Search

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

Meningiomas are intracranial tumors originating from arachnoidal cells. Multiple intracranial meningiomas are rare and occur when two or more lesions occur either simultaneously or sequentially in different locations. We described a case of multiple meningothelial meningioma post forensic autopsy.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Universidade Federal de Uberlândia Ressaltar os processos criativos nas aulas de arte como acontecimentos importantes para a transformação e construção de pensamentos e emoções dos alunos, observando o papel e a formação do professor de arte e sua inserção nesse processo, são objetivos desta pesquisa. Para alcançar os objetivos mencionados, observamos na formação do professor de arte bases importantes a partir das quais surgiram metodologias e dinâmicas de aula que intervieram na realidade de cada escola usada como ambiente deste estudo que, por sua vez, utilizou-se do corpo em ação (performativa) como uma das premissas para a observação do que denominamos “fluxo criativo”. Nesse sentido, buscaram-se experiências de processos criativos usando a pesquisa-ação com alunos do ensino regular, níveis fundamental e médio, da educação pública (prefeitura e estado). Adotando como procedimento técnico a observação participante, apresentaram-se ainda concepções sobre corpo, arte e experiência que permitiram perceber o corpo enquanto instrumento da “experiência em arte” e conectá-las aos sentidos da percepção como forma de lidar com sensações estéticas. Ao trabalhar com alunos em níveis diferentes de aprendizagem na sala de aula, abordaram-se as inteligências e como alcançá-las através da percepção. Por meio de experimentos práticos com o ensino na área de arte, pretendeu-se valorizar aulas que respeitassem as inteligências dos alunos buscando alcançá-los através de um trabalho que proporcionasse o autoconhecimento como produto em confluência com a área da arteterapia. Para tanto, experiências foram propostas com a inteligência emocional, promovendo respeito às diferenças, ressaltando a proposta do professor como interlocutor do processo criativo em arte em que “fluxos criativos” afetaram os sujeitos envolvidos a ponto de transformá-los através da apreciação e fruição na prática performativa. Hacer hincapié en los procesos creativos en las clases de arte como eventos importantes para la transformación y la construcción de los pensamientos y emociones de los estudiantes, señalando la función y la formación de los profesores de arte y su lugar en el proceso, son objetivos de esta investigación. Para lograr estos objetivos, se observa la formación del profesor de arte principales bases de la que surgió metodologías y dinámica de clases que tomaron parte en la realidad de cada escuela se utiliza como medio de este estudio que, a su vez, se utiliza el cuerpo en acción (performativa) como una de las premisas para la observación de lo que llamamos "flujo creativo". En este sentido, trataron de procesos creativos experimentos utilizando la investigación-acción con los estudiantes regulares, nivel de primaria y secundaria, la educación pública (ciudad y estado). La adopción de la observación participante como procedimiento técnico, todavía tenía ideas sobre el arte corporal y la experiencia que les permite percibir el cuerpo como un instrumento de la "experiencia en el arte" y conectarlos a los sentidos de percepción como una forma de hacer frente a las sensaciones estéticas. Cuando se trabaja con los estudiantes en los diferentes niveles de aprendizaje en el aula, dirigida a las inteligencias y cómo llegar a ellos a través de la percepción. A través de experimentos prácticos con la enseñanza en el área de arte, tenía la intención de realzar las lecciones de respetar la inteligencia de los estudiantes que buscan llegar a ellos a través de un trabajo que proporcionaría auto-conocimiento como confluencia producto con el campo de la terapia del arte. Por lo tanto, se propusieron experimentos con la inteligencia emocional, la promoción del respeto a las diferencias, tomando nota de la propuesta del maestro como un socio del proceso creativo en el arte que "flujo creativo" afectó a los involucrados como para transformarlos través de la apreciación y el disfrute en la práctica performativa. Dissertação (Mestrado)

The study reports the evolution of the demyelinization process based on cholesterol ([CHOL]) levels quantified in median nerve samples and collected at different times-from death from both right and left wrists. The statistical data show that the phenomenon evolves differently in the right and left nerves. Such a difference can reasonably be attributed to a different multicenter evolution of the demyelinization. For data analysis, the enrolled subjects were grouped by similar postmortem intervals (PMIs), considering 3 intervals: PMI < 48 hours, 48 hours < PMI < 78 hours, and PMI > 78 hours. Data obtained from tissue dissected within 48 hours of death allowed for a PMI estimation according to the following equations: PMI = 0.000 + 0.7623 [CHOL]right (R = 0.581) for the right wrist and PMI = 0.000 + 0.8911 [CHOL]left (R = 0.794) for the left wrist.At present, this correlation cannot be considered to be definitive because of the limitation of the small size of the samples analyzed, because the differences in the sampling time and the interindividual and intraindividual variation may influence the demyelinization process.

OBJECTIVES: The study aimed to give a first data set of bisphenol A (BPA) levels in the peritoneal fluid of patients suffering from endometriosis and to investigate the relationship between BPA exposure and endometriosis. METHODS: A questionnaire investigating the occupational context, life environment, and habits was administered to 68 patients suffering from endometriosis and 60 endometriosis-free subjects (control group). Urine and peritoneal fluids samples were collected and analysed by GC/MSMS for BPA dosage. RESULTS: Some of the investigated environmental/lifestyle risk factors (closeness to industries/activities at risk) were associated with an increase in endometriosis; smoking resulted as protective factor; others (use of food plastic boxes) did not seem to influence the onset of pathology. The association between the occupational exposure summarising all examined risk factors (working activity, personal protective equipment, seniority) and endometriosis was statistically significant (χ 2 = 5.252, p = 0.02). Contrasting results were obtained when specific activities were examined. Detectable urinary BPA levels were found in all analysed samples (patients: 1.17-12.68 pg/µl; mean ± SD, 5.31 ± 3.36 pg/µl; control group: 1.28-2.35 pg/µl; mean ± SD, 1.64 ± 0.49 pg/µl; median; 1.46 pg/µl), with a statistically significant difference between patients and controls, showing an association between BPA exposure and endometriosis. Only a few subjects from the control group supplied peritoneal fluid; hence, no comparison test with patients (range 0.39-1.46 pg/µl; mean ± SD, 0.67 ± 0.30 pg/µl; median, 0.58 pg/µl) was carried out. CONCLUSIONS: Results highlight the potential association between BPA exposure and endometriosis, as well as the current lack of knowledge regarding occupational exposure to BPA and the need of epidemiological studies focused on single activities/occupations, such as housewives, cleaners, students. Objectives: The study aimed to give a first data set of bisphenol A (BPA) levels in the peritoneal fluid of patients suffering from endometriosis and to investigate the relationship between BPA exposure and endometriosis. Methods: A questionnaire investigating the occupational context, life environment, and habits was administered to 68 patients suffering from endometriosis and 60 endometriosis-free subjects (control group). Urine and peritoneal fluids samples were collected and analysed by GC/MSMS for BPA dosage. Results: Some of the investigated environmental/lifestyle risk factors (closeness to industries/activities at risk) were associated with an increase in endometriosis; smoking resulted as protective factor; others (use of food plastic boxes) did not seem to influence the onset of pathology. The association between the occupational exposure summarising all examined risk factors (working activity, personal protective equipment, seniority) and endometriosis was statistically significant (χ2 = 5.252, p = 0.02). Contrasting results were obtained when specific activities were examined. Detectable urinary BPA levels were found in all analysed samples (patients: 1.17–12.68 pg/µl; mean ± SD, 5.31 ± 3.36 pg/µl; control group: 1.28–2.35 pg/µl; mean ± SD, 1.64 ± 0.49 pg/µl; median; 1.46 pg/µl), with a statistically significant difference between patients and controls, showing an association between BPA exposure and endometriosis. Only a few subjects from the control group supplied peritoneal fluid; hence, no comparison test with patients (range 0.39–1.46 pg/µl; mean ± SD, 0.67 ± 0.30 pg/µl; median, 0.58 pg/µl) was carried out. Conclusions: Results highlight the potential association between BPA exposure and endometriosis, as well as the current lack of knowledge regarding occupational exposure to BPA and the need of epidemiological studies focused on single activities/occupations, such as housewives, cleaners, students. © 2016, Springer-Verlag Berlin Heidelberg.

The presence of macrophages and their activation on the pulmonary tissues of 21 subjects deceased after CO intoxication has been studied. A notable number of activated macrophages, especially in the interstitial level, have been evidenced, and such phenomenon supports the hypothesis of a possible association between CO intoxication and pulmonary macrophages activity. The highlighted association could be mediated by changes of the surfactant, by impairing of mitochondrial respiration and by release of pro-inflammatory cytokines.

According to the Italian laws, some categories of workers entrusted with duties possibly constituting a threat to security, physical safety, and health of third parties have to be screened to exclude the use/abuse of the following drugs of abuse: opiates, cocaine, cannabinoids, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methadone, and buprenorphine. Toxicological tests can be performed with urinary on-site rapid screening devices, provided that sensitivities up to specified cutoffs are ensured. The present study reports performances, in terms of sensitivity, specificity, and accuracy, of an automatic on-site test and of an FPIA-based device, using gas chromatography/mass spectrometry (GC/MS) as a reference methodology. Three levels of concentration were tested, corresponding to the cutoff and to 2 and 3 times the limits, respectively. In terms of sensitivities, neither the on-site nor the benchtop instrumentations gave positive results, since values of zero percentage were obtained for concentrations up to 2-fold the limits. Even if good results were obtained in terms of specificity and accuracy by both devices, none of them seem to be adequate for the current application to the toxicological screening at workplaces. In fact, a rapid screening device can be used for drug tests provided that it ensures sensitivity at the prescribed cutoffs. Data showed that such is completely rejected and a more sensitive instrumentation should be preferred.

The measurement of blood concentration of immunosuppressive drugs is strongly recommended because of the narrow therapeutic range. An important aspect in the therapeutic monitoring of a drug is its possible degradation. This paper is aimed at investigating the stability of two widely-used immunosuppressants, sirolimus and everolimus. Short (storage at 30 degrees C for 3 or 7 days) and long term (storage at -20 degrees C for 0-90 days with a single freeze-thaw cycle) stability of sirolimus and everolimus in whole blood samples from kidney transplant patients were examined by using MEIA and FPIA. Sirolimus and everolimus samples stored at 30 degrees C in light for up to a week showed a decrement in concentration of 5.2 percent and 6.1 percent, respectively. Our findings on long term stability for both sirolimus and everolimus highlight the possibility of storing samples at -20 degrees C for up to 90 days, without the need to use lower storage temperatures. The results have important implications for patients living far from laboratories where drug concentration is measured or when the storing of blood samples is needed for pharmacokinetic studies.

Cholesterol levels in the median nerve were studied at various post-mortem intervals (PMIs). Single median nerve samples were collected from the wrists of 36 subjects during forensic autopsies of subjects with known circumstances and times of death. Although the absolute values varied, increments in cholesterol concentration were recorded. Subsequently, 16 subjects who did not suffer of any neurological and/or metabolic diseases with known times and circumstances of death were enrolled. For each enrolled subject, two samples were collected from the wrist at an interval of approximately two hours (t1 and t2). The obtained results revealed a gradual increase in cholesterol level with increasing time since death. The cholesterol concentration data obtained for each subject at t1 and t2 were correlated with the time since death, a linear interpolation was applied, and the PMI was back-calculated. Similar trends were obtained for the samples collected at similar PMIs; thus, three groups were considered: PMI78h. Good correlation coefficients were obtained, especially for the first group (R(2)=0.9362) for which the PMI could be calculated with an error that ranged from -4 to 5.9h. Although it requires further confirmation via analyses of larger numbers of samples, the method proposed here can currently be applied to PMI determinations.

Rapamycin is a potent immunosuppressive drug capable of significantly reducing acute graft rejection in kidney, liver and heart transplant patients. Its immunosuppressive activity and adverse effects have been related to rapamycin concentration, and therapeutic drug monitoring of the drug is deemed appropriate. This work was aimed at developing a new quantification method based on the isolation of the [M+Na]+ ion as precursor and its further fragmentation through an ion trap mass spectrometer equipped with an electrospray ionization source. A limit of detection (LOD) of 0.7 ng/mL was obtained, while the lower limit of quantification (LLOQ) was 2.4 ng/mL. The accuracy and reproducibility of the responses were evaluated and compared with results obtained when the [M+NH4]+ ion was chosen as the precursor in a triple quadrupole mass spectrometer. In this case the LOD was 0.5 ng/mL and the LLOQ 1.7 ng/mL. Data showed that it would be possible to use the quantification of the sodiated species for the routine determination of rapamycin, as an alternative to the commonly adopted method based on the ammoniated complex. Copyright © 2005 John Wiley & Sons, Ltd.

In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine) m (pyrimidine) m -3′. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3′-3′ phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine)m(pyrimidine)m-3′. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine–pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson–Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3′–3′ phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P=phosphate and X=1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.

The thermodynamic characterization of the stability of two DNA triple helices of alternate sequence was obtained by differential scanning calorimetry (DSC). The conformational properties of these triple helices were investigated by circular dichroism (CD) and molecular mechanics. The triplexes under investigation form by way of major groove Hoogsteen association of a Watson–Crick 16-mer duplex with an all pyrimidine 16-mer or 15-mer third strand. The target duplex is composed of two adjacent oligopurine–oligopyrimidine domains where oligopurine sequences alternate on the two duplex strands. Both the third strands contain a 3′,3′-phosphodiester junction, which introduces the appropriate inversion of polarity and let the switch from one oligopurine strand of the duplex to the other. The two-third strands differ for the lack of a cytidine monophosphate in the junction region. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex with increasing temperature. Transition enthalpies, entropies and free energies were derived from DSC measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region. The thermodynamic data were discussed in relation to structural models.

Mammalian Target Of Rapamycin (mTOR) inhibitors represent a new class of immunosuppressant drugs extensively used for the prevention and the treatment of graft rejection in organ transplant recipients. Their current use is due to referred low nephrotoxic effects, particularly important in kidney transplanted and/or patients with renal failure. The most representative drugs of such class are Sirolimus (Siro) and Everolimus (Rad). Both drugs show a narrow therapeutic window, therefore, monitoring of whole-blood drug levels is recommended in order to optimize the therapy. Among the available assays, Liquid Chromatography coupled with UltraViolet or Electrospray Tandem Mass Spectrometry methods (LC/UV or LC/ESI-MSMS) are the most accurate and specific ones. A reliable alternative is represented by immunoassays, which offer the opportunity to minimize sample pre-treatment, thus reducing the time between drawing blood sample and measuring the drug concentration, an important aspect in high-throughput analyses. Despite this, a limitation in the use of immunoassays for therapeutic drug monitoring is the lower specifity compared with the chromatographic methods when analysing structurally-related drugs. New insights to optimize mTOR inhibitors regimens seem to be offered by the evaluation of CYP450 3A activity by using the probe drug approach. To such purpose, there are a number of major probe drugs used for in vivo studies including: midazolam, cortisol, lidocaine, nifedipine, dextromethorphan, erythromycin, dapsone and alfentanil. The aim of the present paper is to report the most recent knowledge concerning this issue, supplying a critical and comprehensive review for whom are involved both in the clinical and analytical areas.

Doxorubicin (DOXO) is an anthracycline antibiotic which is used in the treatment of human malignancies such as leukemia, lymphoma and a number of solid tumors, particularly breast cancer. Anthracyclines have been reported to contaminate chemotherapy workstation surfaces as well as other workplaces surfaces. The occupational exposure to these drugs could occur in hospitals, for nurses involved in anthracyclines preparation and administration, in chemical industries during the commercial formulate syntheses, and in analytical laboratories. Numerous studies investigated cutaneous effects related to DOXO administration, on the contrary few literature data are available about effects on the skin due to the direct contact with the drug. The present study investigated the DOXO permeability of three commercially available gloves’ types used to protect skin in occupational contexts, as well as the effects of DOXO on human keratinocyte cell line (HaCaT). The results suggest that the DOXO permeability of gloves depends not only on glove material but also on DOXO solutions’ pH, in fact nitrile gloves can be penetrated by acid solutions, while neither natural rubbers nor nitrile gloves are permeable to neutral solutions. Moreover, DOXO solutions, even at low concentration, cause apoptosis in epithelial cells, through activation of intrinsic pathway p53- independent.

Bacterial infection is a frequent event in renal transplant recipients and often requires the use of antimicrobial agents. In this paper it is reported an evidence of pharmacokinetic interaction between clarithromycin and sirolimus in a kidney transplanted woman, suffering from pulmonary infection sustained by a bacterial pathogen, in particular Hemophilus Influenzae. In the present case report, the concomitant administration of clarithromycin and sirolimus determined impressive increase of sirolimus trough blood concentrations from 6.2 up to 54 ng/mL and this increase was associated with an acute impairment of renal function, almost completely reversed upon both drugs discontinuation. This drug-drug interaction is due to a likely inhibition of activity of both cytochrome P450 3A4 and P-glycoprotein. Although this interaction could be predicted, it represents the first reported clinical evidence.

Hospital personnel involved in antineoplastic drug preparation and administration to patients are exposed to large amounts of these drugs. Labour legislation indicates the necessity of planning monitoring strategies aimed at prevention and/or reduction of drug exposure. Monitoring strategies consist of quantitative determinations of indicators, present in environmental and biological matrices. Among the antineoplastic drugs widely used, cyclophosphamide (CP) has been identified as a suitable indicator of potential exposure to mixtures of antineoplastic drugs. Many literature methods for quantitative analysis of CP involve either liquid (LC) or gas chromatography (GC) with mass spectrometry (MS), both of which require use of a suitable internal standard. The present work focuses on the synthesis of mono- and diiodocyclophosphamide (CPI and CPI2) to be used as internal standard. These compounds were analyzed by GC/EI-MS/MS and LC/ESI-MSn using ion trap mass spectrometry. The product ion mass spectra are interpreted in terms of proposed structures of fragment ions. Iodine-chlorine substitution resulted in a weakening of the carbon–halogen bond with a noteworthy influence on the ion fragmentation processes. The proposed suitability of CPI and CPI2 as internal standards was based on similarities to CP as regards ionization and fragmentation processes. The results obtained suggest that CPI could be used as internal standard for CP quantification by LC/ESI-MS/MS, and CPI2 for GC/EI-MS/MS analyses. Copyright © 2005 John Wiley & Sons, Ltd.

The possible cross-reactivity of immunoassays with structurally-related drugs was investigated. Innofluor® Certican® (FPIA) calibrators were measured by using IMx® Sirolimus assay (MEIA) and MEIA Sirolimus calibrators were analysed by using FPIA Certican® assay. Drug concentrations were measured in 95 and 100 samples from renal transplanted patients (RTP) on sirolimus or everolimus treatment by using immunoassays and LC/ESI-MSMS. A high cross-reactivity was found both for MEIA and FPIA. High correlation degrees, confirmed by the Bland-Altman and the Eksborg tests, were found between drug concentrations measured in real samples by both immunoassays (r=0.909 and r=0.970, respectively). LC/ESI-MSMS analysis of samples containing sirolimus showed no positivity for everolimus. Similarly, samples from patients on treatment with everolimus resulted negative as far as regards sirolimus. MEIA and FPIA could be considered mutually reliable and accurate alternatives for the specific-drug immunoassay. It should be noticed that in patients switching from one drug to the other unreal overestimation of the blood levels of the current administered immunosuppressant can occur.