Influence of Brewing Process on the Profile of Biogenic Amines in Craft Beers.

The evaluation of the biogenic amines (BAs) profile of different types of craft beers is herein presented. A previously developed and validated analytical method based on ion-pair chromatography coupled with potentiometric detection was used to determine the presence of 10 BAs. Good analytical features were obtained for all amines regarding linearity (R2 values from 0.9873 ± 0.0015 to 0.9973 ± 0.0015), intra- and inter-day precision (RSD lower than 6.9% and 9.7% for beer samples, respectively), and accuracy (recovery between 83.2-108.9%). Detection and quantification limits range from 9.3 to 60.5 and from 31.1 to 202.3 µg L-1, respectively. The validated method was applied to the analysis of four ale beers and one lager craft beer. Ethylamine, spermidine, spermine, and tyramine were detected in all analyzed samples while methylamine and phenylethylamine were not detected. Overall, pale ale beers had a significantly higher total content of BAs than those found in wheat pale and dark samples. A general least square regression model showed a good correlation between the total content of BAs and the brewing process, especially for Plato degree, mashing, and fermentation temperatures. Knowledge about the type of ingredients and manufacturing processes that contribute to higher concentrations of these compounds is crucial to ensuring consumer safety.

Supramolecular Atropine Potentiometric Sensor.

A supramolecular atropine sensor was developed, using cucurbit[6]uril as the recognition element. The solid-contact electrode is based on a polymeric membrane incorporating cucurbit[6]uril (CB[6]) as an ionophore, 2-nitrophenyl octyl ether as a solvent mediator, and potassium tetrakis (4-chlorophenyl) borate as an additive. In a MES-NaOH buffer at pH 6, the performance of the atropine sensor is characterized by a slope of (58.7 ± 0.6) mV/dec with a practical detection limit of (6.30 ± 1.62) × 10-7 mol/L and a lower limit of the linear range of (1.52 ± 0.64) × 10-6 mol/L. Selectivity coefficients were determined for different ions and excipients. The obtained results were bolstered by the docking and spectroscopic studies which demonstrated the interaction between atropine and CB[6]. The accuracy of the potentiometric analysis of atropine content in certified reference material was evaluated by the t-Student test. The herein proposed sensor answers the need for reliable methods providing better management of this hospital drug shelf-life while reducing its flush and remediation costs.

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay.

Glycogen phosphorylase (GP) is a key enzyme in the glycogenolysis pathway and a potential therapeutic target in the management of type 2 diabetes. It catalyzes a reversible reaction: the release of the terminal glucosyl residue from glycogen as glucose 1-phosphate; or the transfer of glucose from glucose 1-phosphate to glycogen. A colorimetric method to follow in vitro the activity of GP with usefulness in structure-activity relationship studies and high-throughput screening capability is herein described. The obtained results allowed the choice of the optimal concentration of enzyme of 0.38 U/mL, 0.25 mM glucose 1-phosphate, 0.25 mg/mL glycogen, and temperature of 37 °C. Three known GP inhibitors, CP-91149, a synthetic inhibitor, caffeine, an alkaloid, and ellagic acid, a polyphenol, were used to validate the method, CP-91149 being the most active inhibitor. The effect of glucose on the IC50 value of CP-91149 was also investigated, which decreased when the concentration of glucose increased. The assay parameters for a high-throughput screening method for discovery of new potential GP inhibitors were optimized and standardized, which is desirable for the reproducibility and comparison of results in the literature. The optimized method can be applied to the study of a panel of synthetic and/or natural compounds, such as polyphenols.

Evaluation of a flavonoids library for inhibition of pancreatic α-amylase towards a structure-activity relationship.

α-Amylase has been considered an important therapeutic target for the management of type 2 diabetes mellitus (T2DM), decreasing postprandial hyperglycaemia (PPHG). In the present work, a panel of 40 structurally related flavonoids was tested, concerning their ability to inhibit α-amylase activity, using a microanalysis screening system, an inhibitory kinetic analysis and molecular docking calculations. From the obtained results, it was possible to observe that the flavone with a -Cl ion at 3-position of C-ring, an -OH group at 3'- and 4'- positions of B-ring and at 5- and 7- positions of A-ring and the C2 = C3 double bond, was the most active tested flavonoid, through competitive inhibition. In conclusion, some of the tested flavonoids have shown promising inhibition of α-amylase and may be considered as possible alternatives to the modulation of T2DM.

The biocompatibility and bioactivity of hemodialysis membranes: their impact in end-stage renal disease.

End-stage renal disease is a growing health problem with increasing prevalence and high health care costs. Patients suffering from end-stage renal disease exhibit higher morbidity and mortality rates compared to the general population. These patients, who are treated using hemodialysis, typically suffer from anemia, inflammation, and oxidative stress. Inadequate dialyzer membrane biocompatibility exacerbates these negative side effects. Modifications of the composition of hemodialysis membranes have improved their biocompatibility and improve the patients' quality of life. Recently, the use of dialyzer membranes coated with bioactive compounds has also been proposed to further ameliorate dialysis-associated problems. Based on a survey of the current literature, application of bioactive membranes decreases the inflammation and oxidative stress of patients treated with hemodialysis.

Pilot safety study of intrabronchial instillation of bone marrow-derived mononuclear cells in patients with silicosis.

BACKGROUND: Silicosis is an occupational disease for which no effective treatment is currently known. Systemic administration of bone marrow-derived mononuclear cells (BMDMCs) has shown to be safe in lung diseases. However, so far, no studies have analyzed whether bronchoscopic instillation of autologous BMDMCs is a safe route of administration in patients with silicosis. METHODS: We conducted a prospective, non-randomized, single-center longitudinal study in five patients. Inclusion criteria were age 18-50 years, chronic and accelerated silicosis, forced expiratory volume in 1 s <60 % and >40 %, forced vital capacity ≥60 % and arterial oxygen saturation >90 %. The exclusion criteria were smoking, active tuberculosis, neoplasms, autoimmune disorders, heart, liver or renal diseases, or inability to undergo bronchoscopy. BMDMCs were administered through bronchoscopy (2 × 10(7) cells) into both lungs. Physical examination, laboratory evaluations, quality of life questionnaires, computed tomography of the chest, lung function tests, and perfusion scans were performed before the start of treatment and up to 360 days after BMDMC therapy. Additionally, whole-body and planar scans were evaluated 2 and 24 h after instillation. RESULTS: No adverse events were observed during and after BMDMC administration. Lung function, quality of life and radiologic features remained stable throughout follow-up. Furthermore, an early increase of perfusion in the base of both lungs was observed and sustained after BMDMC administration. CONCLUSION: Administration of BMDMCs through bronchoscopy appears to be feasible and safe in accelerated and chronic silicosis. This pilot study provides a basis for prospective randomized trials to assess the efficacy of this treatment approach. CLINICAL TRIALS. GOV IDENTIFIER: NCT01239862 Date of Registration: November 10, 2010.

Vortex-assisted liquid-liquid micro-extraction and high-performance liquid chromatography for a higher sensitivity methyl methacrylate determination in biological matrices.

A vortex-assisted liquid-liquid micro-extraction coupled with high-performance liquid chromatography, with UV-vis, is proposed to pre-concentrate methyl methacrylate and to improve separation in biological matrices. The use of 1-octanol as extracting phase, its volume, the need for a dispersant agent, the agitation conditions and the cooling time before phase separation were evaluated. In optimum conditions, enrichment factors of 20 (±0.5) and enrichment recovery of 99% were obtained. The straightforward association of this extraction process with the HPLC method, previously regulated by the International Organization for Standardization, afforded a detection limit of 122 ng/mL and a quantification limit of 370 ng/mL. The within-batch precision, relative standard deviation, was 3% for a sample with 1.49 µg/mL and 4% for a sample with 13.4 µg/mL. The results showed a between batch-precision of 21% for experiments performed on five different days, for a sample with a concentration of 1.10 µg/mL in methyl methacrylate.

Development of an inexpensive and rapidly preparable enzymatic pencil graphite biosensor for monitoring of glyphosate in waters.

Glyphosate (GLY) is the most widely used non-selective broad-spectrum herbicide worldwide under well-reported side effects on the environment and human health. That's why it's necessary to control its presence in the environment. This work describes the development of an affordable, simple, and accurate electrochemical biosensor using a pencil graphite electrode as support, a horseradish peroxidase enzyme immobilized on a polysulfone membrane doped with multi-walled carbon nanotubes. The developed electrochemical sensor was used in the determination of GLY in river and drinking water samples. Cyclic voltammetry and amperometry were used as electrochemical detection techniques for the characterization and analytical application of the developed biosensor. The working mechanism of the biosensor is based on the inhibition of the peroxidase enzyme by GLY. Under optimal experimental conditions, the biosensor showed a linear response in the concentration range of 0.1 to 10 mg L-1. The limits of detection and quantification are 0.025 ± 0.002 and 0.084 ± 0.007 mg L-1, respectively, which covers the maximum residual limit established by the EPA for drinking water (0.7 mg L-1). The proposed biosensor demonstrated high reproducibility, excellent analytical performance, repeatability, and accuracy. The sensor proved to be selective against other pesticides, organic acids, and inorganic salts. Application on real samples showed recovery rates ranging between 98.18 ± 0.11 % and 97.32 ± 0.23 %. The analytical features of the proposed biosensor make it an effective and useful tool for the detection of GLY for environmental analysis.

Prevalence and associated factors of experimentation with and current use of water pipes and electronic cigarettes among medical students: a multicentric study in Brazil.

OBJECTIVE: To evaluate the prevalence of and factors associated with experimentation with and current use of water pipes and e-cigarettes among medical students. METHODS: This was a cross-sectional multicentric study involving a convenience sample of students from medical schools in most Brazilian geographic regions. Information about experimentation with and current use of conventional cigarettes, water pipes, and e-cigarettes; beliefs and attitudes toward tobacco products; religiosity; and demographics were collected by means of an online structured questionnaire. We used descriptive statistics and logistic regression to analyze the association of those factors. RESULTS: Our sample comprised 700 individuals from four Brazilian regions. Prevalence of experimentation with and current use of cigarettes, water pipes, and e-cigarettes were, respectively, 39.1% and 7.9%; 42.6% and 11.4%; and 13.1% and 2.3%. Water pipe experimentation was higher among those who had a sibling (adjusted OR = 2.64; 95% CI, 1.24-5.61) or friends (adjusted OR = 2.33; 95% CI, 1.63-3.31) who smoke. The same occurred regarding e-cigarette experimentation: siblings (adjusted OR = 2.76; 95% CI, 1.17-6.50) and friends (adjusted OR = 2.47; 95% CI, 1:45-4.22). Curiosity and scent/taste were the major reasons for water pipe use and e-cigarette experimentation. Although 93% of the responders learned about health damages of smoking during medical school classes, 51.4% reported having experimented with at least one of these tobacco products. Most responders who reported feeling the presence of God/the Holy Spirit in their lives were never experimenters of water pipes (59.2%) or e-cigarettes (55.3%). CONCLUSIONS: There is a high prevalence of experimentation with tobacco products among medical students whose siblings or friends smoke, despite their knowledge about smoking harms.

Polysulfone Membranes Doped with Human Neutrophil Elastase Inhibitors: Assessment of Bioactivity and Biocompatibility.

The use of polysulfone (PSU) hemodialysis (HD) membranes modified with bioactive compounds has gained relevance in chronic kidney disease (CKD) management. Compounds based on the 4-oxo-ß-lactam scaffold have outstanding inhibitory ability and selectivity for human neutrophil elastase (HNE). The present work aimed to evaluate the bioactivity and biocompatibility of PSU-based HD membranes doped with HNE inhibitors (HNEIs). For this, two 4-oxo-ß-lactam derivates (D4L-1 and D4L-2) synthesized in house were used, as well as a commercial HNEI (Sivelestat), for comparison purposes. Their HNE inhibition efficacy was evaluated in in vitro and ex vivo (incubations with human plasma) assay conditions. All biomaterials were bioactive and hemocompatible. The inhibitory capacity of the HNEIs and HNEI-PSU membranes in vitro was D4L-1 > D4L-2 > Sivelestat and D4L-2 > Sivelestat > D4L-1, respectively. In ex vivo conditions, both HNEIs and HNEI-PSU materials presented the same relative inhibitory ability (D4L-1 > D4L-2 > Sivelestat). The difference observed between in vitro and ex vivo conditions is most likely due to the inherent lipophilicity/hydrophobicity of each HNEI influencing their affinity and accessibility to HNE when trapped in the membrane. Compared to Sivelestat, both D4L-1 and D4L-2 (and the respective doped membranes) have more potent inhibition capabilities. In conclusion, this work reports the successful development of PSU membranes functionalized with HNEIs.

Application of a novel hybrid MIL-53(Al)@rice husk for the adsorption of glyphosate in water: Characteristics and mechanism of the process.

The development of new materials that have a high capacity to remove pollutants in water-based media is becoming increasingly important because of the serious contamination of water and the negative impact on biodiversity and public health. The presence of glyphosate in water, the most widely used herbicide worldwide, has triggered alerts owing to the collateral effects it may cause on human health. The main objective of the present study was to investigate the potential of the hybrid material MIL-53(Al)@RH for the adsorption of glyphosate in aqueous solution. The material was obtained following the methodology of MIL-53(Al) synthesis in the presence of hydrolyzed rice husk assisted by microwave. Batch adsorption experiments were carried out to evaluate the adsorbent dosage, pH0 solution effect, contact time, adsorbate concentration, and temperature effect. The results demonstrated that a maximum adsorption capacity of 296.95 mg g-1, at pH0 4 with a ratio of 0.04 g MIL-53(Al)@RH/50 mL of solution, was achieved in 30 min. The Avrami and pseudo-second order models appropriately described the adsorption kinetics and the equilibrium by Langmuir and Sips models. The enthalpy changes (ΔH°) determined propose an endothermic reaction governed by chemisorption, corroborating the kinetic and equilibrium settings. Hydrogen bonds, π*-π interactions, and complexation between the metal centers of MIL-53(Al) and the anionic groups of glyphosate were postulated to be involved as adsorption mechanisms. Finally, for practical application, MIL-53(Al)@RH was packed in a column for a fixed-bed test which revealed that the hybrid can remove glyphosate with an adsorption capacity of 76.304 mg L-1, utilizing 90% of the bed.

HPLC-potentiometric method for determination of biogenic amines in alcoholic beverages: A reliable approach for food quality control.

Determination of ten biogenic amines in alcoholic beverages by HPLC coupled to a potentiometric detector for food quality control is herein presented. Biogenic amines were separated by ion-pair chromatography on a C18 column using a gradient mobile phase of acetic acid, acetonitrile, and butane-sulfonic acid. Detection was accomplished by a miniaturized amine-selective electrode. The method was validated following ICH and Eurachem guidelines. Linear regression models provided R2 values from 0.9870 ± 0.0019 to 0.9991 ± 0.0014 for tyramine and cadaverine, respectively. Detection and quantification limits depend on the molecular weight of BAs, ranging from 9.3 to 60.5 and from 31.1 to 202.3 µg L-1 for methylamine and spermine, respectively. Repeatability and intermediate precision showed RSD values lower than 5.8 and 8.3%, respectively. Accuracy of assays yielded recovery values from 86.4 to 109.9%. The biogenic amines content in red wine, white wine, and beer samples were 7.54, 5.24, and 4.58 mg L-1, respectively.

Minimizing the Silver Free Ion Content in Starch Coated Silver Nanoparticle Suspensions with Exchange Cationic Resins.

This work describes the optimization of a methodology for the reduction of silver ions from silver nanoparticle suspensions obtained from low-yield laboratory procedures. The laboratory synthesis of silver nanoparticles following a bottom-up approach starting from silver nitrate, originates silver ions that were not reduced to their fundamental state for nanoparticles creation at the end of the process. However, it is well known that silver ions can easily influence chemical assays due to their chemical reactivity properties and can limit biological assays since they interfere with several biological processes, namely intracellular ones, leading to the death of living cells or organisms. As such, the presence of silver ions is highly undesirable when conducting biological assays to evaluate the influence of silver nanoparticles. We report the development of an easy, low-cost, and rapid methodology that is based on cation exchange resins to minimize the silver ion content in a raw suspension of silver nanoparticles while preserving the integrity of the nanomaterials. This procedure preserves the physical-chemical properties of the nanoparticles, thus allowing the purified nanoparticulate systems to be biologically tested. Different types of cationic resins were tested, and the developed methodology was optimized by changing several parameters. A reduction from 92% to 10% of free silver/total silver ratio was achieved when using the Bio-Rad 50W-X8 100-200 mesh resin and a contact time of 15 min. Filtration by vacuum was used to separate the used resin from the nanoparticles suspension, allowing it to be further reused, as well as the purified AgNPs suspension.

Inhibition of the carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase by hydroxylated xanthones.

Xanthones are oxygen-containing heterocyclic compounds that exhibit a wide range of biological and pharmacological properties. Some natural and synthetic derivatives have been identified for their antidiabetic profile, mainly as α-glucosidase inhibitors. However, studies concerning the inhibition of both carbohydrate-hydrolyzing enzymes α-amylase and α-glucosidase are scarce. Thus, in order to identify some of these dual-target antidiabetic agents, a series of new synthetic xanthones were evaluated together with their commercial parents mangiferin (4), α-mangostin (5) and γ-mangostin (6). The results showed that xanthones exhibited a systematic stronger inhibition against α-glucosidase rather than for α-amylase. Derivatives 2c, 3a and 3b, bearing one catechol moiety, were the most active inhibitors of α-amylase, while xanthones 2c, 3b and 3c were the most active against α-glucosidase activity, with IC50 values lower than 10 µM. These findings suggest that the substitution pattern of the xanthone scaffold modulated the inhibitory activity of these compounds, and some structure-activity relationships could be established for both assays. In addition, the type of inhibition was also studied, and the results indicate a competitive type of inhibition for α-amylase activity by xanthones 2c, 3b, 3c and γ-mangostin (6). On the other hand, non-competitive inhibition mechanisms can be ascribed for all xanthones 1-6 against α-glucosidase. The present work can open a promising area of research based on the design of novel xanthone derivatives, based on natural ones, for targeting key enzymes involved in glucose metabolism and therefore in the management of type 2 diabetes mellitus.

A combined experimental and computational study to discover novel tyrosinase inhibitors.

Depigmenting properties of tyrosinase inhibitors (TAi) boosted the search for new compounds applicable in cosmetics. Kojic acid, a 3-hydroxy-4-pyrone, is the most studied tyrosinase inhibitor but undesirable side effects, like dermatitis, and unspecified mechanism led to its exclusion in several countries. To discover safer and more efficient TA, we evaluated tyrosinase inhibitory effect of twelve 3-hydroxy-4-pyridinones (3,4-HPO) in vitro and considering the two reaction steps of inhibition in mushroom tyrosinase enzyme. In parallel we performed molecular docking studies in human and mushroom enzymes. Ligands I6 and I11 were the most effective compounds considering their inhibitory activity in both reaction steps. Our studies revealed that I6 has a non-competitive and mixed type of inhibition for monophenolase and diphenolase activity, while ligand I11 showed a mixed and competitive inhibition type for each reaction step. Molecular Docking results indicated that ligands tend to bind the enzyme by coordinating directly with the binuclear cooper centre and highlighted the relevance of voluminous and non-polar substituents at R2 to avoid the binding of the ligands to the enzyme. The work clarifies the type of inhibition established for kojic acid and points out the differences found for the set of 3,4-HPO chelators studied as prospective tyrosinase inhibitors.

Challenges in the design of electrochemical sensor for glyphosate-based on new materials and biological recognition.

Glyphosate (GLY) is the main ingredient in the weed killer Roundup and the most widely used pesticide in the world. Studies of the harmful effects of GLY on human health began to become more wide-ranging after 2015. GLY is listed by the International Agency for Research on Cancer (IARC) as a carcinogenic hazard to humans. Moreover, GLY has the property to complex with transition metals and are stable for long periods, being considered a high-risk element for different matrices, such as environmental (soil and water) and food (usually genetically modified crops). Since that, it was noticed an increment in the development of new analytical methods for its determination in different matrices like food, environmental and biological fluids. Noteworthy, the application of electrochemical techniques for downstream detection sparked interest due to the ability to minimize or eliminate the use of polluting chemicals, using simple and affordable equipment. This work aims to review the contribution of the electroanalytical methods for the determination of GLY in different food and environmental matrices. Parameters such as the electrochemical transduction techniques based on the electrical measurement signals, receptor materials for electrodes preparation, and the detection mechanisms are described in this review. The literature review shows that the electrochemical sensors are powerful detection system that can be improved by their design and by their portability to fulfil the needs of the GLY determination in laboratory benches, or even in situ analysis.

Pyrazoles as novel protein tyrosine phosphatase 1B (PTP1B) inhibitors: An in vitro and in silico study.

Type 2 diabetes mellitus (DM) is a complex chronic disorder and a major global health problem. Insulin resistance is the primary detectable abnormality and the main characteristic feature in individuals with type 2 DM. Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of the insulin signaling pathway, which dephosphorylates insulin receptor and insulin receptor substrates, suppressing the insulin signaling cascade. Therefore, the inhibition of PTP1B has become a potential strategy in the management of type 2 DM. In this study, a library of 22 pyrazoles was evaluated here for the first time against human PTP1B activity, using a microanalysis screening system. The results showed that 5-(2-hydroxyphenyl)-3-{2-[3-(4-nitrophenyl)-1,2,3,4-tetrahydronaphthyl]}-1-phenylpyrazole 20 and 3-(2-hydroxyphenyl)-5-{2-[3-(4-methoxyphenyl)]naphthyl}pyrazole 22 excelled as the most potent inhibitors of PTP1B, through noncompetitive inhibition mechanism. These findings suggest that the presence of additional benzene rings as functional groups in the pyrazole moiety increases the ability of pyrazoles to inhibit PTP1B. The most active compounds showed selectivity over the homologous T-cell protein tyrosine phosphatase (TCPTP). Molecular docking analyses were performed and revealed a particular contact signature involving residues like TYR46, ASP48, PHE182, TYR46, ALA217 and ILE219. This study represents a significant beginning for the design of novel PTP1B inhibitors.

Nanostructured pencil graphite electrodes for application as high power biocathodes in miniaturized biofuel cells and bio-batteries.

This work describes a simple method for the fabrication of an enzymatic electrode with high sensitivity to oxygen and good performance when applied as biocathode. Pencil graphite electrodes (PGE) were chosen as disposable transducers given their availability and good electrochemical response. After electrochemical characterization regarding hardness and surface pre-treatment suited modification with carbon-based nanostructures, namely with reduced graphene, MWCNT and carbon black for optimal performance was proceeded. The bioelectrode was finally assembled through immobilization of bilirubin oxidase (BOx) lashed on the modified surface of MWCNT via π-π stacking and amide bond functionalization. The high sensitivity towards dissolved oxygen of 648 ± 51 µA mM-1 cm-2, and a LOD of 1.7 µM, was achieved for the PGE with surface previously modified with reduced graphene (rGO), almost the double registered for direct anchorage on the bare PGE surface. Polarization curves resulted in an open circuit potential (OCP) of 1.68 V (vs Zn electrode) and generated a maximum current density of about 650 µA cm-2 in O2 saturated solution.

Doping Polysulfone Membrane with Alpha-Tocopherol and Alpha-Lipoic Acid for Suppressing Oxidative Stress Induced by Hemodialysis Treatment.

The reduction of free radicals by bioactive membranes used for hemodialysis treatment is an important topic due to the constant rise of oxidative stress-associated cardiovascular mortality by hemodialysis patients. Therefore, it is urgent to find an effective solution that helps to solve this problem. Polysulfone membranes enriched with α-lipoic acid, α-tocopherol, and with both components are fabricated by spin coating. The antioxidant properties of these membranes are evaluated in vitro by determining the lipid-peroxidation level and the total antioxidant status of the blood plasma. The biocompatibility is assessed by quantifying the protein adsorption, platelet adhesion, complement activation, and hemolytic effect. All types of membranes show in vitro antioxidant activity and a trend to reduce oxidative stress in vivo; the best results show membranes prepared with a combination of both compounds and prove to be nonhemolytic and hemocompatible. Moreover, the membrane specific separation ability for the main waste products is not affected by antioxidants incorporation.

Consensus Document on Medical Faculty Education on the Treatment of Smoking. / Documento de consenso sobre la docencia del tabaquismo en las facultades de Medicina.

We report the results of a consensus reached by an expert group of representatives from different medical societies in Latin America on the objectives, competencies (knowledge, and skills), content, and duration of smoking cessation education in Latin American medical schools. The document discusses the following aspects: epidemiology, nicotine dependence, factors for initiation and maintenance of tobacco use, smoking-related disorders, diagnosis, minimal intervention, non-pharmacological and pharmacological interventions for smoking cessation, and prevention of smoking.