivered in three randomized sessions of stimulations as anodal, cathodal and sham as a control. The task performed pre- and post-tDCS stimulation, was to point as fast and as precise as possible with the big toe to targets having different sizes (2 and 8 cm; Width) and positioned at different distances (20 and 60 cm; Distance). An optoelectronic motion capture system was used to collect the kinematics of movement. The result indicates that individuals after receiving anodal stimulation decreased their movement time and increased their movement speed, while the opposite happened after receiving a cathodal stimulation. The scarcity of studies in this area invites us to plan a research that aims at the trade-off especially in the clinical settings.

Polysubstance Use Patterns Among High Dose Benzodiazepine Users: A Latent Class Analysis and Differences Between Male and Female Use.

Benzodiazepines (BZDs) represent one of the most widely used groups of pharmaceuticals, but if used for long periods of time they are associated with dependence and an increased risk of harmful effects. High-dose (HD) BZD dependence is a specific substance use disorder associated with a poor quality of life. It is especially important to pinpoint differences in HD BZD addict subgroups in order to tailor treatment to the individual's specific needs, also considering possible comorbidities with other substance use disorders. We conducted a study to evaluate HD BZD dependence (converted doses to diazepam equivalents, mg) in an Italian sample of 1,354 participants. We also investigated if and to which extent participants co-used other substances (alcohol, tobacco, cannabis/cannabinoids, cocaine, and heroin). We then performed latent class analysis (LCA) to identify the use patterns of these substances, finding three classes: participants in Class 1 (4.3% of the sample) had the highest probability of also using cocaine and alcohol (Polysubstance BZD users); Class 2 comprised subjects with the highest probability of being former heroin, cocaine, THC, and alcohol users (Former polysubstance BZD users); Class 3 represented mono-dependence BZD users (78.5% of the sample) and was the most prevalent among women, while young men were most prevalent in Class 1. The present study underlines different characteristics in HD BZD users both concerning other addictions and sex, and also highlights the need for a stricter control of BZD use, ranging from prescriptions to sales.

The effect of benzodiazepines on exercise in healthy adult participants: A systematic review.

The use of benzodiazepines among athletes is a new and growing phenomenon according to the recent case reports published. Therefore, there is an urgent need to identify if there is rationale for the use of benzodiazepines and its effects on exercise. This review aims to provide an overview of the effects of benzodiazepine in exercise among healthy adult participants and if they might have an additional ergogenic or ergolytic effect. Electronic searches were conducted in Pubmed, Scopus and Web of Science databases up to December 2020. Search terms covered all active substance names of benzodiazepine class and search terms about sport, exercise performance and athletes. We used the Physiotherapy Evidence Database (PEDro) to assess the methodological quality of the studies included in the qualitative synthesis. The methods and planned analyses of this systematic review were pre-registered at Open Science Framework (OSF: https://osf.io/uq6j8). A total of thirty-one full articles were assessed for eligibility and ten of them were included in the qualitative analysis. We found 7 studies which investigated benzodiazepine effects after an acute dose administered, whereas only 3 studies studied long-term effects after several doses used. According to our findings it does not seem that benzodiazepines might have an ergogenic or ergolytic effect on exercise performance. The small number of articles included (n = 10) with a relatively low sample of participants (N = 16, range = 6-58) does invite us to take our results with caution. This review evidences valuable insights into the use of benzodiazepines from a physical performance point of view. Our findings highlight the unclear effects benzodiazepines might have on exercise performance and its possible mechanisms of actions. Hence, the need to conduct new studies to understand its possible effects becomes essential to protect the health of athletes of all levels.

Benzodiazepines in sport, an underestimated problem: Recommendations for sports medicine physicians' practice.

In the last years, only few studies in literature have focused on the use and abuse of benzodiazepines (BZDs) in sport. Benzodiazepine-related problems include misuse, addiction, driving impairments, and morbidity and mortality related to overdose and withdrawal. Two clinical cases regarding elite endurance athletes evidenced that they had started to use BZDs to counteract insomnia, to recover faster from training sessions and to manage muscle pain. One of the important points that emerged from their stories was that their sports doctors did not recognize the drugs' addictive properties, and did not intervene to gradually reduce the dosage. Experts have previously provided recommendations for BZD therapy management in clinical practice. In this article, we would like to address sports medicine physicians specifically and provide guidelines to help them manage situations involving BZD prescription, the recognition of addiction, and intervention strategies.

Therapeutic alliance impact on analgesic outcomes in a real-world clinical setting: An observational study.

A good therapeutic alliance is relevant for healthcare providers exposed to patients' suffering, especially since patients and physicians may understand the painful experience differently. Our aim was to explore the impact of therapeutic alliance on analgesic outcomes in a real-world interdisciplinary pain unit (PU). A cross-sectional observational study was conducted on outpatients (n = 69) using opioids on a long-term basis for the treatment of chronic non-cancer pain, where clinical pharmacologists and pharmacists advised patients about their opioid treatment. Responses to the patient-doctor relationship questionnaire (PDRQ), sociodemographic and clinical information (pain level, quality of life and hospital use) were collected, whereas pharmacology data (analgesic prescription, adverse events, and compliance) were obtained from electronic health records. Patients were predominantly middle-aged (75 % women, 72 % retired), experiencing moderate pain (VAS 40-70 mm) on average, and under a high morphine equianalgesic dosage (95 ± 88 mg per day, mainly tapentadol or fentanyl). Patients with better PDRQ outcomes, and therefore better therapeutic alliance, showed lower pain intensity than patients with worse PDRQ outcomes (pain intensity: high scores 60 ± 47 mm and medium scores 60 ± 45 mm vs. low scores 80 ± 75 mm, p < 0.01). Along with this, pain intensity was lower when patients affirmed that, thanks to the health-care providers, they "gained new insight", "felt better", or "felt content with their doctor's treatment". What´s more, patients who affirmed "I benefit from the treatment" experienced increased pain relief (benefit 40 ± 30 vs. non-benefit 19 ± 26 mm, p = 0.010) and improved quality of life (benefit 33 ± 25 vs. non-benefit 18 ± 16 mm, p = 0.031). However, there was a percentage of patients who did not fully understand the provided information, which is something to be taken into account to improve in clinical routine. Therapeutic alliance supported by pharmacist experts on pain management can be an effective strategy to improve analgesic outcomes. Further efforts are needed to improve communication strategies for pain management. Future directions of research should include the analysis of the role of the pharmacist in poly-professional consultations as related to the advice of patients about their medication, and the mutual trust with the patients.

Promoting Media Literacy Online: An Intervention on Performance and Appearance Enhancement Substances with Sport High School Students.

The outbreak of coronavirus required adjustment regarding the delivery of interventions. Media literacy interventions are necessary to help people acquire relevant skills to navigate the complexities of media communications, and to encourage health-promoting behaviors. The present study aimed to promote a media literacy intervention regarding performance and appearance enhancement substances use in sports high school students. The COVID-19 contingency allowed us to evaluate whether online sessions can effectively promote greater awareness of media influence, a stronger sense of confidence in persuading others to deal with media messages, and healthier attitudes about PAES use among high school students. The study relied on an "intervention group" comprising 162 students (31.5% female) and a "control group" comprising 158 students (42% female). Data were analyzed through repeated measures of Group X Time MANOVA and ANOVA, demonstrating some degree of efficacy of the media literacy intervention. The "intervention group" reported higher awareness of potential newspapers' influence and a significant increase in their sense of confidence in dealing with media influence compared to the "control group". Findings support the efficacy of online media literacy programs to prevent doping consumption in adolescents.

A virtual reality study on postretrieval extinction of smoking memory reconsolidation in smokers.

Exposure to smoking-related stimuli may induce the reconsolidation of smoking-related memories in smokers. Research has proposed that extinction applied after the retrieval of a smoking memory may inhibit reconsolidation and prevent craving. The aim of this study was to test the effect of postretrieval extinction (PRE) on the reconsolidation of smoking memory by using a virtual reality (VR) simulation in smokers. On the day 1 session, the study exposed 46 smokers to a neutral and then to a smoking VR scenario under a fixed-block protocol. On day 2, the study randomized participants into three groups (G) and exposed them to a 15-s VR immersion in smoking (G1, G3) or neutral (G2) scenario for memory retrieval. After 15 min, the study exposed G1 and G2 to a VR PRE during the temporal window of memory vulnerability, whereas the study exposed G3 to extinction immediately after retrieval. On day 3, the study exposed all groups to neutral and smoking scenarios similar to day 1. All groups significantly increased craving for cigarettes after exposure to the smoking scenario on day 1 (p < 0.01). On day 3, VR PRE after a 15-second VR smoking memory retrieval was able to inhibit reconsolidation in G1, but not in G3 exposed to PRE before the window of vulnerability, or in G2 not exposed to the smoking memory retrieval. These findings show the superiority of VR PRE after smoking memory retrieval compared to a standard extinction procedure.

Smokers "Context Reactivity" in Virtual Domestic Environments.

INTRODUCTION: Although the effects of proximal smoking cues have been widely studied in smokers, little is known on the features associated with background spatial context effect, that is, "context reactivity." The aim of this study was to investigate context reactivity exhibited by smokers in virtual cue-free domestic scenarios. METHODS: Sixty-nine participants divided in 2 cohorts (33 smokers and 36 non-smokers) were exposed to a virtual reality session with 4 domestic room scenarios presented in a balanced order: bedroom, bathroom, kitchen, and living room. RESULTS: We showed that (i) it is possible to elicit smoking craving in smokers in virtual reality, and (ii) these effects are room dependent and (iii) associated with a lower sense of presence; furthermore, (iv) smokers reported higher craving scores for alcohol and food in a room-dependent fashion compared to non-smokers. CONCLUSION: Our study provides an experimental paradigm for assessing context reactivity in smokers and suggests a potential use for the identification of non-pharmacological interventions as a co-adjuvant of smoking cessation treatment.