A molecularly imprinted sensor for sensitive detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative stress biomarker

6th Graduate Student Symposium on Molecular Imprinting

A biomimetic sensor for monitoring oxidative stress biomarker in point-of-care

1st ASPIC International Congress

Avalia����o do stress oxidativo na disfun����o er��til

As sequelas fisiopatol��gicas do stress oxidativo s��o dif��ceis de quantificar. Apesar dos obst��culos, a relev��ncia m��dica do stress oxidativo tem vindo a ser cada vez mais reconhecida, sendo hoje em dia encarado como um componente chave de virtualmente todas as doen��as. A disfun����o er��til (DE) surge neste contexto como uma esp��cie de bar��metro da fun����o endotelial e do dano oxidativo. A quantifica����o de biomarcadores de stress oxidativo poder�� apresentar um enorme impacto na avalia����o de pacientes com DE. O r��cio glutationa reduzida/oxidada (GSH/GSSG) e a nitrotirosina (3-NT) t��m vindo a demonstrar relev��ncia cl��nica. A considera����o de polimorfismos gen��ticos constitui ainda uma abordagem promissora na avalia����o destas rela����es no futuro. Um m��todo altamente sens��vel de cromatografia l��quida de alta performance (HPLC) foi desenvolvido para a determina����o de 3-NT em plasma humano. As concentra����es de 3-NT medidos em indiv��duos com DE foram 6,6��2,1��M (m��dia��S.D., n = 46). A medi����o da concentra����o plasm��tica de 3-NT poder�� revelar-se ��til como marcador de dano oxidativo dependente do ��xido n��trico (NO). O n��vel de stress oxidativo pode tamb��m ser quantificado atrav��s da medi����o do decr��scimo do r��cio GSH/GSSG, que tem mostrado altera����es numa mir��ade de patologias, como a DE e a diabetes mellitus. O m��todo proposto para a quantifica����o do r��cio GSH/GSSG em HPLC apresenta a vantagem de avalia����o concomitante dos dois par��metros em apenas uma corrida. O valor do r��cio GSH/GSSG obtido a partir de sangue de indiv��duos com DE foi 11,9��9,8 (m��dia��S.D., n = 49). Os resultados estat��sticos revelaram diferen��as significativas (p<0,001) entre ambos a concentra����o plasm��tica de 3-NT e o r��cio GSH/GSSG de sangue de indiv��duos com DE e as respetivas medi����es em indiv��duos saud��veis. Observaram-se ainda diferen��as estatisticamente significativas (p���0,027) entre o r��cio GSH/GSSG do sangue de pacientes apenas com diagn��stico de DE e a medi����o respetiva em indiv��duos com DE e comorbilidades cardiovasculares. Estes resultados enfatizam o papel do dano oxidativo na biopatologia da DE, elucidado com o aux��lio destas duas metodologias, que poder��o ter um amplo campo de aplica����o no futuro, dado que se mostraram simples, n��o dispendiosas e r��pidas, podendo eventualmente adequar-se a estudos de rastreio em larga escala.

Fractional dynamics in DNA

This paper addresses the DNA code analysis in the perspective of dynamics and fractional calculus. Several mathematical tools are selected to establish a quantitative method without distorting the alphabet represented by the sequence of DNA bases. The association of Gray code, Fourier transform and fractional calculus leads to a categorical representation of species and chromosomes.

Mitochondria are the main source and one of the targets of Pb (lead)-induced oxidative stress in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model organism for studying lead (Pb) toxicity. Yeast cells of a laboratory S. cerevisiae strain (WT strain) were incubated with Pb concentrations up to 1,000 ��mol/l for 3 h. Cells exposed to Pb lost proliferation capacity without damage to the cell membrane, and they accumulated intracellular superoxide anion (O2 .���) and hydrogen peroxide (H2O2). The involvement of the mitochondrial electron transport chain (ETC) in the generation of reactive oxygen species (ROS) induced by Pb was evaluated. For this purpose, an isogenic derivative ��0 strain, lacking mitochondrial DNA, was used. The ��0 strain, without respiratory competence, displayed a lower intracellular ROS accumulation and a higher resistance to Pb compared to the WT strain. The kinetic study of ROS generation in yeast cells exposed to Pb showed that the production of O2 .��� precedes the accumulation of H2O2, which is compatible with the leakage of electrons from the mitochondrial ETC. Yeast cells exposed to Pb displayed mutations at the mitochondrial DNA level. This is most likely a consequence of oxidative stress. In conclusion, mitochondria are an important source of Pb-induced ROS and, simultaneously, one of the targets of its toxicity.

Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2���-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction

Background Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. Methods Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and ��-smooth muscle actin (��-SMA) and eNOS�����-SMA. Results There was a significant increase in urinary H2O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. Conclusions The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.

Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line

Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoaging. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1 ��g/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on 1O2 mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 ��g/mL. This effect was found to be related to a direct antioxidant effect (involving 1O2) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1 ��g/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin.