Dominik Lenz

Intracellular FRET analysis of lipid/DNA complexes using flow cytometry and fluorescence imaging techniques

Gene therapy is a promising therapeutic concept for a large number of incurable diseases. Lipid/DNA complexes (lipoplexes) are used to deliver genes into cells. However, while large efforts have been made to investigate the fate of lipoplexes once inside the cell, the rate of intracellular dissociation is still largely unknown. Analysis of the dissociation rates of DNA from lipid/DNA complexes is crucial for the evaluation of a gene delivery systems efficiency. This study introduces a new fluorescence resonance energy transfer (FRET) approach for the intracellular dissociation analysis of lipid/DNA complexes. Here, the labeling of both complex components, DNA as well as lipid, reveals whether DNA is still associated with the lipid or has dissociated. In this study the kinetic properties of complex dissociation were consistently measured with flow cytometry and fluorescence microscopy, and indicated that most complexes were dissociated after 24h in A-10 cells.

Testosterone induces apoptosis in cardiomyocytes by increasing proapoptotic signaling involving tumor necrosis factor-α and renin angiotensin system

Anabolic androgenic steroids lead to cardiac complications and have been shown to exhibit proapoptotic effects in cardiac cells; however, the mechanism involved in those effects is unclear. The aim of this study was to assess whether apoptosis and the activation of caspase-3 (Casp-3) induced by testosterone in high concentrations involves increments in tumor necrosis factor-α (TNF-α) concentrations and angiotensin-converting enzyme (ACE) activity in cardiomyocytes (H9c2) cell cultures. Cardiomyocytes were treated with testosterone (5 × 10−6 mol/L), doxorubicin (9.2 × 10−6 mol/L), testosterone + etanercept (Eta; 6.67 × 10−5 mol/L), testosterone + losartan (Los; 10−7 mol/L), and testosterone + AC-DEVD-CHO (10−5 mol/L; Casp-3 inhibitor). Apoptosis was determined by flow cytometry and by the proteolytic activity of Casp-3. We demonstrated that incubation of H9c2 cells for 48 h with testosterone causes the apoptotic death of 60–70% of the cells and co-treatments with Eta, Los, or AC-DEVD-CHO reduced this effect. Testosterone also induces apoptosis (concentration dependent) and increases the proteolytic activity of Casp-3, which were reduced by co-treatments. TNF-α and ACE activities were elevated by testosterone treatment, while co-treatment with Los and Eta reduced these effects. We concluded that an interaction between testosterone, angiotensin II, and TNF-α induced apoptosis and Casp-3 activity in cultured cardiomyocytes, which contributed to the reduced viability of these cells induced by testosterone in toxic concentrations.

Essential oil from the resin of Protium heptaphyllum: Chemical composition, cytotoxicity, antimicrobial activity, and antimutagenicity

Background: Protium heptaphyllum (Aubl.) March is popularly used as an analgesic and anti-inflammatory agent. Objective: This study aimed to evaluate the chemical composition of P. heptaphyllum essential oil, its cytotoxicity in a breast cancer cell line (MCF-7), antimicrobial activity, and its antimutagenicity in vivo. Materials and Methods: The chemical composition of the essential oil collected in three 3 years was determined by gas chromatography-mass spectrometry. The cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Annexin V conjugated with fluorescein isothiocyanate, caspase-3, and tumor necrosis factor-alpha (TNF-α) assays were performed to evaluate apoptosis and inflammatory events. The antimutagenic activity at doses of 25, 50, and 100 mg/kg was determined using a micronucleus test in murine bone marrow. Results: The essential oil showed a predominance of monoterpene compounds, being the terpinolene, p-cymene-8-ol, and p-cymene, present in the essential oil extracted in the 3 years. The essential oil showed a protection against cyclophosphamide-induced genotoxicity, and the cytotoxicity index polychromatic erythrocytes/normochromatic erythrocytes ratio in animals treated with oil at all doses (1.34 ± 0.33; 1.15 ± 0.1; 1.11 ± 0.13) did not differ from the negative control animal (1.31 ± 0.33), but from the cyclophosphamide group (0.61 ± 0.12). Cytotoxicity, at a concentration of 40.0 μg/mL, and antimicrobial activity were not observed for the essential oil (minimum inhibitory concentration ≥0.5 mg/mL). The essential oil did not change the levels of caspase-3 in the TNF-α level. Conclusion: The essential oil showed antimutagenic activity due to its chemical composition.

Hepatitis C in prisoners and non-prisoners in Colatina, Espírito Santo, Brazil

The aim of the present work was to compare hepatitis C prevalence, genotypes, and risk factors between prisoners and non-prisoners in the city of Colatina, Espirito Santo, Brazil. This cross-sectional study involved approximately 1,600 residents and 730 prisoners, all of whom were living in Colatina. The percentage of individuals who tested positive for anti-HCV was 0.1% (2/1,600) in the non-prisoner group and 1.0% (7/730) in the prisoner group, confirming a higher risk of hepatitis C in the latter group. The percentage of subjects who progressed to HCV-RNA negative was 11.1% (1/9), confirming the high probability of evolution to chronicity. Genotype 1 was the most predominant genotype found. Factors associated with increased risk of hepatitis C were being male, being institutionalized, having an income of less than three minimum wages, having low educational attainment, and using injected drugs. Alcohol use, pain in the liver, migraine, and reported history of hepatitis were markedly associated with hepatitis C. The prison population tested positive for anti-HCV at a higher rate than the non-prison population.

Scientific production in pharmaceutical care: comparison between Brazil, USA and Spain

The aim of this study was to estimate the evolution of the field of Pharmaceutical Care (PC) by measuring the quality and quantity of the scientific production on the topic of PC in Brazil compared to two pioneering countries in the field, the United States of America (USA) and Spain. The databases Web of Science, Scopus, Medline, Lilacs and SciELO were used as sources for the literature search. Pharmaceutical Care, or the appropriate translations, was used as the search term for the literature search, which was limited to articles published between 1990 and 2009. A score of quality (SQ) was calculated using variables such as impact factor and the frequency of the citations. We included 3265 articles published in 544 journals. We found that there was a steady increase in scientific production since 1990 and that the USA had a higher quality of scientific production than Spain, whereas the Spain produced the highest quantity of articles. In comparison, the Brazilian production of scientific publications on PC is low in terms of both quality and quantity but has increased steadily since 2002. Nevertheless, Brazil has not yet reached the level of the USA or Spain. In conclusion, Brazils scientific production has evolved over the second decade studied in this work, with particularly high levels of production in the last five years. However, an increase in the quantity and quality of the publications should be encouraged.

Evaluation of the use of topic nasal decongestants in university students from health sciences courses

Nasal decongestants in Brazil are available without prescription. They are one of the most requested medications in Brazil. The use of nasal decongestants, however, may lead to serious consequences. For this study, university students majoring in the health field were evaluated in terms of their profile regarding the use of nasal decongestants. Additionally, health sciences seniors were evaluated regarding their level of knowledge concerning the correct and proper use of nasal decongestants. Among the 71.8% of respondents who had already used topic nasal decongestants, 64.3% had used this medication for at least 15 days and 64.6% had started using it due to nasal obstruction. The gathered data indicate that the pharmacist was the main health professional reported as having provided patients with guidance on how to use this medication. A five-point Likert Type intensity scale (3.6 ± 0.08) shows that a regular level of knowledge has been observed among the students. Most of the students polled were concluded to incorrectly use topic nasal decongestants. Furthermore, the results of this study indicate that the future health professionals are not well prepared to promote health education for patients concerning the rational use of nasal decongestants.

In vivo hypotensive effect and in vitro inhibitory activity of some Cyperaceae species

In 1820, French naturalist August Saint Hillaire, during a visit in Espirito Santo (ES), a state in southeastern Brazil, reported a popular use of Cyperaceae species as antidote to snake bites. The plant may even have a hypotensive effect, though it was never properly researched. The in vitro inhibitory of the angiotensin converting enzyme (ACE) activity of eigth ethanolic extracts of Cyperaceae was evaluated by colorimetric assay. Total phenolic and flavonoids were determined using colorimetric assay. The hypotensive effect of the active specie (Rhychonospora exaltata, ERE) and the in vivo ACE assay was measured in vivo using male Wistar Kyoto (ERE, 0.01-100mg/kg), with acetylcholine (ACh) as positive control (5 µg/kg, i.v.). The evaluation of ACE in vivo inhibitory effect was performed comparing the mean arterial pressure before and after ERE (10 mg/kg) in animals which received injection of angiotensin I (ANG I; 0,03, 03 and 300 µg/kg, i.v.). Captopril (30 mg/kg) was used as positive control. Bulbostylis capillaris (86.89 ± 15.20%) and ERE (74.89 ± 11.95%, ERE) were considered active in the in vitro ACE inhibition assay, at 100 µg/mL concentration. ACh lead to a hypotensive effect before and after EREs curve (-40±5% and -41±3%). ERE showed a dose-dependent hypotensive effect and a in vivo ACE inhibitory effect. Cyperaceae species showed an inhibitory activity of ACE, in vitro, as well as high content of total phenolic and flavonoids. ERE exhibited an inhibitory effect on both in vitro and in vivo ACE. The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.