Third-generation electrochemical biosensor based on nitric oxide reductase immobilized in a multiwalled carbon nanotubes/1-n-butyl-3-methylimidazolium tetrafluoroborate nanocomposite for nitric oxide detection

Nitric oxide (NO) has a crucial role in signaling and cellular physiology in humans. Herein, a novel third-generation biosensor based on the Marinobacter hydrocarbonoclasticus metalloenzyme (nitric oxide reductase (NOR)), responsible for the NO reduction in the denitrifying processes, was developed through the direct adsorption of a new nanocomposite (multiwalled carbon nanotubes (MWCNTs)/1-n-butyl-3-methylimidazolium tetrafluoroborate (BMIMBF4)/NOR) onto a pyrolytic graphite electrode (PGE) surface. The NOR direct electron transfer behavior (formal potential of -0.255 ± 0.003 V vs. Ag/AgCl) and electrocatalysis towards NO reduction (−0.68 ± 0.03 V vs. Ag/AgCl) of the PGE/[MWCNTs/BMIMBF4/NOR] biosensor were investigated in phosphate buffer at pH 6.0. Large enzyme loading (2.04 × 10−10 mol/cm2), acceptable electron transfer rate between NOR and the PGE surface (ks = 0.35 s-1), and high affinity for NO (Km = 2.17 μmol L-1) were observed with this biosensor composition. A linear response to NO concentration (0.23–4.76 μmol L-1) was perceived with high sensitivity (0.429 μA/μmolL-1), a detection limit of 0.07 μmol L-1, appropriate repeatability (9.1% relative standard deviations (RSD)), reproducibility (6.0–11% RSD) and 80–102% recoveries. The biosensor was stable during 1 month retaining 79–116% of its initial response. These data confirmed that NOR incorporated in the MWCNTs/BMIMBF4 nanocomposite can efficiently maintain its bioactivity paving a new and effective way for NO biosensing.FG thanks FCT/MCTES for the fellowship grant SFRH/BD/52502/2014, which is financed by national funds and co-financed by FSE. LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract. This work was supported by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES) and also by the Associate Laboratory Research Unit for Green Chemistry - Technologies and Processes Clean – LAQV, financed by national funds from FCT/MEC (UID/QUI/50006/2019) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged. The financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e Tecnologia-FCT) through project PTDC/ASP-PES/29547/2017, by FCT/MEC with national funds and co-funded by FEDER, is also acknowledged.info:eu-repo/semantics/publishedVersio

Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, -0.108 ± 0.008, -0.328 ± 0.001 and -0.635 ± 0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ± 1.84 × 10-9 A/μM and - 2.71 × 10-8 ± 1.44 × 10-9 A/μM for NO and O2, respectively), limit of detection (0.5 μM for NO and 1.0 μM for O2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors.FG and LBM thank FCT/MCTES for the fellowship grants SFRH/BD/52502/2014 and SFRH/BPD/111404/2015, respectively, which are financed by national funds and co-financed by FSE. CMC acknowledges FCT-MCTES funding through project PTDC/BBB-BQB/0129/2014 (FCT/MCTES). This work was supported by the REQUIMTE, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013 and UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265 and POCI-01-0145-FEDER-007728), and also by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES). Funding through REQUIMTE project entitled “NOR-based biosensor for nitric oxide detection in biological and environmental samples” is also acknowledged.info:eu-repo/semantics/publishedVersio

Chemical composition, antinociceptive and anti-inflammatory effects in rodents of the essential oil of Peperomia serpens (Sw.) Loud

AbstractEthnopharmacological relevancePeperomia serpens (Piperaceae), popularly known as “carrapatinho”, is an epiphyte herbaceous liana grown wild on different host trees in the Amazon rainforest. Its leaves are largely used in Brazilian folk medicine to treat inflammation, pain and asthma.Aim of the studyThis study investigated the effects of essential oil of Peperomia serpens (EOPs) in standard rodent models of pain and inflammation.Materials and methodsThe antinociceptive activity was evaluated using chemical (acetic acid and formalin) and thermal (hot plate) models of nociception in mice whereas the anti-inflammatory activity was evaluated by carrageenan- and dextran-induced paw edema tests in rats croton oil-induced ear edema, as well as cell migration, rolling and adhesion induced by carrageenan in mice. Additionally, phytochemical analysis of the EOPs has been also performed.ResultsChemical composition of the EOPs was analyzed by gas chromatography and mass spectrometry (GC/MS). Twenty-four compounds, representing 89.6% of total oil, were identified. (E)-Nerolidol (38.0%), ledol (27.1%), α-humulene (11.5%), (E)-caryophyllene (4.0%) and α-eudesmol (2.7%) were found to be the major constituents of the oil. Oral pretreatment with EOPs (62.5–500mg/kg) significantly reduced the writhing number evoked by acetic acid injection, with an ED50 value of 188.8mg/kg that was used thereafter in all tests. EOPs had no significant effect on hot plate test but reduced the licking time in both phases of the formalin test, an effect that was not significantly altered by naloxone (0.4mg/kg, s.c.). EOPs inhibited the edema formation induced by carrageenan and dextran in rats. In mice, EOPs inhibited the edema formation by croton oil as well as the leukocyte and neutrophil migration, the rolling and the adhesion of leukocytes.ConclusionsThese data show for the first time that EOPs has a significant and peripheral antinociceptive effect that seems unrelated to interaction with the opioid system. EOPs also displays a significant anti-inflammatory effect in acute inflammation models. This effect seems to be related to components which inhibit the production of several inflammatory mediators. These results support the widespread use of Peperomia serpens in popular medicine to treat inflammation and pain

Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocomposite

An enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10-4 cm.s-1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 μM), wide linear range (0.44-9.09 μM), low detection limit (0.13 μM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5 weeks). Selectivity tests towards L-arginine, ascorbic acid, sodium nitrate, sodium nitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ± 9.3%-98.4 ± 5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.FG thanks to Fundacão para a Ciência e a Tecnologia, MCTES (FCT/MCTES) for the fellowship grant SFRH/BD/52502/2014, which is financed by national funds and co-financed by FSE. LBM thanks to FCT/MCTES for the CEEC-Individual 2017 Program Contract. This work was supported by the PTDC/BB-BQB/0129/2014 project (FCT/MCTES), by FCT/MEC with national funds and co-funded by FEDER, and also by the Associate Laboratory Research Unit for Green Chemistry - Technologies and Processes Clean – LAQV, financed by national funds from FCT/MCTES (UID/QUI/50006/2019). The financial support from the European Union (FEDER funds through COMPETE) and National Funds (Fundação para a Ciência e Tecnologia-FCT) through project PTDC/ASP-PES/29547/2017, by FCT/MEC with national funds and co-funded by FEDER, is also acknowledged. J.A. Loureiro post-doc grant was supported by NORTE-01-0145-FEDER-000005 – LEPABE-2 ECO-INNOVATION, from North Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).info:eu-repo/semantics/publishedVersio

Intravitreal bevacizumab (Avastin®) for diabetic retinopathy at 24-months: The 2008 Juan Verdaguer-planas lecture

Diabetic retinopathy (DR) remains the major threat to sight in the working age population. Diabetic macular edema (DME) is a manifestation of DR that produces loss of central vision. Proliferative diabetic retinopathy (PDR) is a major cause of visual loss in diabetic patients. In PDR, the growth of new vessels is thought to occur as a result of vascular endothelial growth factor (VEGF) release into the vitreous cavity as a response to ischemia. Furthermore, VEGF increases vessel permeability leading to deposition of proteins in the interstitium that facilitate the process of angiogenesis and macular edema. This review demonstrates multiple benefits of intravitreal bevacizumab (IVB) on DR including DME and PDR at 24 months of follow up. The results indicate that IVB injections may have a beneficial effect on macular thickness and visual acuity (VA) in diffuse diabetic macular edema. Therefore, in the future this new therapy could replace or complement focal/grid laser photocoagulation in DME. In PDR, this new option could be an adjuvant agent to pan-retina photocoagulation so that more selective therapy may be applied. In addition, we report a series of patients in which tractional retinal detachment developed or progressed after adjuvant preoperative IVB in severe PDR. © 2010 Bentham Science Publishers Ltd

Macrófitas aquáticas do sistema lacustre do Vale do Rio Doce, Minas Gerais, Brasil

Resumo Esta pesquisa trata da composição e da ocorrência de espécies de macrófitas aquáticas em área de proteção ambiental e áreas não protegidas, que compõem o conjunto de lagos do Vale do Rio Doce em Minas Gerais, terceiro maior sistema lacustre brasileiro. As informações foram levantadas a partir de publicações, material depositado em herbários e coletas botânicas entre os anos de 2007 e 2010, em ambientes aquáticos localizados no Parque Estadual do Rio Doce (PERD) e zona de amortecimento. Foram registradas 184 espécies pertencentes a distintos grupos taxonômicos, hábitos e formas biológicas, sendo aqui proposta a criação de uma nova categoria destas, designada embalsada, para contemplar plantas que se estabelecem em ilhas flutuantes. A pesquisa contribuiu com 152 novas citações para o Vale do Rio Doce em Minas Gerais, com dois primeiros registros nesse estado e com a descrição de uma espécie inédita para a ciência. A similaridade florística entre áreas protegidas e não protegidas indicou que o PERD guarda 74% das espécies de macrófitas aquáticas encontradas. Entretanto, 26% do total de espécies estão desprotegidas, pois não ocorrem nessa unidade de conservação