Education and Social Exclusion of Children with Chronic Diseases: The School Reintegration

This research attempts to identify the conditions under which children with chronic diseases are reintegrated into formal education after they have been hospitalized for a long period. In addition, it attempts to probe the conditions under which the children are being taught during the home schooling period. Educational inequality is a thorny issue that concerns every education system. Many important social vulnerabilities affect the equal distribution of educational opportunities, but the education of children with chronic illnesses creates a double vulnerability, the results of which are most often truly tragic for the affected students and their families.  The research data was collected after a series of interviews with parents whose children had been hospitalized with a serious illness and teachers who had worked in hospital schools and in formal education. The findings of the research show that the reintegration of hospitalized children who have been absent for a long time from school is difficult and often causes anxiety due to the abusive behaviors they receive from their classmates. It is also emphasizes that the teachers of the formal education have serious lack of training related to the reintegration of children with chronic diseases as well as a holistic cooperation plan that should prevent the stigmatization related to the disease is necessary. Keywords: Social exclusion, School reintegration, Chronic diseases, Hospital education, Home schooling DOI: 10.7176/JEP/14-16-05 Publication date:June 30th 202

Study of the chemical composition of plant extracts for the presence of their bioactive compounds using modern hyphenated techniques of instrumental analysis

Natural environment and especially plants continue to provide a dynamic research area in the field of drug discovery, since phytochemicals can be possible starting materials of novel bioactive compounds. In the present drug discovery programmes, natural products or compounds derived from natural products account for > 40% of the new registered drugs. In order to discover new bioactive compounds, extracts from plants or other natural sources should be submitted at the same time to chemical screening and various biological or pharmacological targets. Due to the complexity of the plant extracts, it is rather difficult to characterize them chemically in one step. Usually, their study includes fractionation of the complex mixture, separation and isolation of the individual components with liquid chromatography, and structure elucidation using various spectroscopic methods (UV, IR, MS, NMR). Our experience in studying antioxidants that are present in plant extracts revealed that 1D and 2D NMR spectroscopy can be used as an alternative analytical technique to provide insight into mixtures without any previous separation of the individual components. The aim of this thesis was to use different 1D and 2D NMR methodologies for the characterization of different classes of secondary metabolites in plant extracts without any previous separation and isolation of the individual components. The crude plant extracts, belonging in the Lamiaceae, Oleaceae and Apocynaceae families, were dissolved in deuterated solvents (CD3CN and pyridine-d5) and an attempt was made for the identification, and in some cases, the quantification of their main components, belonging to the classes of flavonoids and phenolic acids, diterpenes and triterpenoids. Ground leaves of Rosmarinus officinalis, Salvia officinalis, Thymus vulgaris, Origanum majorana, Olea europaea and Nerium oleander were subsequently extracted with n-hexane and ethyl acetate. The extracts were concentrated in rotary evaporator and kept into sealed flasks. As a first step, extracts were investigated for their radical scavenging activity using DPPH∙ assay and the content of total phenolic compounds and flavonoids was measured in plant extracts. Because of their strong radical scavenging activity (of most of the extracts) and the high amounts of total phenolic compounds and flavonoids of the ethyl acetate extracts, their composition was further investigated. All crude extracts were dissolved in CD3CN and the 1H- NMR, 1Η- 1Η COSY, 1H-13C HSQC and HMBC spectra were acquired. In the 1H- NMR spectrum of the extracts a very characteristic region between 11 to 13 ppm, which is of high diagnostic value for the presence of flavonoids, was observed. The highly deshielded signals in this region are attributed to the hydroxyl protons OH(5), which participate in a strong intramolecular hydrogen bond, formed between the hydroxyl and the carbonyl group at CO(4). As a rule the 1H- NMR resonances of the –OH groups appear at room temperature as broad signals especially in protic solvents, owing to the mobility of the hydrogen and its fast exchange, on the NMR time scale, with the protons of the solvent. However, by decreasing the temperature, the proton exchange rate is reduced and the –OH peaks are revealed as sharp peaks. With the use of proper temperature and by spiking with standards, certain flavones, flavonols and flavanones were detected in ethyl acetate extracts. Also in the 1H- NMR spectrum of the ethyl acetate extracts of the Lamiaceae family two doublets resonating at ~ 6.2 and ~ 7.5 ppm respectively (J =15.8 Hz), were observed. Their coupling constant indicates the presence of the trans-isomer of the hydroxycinammic acid derivative. In 1Η- 1Η COSY spectrum the strongest cross-peaks (Η3a, H2a), (Η3a, H3β) και (Η2, Η3α,β) are diagnostic of the caffeic acid moiety. Moreover in the 1H-13C HSQC spectrum certain very strong cross-peaks indicate a coupling network that can be attributed to rosmarinic acid. Both proton and carbon-13 chemical shifts of rosmarinic and caffeic acid are almost the same, but there is a significant difference in the 13C shielding and the number of cross-peaks of the –COOR and –COOH carbons, in the 1H-13C HMBC spectrum, which offers the solution to the problem of the unequivocal assignment of them. The high resolution of the 2D 1H-13C HMBC spectrum allowed us to determine unambiguously the individual cross-peaks of the spin system of rosmarinic acid r(H3a, C1a), r(H2a, C1a), even at room temperature. In addition, in the hexane and ethyl acetate extracts of sage and rosemary, using a combination of 1H-13C HSQC and 1H-13C HMBC NMR spectroscopy, the diterpenes carnosic acid and carnosol were identified. In order to confirm the results from the identification of compounds with 1D and 2D NMR spectroscopy and to ascertain the complexity or not of the composition of crude plant extracts, they were dissolved in methanol and analyzed with LC-ESI-MS. Various compounds were tentatively identified and for some of them using standards were identified, belonging to the classes of flavonoids and phenolic acid (rosmarinic acid), diterpenes, simple phenols and triterpenic acids. The presence of compounds identified using 1D and 2D NMR spectroscopy was confirmed with the LC-ESI-MS analysis. The quantification of the standard compounds that were commercially available was achieved using seven point calibration curves. The levels of rosmarinic acid in the extracts were also examined by the use of 1H- NMR spectroscopy and were in good agreement with those obtained by HPLC. Finally, a novel strategy for NMR analysis of mixtures of oleanolic (OA) and ursolic acids (UA) that occur in natural products was described. The combination of 1H-13C HSQC and 1H-13C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of OA and UA without resorting to physicochemical separation of the components. The methodology was extended and to other triterpenoids (betulinic acid, betulin, erythrodiol and uvaol) that were identified in some of the above extracts. The quantitative results provided by 2D 1H-13C HSQC NMR data were obtained within a short period of time (~ 14 min) and were in excellent agreement with those obtained by HPLC (using modified cyclodextrins in the mobile phase), which supported the efficiency of the suggested methodology

Analytical and Structural Tools of Lipid Hydroperoxides: Present State and Future Perspectives

Mono- and polyunsaturated lipids are particularly susceptible to peroxidation, which results in the formation of lipid hydroperoxides (LOOHs) as primary nonradical-reaction products. LOOHs may undergo degradation to various products that have been implicated in vital biological reactions, and thus in the pathogenesis of various diseases. The structure elucidation and qualitative and quantitative analysis of lipid hydroperoxides are therefore of great importance. The objectives of the present review are to provide a critical analysis of various methods that have been widely applied, and more specifically on volumetric methods, applications of UV-visible, infrared, Raman/surface-enhanced Raman, fluorescence and chemiluminescence spectroscopies, chromatographic methods, hyphenated MS techniques, NMR and chromatographic methods, NMR spectroscopy in mixture analysis, structural investigations based on quantum chemical calculations of NMR parameters, applications in living cells, and metabolomics. Emphasis will be given to analytical and structural methods that can contribute significantly to the molecular basis of the chemical process involved in the formation of lipid hydroperoxides without the need for the isolation of the individual components. Furthermore, future developments in the field will be discussed

1H-NMR as a Structural and Analytical Tool of Intra- and Intermolecular Hydrogen Bonds of Phenol-Containing Natural Products and Model Compounds

Experimental parameters that influence the resolution of 1H-NMR phenol OH signals are critically evaluated with emphasis on the effects of pH, temperature and nature of the solvents. Extremely sharp peaks (Δν1/2 ≤ 2 Hz) can be obtained under optimized experimental conditions which allow the application of 1H-13C HMBC-NMR experiments to reveal long range coupling constants of hydroxyl protons and, thus, to provide unequivocal assignment of the OH signals even in cases of complex polyphenol natural products. Intramolecular and intermolecular hydrogen bonds have a very significant effect on 1H OH chemical shifts which cover a region from 4.5 up to 19 ppm. Solvent effects on –OH proton chemical shifts, temperature coefficients (Δδ/ΔT), OH diffusion coefficients, and nJ(13C, O1H) coupling constants are evaluated as indicators of hydrogen bonding and solvation state of phenol –OH groups. Accurate 1H chemical shifts of the OH groups can be calculated using a combination of DFT and discrete solute-solvent hydrogen bond interaction at relatively inexpensive levels of theory, namely, DFT/B3LYP/6-311++G (2d,p). Excellent correlations between experimental 1H chemical shifts and those calculated at the ab initio level can provide a method of primary interest in order to obtain structural and conformational description of solute-solvent interactions at a molecular level. The use of the high resolution phenol hydroxyl group 1H-NMR spectral region provides a general method for the analysis of complex plant extracts without the need for the isolation of the individual components

Structural Basis of Artemisinin Binding Sites in Serum Albumin with the Combined Use of NMR and Docking Calculations

Artemisinin is known to bind to the main plasma protein carrier serum albumin (SA); however, there are no atomic level structural data regarding its binding mode with serum albumin. Herein, we employed a combined strategy of saturation transfer difference (STD), transfer nuclear Overhauser effect spectroscopy (TR-NOESY), STD–total correlation spectroscopy (STD-TOCSY), and Interligand Noes for PHArmacophore Mapping (INPHARMA) NMR methods and molecular docking calculations to investigate the structural basis of the interaction of artemisinin with human and bovine serum albumin (HSA/BSA). A significant number of inter-ligand NOEs between artemisinin and the drugs warfarin and ibuprofen as well as docking calculations were interpreted in terms of competitive binding modes of artemisinin in the warfarin (FA7) and ibuprofen (FA4) binding sites. STD NMR experiments demonstrate that artemisinin is the main analyte for the interaction of the A. annua extract with BSA. The combined strategy of NMR and docking calculations of the present work could be of general interest in the identification of the molecular basis of the interactions of natural products with their receptors even within a complex crude extract

Exploration of the Antiplatelet Activity Profile of Betulinic Acid on Human Platelets

Betulinic acid, a natural pentacyclic triterpene acid, presents a diverse mode of biological actions including antiretroviral, antibacterial, antimalarial, and anti-inflammatory activities. The potency of betulinic acid as an inhibitor of human platelet activation was evaluated, and its antiplatelet profile against in vitro platelet aggregation, induced by several platelet agonists (adenosine diphosphate, thrombin receptor activator peptide-14, and arachidonic acid), was explored. Flow cytometric analysis was performed to examine the effect of betulinic acid on P-selectin membrane expression and PAC-1 binding to activated platelets. Betulinic acid potently inhibits platelet aggregation and also reduced PAC-1 binding and the membrane expression of P-selectin. Principal component analysis was used to screen, on the chemical property space, for potential common pharmacophores of betulinic acid with approved antithrombotic drugs. A common pharmacophore was defined between the NMR-derived structure of betulinic acid and prostacyclin agonists (PGI2), and the importance of its carboxylate group in its antiplatelet activity was determined. The present results indicate that betulinic acid has potential use as an antithrombotic compound and suggest that the mechanism underlying the antiplatelet effects of betulinic acid is similar to that of the PGI2 receptor agonists, a hypothesis that deserves further investigation.Esthir Gkani Foundation (Ioannina, Greece)Esthir Gkani Foundation (Ioannina, Greece)Regional Operational Programme of Thessaly-Mainland Greece-Epirus Research and Technological Development in the Region of Epirus Research Program "New Knowledge"Regional Operational Programme of ThessalyMainland GreeceEpirus Research and Technological Development in the Region of Epirus Research Program New Knowledg

Exhaled nitric oxide and exhaled breath condensate pH as predictors of sputum cell counts in optimally treated asthmatic smokers

Background and objective: Smoking is thought to modify the pattern of airway inflammation. Induced sputum provides useful information on cellular phenotype in inflammatory airways disorders; however, it is time-consuming and difficult to implement in everyday clinical practice. The aim of this study was to determine whether exhaled NO (FeNO) and exhaled breath condensate (EBC) pH differed in asthmatic smokers compared with asthmatic non-smokers and healthy subjects, and to evaluate the performance of FeNO and EBC pH for predicting the cellular phenotype of induced sputum. Methods: Asthmatic smokers (n = 40) and nonsmoking asthmatic patients (n = 43) were recruited for the study. Healthy smoking (n = 30) or non-smoking (n = 30) subjects served as controls. FeNO and EBC pH were measured and all subjects underwent sputum induction for assessment of cell counts. Results: EBC pH was significantly lower in asthmatic smokers compared with non-smokers (P < 0.01). FeNO levels were also significantly lower in asthmatic smokers compared with non-smokers (P < 0.001). EBC pH was inversely associated with sputum eosinophils in both asthmatic smokers and non-smokers (P < 0.001), whereas it was inversely associated with sputum neutrophils only in asthmatic smokers (P < 0.001). FeNO was positively associated with sputum eosinophils both in asthmatic smokers and non-smokers (P < 0.001) but was not associated with sputum neutrophils. In asthmatic smokers, FeNOwas a better predictor of sputum eosinophilia, whereas EBC pH was a better predictor of sputum neutrophilia. A combination of FeNO <= 14 ppb together with EBC pH > 7.20 predicted the paucigranulocytic induced sputum phenotype. Conclusions: EBC pH and FeNO levels were significantly lower in asthmatic smokers compared with nonsmokers. Combined specific cut-off levels for FeNO and EBC pH may predict the paucigranulocytic phenotype in asthmatic smokers

Ethyl Acetate Extract of Origanum vulgare L. ssp. hirtum Prevents Streptozotocin-Induced Diabetes in C57BL/6 Mice

Type 1 diabetes (T1D) is an autoimmune disease that develops as a consequence of pancreatic β-cell death induced by proinflammatory mediators. Because Origanum vulgare L. ssp. hirtum (Greek oregano) contains antiinflammatory molecules, we hypothesized that it might be beneficial for the treatment of T1D. An ethyl acetate extract of oregano (EAO) was prepared from the leaves by a polar extraction method. Phytochemical composition was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface (LC/DAD/ESI-MSn). In vitro immunomodulatory effect of EAO was estimated by measuring proliferation (MTT) or cytokine secretion (ELISA) from immune cells. Diabetes was induced by multiple low doses of streptozotocin (MLDS) in male C57BL/6 mice and EAO was administered intraperitoneally for 10 d. Determination of cellular composition (flow cytometry) and cytokine production (ELISA) was performed on 12th d after diabetes induction. EAO suppressed the function of both macrophages and lymphocytes in vitro. In vivo, EAO treatment significantly preserved pancreatic islets and reduced diabetes incidence in MLDS-challenged mice. Besides down-modulatory effect on macrophages, EAO reduced the number of total CD4+ and activated CD4+CD25+ T cells. Furthermore, EAO affected the number of T helper 1 (Th1) and T helper 17 (Th17) cells through downregulation of their key transcription factors T-bet and RORγT. Because EAO treatment protects mice from development of hyperglycemia by reducing proinflammatory macrophage/Th1/Th17 response, this plant extract could represent a basis for future diabetes therapy