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Purpose: To translate and validate a questionnaire that evaluates the theoretical knowledge regarding fundus examination., Methods: A 60-item multiple-choice English questionnaire that investigates various aspects of knowledge regarding fundus examination was translated into Portuguese. The process involved translation, back-translation, and evaluation by an expert committee. The resulting questionnaire was applied to final-year medical students and ophthalmology residents. Each included subject answered the questionnaire twice, with an interval of one week between each application. Internal consistency, test-retest reliability, inter-rater reliability, and percentage agreement were calculated., Results: Thirty participants were included (25 medical students and 5 ophthalmology residents). The pass-fail cutoff was calculated at 46, the theoretical false positives were 8.7% and the theoretical false negatives were 2.8%. The observed false positive and false negative rates were 0%. Among the 60 items, test-retest reliability was strong in 17 items, which one had a negative correlation, moderate in 14 items, which one had a negative correlation, and weak in 29 items; inter-rater reliability of 34 items was under 0.4, 17 items were between 0.4 and 0.6, and 8 items were above 0.6. One item had a negative kappa. Among the percent agreement, 10 items were between 40%-60% agreement, 50 were above 60% agreement, and 18 were above 80%. Cronbach's alpha was calculated as 0.674., Conclusions: The translated questionnaire provided a standard instrument for future research and interventions to improve medical education in ophthalmology.

Rationale: To uncover whether psychedelic drugs attenuate fear memory responses would advance the development of better psychedelic-based treatments for posttraumatic stress disorder (PTSD). Ayahuasca (AYA), a psychedelic brew containing indolamine N, N-dimethyltryptamine (DMT) and β-carbolines, facilitates fear extinction and improves neural plasticity. Upon retrieval, fear memory undergoes labilization and reconsolidation; however, the effects of AYA on this memory stabilization phase are unknown., Objectives: We aimed to investigate the effects of AYA treatment on fear memory reconsolidation., Methods: Fear-conditioned Wistar rats received AYA (60, 120, or 240 mg/kg) or H 2 O orally via gavage o.g. 20 min before, immediately, or 3 h after a short retrieval session. Analysis of AYA through liquid chromatography-tandem mass spectrometry was used to determine the content of DMT and β-carbolines in AYA., Results: AYA impaired fear memory reconsolidation when given 20 min before or 3 h after memory retrieval, with the dose of 60 mg/kg being effective at both moments. This dose of AYA was devoid of anxiolytic effect. Importantly, during retrieval, AYA did not change fear expression. The lack of retrieval abolished the reconsolidation impairing effect of AYA. The effects of AYA treatment 20 min before or 3 h after memory retrieval lasted at least 22 days, suggesting no spontaneous recovery of fear memory. Fear memory impairments induced by AYA treatment, at both moments, do not show reinstatement., Conclusions: Our findings support the view that a low dose of AYA treatment impairs early and late stages of memory reconsolidation instead of facilitating fear extinction., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The objective of this study was to evaluate the behavioral response of ayahuasca in rats submitted to neuroinflammation through the intraperitoneal application of lipopolysaccharide (0.63 mg/kg/mL). Eighty animals, male, about 90 days old, were divided into control and LPS groups and later into prevention and treatment subgroups. The prevention subgroup was administered ayahuasca or saline solution, via gavage, at a dose of 4 mL/kg one hour before applying LPS or saline, while the treatment subgroup received the same dose of the respective substances 24 h after intraperitoneal applications. Behavioral parameters were evaluated using open field (anxiety-like) and forced swimming (depressive-like) tests. A decrease in LPS/AYA rats in the prevention and treatment subgroups regarding anxiety-like behavior was observed. As for the depressive-like behavior, there was a decrease in the group induced to the disease model, both in the prevention subgroup (when compared to the SAL/SAL, SAL/AYA, and LPS/AYA with LPS/SAL groups) and in the treatment (when comparing SAL/SAL and LPS/AYA with LPS/SAL). This study concludes the anxiolytic and antidepressant potential of ayahuasca in an animal model of neuroinflammation, possibly due to the antineuroinflammatory effects already reported of the compound., (Copyright © 2022. Published by Elsevier B.V.)

Background: Smoking is the leading cause of preventable death worldwide. It is responsible for several types of cancer, cardiovascular diseases, and diseases of the reproductive system, among others. Therefore, advances in research are increasingly necessary in order to make smoking cessation treatment more effective. Some studies have investigated the association of the nicotine metabolite ratio (NMR) with general characteristics and treatment outcomes. In the present study, the main aim was to evaluate the NMR in smoking patients from an Assistance Program of a tertiary cardiology hospital. Methodology: Serum samples were collected from 185 patients at T0 (while patients were still smoking and before starting pharmacological treatment). Cotinine and hydroxycotinine analytes were measured using liquid-chromatography tandem mass-spectrometry (LC-MS/MS). By looking at the relationship between hydroxycotinine and cotinine, we can obtain the NMR, with which it is possible to classify patients into slow metabolizers (NMR < 0.31), as well as normal or fast metabolizers (NMR ≥ 0.31). Results: From 185 patients, 55 were considered slow metabolizers and 130 as normal/fast. The metabolite averages were associated with the number of cigarettes smoked per day ( p < 0.001 for cotinine and 0.023 hydroxycotinine). However, we were unable to analyze the association of the NMR with general and clinical characteristics of patients under smoking cessation treatment. Conclusion: We were able to evaluate the NMR, and to observe categories of metabolizers in Brazilian patients under pharmacological treatments. Thus, this study can contribute to the indication of a form of analysis, which might form part of the customization of smoking cessation treatments and, consequently, improve the success rates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tomaz, Gonçalves, Santos, Scholz, Abe, Gaya, Figueiredo, de Faria, Martins, Pego, Bismara, Yonamine, Pereira and Santos.)

Cocaine addiction is a relapsing disorder with loss of control in limiting drug intake. Considering the involvement of acetylcholine in the neurobiology of the disease, our aim was to evaluate whether cocaine induces plastic changes in the hippocampal cholinergic muscarinic system. Male Swiss-Webster mice received saline or cocaine (ip) three times daily (60-min intervals) either acutely or in an escalating-dose binge paradigm for 14 days. Locomotor activity was measured in all treatment days. Dopaminergic and cholinergic muscarinic receptors (D 1 R, D 2 R, M 1 -M 5, mAChRs), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and acetylcholinesterase (AChE) were quantified in the hippocampus by immunoblotting one hour after the last injection (on drug) or after 14 days of abstinence (withdrawal). Escalating-dose group showed cocaine-induced locomotor sensitization from day 2. M 3 mAChR and ChAT significantly increased after the on-drug acute binge treatment. Escalating-dose on-drug group showed increased ChAT, M 1 , M 5 mAChR and D 2 R; and decreased D 1 R. Acute-binge withdrawal group showed increased VAChT, M 2 mAChR, D 1 R, and D 2 R; and decreased M 1 mAChR. Escalating-dose withdrawal group presented increased D 1 R and VAChT and decreased M 1 mAChR and D 2 R. Locomotor activity was negatively correlated with M 1 mAChR and AChE in on-drug group and positively correlated with VAChT in withdrawal group. M 1 mAChR was positively correlated with M 2 mAChR and ChAT in on-drug group, whereas ChAT was positively correlated with M 5 mAChR in withdrawal group. The results indicate that cocaine induced an increase in the hippocampal cholinergic tone in the presence of the drug, whereas withdrawal causes a resetting in the system., (Copyright © 2022. Published by Elsevier Inc.)

Objective: To assess endocannabinoid (anandamide, AEA; 2-arachidonoylglycerol, 2-AG) plasma levels in healthy volunteers and in volunteers with social anxiety disorder (SAD) after a single oral dose of ayahuasca or placebo., Methods: Post hoc analysis of endocannabinoid plasma levels (baseline, 90 and 240 min after drug intake) from two parallel-group, randomized, placebo-controlled trials. In Study 1, 20 healthy volunteers ingested ayahuasca (average 1.58 mg/ml dimethyltryptamine (DMT)) or placebo, and in Study 2, 17 volunteers with SAD received ayahuasca (average 0.680 mg/ml DMT) or placebo., Results: A significant difference was observed in AEA concentrations in Study 2 after ayahuasca intake (Χ 2 (2) = 6.5, p = 0.03, Friedman test), and near significant differences (increases) were observed between baseline and 90 (Z = 0, p = 0.06, Wilcoxon test) and 240 (Z = 10, p = 0.06) minutes after ayahuasca intake., Conclusions: Although our findings suggest that ayahuasca could modulate AEA levels in SAD patients, the high interindividual variability in both trials and the small samples preclude definitive conclusions. More research with larger samples is needed to better understand the effects of ayahuasca and other hallucinogens in the endocannabinoid system., (© 2022 John Wiley & Sons Ltd.)

Accumulating evidence to date suggests that brown rice is superior to white rice in regard to its beneficial impact on a number of risk factors of the metabolic syndrome (MetS). However, little is known about the influence of fermented brown rice beverage on the gut microbiota in humans. We therefore hypothesized that its impact would beneficially alter the gut microbiota composition of patients with MetS. Using a 4-week randomized, single-arm study design, subjects (n = 40) were advised to consume a daily fermented brown rice beverage (BA) or fermented white rice beverage (WA) as a replacement of their main meal. Clinical and anthropometric measurements as well as fecal samples were collected at baseline and immediately after completion of the intervention. Gut microbiota was analyzed using 16S ribosomal RNA sequencing and capillary electrophoresis-time-of-flight mass spectrometry was used to measure plasma short-chain fatty acids. Interestingly, ingestion of BA in contrast to WA resulted in a unique elevation in the abundance of number of beneficial species belonging to the Clostridia class, associated with reduced inflammation, and increased short-chain fatty acid production: Lactobacillales bacterium DJF B280 (P = .005), Butyrate producing bacterium A2 207 (P = .012), and Firmicutes bacterium DJF VP44 (P = .038). This study demonstrates that consumption of BA is effective to beneficially modulate the gut microbiota compared with WA in patients with MetS., Competing Interests: Declaration of Competing Interest There are no conflicts of interest to declare., (Copyright © 2022. Published by Elsevier Inc.)

Anabolic androgenic steroid (AAS) abuse leads to myocardial toxicity. Human studies are conflicting about the myocardial fibrosis in AAS users. We evaluated cardiac tissue characterization, left ventricle (LV) function, and cardiac structure by cardiovascular magnetic resonance (CMR). Twenty strength-trained AAS users (AASU) aged 29±5 yr, 20 strength-trained AAS nonusers (AASNU), and 7 sedentary controls (SC) were enrolled. Native T1 mapping, late-gadolinium enhancement (LGE), extracellular volume (ECV), and myocardial strain were evaluated. AASU showed lower Native T1 values than AASNU (888±162 vs. 1020±179 ms p=0.047). Focal myocardial fibrosis was found in 2 AASU. AASU showed lower LV radial strain (30±8 vs. 38±6%, p<0.01), LV circumferential strain (-17±3 vs. -20±2%, p<0.01), and LV global longitudinal strain (-17±3 vs. -20±3%, p<0.01) than AASNU by CMR. By echocardiography, AASU demonstrated lower 4-chamber longitudinal strain than AASNU (-15±g3 vs. -18±2%, p=0.03). ECV was similar among AASU, AASNU, and SC (28±10 vs. 28±7 vs. 30±7%, p=0.93). AASU had higher LV mass index than AASNU and SC (85±14 vs. 64±8 vs. 58±5 g/m 2 , respectively, p<0.01). AAS abuse may be linked to decreased myocardial native T1 values, impaired myocardial contractility, and focal fibrosis. These alterations may be associated with maladaptive cardiac hypertrophy in young AAS users., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The embryonic stage is the most vulnerable period for congenital abnormalities. Due to its prolonged developmental course, the central nervous system (CNS) is susceptible to numerous genetic, epigenetic, and environmental influences. During embryo implantation, the CNS is more vulnerable to external influences such as environmental tobacco smoke (ETS), increasing the risk for delayed fetal growth, sudden infant death syndrome, and immune system abnormalities. This study aimed to evaluate the effects of in utero exposure to ETS on neuroinflammation in the offspring of pregnant mice challenged or not with lipopolysaccharide (LPS). After the confirmation of mating by the presence of the vaginal plug until offspring birth, pregnant C57BL/6 mice were exposed to either 3R4F cigarettes smoke (Kentucky University) or compressed air, twice a day (1h each), for 21 days. Enhanced glial cell and mixed cell cultures were prepared from 3-day-old mouse pups. After cell maturation, both cells were stimulated with LPS or saline. To inhibit microglia activation, minocycline was added to the mixed cell culture media 24 h before LPS challenge. To verify the influence of in utero exposure to ETS on the development of neuroinflammatory events in adulthood, a different set of 8-week-old animals was submitted to the Autoimmune Experimental Encephalomyelitis (EAE) model. The results indicate that cells from LPS-challenged pups exposed to ETS in utero presented high levels of proinflammatory cytokines such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNFα) and decreased cell viability. Such a proinflammatory environment could modulate fetal programming by an increase in microglia and astrocytes miRNA155. This scenario may lead to the more severe EAE observed in pups exposed to ETS in utero ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Durão, Brandão, Bruno, W. Spelta, Duro, Barreto dos Santos, Paranhos, Zanluqui, Yonamine, Pierre Schatzmann Peron, Munhoz and Marcourakis.)

Alcohol use disorder needs more effective treatments because relapse rates remain high. Psychedelics, such as ayahuasca, have been used to treat substance use disorders. Our study aimed to evaluate the effects of ayahuasca on ethanol-induced behavioral sensitization (EIBS). Swiss mice received 2.2 g/kg ethanol or saline IP injections every other day across nine days (D1, D3, D5, D7, and D9), and locomotor activity was evaluated 10 min after each injection. Then, animals were treated daily with ayahuasca (corresponding to 1.76 mg/kg of N,N-dimethyltryptamine, DMT) or water by oral gavage for eight consecutive days. On the seventh day, mice were evaluated in the elevated plus maze. Then, mice were challenged with a single dose of ethanol to measure their locomotor activity. Dopamine receptors, serotonin receptors, dynorphin, and prodynorphin levels were quantified in the striatum and hippocampus by blot analysis. Repeated ethanol administration resulted in EIBS. However, those animals treated with ayahuasca had an attenuated EIBS. Moreover, ayahuasca reduced the anxiogenic response to ethanol withdrawal and prevented the ethanol-induced changes on 5-HT1a receptor and prodynorphin levels in the hippocampus and reduced ethanol effects in the dynorphin/prodynorphin ratio levels in the striatum. These results suggest a potential application of ayahuasca to modulate the neuroplastic changes induced by ethanol., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Purpose: Synthetic cannabinoid receptor agonists (SCRAs) are a class of varied compounds that mimic the effects of natural cannabinoids found in cannabis. Because they have a wide range of diverse structures, they vary widely in their potency. The abuse of new psychoactive substances (NPS) in prisons was reported in many European countries and in the USA. In the present study, we have described the identification of SCRAs in 56 infused paper sheet samples, seized mainly in Brazilian prisons between 2016 and 2020., Methods: The materials were seized by local or federal law enforcement and analyzed by São Paulo State Police or Brazilian Federal Police using gas chromatography-mass spectrometry, attenuated total reflection-Fourier transform infrared spectroscopy, liquid chromatography-high-resolution mass spectrometry or nuclear magnetic resonance spectrometry., Results: Most of these samples (87.5%) were seized in 2019-2020; seven different SCRAs were identified in samples, and the most frequently identified substances were MDMB-4en-PINACA (23.6%) and 5F-MDMB-PICA (36.4%), the newest SCRAs emerging recently., Conclusions: As observed in Europe and the USA, Brazil also shows the prevalence of indazole-3-carboxamides and indole-3-carboxamides among SCRAs seizures in the prison system. This phenomenon is spreading all over the world at this moment. These data on the prevalence could help to alert judicial authorities to shutting down the introduction of NPS, including SCRAs, into prisons to ensure safety and security for avoiding health risks of prisoners and staff, leading to positive effects in this population. To our knowledge, this is the first demonstration of SCRAs smuggling into prisons in Latin America., (© 2021. Japanese Association of Forensic Toxicology.)

Background: Poisonings resulting from the abuse of drugs currently represent a serious problem for public health. Among the main agents involved, cocaine stands out. It became one of the most abused drugs around the world, and one of the main reasons for visits to the emergency department due to the use of illicit substances. The use of cocaine is primarily in combination with alcoholic beverages. There are few studies that correlate cocaine blood concentration and the severity of clinical manifestations in patients evaluated at Emergency Department. The aim of the present study was to verify the possible relationship between the blood concentration of cocaine and cocaethylene (product of the interaction of cocaine with ethanol) with the severity of the clinical manifestations presented by patients with cocaine intoxication., Methods: Blood levels were measured by high-performance liquid chromatography (HPLC) and the severity of clinical manifestations was assessed using the Stimulant Intoxication Score (SIS). To establish this relationship, Pearson's chi-square statistical test (x 2 ) was used for categorical variables and Student's t for continuous variables, with statistical significance of 5% (p < 0.05)., Results: Of the 81 patients included in the study, 77.8% were men with a mean age of 32.5 years ± 8.5 and mean of SIS 3.4 ± 2.5. Considering the toxicological analysis results, 24.7% of the blood samples were positive. The mean of cocaine and cocaethylene concentrations were 0.34 μg/mL ± 0.45 and 0.38 μg/mL ± 0.34, respectively. The blood concentration of cocaine and cocaethylene has not been shown to be useful information for the treatment and prognosis of patients, but blood levels of these substances at the time of treatment, regardless of their concentration, may be an indicator of severity, showing that any concentrations of these substances should be considered as potentially toxic., Conclusion: The application of the SIS score proved to be an important alternative capable of predicting the severity of the patients due to cocaine intoxication in a fast and simplified way., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Introduction: The presence of anabolic-androgenic steroids (AAS) in illegal commercial products has been pointed as a global threat for public health. Due the correlation with adverse toxicological effects, there is a growing interest in the implementation of straightforward methods for the determination of AAS in seized products. This work exploited the development of a mass spectrometry approach to characterize the illegal oil formulations containing AAS., Methods: The optimization of sample preparation was performed through a simplex-centroid design and the best condition was described as follow: an aliquot of 5 μL of sample were added with 995 μL of acetonitrile and water (75:25, v/v). The solution was vortexed and centrifuged. After that, 10 μL of supernatant were added with 35 μL of acetonitrile and water and internal standard (testosterone-d 3 , 1.25 ng). An aliquot of 5 μL was injected into the analytical system., Results: The method developed was validated and successfully applied in 115 seized samples. Testosterone and its esters had the highest incidence, found in more than 50% of the samples. Besides that, drugs such as boldenone, methandienone, and trenbolone have also been found, where the low quality of the samples was evidenced by the wide variation in the concentration of the drugs, always quantified in sub-doses. Finally, at least one AAS was detected in each sample analyzed. The statistical results were grouped by principal components analysis, to better understand the profile of the seized samples., Conclusion: This work successfully established a fast and simple method for determination of AAS and can be applied to verify the profile of seized samples., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

As medical doctors, we routinely check patient blood chemistry and CBC data to diagnose disease. However, these data and methods of analysis are very rarely used to find pre-disease conditions or treat undiagnosed malaise. Masatoshi Kaneko Ph.D. found that many pre-disease conditions and types of malaise could be detected using his unique method of blood data analysis, and could also be treated using personalized nutritional therapy as an alternative to using drugs. The authors of this article introduce personalized nutritional therapy based on blood data analysis (Kaneko's method), and present and discuss some clinical cases. In total, 253 pre-disease or undiagnosed patients were treated using this nutritional therapy approach, and most of them recovered from their chronic symptoms and pre-disease conditions. This novel nutritional therapy has the potential to help many presymptomatic and undiagnosed patients suffering from malaise.

Dried matrix spots (DMS) has gained the attention of different professionals in different fields, including toxicology. Investigations have been carried out in order to assess the potential of using DMS for the analysis of illicit substances, the main interest of forensic toxicologists. This technique uses minimal volumes of samples and solvents, resulting in simple and rapid extraction procedures. Furthermore, it has proved to increase analyte stability, improving storage and transportation. However, DMS presents some limitations: the hematocrit influencing accuracy and inconsistencies regarding the means of spotting samples and adding internal standard on paper. Thus, we provide an overview of analytical methodologies with forensic applications focusing on drugs of abuse and discussing the main particularities, limitations and achievements.

Background: Ayahuasca is a classic hallucinogen with anxiolytic and antidepressive properties. Anecdotal evidence also suggests that it improves performance (eg, singing, speech). This controlled trial assessed the effects of ayahuasca on speech performance and anxiety in individuals with social anxiety disorder., Methods: Seventeen volunteers with social anxiety disorder participated in a pilot, proof-of-concept, randomized, parallel-group trial. Self-perception of performance during a public-speaking test was assessed with the Self-statements During Public Speaking Scale primary outcome). Secondary outcomes included anxiety/subjective effects (Visual Analog Mood Scale; Bodily Symptoms Scale), recognition of emotions in facial expressions (REFE), tolerability measures (cardiovascular measures, self-reports), and brain-derived neurotrophic factor plasma levels. Effects on anxiety and REFE were assessed again 7, 14, and 21 days postdrug., Findings: Compared with placebo, ayahuasca significantly improved self-perception of speech performance (Self-statements During Public Speaking Scale) and increased somatic symptoms (Bodily Symptoms Scale). There was also a significant time × group interaction in the cognitive deterioration Visual Analog Mood Scale factor and a significant effect of time in the REFE task, especially in reaction time. Other measures were not significantly modified. Ayahuasca was well tolerated, producing mainly nausea, gastrointestinal discomfort, and vomiting., Conclusions: Ayahuasca improved self-perception of speech performance in socially anxious individuals. These effects occurred independent of task-related anxiety and REFE, suggesting that ayahuasca could specifically improve the cognitive aspect of speech performance. Further studies should try to unveil the mechanisms involved in the effects of ayahuasca and to better understand its effects on anxiety., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The high incidence of germline variants in pheochromocytoma and paraganglioma (PPGL) has been reported mainly in Europe, but not among Japanese populations in Asia. We aimed to study the prevalence of germline variants in Japanese PPGL patients and the genotype-phenotype correlation. We examined 370 PPGL probands, including 43 patients with family history and/or syndromic presentation and 327 patients with apparently sporadic (AS) presentation. Clinical data and blood samples were collected, and the seven major susceptibility genes ( MAX , SDHB , SDHC , SDHD , TMEM127 , VHL , and RET ) were tested using Sanger sequencing. Overall, 120/370 (32.4%) patients had pathogenic or likely pathogenic variants, with 81/327 (24.8%) in AS presentation. SDHB was the most frequently mutated gene (57, 15.4%), followed by SDHD (27, 7.3%), and VHL (18, 4.9%). The incidence of metastatic PPGL was high in SDHB carriers (21/57, 36.8%). A few unique recurrent variants ( SDHB c.137G>A and SDHB c.470delT) were detected in this Japanese cohort, highlighting ethnic differences. In summary, almost a quarter of patients with apparently sporadic PPGL in Japan harboured germline variants of the targeted genes. This study reinforces the recommendation in Western guidelines to perform genetic testing for PPGL and genotype-based clinical decision-making in the Japanese population.

Rationale: Previous studies with the serotonergic hallucinogens LSD and psilocybin showed that these drugs induced changes in personality traits, such as increases in Openness. However, results are inconsistent, and the effects of ayahuasca on personality were never investigated in a controlled trial. Objectives: To assess the effects of ayahuasca on personality in two randomized, placebo-controlled trials in healthy volunteers. Methods: Data from two parallel-group, randomized, placebo-controlled trials in healthy volunteers were included. In the first trial, 15 volunteers ingested ayahuasca or placebo, while in the second trial 15 volunteers received placebo+ayahuasca or cannabidiol (CBD)+ayahuasca. Personality was assessed with the NEO-Five Factor Inventory (NEO-FFI) at baseline and 21 days post-treatment. Results: There were significant differences between groups in baseline Openness scores, but not on day 21. A significant increase in Openness scores was observed in the placebo + ayahuasca group in study 2. No other within-group differences were observed for any other domain. Conclusions: Ayahuasca produced inconsistent effects on personality since it induced significant increase in Openness 21 days post-drug intake only in one of the trials. The absence of significant differences in the other ayahuasca groups could be due to small sample sizes and baseline differences among groups. The effects of ayahuasca and other serotonergic hallucinogens on personality should be further investigated in clinical samples., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mendes Rocha, Rossi, Osório, Bouso Saiz, Silveira, Yonamine, Crevelin, Queiroz, Cecílio Hallak and Dos Santos.)

The prevalence of nonalcoholic fatty liver disease (NAFLD) has been rapidly increasing worldwide. A choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) has been used to create a mouse model of nonalcoholic steatohepatitis (NASH). There are some reports on the effects on mice of being fed a CDAHFD for long periods of 1 to 3 months. However, the effect of this diet over a short period is unknown. Therefore, we examined the effect of 1-week CDAHFD feeding on the mouse liver. Feeding a CDAHFD diet for only 1-week induced lipid droplet deposition in the liver with increasing activity of liver-derived enzymes in the plasma. On the other hand, it did not induce fibrosis or cirrhosis. Additionally, it was demonstrated that CDAHFD significantly impaired mitochondrial respiration with severe oxidative stress to the liver, which is associated with a decreasing mitochondrial DNA copy number and complex proteins. In the gene expression analysis of the liver, inflammatory and oxidative stress markers were significantly increased by CDAHFD. These results demonstrated that 1 week of feeding CDAHFD to mice induces steatohepatitis with mitochondrial dysfunction and severe oxidative stress, without fibrosis, which can partially mimic the early stage of NASH in humans.

Background: Pheochromocytoma and paraganglioma caused by succinate dehydrogenase gene mutations is called hereditary pheochromocytoma/paraganglioma syndrome. In particular, succinate dehydrogenase subunit B mutations are important because they are strongly associated with the malignant behavior of pheochromocytoma and paraganglioma . This is a case report of a family of hereditary pheochromocytoma/paraganglioma syndrome carrying a novel mutation in succinate dehydrogenase subunit B., Case Presentation: A 19-year-old Japanese woman, whose father died of metastatic paraganglioma, was diagnosed with abdominal paraganglioma, and underwent total resection. Succinate dehydrogenase subunit B genetic testing detected a splice-site mutation, c.424-2delA, in her germline and paraganglioma tissue. Afterwards, the same succinate dehydrogenase subunit B mutation was detected in her father's paraganglioma tissues. In silico analysis predicted the mutation as "disease causing." She is under close follow-up, and no recurrence or metastasis has been observed for 4 years since surgery., Conclusions: We detected a novel succinate dehydrogenase subunit B mutation, c.424-2delA, in a Japanese family afflicted with hereditary pheochromocytoma/paraganglioma syndrome and found the mutation to be responsible for hereditary pheochromocytoma/paraganglioma syndrome. This case emphasizes the importance of performing genetic testing for patients with pheochromocytoma and paraganglioma suspected of harboring the succinate dehydrogenase subunit B mutation (that is, metastatic, extra-adrenal, multiple, early onset, and family history of pheochromocytoma and paraganglioma) and offer surveillance screening to mutation carriers.

The effects of exercise on nutrient digestion and absorption in the intestinal tract are not well understood. A few studies have reported that exercise training increases the expression of molecules involved in carbohydrate digestion and absorption. Exercise was also shown to increase the blood concentration of glucagon-like peptide-2 (GLP-2), which regulates carbohydrate digestion and absorption in the small intestine. Therefore, we investigated the effects of exercise on the expression of molecules involved in intestinal digestion and absorption, including GLP-2. Six-week-old male mice were divided into a sedentary (SED) and low-intensity exercise (LEx) group. LEx mice were required to run on a treadmill (12.5 m/min, 1 h), whereas SED mice rested. All mice were euthanized 1 h after exercise or rest, and plasma, jejunum, ileum, and colon samples were collected, followed by analysis via IHC, EIA, and immunoblotting. The levels of plasma GLP-2 and the jejunum expression of the GLP-2 receptor, sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) were higher in LEx mice. Thus, we showed that acute low-intensity exercise affects the expression of molecules involved in intestinal carbohydrate digestion and absorption via GLP-2. Our results suggest that exercise might be beneficial for small intestine function in individuals with intestinal frailty.

Background: The recognition of emotions in facial expressions (REFE) is a core aspect of social cognition. Previous studies with the serotonergic hallucinogens lysergic acid diethylamide and psilocybin showed that these drugs reduced the recognition of negative (fear) faces in healthy volunteers. This trial assessed the acute and prolonged effects of a single dose of ayahuasca on the REFE., Methods: Twenty-two healthy volunteers participated in a pilot, proof-of-concept, randomized trial. Study variables included a REFE task performed before and 4 hours after drug intake, subjective effects (self-reports/observer impressions), tolerability measures (cardiovascular measures, self-reports), and brain-derived neurotrophic factor plasma levels. The REFE task was applied again 1, 7, 14, and 21 days and 3 months after drug intake. Stability of ayahuasca alkaloids during the study was also assessed (room temperature, 18 months)., Findings: Compared with placebo, ayahuasca did not modify the REFE. No significant effects were observed on cardiovascular measures and brain-derived neurotrophic factor levels. Volunteers reported visual effects, tranquility/relaxation, and well-being, with few reports of transient anxiety/confusion. Ayahuasca was well tolerated, producing mainly nausea, gastrointestinal discomfort, and vomiting. A significant time-dependent deterioration of alkaloids was observed, especially for dimethyltryptamine., Conclusions: Absence of significant effects on the REFE task could be due to lack of effects of ayahuasca (at the doses used), alkaloid degradation, learning effects, and the high educational level of the sample. Further trials with different samples are needed to better understand the effects of ayahuasca and other serotonergic hallucinogens on the REFE. Future trials should improve methods to guarantee the stability of ayahuasca alkaloids., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The present study describes the development of a novel solvent-free vortex-assisted dispersive liquid-liquid microextraction alternative based on a natural essential oil as extracting solvent (VA-EO-DLLME) for the determination of N,N-dimethyltryptamine (DMT), harmine (HRM), harmaline (HRL) and tetrahydroarmine (THH) (compounds found in the ayahuasca tea, a psychedelic plant preparation) in human plasma. After optimization through full factorial and Box-Behnken experimental designs, this VA-EO-DLLME followed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was completely validated and applied to authentic plasma specimens. Sample preparation consisted in the addition of 60 mg of NaCl, 100 μL of borate buffer and 100 μL of Eucalyptus globulus essential oil to a 200 μL aliquot of human plasma. After 30 s of vortex agitation followed by 5 min of centrifugation (10,000 rpm), 80 μL of the oil supernatant was dried and resuspended in mobile phase prior to injection into the UHPLC-MS/MS system. Once optimized, the validated method yielded LoDs ≤1.0 ng mL -1 for all analytes. LoQ was 1.0 ng mL -1 for DMT, HRL and HRM and 2.0 ng mL -1 for THH. The method has shown to be linear over the range of LoQ up to 150 ng mL -1 (r 2  ≥ 0.9926). Intra/inter-day precision and accuracy met the acceptance criteria at three quality control (QC) levels. An additional intermediate precision study demonstrated that, except for THH and HRL at low and medium QCs, the overall method performance was similar for the three different oil sources. Matrix effect evaluation showed predominant ion suppression, ranging from 56% to 83%. Recovery varied from 33 up to 101% with an average of 50 ± 15.8%. Selectivity studies showed no interferences. Analysis of 13 authentic samples proved method feasibility. Finally, we believe that our novel VA-EO-DLLME approach offers a very simple, fast, cost-effective and eco-friendly alternative based on the use of an easily accessible and entirely green material as an extracting solvent. This may represent an incentive for researchers to investigate novel and creative alternatives, such as essential oils, as substitutes of organic solvents for microextraction methods in forensic and clinical contexts., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ayahuasca tea is an entheogen hallucinogenic beverage used for shamanic and spiritual purposes, prepared by the decoction of different Amazonian plants containing N,N-dimethyltryptamine (DMT) and harmala alkaloids. Since the therapeutic potential of this tea has been broadly studied in recent years, mainly for the treatment of psychiatric disorders, the determination of the ayahuasca tea components in human and animal matrices is of utmost importance. In order to avoid the use of large amounts of toxic solvents, typically employed in traditional sample preparation methods, hollow fiber liquid-phase microextraction (HF-LPME) presents a greener and time-saving alternative. The present study aims to fully develop and apply an HF-LPME method for the determination of DMT, harmine (HRM), harmaline (HRL), and tetrahydroharmine (THH) in human urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fractional factorial and Box-Behnken designs were used to identify and optimize significant method variables. Once optimized, validation has shown a limit of detection (LoD) of 1.0 ng/ml for DMT and 2.0 ng/ml for the harmala alkaloid. The limit of quantification (LoQ) was of 5.0 ng/ml for all analytes. The method has shown to be linear over a concentration range of 5-200 ng/ml ( r 2 ≥ 0.99). Intra/inter-day precision and accuracy met the acceptance criteria at the three quality control (QC) levels studied (15.0, 90.0, and 170.0 ng/ml, n = 6, each). Matrix effect evaluation showed predominant ion enhancement and recovery values were above 80%. Dilution factors of 10- and 20-fold have shown acceptable values of accuracy. Selectivity studies showed no interferences. Analysis of eight authentic samples collected from four subjects proved method feasibility. A simple, time-saving and green alternative for the analysis of DMT and harmala alkaloids in human urine samples was developed, optimized using design of experiments, fully validated and applied to authentic samples., (Copyright © 2020 Silveira, Lourenço, Bruno and Yonamine.)

Purpose: Extra virgin olive oil (EVOO) and flaxseed oil (FO) contain a variety of constituents beneficial for chronic inflammation and cardio-metabolic derangement. However, little is known about the impact of EVOO and FO on dysbiosis of gut microbiota, intestinal immunity, and barrier. We, therefore, aimed to assess the impact of EVOO and FO on gut microbiota, mucosal immunity, barrier integrity, and metabolic health in mice., Methods: C57BL/6 J mice were exposed to a low-fat (LF), lard (HF), high fat-extra virgin olive oil (HF-EVOO), or high fat-flaxseed oil (HF-FO) diet for 10 weeks. Gut microbiota assessment was undertaken using 16S rRNA sequencing. Levels of mRNA for genes involved in intestinal inflammation and barrier maintenance in the intestine and bacterial infiltration in the liver were measured by qPCR., Results: HF-EVOO or HF-FO mice showed greater diversity in gut microbiota as well as a lower abundance of the Firmicutes phylum in comparison with HF mice (P < 0.05). The qPCR analyses revealed that mRNA level of FoxP3, a transcription factor, and IL-10, an inducer of regulatory T cells, was significantly elevated in the intestines of mice-fed HF-EVOO in comparison with mice-fed HF (P < 0.05). The mRNA level of the antimicrobial peptide, RegӀӀӀγ, was markedly elevated in the intestines of HF-EVOO and HF-FO compared with HF group (P < 0.05)., Conclusions: Our data suggest that the consumption of EVOO or FO can beneficially impact gut microbiota, enhance gut immunity, and assist in the preservation of metabolic health in mice.

Direct ambient ionization techniques have been developed with the aim to reduce the complexity of mass spectrometry analysis by minimizing sample preparation and chromatographic separation. In this context, paper spray-MS (PS-MS) is an innovative approach that provides faster and cheaper analysis of biofluids by the addition of the sample directly to a paper. In forensic toxicology, the analytical workflow for the detection and quantification of drugs of abuse is onerous, including sample treatment, extraction and clean up, especially regarding complex biological matrices. PS-MS allows the detection of analytes of toxicological interest in blood, plasma and urine using low sample volume. This review aims to discuss the potential use, advances and challenges of PS-MS in forensic toxicology.

Introduction: Ayahuasca is a beverage composed by a mixture of herbs which contain the compound N,N-dimethyltriptamine (DMT) and the β-carbolines. Although its use is legalized in Brazil only for religious and spiritual ceremonies, there is a growing black market specialized in the distribution of these compounds in form of herbal material through internet and mail. The purpose of this work was the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of ayahuasca alkaloids and its application in seized ayahuasca products., Methods: An aliquot of seized products was weighted and diluted with methanol. An aliquot of this solution was added with internal standard (DMT-d 6 ), followed by injection in the analytical system., Results: The limit of quantitation was 10ng/mL for DMT and 25ng/mL for harmine, harmaline and tetrahydroharmine. The concentration ranges used were 10-100ng/mL for DMT, harmine and harmaline and all analytes presented a coefficient of determination (r 2 )≥0,99. Analysis of four seized samples presented concentrations of DMT ranging between 31.5 and 46.5mg/g. Presence of β-carbolines was not detected in the products. The variability of DMT concentrations can be correlated with the potential intoxications described in the literature., Conclusion: This work successfully established a determination method for ayahuasca alkaloids in herbal material. In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of psychoactive compounds in seized materials, leading to further investigation of ayahuasca ritualistic or recreational exposure., Competing Interests: Declaration of Competing Interest There are no financial or other relations that could lead to a conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The number of patients using cannabis for therapeutic purposes is growing worldwide. While research regarding the treatment of certain diseases/disorders with cannabis and cannabinoids is also expanding, only a few longitudinal studies have assessed the mid-term impacts of medical cannabis use on psychological variables and quality of life (QoL). The aim of the study was to assess the psychological safety and QoL of patients with chronic diseases who self-medicate with cannabis over time. We recruited patients with various chronic diseases who use cannabis and collected data regarding patterns of cannabis use as well as mental health, personality and QoL. Participants were followed-up at baseline, 4, 8 and 12 months. Hair analysis was conducted to confirm the presence of cannabinoids. Personality assessment showed a consistent decrease in self-transcendence and self-directedness scores. Neither cognitive nor psychopathological deterioration was found. There were also no variations in QoL. Mid-term use of medical cannabis seems to show adequate tolerability regarding cognitive and psychopathological abilities, and it may help patients with chronic diseases to maintain an acceptable QoL., (© 2020 John Wiley & Sons, Ltd.)

Multivariate analysis techniques could be used to identify possible intercorrelations in intoxications cases. The statistical analyses used were a multiple logistic regression, multiple correspondence analysis, principal component and hierarchical cluster analysis. Of the 320 samples analysed, 192 samples were positive for some of the investigated toxic agents, of which 100 were positive for ethanol and 131 were positive for other substances. It was possible to group the patients into 3 clusters, which appears 66.5% of this information in the three first factorial axes. On the first axis, the male patients were separated from the female patients. Patients exposed to drugs, between 30 and 39 years old were grouped in the same cluster. On the second factorial axis, patients who were intoxicated with ethanol and who became intoxicated with diazepam were grouped. This work contributed to the mapping of intoxication cases at the Poison Control Centre of the São Paulo city, Brazil (CCI-SP) and serves as an initial study for the creation of a database that could be updated constantly and thus could provide a toxicovigilance system for educational policies.

Ayahuasca tea is a hallucinogenic beverage used for religious purposes in Brazil and many other countries that has therapeutic potential in the treatment of some mental health disorders. In the context of psychedelic research, quantification of the tea's main alkaloids prior to its administration in animal or human studies is essential. For this reason, this study aims to provide information regarding the stability of the main ayahuasca alkaloids (dimethyltryptamine, DMT; harmine, HRM; tetrahydroharmine, THH; harmaline, HRL) in three different conditions: (1) A year stored in a refrigerator either in plastic or glass containers, (2) seven days at 37 °C to reproduce usual mail transportation, and (3) after three freeze-thaw cycles. Samples were quantified after a dilute-and-shoot procedure using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). There was no significant degradation of DMT concentration over time in all tested conditions. Harmala alkaloids (THH, HRL, and HRM) showed important variations after long-term and high-temperature storages. Although DMT has proven to be stable in all studied conditions, the harmala alkaloids revealed intense degradation and even concentration increment. This may be caused by degradation, alkaloid inter-conversion, and leaching from tea precipitate material. Therefore, ayahuasca quantification before administration in controlled sets is mandatory.

Introduction: Despite the enhancing effects of hyaluronidase (HYAL) over duration of anesthesia, this enzyme could cause adverse effects when injected concomitantly with local anesthetics in dental blocks., Objective: This study aimed to assess the tissue alterations caused by a local anesthetic protocol consisting of a late HYAL injection and confirm its functional effectiveness., Materials and Methods: The protocol efficacy was proved by evaluating sensory and motor functions in rats. The sciatic nerve was blocked with 2% lidocaine (LID) with epinephrine (n = 25). Thirty minutes later, 75 TRU/ml HYAL was injected into the same site (experimental group, LID/HYAL). One week later, this protocol was repeated in the contralateral hindlimb, injecting only HYAL's vehicle (control group, LID/vehicle [LID/V]). To observe the integrity of the local tissues, histological specimens were obtained 1, 24, 48, and 72 h after treatment with LID/HYAL or LID/V (n = 16 each) and stained with hematoxylin/eosin and picrosirius red., Results: Local inflammation was similar in both groups. The integrity of the nerve fibers was preserved, in spite of some inflammation-associated injuries in the surrounding tissues. The reversible tissue disorganization caused by HYAL, probably facilitated the diffusion of the residual anesthetic to the nerve, resulting in a prolonged anesthetic effect (P < 0.05)., Conclusions: No irreversible morphological alterations are caused by the administration of HYAL prior the end of the LID-induced block. Moreover, this protocol prolongs LID's anesthetic effect., Competing Interests: None

A fast and simple approach to overcome challenges in emergency toxicological analysis, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has been developed, for the detection of analytes in blood and urine samples from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants, drugs of abuse, and pesticides. These substances are relevant in the context of emergency toxicology in Brazil. The sample preparation procedure was relatively easy and fast to perform. The method was fully validated giving limits of in the range of 0.5 and 20 ng mL -1 for blood and urine samples. The intraday and interday precision and accuracy were considered adequate for all analytes once the relative standard deviation (RSD) (%) was lower than 20% for quality control (QC) low and lower than 15% for CQ medium and high. The developed method was successfully applied to 320 real samples collected at the Poison Control Center of São Paulo, and 89.1% have shown to be positive for some of the analytes. This confirms its applicability and importance to emergency toxicological analysis, and it could be very useful in both fields of clinical and forensic toxicology., (© 2019 John Wiley & Sons, Ltd.)

Background and Aims: Anabolic androgenic steroids (AAS) have been associated with coronary artery disease (CAD). AAS abuse leads to a remarkable decrease in high-density lipoprotein (HDL) plasma concentration, which could be a key factor in the atherosclerotic process. Moreover, not only the concentration of HDL, but also its functionality, plays a pivotal role in CAD. We tested the functionality of HDL by cholesterol efflux and antioxidant capacity. We also evaluated the prevalence of CAD in AAS users., Methods: Twenty strength-trained AAS users (AASU) age 29 ± 5 yr, 20 age-matched strength-trained AAS nonusers (AASNU), and 10 sedentary controls (SC) were enrolled in this cross-sectional study. Functionality of HDL was evaluated by 14 C-cholesterol efflux and the ability of HDL in inhibiting LDL oxidation. Coronary artery was evaluated with coronary computed tomography angiography., Results: Cholesterol efflux was lower in AASU compared with AASNU and SC (20 vs. 23 vs. 24%, respectively, p < 0.001). However, the lag time for LDL oxidation was higher in AASU compared with AASNU and SC (41 vs 13 vs 11 min, respectively, p < 0.001). We found at least 2 coronary arteries with plaques in 25% of AASU. None of the AASNU and SC had plaques. The time of AAS use was negatively associated with cholesterol efflux., Conclusions: This study indicates that AAS abuse impairs the cholesterol efflux mediated by HDL. Long-term AAS use seems to be correlated with lower cholesterol efflux and early subclinical CAD in this population., (Copyright © 2019. Published by Elsevier B.V.)

Disturbed shear rate (SR), characterized by increased retrograde and oscillatory SR in the brachial artery, is associated with inflammation, atherosclerosis, endothelial dysfunction, and sympathetic hyperactivity. Young subjects do not have disturbed SR; however, elderly subjects do, which seems to be associated with sympathetic hyperactivity. Anabolic androgenic steroids (AAS) abuse in young is associated with increased muscle sympathetic nerve activity (MSNA). We hypothesized that AAS users might have disturbed SR. We tested the association between retrograde and oscillatory SR with MSNA. In addition, we measured the high-sensitivity C-reactive protein (hs-CRP). We evaluated 10 male AAS users, age 27 ± 4 years, and 10 age-matched AAS nonusers, age 29 ± 5 years. At rest, retrograde and oscillatory SR were evaluated by Doppler ultrasound, MSNA was measured with microneurography, and hs-CRP was measured in blood sample. Flow-mediated dilation (FMD) was also assessed. AAS users had higher retrograde SR (24.42 ± 17.25 vs 9.15 ± 6.62 s - 1 , P = 0.01), oscillatory SR (0.22 ± 0.13 vs 0.09 ± 0.07 au P = 0.01), and MSNA (42 ± 9 vs 32 ± 4 bursts/100 heart beats, P = 0.018) than nonusers. MSNA (bursts/100 heart beats) was correlated with retrograde SR (r = 0.50, P = 0.050) and oscillatory SR (r = 0.51, P = 0.042). AAS users had higher hs-CRP [1.17 (0.44-3.63) vs 0.29 (0.17-0.70) mg/L, P = 0.015] and decreased FMD (6.42 ± 2.07 vs 8.28% ± 1.53%, P = 0.035) than nonusers. In conclusion, AAS abuse is associated with retrograde and oscillatory SR which were associated with augmented sympathetic outflow. In addition, AAS seems to lead to inflammation characterized by increased hs-CRP. These alterations may have the potential of increasing the early risk of atherosclerotic disease in young AAS users., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Recent open-label trials show that psychedelics, such as ayahuasca, hold promise as fast-onset antidepressants in treatment-resistant depression., Methods: To test the antidepressant effects of ayahuasca, we conducted a parallel-arm, double-blind randomized placebo-controlled trial in 29 patients with treatment-resistant depression. Patients received a single dose of either ayahuasca or placebo. We assessed changes in depression severity with the Montgomery-Åsberg Depression Rating Scale (MADRS) and the Hamilton Depression Rating scale at baseline, and at 1 (D1), 2 (D2), and 7 (D7) days after dosing., Results: We observed significant antidepressant effects of ayahuasca when compared with placebo at all-time points. MADRS scores were significantly lower in the ayahuasca group compared with placebo at D1 and D2 (p = 0.04), and at D7 (p &lt; 0.0001). Between-group effect sizes increased from D1 to D7 (D1: Cohen's d = 0.84; D2: Cohen's d = 0.84; D7: Cohen's d = 1.49). Response rates were high for both groups at D1 and D2, and significantly higher in the ayahuasca group at D7 (64% v. 27%; p = 0.04). Remission rate showed a trend toward significance at D7 (36% v. 7%, p = 0.054)., Conclusions: To our knowledge, this is the first controlled trial to test a psychedelic substance in treatment-resistant depression. Overall, this study brings new evidence supporting the safety and therapeutic value of ayahuasca, dosed within an appropriate setting, to help treat depression. This study is registered at http://clinicaltrials.gov (NCT02914769).

The sample preparation is the most critical step involved in the bioanalytical process. When dealing with green analytical chemistry, sample preparation can be even more challenging. To fit the green analytical chemistry principles, efforts should be made toward the elimination or reduction of the use of toxic reagents and solvents, minimization of energy consumption and increased operator safety. The simplest sample preparations are more appropriate for liquid biological matrices with little interfering compounds such as urine, plasma and oral fluid. The same does not usually occur with complex matrices that require more laborious procedures. The present review discusses green analytical approaches for the analyses of drugs of abuse in complex biological matrices, such as whole blood, breast milk, meconium and hair.

Aims/introduction: There is controversy as to whether hyperuricemia is an independent risk factor for cardiometabolic diseases. The serum level of uric acid is affected by a wide variety of factors involved in its production and excretion. In contrast, evidence has accumulated that locally- and systemically-activated xanthine oxidase (XO), a rate-limiting enzyme for production of uric acid, is linked to metabolic derangement in humans and rodents. We therefore explored the clinical implication of plasma XO activity in patients with type 2 diabetes mellitus and metabolic syndrome (MetS)., Materials and Methods: We enrolled 60 patients with type 2 diabetes mellitus and MetS. MetS was defined according to the 2005 International Diabetes Federation guidelines. Plasma XO activity was measured by highly-sensitive fluorometric assay measuring the conversion of pterin to isoxanthopterin, and explored associations between the value of plasma XO activity and metabolic parameters., Results: The value of plasma XO activity was correlated with indices of insulin resistance and the level of circulating liver transaminases. In contrast, the level of serum uric acid was not correlated with indices of insulin resistance. The value of plasma XO activity was not correlated with the serum uric acid level., Conclusions: Plasma XO activity correlates with indices of insulin resistance and liver dysfunction in Japanese patients with type 2 diabetes mellitus and MetS. Through assessing the plasma XO activity, patients showing normal levels of serum uric acid with higher activity of XO can be screened, thereby possibly providing a clue to uncovering metabolic risks in type 2 diabetes mellitus and MetS patients., (© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Objective: Truck drivers represent a group that is susceptible to the use of stimulant substances to reduce the symptoms of fatigue, which may be caused by a stressful and exhausting work environment. The use of psychoactive substances may increase the risk for involvement in road traffic crashes. Previous studies have demonstrated that amphetamine, cocaine, and cannabis are the 3 main drugs used by Brazilian truck drivers. We studied the prevalence of amphetamine, benzoylecgonine (indicating use of cocaine), and Δ-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH; indicating use of cannabis) in urine samples from truck drivers in the state of São Paulo, Brazil, using the same methodology during 8 years (2009-2016)., Methods: Samples were collected during a health program supported by the Federal Highway Police. Toxicological analyses were performed using immunoassays and gas chromatography-mass spectrometry., Results: The total prevalence of illicit drugs was 7.8%. Benzoylecgonine was the most prevalent substance (3.6%), followed by amphetamine (3.4%) and THC-COOH (1.6%). We found the highest drug prevalence in 2010 (11.3%) and the lowest in 2011 (6.1%). We could detect a slight change in the pattern of stimulant use: until 2010, amphetamine was the most prevalent substance; however, in 2011 benzoylecgonine became the most frequently detected substance. This lasted until 2015, probably due to changes in Brazilian legislation regarding appetite suppressants; the most common one is metabolized to amphetamine., Conclusion: These data show that the use of psychoactive substances by truck drivers in Brazil did not decrease during the study period. This reinforces the need for further preventive measures to reduce drug use among drivers, which could lead to a decrease in traffic crashes in Brazil.

Chronic overconsumption of animal fats causes a variety of health problems, including diabetes mellitus and obesity. Underlying molecular mechanisms encompass leptin resistance, a decrease in rewarding effects of physical activities, xanthine oxidase-induced oxidative stress in vasculature and peripheral tissue, impaired activation of incretin signaling, deviation in food preference, and dysbiosis of gut microbiota. Based on our clinical observation that daily intake of brown rice effectively ameliorates bodyweight gain, impaired glucose tolerance/insulin resistance and dependence on fatty foods in obese, prediabetes men, a line of research on brown rice (rice bran)-derived γ-oryzanol in mice experiments, cultured cells and human clinical trials is underway in our laboratory. Our works in mice showed that γ-oryzanol, an ester mixture of ferulic acid and several kinds of phytosterols, acts as a molecular chaperone, thereby attenuating the strong preference for animal fats through suppression of endoplasmic reticulum stress in the hypothalamus. In pancreatic islets from both high-fat diet-induced and streptozotocin-induced diabetic mice, γ-oryzanol ameliorates endoplasmic reticulum stress and protects β-cells against apoptosis. Noticeably, γ-oryzanol also acts as a potent inhibitor against deoxyribonucleic acid methyltransferases in the brain reward system (striatum) in mice, thereby attenuating, at least partly, the preference for a high-fat diet through the epigenetic modulation of striatal dopamine D2 receptor. Because dopamine D2 receptor signaling in the brain reward system is considerably attenuated in obese humans and rodents, γ-oryzanol might represent a unique property to ameliorate both hedonic and metabolic dysregulation of feeding behavior, highlighting a promising prophylactic avenue to protect against metabolic derangement., (© 2018 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

This work describes a simple approach to overcome challenges in emergency toxicological analysis, using liquid-liquid extraction and high-performance liquid chromatography coupled with a diode-array detector (HPLC-DAD). A rapid procedure has been developed, for the extraction and detection of 19 analytes from the following drug classes: analgesics, benzodiazepines, antidepressants, anticonvulsants and drugs of abuse. These substances are relevant in the context of emergency toxicology in Brazil. The method has been validated according to international guidelines by establishing parameters such as lower limit of quantification, sensitivity, linearity, accuracy and precision. The intra and inter-day precision values, at the lowest concentration levels, have always been less than 20% considering its relative standard deviation. As for accuracy values, these have also been satisfactory (above 81.3%). This method was successfully applied in 201 blood samples from patients with suspected poisoning of the Poison Control Center of São Paulo (PCC-SP), Brazil. Finally, the developed method has shown to be relevant for emergency toxicology due to its high sensitivity and it could be also very useful in both fields of clinical and forensic toxicology., (© 2018 John Wiley & Sons, Ltd.)

Violence is a dreadful phenomenon spread throughout the world, resulting in unfortunate events that can ultimately cause death. It is known that some countries play a much worrying role in this scenario than others. Brazil is one of them. The present work has focused on identifying the use of cocaine in 105 postmortem cases arriving at the Institute of Legal Medicine of Sao Paulo (IML-SP), the largest Brazilian city. Both blood and hair samples have been analyzed through ultra-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) in order to distinguish between recent or chronic cocaine use. The purpose of this work was to evaluate the proportion of cocaine use amongst violent individuals whose violence has ultimately led to their death. In order to do so, two previous methods, validated in-house, based on methanolic extraction for hair and protein precipitation for blood, have been used for this purpose and the final residue was analyzed through UPLC-ESI-MS/MS system. When looking at the demographics from the 105 postmortem cases analyzed, the results have shown the most critical age range to be between 18 and 25 years old and the least frequent between 37 and 45 years old. Gender wise, a rather extreme difference was found as 97 of the individuals were men and finally, considering the manner of death, the four-category criteria established appear to be fairly similar with 34 cases related to general violence and risk behavior, 26 to drug abuse suspicion, 23 to homicide resulting from opposition to police intervention and 22 to possible suicide., (Copyright © 2018 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

Purpose: Increased resting muscle sympathetic nerve activity (MSNA) and lower forearm blood flow (FBF) were observed in young men who use anabolic androgenic steroids (AAS). However, the response of MSNA and FBF in AAS users triggered by muscle mechanoreflex and central command has never been tested. In addition, we evaluated the blood pressure (BP) and heart rate (HR) responses during these maneuvers., Methods: Nineteen AAS users (AASU) 31 ± 6 yr of age and 18 AAS nonusers (AASNU) 29 ± 4 yr of age were recruited. All participants were involved in strength training. AAS use was determined using a urine test (liquid chromatography with tandem mass spectrometry). MSNA was measured using the microneurography technique. FBF was measured by using venous occlusion plethysmography. BP was measured using an automatic oscillometric device. HR was recorded continuously through ECG. Isometric handgrip exercise was performed at 30% of the maximal voluntary contraction for 3 min, and mental stress was elicited by the Stroop color-word test for 4 min., Results: The MSNA and FBF responses during exercise were similar between AASU and AASNU, with a trend toward higher MSNA (bursts per minute; P = 0.084) and lower forearm vascular conductance (FVC; units; P = 0.084) in AASU than in AASNU. During mental stress, AASU showed a significantly higher MSNA (P < 0.05) and lower FBF (P < 0.05) compared with AASNU. During both maneuvers, HR and BP increased linearly in both groups; however, AASU showed a significantly higher HR compared with AASNU., Conclusions: During muscle mechanoreflex activation (isometric exercise), AASU have normal MSNA and FBF responses, whereas during central command (mental stress) stimulation, AASU have exacerbated MSNA and blunted vasodilation. Therefore, mental stress seems to exacerbate neurovascular control throughout stress reaction situations in AASU.

Cannabis misuse during pregnancy is associated with severe impacts on the mother and baby health, such as newborn low birth weight, growth restriction, pre-term birth, neurobehavioral and developmental deficits. In most of the cases, drug abuse is omitted or denied by the mothers. Thus, toxicological analyzes using maternal-fetal matrices takes place as a suitable tool to assess drug use. Herein, meconium was the chosen matrix to evaluate cannabis exposure through identification and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic (THCCOOH). Accelerated solvent extraction (ASE) was applied for sample preparation technique to simultaneously extract and hydrolyze conjugated THCCOOH from meconium, followed by a solid-phase extraction (SPE) procedure. The method was developed and validated for gas chromatography-mass spectrometry (GC-MS), reaching hydrolysis efficiency of 98%. Limits of detection (LOD) and quantification (LOQ) were, respectively, 5 and 10 ng/g. The range of linearity was LOQ to 500 ng/g. Inter and intra-batch coefficients of variation were <8.4% for all concentration levels. Accuracy was in 101.7-108.9% range. Recovery was on average 60.3%. Carryover effect was not observed. The procedure was applied in six meconium samples from babies whose mothers were drug users and showed satisfactory performance to confirm fetal cannabis exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N , N -dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L ( n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.

Traffic accidents are responsible for 1.25 million deaths worldwide and are the most common cause of death among those aged 15-29 years. In Brazil, traffic accidents caused more than 44,000 deaths in 2014. The use of psychoactive drugs is an important risk factor for being involved in traffic accidents. Previous studies have found that psychoactive substances are commonly used by truck drivers in Brazil to maintain their extensive work schedule and stay awake while driving during nighttime hours. The state of Sao Paulo is one of the most important states regarding goods transportation. Important highways cross through Sao Paulo to other regions from Brazil and to other countries in Latin America. This study aims to determine the prevalence of illicit drug use by truck drivers in the state of Sao Paulo through toxicological analyses of oral fluid. Truck drivers were randomly stopped by police officers on federal roads during morning hours. Oral fluid samples were collected using the Quantisal™ device. In addition, a questionnaire concerning sociodemographic characteristics and health information was administered. Oral fluid samples were screened for amphetamine, cocaine, and tetrahydrocannabinol (Δ9-THC) by ELISA and the confirmation was performed using ultra performance liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS). Of the 764 drivers stopped, 762 agreed to participate. The participants were driving an average of 614km and 9.4h a day. Of the total samples, 5.2% (n=40) tested positive for drugs. Cocaine was the most frequently found drug (n=21), followed by amphetamine (n=16) and Δ9-THC (n=8). All drivers were men with an average age of 42.5 years. With these results we were able to verify that many truck drivers were still consuming psychoactive drugs while driving, and cocaine was the most prevalent one. This reinforces the need for preventive measures aimed at controlling the use of illicit drugs by truck drivers in Brazil., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Crack cocaine has a high potential to induce cocaine addiction and its smoke contains cocaine's pyrolysis product anhydroecgonine methyl ester (AEME), a partial agonist at M 1 - and M 3 -muscarinic acetylcholine receptor and an antagonist at the remaining subtypes. No reports have assessed AEME's role in addiction. Adult male Wistar rats were intraperitoneally administered with saline, 3mg/kg AEME, 15mg/kg cocaine, or a cocaine-AEME combination on every other day during a period of 9 days. After a 7-days withdrawal period, a challenge injection of the respective drugs was performed on the 17th day. The locomotor activity was evaluated on days 1, 3, 5, 7, 9 and 17, as well as dopamine levels (9th day) and dopaminergic receptors proteins (D 1 R and D 2 R on the 17th day) in the caudate-putamen (CPu) and nucleus accumbens (NAc). AEME was not able to induce the expression of behavioral sensitization, but it substantially potentiates cocaine-effects, with cocaine-AEME combination presenting higher expression than cocaine alone. An increase in the dopamine levels in the CPu in all non-saline groups was observed, with the highest levels in the cocaine-AEME group. There was a decrease in D 1 R protein level in this brain region only for cocaine and cocaine-AEME groups. In the NAc, an increase in the dopamine levels was only observed for cocaine and cocaine-AEME groups, with no changes in both D 1 R and D 2 R protein levels. These behavioral and neurochemical data indicate that AEME alone does not elicit behavioral sensitization but it significantly potentiates cocaine effects when co-administered, resulting in dopamine increase in CPu and NAc, brain regions where dopamine release is mediated by cholinergic activity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)