Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET

<p>Abstract</p> <p>Background</p> <p>Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer <it>in vivo</it>.</p> <p>Results</p> <p>In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.</p> <p>Conclusion</p> <p>The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future <it>in vivo </it>applications.</p

Sistema dinâmico combinado para balística/absorção de ondas de choque

Mestrado Engenharia MecânicaPropõe-se neste trabalho apresentar uma solução para acomodar a onda de pressão que resulta da rápida libertação de energia armazenada em gás (ar) comprimido, num canhão balístico. Esta solução é necessária para garantir a segurança do utilizador e das infra-estruturas circundantes. A solução adoptada consiste num acréscimo de volume ao canhão, conferido por um reservatório sob pressão que se designou por reservatório de expansão são. Foram abordados fundamentos termodinâmicos para obtenção do volume mínimo necessário ao reservatório de expansão. Foram implementadas metodologias adicionais (método dos elementos finitos) para análise do perfil de pressões ao longo da expansão (Abordagem Lagrangiana e Euleriana) e para análise e determinação de uma configuração ideal para a geometria do reservatório. Analisaram-se normas de projecto e seleccionou-se uma norma preferencial (EN 13445-3) para definição da geometria do reservatório. Para este procedimento foi ainda implementada uma rotina de cálculo. Com base na norma EN 13445-3, definiram-se os sistemas anexos necessários: tubuladuras, apoios, olhais. Foram ainda considerados elementos de medição e controlo, atendendo a especificações do fabricante, e definidas as ligações necessárias para acoplamento do sistema. Os resultados obtidos, não estando validados experimentalmente, servem de base à construção de um reservatório que permita, numa primeira abordagem, o funcionamento do sistema.It is proposed to provide a solution to reduce pressure-wave e_ects due to the rapid expansion of a compressed gas behind a piston, on a ballistic test cannon. This solution is needed to ensure the safety of user and infrastructure. The solution adopted consists in a volume increase to the cannon barrel, adding a pressure vessel, known as expansion tank. Thermodynamic fundamentals are discussed to obtain the minimum volume needed for the expansion tank. Additional methodologies were implemented (_nite element) to analyse the pressure pro_le during the expansion (Lagrangian and Eulerian approach) and for the analysis and determination of the optimum con_guration for the geometry of the tank. Design standards were analysed to de_ne the geometry of the expansion tank and a preferred one was selected: EN 13445-3. For this procedure, a calculation routine was also implemented. Based on EN 13445-3, attachment systems such as nozzles, sadle supports and lifting lugs were also designed. Measurement and control elements were also considered regarding the manufacturer's speci_cations and so were the necessary connections for coupling the system. The resulting geometry for the expansion tank is to be taken as basis for construction

Characterization of DNA/Lipid Complexes by Fluorescence Resonance Energy Transfer

AbstractFluorescence resonance energy transfer (FRET) is a potential method for the characterization of DNA-cationic lipid complexes (lipoplexes). In this work, we used FRET models assuming a multilamellar lipoplex arrangement. The application of these models allows the determination of the distance between the fluorescent intercalator on the DNA and a membrane dye on the lipid, and/or the evaluation of encapsulation efficiencies of this liposomal vehicle. The experiments were carried out in 1,2-dioleoyl-3-trimethylammonium-propane/pUC19 complexes with different charge ratios. We used 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and 2-(4,4-difluoro-5-octyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-PC) as membrane dyes, and ethidium bromide (EtBr) and BOBO-1 as DNA intercalators. In cationic complexes (charge ratios (+/−)≥2), we verified that BOBO-1 remains bound to DNA, and FRET occurs to the membrane dye. This was also confirmed by anisotropy and lifetime measurements. In complexes with all DNA bound to the lipid (charge ratio (+/−)=4), we determined 27Å as the distance between the donor and acceptor planes (half the repeat distance for a multilamellar arrangement). In complexes with DNA unbound to the lipids (charge ratio (+/−)=0.5 and 2), we calculated the encapsulation efficiencies. The presented FRET methodology is, to our knowledge, the first procedure allowing quantification of lipid-DNA contact

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-1

and in Tris-HCl, followed by incubation in PBS (C,D). Fitting curves using Eqs. 2–3 are also shown.<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-3

Tris-HCl, pH 7.4 (-○-); Lipoplexes prepared in PBS (-◆-); Lipoplexes prepared in Tris-HCl and incubated on PBS (-△-); Lipoplexes prepared in Tris-HCl, incubated on PBS, followed by incubation on FBS (-□-).<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-0

Onor probe (d) on DNA (BOBO-1).<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET-2

, incubated in PBS, followed by incubation in FBS for 30 min. (open symbols) and 2 h (filled symbols). Fitting curves using Eqs. 2–3 are also shown.<p><b>Copyright information:</b></p><p>Taken from "Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET"</p><p>http://www.biomedcentral.com/1472-6750/8/20</p><p>BMC Biotechnology 2008;8():20-20.</p><p>Published online 26 Feb 2008</p><p>PMCID:PMC2275333.</p><p></p

Características clínicas e prática de exercício físico de pacientes acometidos por COVID-19 atendidos em uma Clínica Escola

Introduction: With the Sars-CoV-2 pandemic, social isolation favored a sedentary lifestyle, causing individuals to abandon physical exercise, which favored clinical complications in those affected by the disease. Objective: To verify the clinical characteristics and physical exercise practice of patients who had COVID-19 treated at a Teaching Clinic. Method: Cross-sectional study carried out with 81 individuals treated in the Nutrition sector of a School Clinic that serves patients from the Unified Health System (SUS). Individuals of both sexes, over 19 years of age and who were diagnosed with COVID-19, were included. Data were collected from January to September 2023. All individuals were asked to fill out a questionnaire that assessed socioeconomic, demographic, clinical and lifestyle data. Data were analyzed using Stata® version 16.0. This research was approved by the Research Ethics Committee (CEP) of the Centro Universitário do Maranhão – UNICEUMA under substantiated opinion number no. 4,657,208. Results: Of the patients interviewed, 81.48% were female and 46.91% were aged 40 to 59 years. Only 6.17% of patients reported being asymptomatic, of which 60% exercised. It was observed that 49.38% had shortness of breath as a symptom and that 67.50% of these did not exercise (p=0.003). Conclusion: The practice of physical exercise and COVID-19 are related, those who did not have this habit were more susceptible to symptoms.Introdução: Com a pandemia do Sars-CoV-2, o isolamento social favoreceu o sedentarismo, fazendo com que os indivíduos deixassem de lado a prática de exercício físico, o que favoreceu as complicações clínicas daqueles que foram acometidos pela doença. Objetivo: Verificar as características clínicas e a prática de exercício físico de pacientes que tiveram COVID-19 atendidos em uma Clínica-Escola. Método: Estudo transversal realizado com 81 pacientes atendidos no setor de Nutrição de uma Clínica Escola que atende usuários do Sistema Único de Saúde (SUS). Foram incluídos pacientes de ambos os sexos, maiores que 19 anos e que apresentaram diagnóstico para COVID-19. Os dados foram coletados nos meses de janeiro a setembro de 2023. Todos os indivíduos foram submetidos ao preenchimento do questionário que avaliou os dados socioeconômicos, demográficos, clínicos e estilo de vida. Os dados foram analisados no programa Stata® versão 16.0. Resultados: Dos pacientes entrevistados, 81,48% eram do sexo feminino e 46,91% possuíam idade de 40 a 59 anos. Apenas 6,17% dos pacientes relataram ser assintomáticos, desses 60% faziam exercício físico. Observou-se que 49,38% apresentavam falta de ar como sintoma e que destes 67,50% não faziam exercício físico (p=0,003); Conclusão: A  prática de exercício físico e o acometimento pela COVID-19 possui relação, aqueles que não possuíam esse hábito estavam mais suscetíveis ao quadro de sintomais