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Rodrigo Franco Lafetá Queiroz,1,2 Danilo Varela Kniggendorf,1,2 André Lins de Medeiros,1,2 Wilson Takashi Hida,1–4 Celso Takashi Nakano,1,2 Pedro Carlos Carricondo,3 Walton Nosé,4 Andre Gustavo Rolim,1,2 Antonio Francisco Pimenta Motta1,21Hospital Oftalmológico de Brasília, Brasília, DF, Brazil; 2Centro de Estudos Oftalmológicos Renato Ambrósio, Brasília, DF, Brazil; 3Universidade de São Paulo, SP, Brazil; 4Universidade Federal de São Paulo, São Paulo, SP, BrazilPurpose: The aim of this study was to evaluate the influence of four different types of speculums on aberrometry reading (OPD SCAN III [OPD]) and on intraoperative aberrometry reading (optiwave response analyzer, ORA).Patients and methods: This prospective, controlled, comparative study of consecutive cases included the evaluation of five eyes of five patients with monofocal intraocular lens (IOL) implantation. Seventeen measures were performed on each patient: for each speculum, there were two measurements on the OPD and another two on ORA with four different types of blepharostats. A control measure was performed on the without blepharostat in the dominant eye of each patient, therefore totalizing 85 measurements. The measures with the blepharostats were as follows: without pressure (WF) or passive measure and after pressure (AF) or active measure to close the eye. The speculum used in all patients was as follows: open-edged wire (Barraquer); threaded with open blade (Lieberman), with 21 mm aperture; wired with solid blade (Barraquer); and threaded with solid blade (Lieberman) with 21 mm opening. An evaluation of the objective refractive data from the OPD and ORA and the corneal astigmatism from the OPD was performed.Results: Spherical equivalent (SE) of the OPD with the use of blepharostat compared to the OPD without speculum presented only 37.5% of results without statistical significance. Regarding the SE of ORA with speculum usage, compared to the OPD without blepharostat, only 12.5% were not significant. Regarding the accuracy of the ORA refractive axis with the use of blepharostats, all results presented statistical significance.Conclusion: Thus, in the present study, we reached the conclusion between the studied blepharostats that the most suitable for use in the aphakic and pseudophakic capture of the ORA is the open blade threaded blepharostat (Lieberman).Keywords: cataract, surgery, astigmatism, corneal topography, cornea

Selenium-containing compounds have emerged as promising treatment for redox-based and inflammatory diseases. This study aimed to investigate the in vitro and in vivo anti-inflammatory activity of a novel diselenide named as dibenzyl[diselanediyIbis(propane-3-1diyl)] dicarbamate (DD). DD reacted with HOCl ( k  = 9.2 x 10 7 M -1 s -1 ), like glutathione ( k  = 1.2 x 10 8 M -1 s -1 ), yielding seleninic and selenonic acid derivatives, and it also decreased HOCl formation by activated human neutrophils (IC 50 =4.6 μM) and purified myeloperoxidase (MPO) (IC 50 =3.8 μM). However, tyrosine, MPO-I and MPO-II substrates, did not restore HOCl formation in presence of DD. DD inhibited the oxidative burst in d HL-60 cells with no toxicity up to 25 µM for 48h. Next, an intraperitoneal administration of 25, 50, and 75 mg/kg DD decreased total leukocyte, neutrophil chemotaxis, and inflammation markers (MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α, IL-1β, CXCL1/KC, and CXCL2/MIP-2) on a murine model of carrageenan-induced peritonitis. Likewise, 50 mg/kg DD (i.p.) decreased carrageenan-induced paw edema over 5h. Histological and immunohistochemistry analyses of the paw tissue showed decreased neutrophil count, edema area, and MPO, carbonylated, and nitrated protein staining. Furthermore, DD treatment decreased the fMLP-induced chemotaxis of human neutrophils (IC 50 =3.7 μM) in vitro with no toxicity. Lastly, DD presented no toxicity in a single-dose model using mice (50 mg/kg, i.p.) over 15 days and in Artemia salina bioassay (50 to 2000 µM), corroborating findings from in silico toxicological study. Altogether, these results demonstrate that DD attenuates carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting damage from MPO-mediated oxidative burst.

We investigated the magnitude of exercise-induced changes in muscular bioenergetics, redox balance, mitochondrial function, and gene expression within 24 h after the exercise bouts performed with different intensities, durations, and execution modes (continuous or with intervals). Sixty-five male Swiss mice were divided into four groups: one control (n = 5) and three experimental groups (20 animals/group), submitted to a forced swimming bout with an additional load (% of animal weight): low-intensity continuous (LIC), high-intensity continuous (HIC), and high-intensity interval (HII). Five animals from each group were euthanized at 0 h, 6 h, 12 h, and 24 h postexercise. Gastrocnemius muscle was removed to analyze the expression of genes involved in mitochondrial biogenesis (Ppargc1a), fusion (Mfn2), fission (Dnm1L), and mitophagy (Park2), as well as inflammation (Nos2) and antioxidant defense (Nfe2l2, GPx1). Lipid peroxidation (TBARS), total peroxidase, glutathione peroxidase (GPx), and citrate synthase (CS) activity were also measured. Lactacidemia was measured from a blood sample obtained immediately postexercise. Lactacidemia was higher the higher the exercise intensity (LIC < HIC < HII), while the inverse was observed for TBARS levels. The CS activity was higher in the HII group than the other groups. The antioxidant activity was higher 24 h postexercise in all groups compared to the control and greater in the HII group than the LIC and HIC groups. The gene expression profile exhibited a particular profile for each exercise protocol, but with some similarities between the LIC and HII groups. Taken together, these results suggest that the intervals applied to high-intensity exercise seem to minimize the signs of oxidative damage and drive the mitochondrial dynamics to maintain the mitochondrial network, similar to low-intensity continuous exercise.

This study aimed to evaluate the effect of a methoxylated fraction from Vellozia dasypus Seub on myeloperoxidase (MPO)-chlorinating activity and subsequent in silico assays for binding profile prediction. Therefore, the ethyl acetate extract of aerial parts from Vellozia dasypus Seub was fractionated on open-column chromatography containing SiO 2 and eluted with solvent in crescent polarity to yield a fraction with a mixture of flavonols quercetin 3- O -methyl ether ( 1 ) and 6-C-methyl quercetin 3- O -methyl ether ( 2 ). Their chemical structures were proposed by HPLC coupled to photodiode array (HPLC-DAD) and mass spectrometer using electrospray ionization multistage analysis (HPLC-MS/MS). The fraction enriched with compounds 1 and 2 inhibited more efficiently the in vitro MPO-chlorinating activity (IC 50 = 40 µg/mL) than the ethyl acetate extract (IC 50 = 64.0 µg/mL). Molecular docking studies revealed that these compounds interact with MPO active pocket similarly to trifluoromethyl-substituted aromatic hydroxamate, a well-known MPO inhibitor, co-crystallized at the MPO binding site (PDB ID: 4C1M). Molecular dynamics trajectories confirmed that these two molecules interact with the MPO binding site with a similar energetic pattern when compared to the crystallographic ligand. Taken together, these data expand the sources of phenolic natural compounds that may be further investigated against inflammation-related diseases. Communicated by Ramaswamy H. Sarma.

Cisplatin is an effective antineoplastic agent, but its use is limited by its nephrotoxicity caused by the oxidative stress in tubular epithelium of nephrons. On the other hand, regular exercise provides beneficial adaptations in different tissues and organs. As with many drugs, dosing is extremely important to get the beneficial effects of exercise. Thus, we aimed to investigate the influence of exercise intensity and frequency on cisplatin-induced (20 mg/kg) renal damage in mice. Forty male Swiss mice were divided into five experimental groups (n=8 per group): 1) sedentary; 2) low-intensity forced swimming, three times per week; 3) high-intensity forced swimming, three times per week; 4) low-intensity forced swimming, five times per week; and 5) high-intensity forced swimming, five times per week. Body composition, renal structure, functional indicators (plasma urea), lipid peroxidation, antioxidant enzyme activity, expression of genes related to antioxidant defense, and inflammatory and apoptotic pathways were evaluated. Comparisons considered exercise intensity and frequency. High lipid peroxidation was observed in the sedentary group compared with trained mice, regardless of exercise intensity and frequency. Groups that trained three times per week showed more benefits, as reduced tubular necrosis, plasma urea, expression of CASP3 and Rela (NFkB subunit-p65) genes, and increased total glutathione peroxidase activity. No significant difference in Nfe2l2 (Nrf2) gene expression was observed between groups. Eight weeks of regular exercise training promoted nephroprotection against cisplatin-mediated oxidative injury. Exercise frequency was critical for nephroprotection.

Regular physical training and cigarette smoke exposure (CSE) have opposite effects on physical performance, antioxidant, and inflammatory profile. However, the interaction between these events is not well studied. We aimed to investigate how regular physical training and CSE interact, and in what is the outcome of this interaction on the physical performance, skeletal muscle antioxidant defense and molecular profile response of pro and anti-inflammatory cytokines. Male C57BL/6 mice were randomly divided into 4 groups (n = 8/group): 1) Sedentary group (SED); 2) 4 weeks of control, followed by 4 weeks of CSE (SED + CSEG); 3) Physically active (PA) along 8 weeks (forced swim training, 5 times a week); 4) Physically active and exposed to the cigarette smoke (PA + CSEG), group submitted to forced swim training for 4 weeks, followed by 4 weeks of concomitant training and CSE. Physical performance was evaluated before and after the experimental period (8 weeks), total peroxidase and glutathione peroxidase (GPx) activities, expression of genes encoding TNF-α, MCP-1, IL1β, IL-6, IL-10, TGF-β, HO-1 and the TNF-α/IL-10 ratio were determined from gastrocnemius muscle at the end of experimental period. The CSE attenuated the aerobic capacity adaptation (time to exhaustion in swimming forced test) promoted by physical training and inhibit the improvement in local muscle resistance (inverted screen test). The regular physical training enhanced the antioxidant defense, but the CSE abrogated this benefit. The CSE induced a harmful pro-inflammatory profile in skeletal muscle from sedentary animals whereas the regular physical training induced an opposite adaptation. Likewise, the CSE abolished the protective effect of physical training. Together, these results suggest a negative effect of CSE including, at least in part, the inhibition/attenuation of beneficial adaptations from regular physical training., (Copyright © 2022 Elsevier B.V. All rights reserved.)

During systemic inflammation, indoleamine 2,3-dioxygenase 1 (IDO1) becomes expressed in endothelial cells where it uses hydrogen peroxide (H 2 O 2 ) to oxidize L-tryptophan to the tricyclic hydroperoxide, cis-WOOH, that then relaxes arteries via oxidation of protein kinase G 1α. Here we show that arterial glutathione peroxidases and peroxiredoxins that rapidly eliminate H 2 O 2 , have little impact on relaxation of IDO1-expressing arteries, and that purified IDO1 forms cis-WOOH in the presence of peroxiredoxin 2. cis-WOOH oxidizes protein thiols in a selective and stereospecific manner. Compared with its epimer trans-WOOH and H 2 O 2 , cis-WOOH reacts slower with the major arterial forms of glutathione peroxidases and peroxiredoxins while it reacts more readily with its target, protein kinase G 1α. Our results indicate a paradigm of redox signaling by H 2 O 2 via its enzymatic conversion to an amino acid-derived hydroperoxide that 'escapes' effective reductive inactivation to engage in selective oxidative activation of key target proteins., (© 2021. The Author(s).)

Pseudomonas aeruginosa is an opportunistic bacterium in patients with cystic fibrosis and hospital acquired infections. It presents a plethora of virulence factors and antioxidant enzymes that help to subvert the immune system. In this study, we identified the 2-Cys peroxiredoxin, alkyl-hydroperoxide reductase C1 (AhpC1), as a relevant scavenger of oxidants generated during inflammatory oxidative burst and a mechanism of P. aeruginosa (PA14) escaping from killing. Deletion of AhpC1 led to a higher sensitivity to hypochlorous acid (HOCl, IC 50 3.2 ± 0.3 versus 19.1 ± 0.2 μM), hydrogen peroxide (IC 50 91.2 ± 0.3 versus 496.5 ± 6.4 μM) and the organic peroxide urate hydroperoxide. ΔahpC1 strain was more sensitive to the killing by isolated neutrophils and less virulent in a mice model of infection. All mice intranasally instilled with ΔahpC1 survived as long as they were monitored (15 days), whereas 100% wild-type and ΔahpC1 complemented with ahpC1 gene (ΔahpC1 attB:ahpC1) died within 3 days. A significantly lower number of colonies was detected in the lung and spleen of ΔahpC1-infected mice. Total leucocytes, neutrophils, myeloperoxidase activity, pro-inflammatory cytokines, nitrite production and lipid peroxidation were much lower in lungs or bronchoalveolar liquid of mice infected with ΔahpC1. Purified AhpC neutralized the inflammatory organic peroxide, urate hydroperoxide, at a rate constant of 2.3 ± 0.1 × 10 6  M -1 s -1 , and only the ΔahpC1 strain was sensitive to this oxidant. Incubation of neutrophils with uric acid, the urate hydroperoxide precursor, impaired neutrophil killing of wild-type but improved the killing of ΔahpC1. Hyperuricemic mice presented higher levels of serum cytokines and succumbed much faster to PA14 infection when compared to normouricemic mice. In summary, ΔahpC1 PA14 presented a lower virulence, which was attributed to a poorer ability to neutralize the oxidants generated by inflammatory oxidative burst, leading to a more efficient killing by the host. The enzyme is particularly relevant in detoxifying the newly reported inflammatory organic peroxide, urate hydroperoxide., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The L-tryptophan-derived tricyclic hydroperoxide cis-WOOH was recently identified as a novel and biologically important factor for regulating vascular tone and blood pressure under inflammatory conditions and potentially other cellular redox signaling events. cis-WOOH is highly labile and currently not available commercially. In this protocol, we provide procedures for the synthesis, purification, quantification and characterization of cis-WOOH, its epimer trans-WOOH and their respective alcohols (cis-WOH and trans-WOH). Photo-oxidation of L-tryptophan (L-Trp) results in a mixture containing cis-WOOH and trans-WOOH, which are separated and purified by semi-preparative HPLC. cis-WOH and trans-WOH are then produced by sodium borohydride reduction and purified by semi-preparative HPLC. Characterization of cis-WOOH and trans-WOOH and the reduced alcohol variants is achieved using HPLC, fluorescence, NMR and liquid chromatography-tandem mass spectrometry. The protocol provides instructions for storage and quantification, as well as ways to test the stability of these hydroperoxides in commonly used buffers and media. Finally, we describe examples of how to monitor the formation of cis-WOOH in biological samples. The protocol ensures reasonable yield (11%) and purity (>99%) of cis-WOOH and control compounds in 5-6 d and outlines conditions under which cis-WOOH is stable for several months.

Cigarette smoke is a complex mixture capable of triggering inflammation and oxidative damage in animals at pulmonary and systemic levels. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) reduces tissue injury associated with inflammation in vivo by mechanisms that are not completely understood. Here we evaluated the effect of tempol on inflammation and oxidative damage induced by acute exposure to cigarette smoke in vivo. Male C57BL/6 mice (n = 32) were divided into 4 groups (n = 8 each): 1) control group exposed to ambient air (GC), 2) animals exposed to cigarette smoke for 5 days (CSG), mice treated 3) prior or 4) concomitantly with tempol (50 mg/kg/day) and exposed to cigarette smoke for 5 days. The results showed that the total number of leukocytes and neutrophils increased in the respiratory tract and lung parenchyma of mice exposed to cigarette smoke. Likewise, MPO levels and activity as well as lipid peroxidation and lung protein nitration and carbonylation also increased. Administration of tempol before or during exposure to cigarette smoke inhibited all the above parameters. Tempol also reduced the pulmonary expression of the inflammatory cytokines Il-6, Il-1β and Il-17 to basal levels and of Tnf-α by approximately 50%. In contrast, tempol restored Il-10 and Tgf-β levels and enhanced the expression of Nrf2-associated genes, such as Ho-1 and Gpx2. Accordingly, total GPx activity increased in lung homogenates of tempol-treated animals. Taken together, our results show that tempol protects mouse lungs from inflammation and oxidative damage resulting from exposure to cigarette smoke, likely through reduction of leukocyte infiltration and increased transcription of some of the Nrf2-controlled genes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: To understand how nursing workers perceive care of hypertension (HBP) in older adult within the scope of the Family Health Strategy., Method: A qualitative descriptive study carried out in the city of Natal/RN, with 20 nursing workers providing care to older adults with HBP. The data were obtained through a semi-structured interview and analysed by the Thematic Analysis, based on the theoretical support of the integrality, using Atlas.ti 7.0 software., Results: The elements found as facilitators were: territorialization, partnerships, professional proactivity and the user's bond with the team. Among those found as barriers were: disease-centered care; academic education based on the biomedical model; lack of inter-sectorality and discontinuity of care in the care network., Final Considerations: Nursing workers perceive that health institutions lack articulated and innovative practices that incorporate new paradigms focused on integrality of care.

Objective: determine the coordination of Primary Health Care with community resources in the clinical management of tuberculosis., Method: descriptive study, of quantitative approach, with participation of one hundred health professionals. Part of a questionnaire was used in evaluating local institutional capacity for the model of chronic conditions care, adapted for tuberculosis care., Results: the coordination between health units, individuals with tuberculosis, and community organizations; the partnerships between institutions and local health councils/committees showed limited capacity. On the other hand, the component for the participation of the Community Health Agent presented more favorable capacity., Conclusion: the municipality has unfavorable capacity for coordination of health units and the community. It is reinforced the need to promote these coordinations in search of symptomatic cases in the communities, in the directly observed treatment, and in promoting the association between different social actors.

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 10 7 -10 8 M -1 ⋅s -1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 10 7 M -1 ⋅s -1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H 2 O 2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions., Competing Interests: The authors declare no conflict of interest.

Mansoa hirsuta (Bignoniaceae) is a native plant from caatinga in Brazilian semiarid. This plant has been locally used as antimicrobial and hypoglycemiant agents, but their action mechanisms and toxicity remain largely unknown. Therefore, we evaluated the composition and antioxidant, cytoprotective and hypoglycemiant effects of raw extract, fractions and compounds from leaves of M. hirsuta. The cytogenotoxic effects of ursolic and oleanolic acids, the main phytotherapic components of this plant, were assessed. The raw extract and fractions presented steroids, saponins, flavonols, flavanonols, flavanones, xanthones, phenols, tannins, anthocyanins, anthocyanidins and flavonoids. The ethyl acetate fraction inhibited efficiently the cascade of lipid peroxidation while the hydroalcoholic fraction was richer in total phenols and more efficient in capturing 2,2-diphenyl-1-picrylhydrazyl (·DPPH) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS·+) radicals. The isolated fraction of M. hirsuta also inhibited the α-amylase activity. Cytotoxic effects were absent in both raw extract and fractions while ursolic+oleanolic acids were efficient in protecting cells after exposure to hydrogen peroxide. Moreover, this mixture of acid shad no significant interference on the mitotic index and frequency of nuclear and/or chromosomal abnormalities in Allium cepa test. Therefore, M. hirsuta represents a potential source of phytochemicals against inflammatory and oxidative pathologies, including diabetes.

Geopropolis is a special type of propolis produced by stingless bees. Several pharmacological properties have been described for different types of geopropolis, but there have been no previous studies of the geopropolis from Melipona mondury. In this study, we investigated the antioxidant, antibacterial, and antiproliferative activities of M. mondury geopropolis, and determined its chemical profile. The antioxidant activity was determined using in vitro ABTS·+, ·DPPH, and β-carotene/linoleic acid co-oxidation methods. The antibacterial activity was determined using a microdilution method with Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant S. aureus. The antiproliferative effect was determined in tumor cell lines using the Alamar Blue assay. The chemical profile was obtained using UHPLC-MS and UHPLC-MS/MS. The butanolic fraction had the highest concentration of phenolic compounds and more potent antioxidant properties in all assays. This fraction also had bacteriostatic and bactericidal effects against all bacterial strains at low concentrations, especially S. aureus. The hexane fraction had the highest antiproliferative potential, with IC50 values ranging from 24.2 to 46.6 µg/mL in HL-60 (human promyelocytic leukemia cell) and K562 (human chronic myelocytic leukemia cell), respectively. Preliminary chemical analysis indicates the presence of terpenes and gallic acid in the geopropolis. Our results indicate the therapeutic potential of geopropolis from M. mondury against inflammatory, oxidative, infectious, and neoplastic diseases.

Background: Accurate diagnostic techniques for schistosomiasis are essential for prevalence determination and identification of positive patients. A point-of-care test for detecting schistosome circulating cathodic antigen (POC-CCA) has been evaluated for its accuracy in different endemic regions. This reagent strip/dipstick based assay has showed high sensitivity for individuals with high or moderate worm burden, but the interpretation of light infections is less clear, especially for trace readings., Methodology/principal Findings: We introduced a urine lyophilization step to the POC-CCA assay to improve its sensitivity and clarify the interpretation of traces. We evaluated POC-CCA sensitivity and specificity within individuals with low parasite burdens in a Brazilian endemic area where a high number of traces were detected. Patients that were positive for other helminths were also evaluated for cross reactions. In all cases, a combined parasitological diagnosis using Kato-Katz (24 slides) and Saline Gradient (1 g of feces) were used as reference. At baseline, diagnosis by POC-CCA (1-2 cassettes) showed 6% sensitivity, inaccurately predicting a low prevalence of Schistosoma mansoni infections (2 POC-CCA positives/32 egg positives). After urine lyophilization, the sensitivity was increased significantly (p < 0.05). Prevalence rates changed from 2% to 32% (27 POC-CCA positives/32 egg positives), equivalent to parasitological techniques. Most of the trace readings changed to positive after lyophilization while some negatives turned into traces. Cross reaction analysis confirmed the specificity of POC-CCA., Conclusions/significance: Trace readings cannot be primarily defined as positive or negative cases. It is critical to verify case-by-case by concentrating urine 10 fold by lyophilization for the diagnosis. Following lyophilization, persistent trace readings should be read as negatives. No trained technician is needed and cost is restricted to the cost of a lyophilizer and the electricity to run it.

Despite extensive investigation of the irreversible oxidations undergone by proteins in vitro and in vivo, the products formed from the oxidation of Trp residues remain incompletely understood. Recently, we characterized a ditryptophan cross-link produced by the recombination of hSOD1-tryptophanyl radicals generated from attack of the carbonate radical produced during the bicarbonate-dependent peroxidase activity of the enzyme. Here, we examine whether the ditryptophan cross-link is produced by the attack of the carbonate radical on proteins other than hSOD1. To this end, we treated hen egg white lysozyme with photolytically and enzymatically generated carbonate radical. The radical yields were estimated and the lysozyme modifications were analyzed by SDS-PAGE, western blot, enzymatic activity and MS/MS analysis. Lysozyme oxidation by both systems resulted in its inactivation and dimerization. Lysozyme treated with the photolytic system presented monomers oxidized to hydroxy-tryptophan at Trp(28) and Trp(123) and N-formylkynurenine at Trp(28), Trp(62) and Trp(123). Lysozyme treated with the enzymatic system rendered monomers oxidized to N-formylkynurenine at Trp(28). The dimers were characterized as lysozyme-Trp(28)-Trp(28)-lysozyme and lysozyme-Trp(28)-Trp(32)-hSOD1. The results further demonstrate that the carbonate radical is prone to causing biomolecule cross-linking and hence, may be a relevant player in pathological mechanisms. The possibility of exploring the formation of ditryptophan cross-links as a carbonate radical biomarker is discussed., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lipid peroxidation is a well-known process that has been implicated in many diseases. Recent evidence has shown that mitochondrial cholesterol levels are increased under specific conditions, making it an important target for peroxidation inside the mitochondria. Cholesterol peroxidation generates, as primary products, several hydroperoxides (ChOOH), which can react with transition metals and metalloproteins. In this sense, cytochrome c (CYTC), a heme protein largely found in the mitochondria, becomes a candidate to react with ChOOH. Using CYTC associated with SDS micelles to mimic mitochondrial conditions, we show that ChOOH induces dose-dependent CYTC Soret band bleaching, indicating that it is using ChOOH as a substrate. This reaction leads to protein oligomerization, suggesting the formation of a protein radical that, subsequently, recombines, giving dimers, trimers, and tetramers. EPR experiments confirmed the production of carbon-centered radicals from both protein and lipid in the presence of ChOOH. Similar results were obtained with linoleic acid hydroperoxides (LAOOH). In addition, replacing SDS micelles by cardiolipin-containing liposomes as the mitochondrial mimetic led to similar results with either ChOOH or LAOOH. Importantly, kinetic experiments show that CYTC bleaching is faster with ChOOH than with H2O2, suggesting that these hydroperoxides could be relevant substrates for CYTC peroxidase-like activity in biological media. Altogether, these results show that CYTC induces homolytic cleavage of lipid-derived hydroperoxides, producing lipid and protein radicals.

Human schistosomiasis, caused mainly by Schistosoma mansoni, S. japonicum, and S. hematobium, remains a prevalent and serious parasitic disease worldwide. Although it is a debilitating disease, a lack of sensitive methods for the precise diagnosis of active infection cases is important to prevent morbidity. The optimization of new diagnostic approaches may be accomplished by the selection of specific markers. In that manner, markers can be satisfactorily used for detection of different phases of infection, as acute and chronic phases, pre-patent and post-patent phases and after chemotherapy, improving the efficiency of methods. For that purpose, proteomics and glycomics analyses have been performed in schistosomes, in particular S. mansoni, using powerful high-throughput methodologies. These investigations have not only chartered protein, o-glycan and n-glycan profiles across developmental stages within mammalian host, but are also leading to the characterization of features of the surface tegument, the eggshell and excretory-secretory proteomes of schistosomes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity., Methodology/principal Findings: We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure., Conclusions/significance: The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.

Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of various diseases via mechanisms that are not completely understood. Recently, we reported that high doses of tempol moderately increased survival in a rat model of ALS (amyotrophic lateral sclerosis) while decreasing the levels of oxidized hSOD1 (human Cu,Zn-superoxide dismutase) in spinal cord tissues. To better understand such a protective effect in vivo, we studied the effects of tempol on hSOD1 oxidation in vitro. The chosen oxidizing system was the bicarbonate-dependent peroxidase activity of hSOD1 that consumes H2O2 to produce carbonate radical, which oxidizes the enzyme. Most of the experiments were performed with 30 μM hSOD1, 25 mM bicarbonate, 1 mM H2O2, 0.1 mM DTPA (diethylenetriaminepenta-acetic acid) and 50 mM phosphate buffer at a final pH of 7.4. The results showed that tempol (5-75 μM) does not inhibit hSOD1 turnover, but decreases its resulting oxidation to carbonylated and covalently dimerized forms. Tempol acted by scavenging the carbonate radical produced and by recombining with hSOD1-derived radicals. As a result, tempol was consumed nearly stoichiometrically with hSOD1 monomers. MS analyses of turned-over hSOD1 and of a related peptide oxidized by the carbonate radical indicated the formation of a relatively unstable adduct between tempol and hSOD1-Trp32•. Tempol consumption by the bicarbonate-dependent peroxidase activity of hSOD1 may be one of the reasons why high doses of tempol were required to afford protection in an ALS rat model. Overall, the results of the present study confirm that tempol can protect against protein oxidation and the ensuing consequences.

Current schistosomiasis control strategies are largely based on chemotherapeutic agents and a limited number of drugs are available today. Praziquantel (PZQ) is the only drug currently used in schistosomiasis control programs. Unfortunately, this drug shows poor efficacy in patients during the earliest infection phases. The effects of PZQ appear to operate on the voltage-operated Ca2+ channels, which are located on the external Schistosoma mansoni membrane. Because some Ca2+ channels have dihydropyridine drug class (a class that includes nifedipine) sensitivity, an in vitro analysis using a calcium channel antagonist (clinically used for cardiovascular hypertension) was performed to determine the antischistosomal effects of nifedipine on schistosomula and adult worm cultures. Nifedipine demonstrated antischistosomal activity against schistosomula and significantly reduced viability at all of the concentrations used alone or in combination with PZQ. In contrast, PZQ did not show significant efficacy when used alone. Adult worms were also affected by nifedipine after a 24 h incubation and exhibited impaired motility, several lesions on the tegument and intense contractility. These data support the idea of Ca2+ channels subunits as drug targets and favour alternative therapeutic schemes when drug resistance has been reported. In this paper, strong arguments encouraging drug research are presented, with a focus on exploring schistosomal Ca2+ channels.

Schistosomiasis diagnosis is based on the detection of eggs in the faeces, which is laborious and lacks sensitivity, especially for patients with a low parasite burden. Immunological assays for specific antibody detection are available, but they usually demonstrate low sensitivity and/or specificity. In this study, two simple immunological assays were evaluated for the detection of soluble Schistosoma mansoni adult worm preparation (SWAP) and egg-specific IgGs. These studies have not yet been evaluated for patients with low parasite burdens. Residents of an endemic area in Brazil donated sera and faecal samples for our study. The patients were initially diagnosed by a rigorous Kato-Katz analysis of 18 thick smears from four different stool samples. The ELISA-SWAP was successful for human diagnosis with 90% sensitivity and specificity, confirming the Kato-Katz diagnosis with nearly perfect agreement, as seen by the Kappa index (0.85). Although the ELISA-soluble S. mansoni egg antigen was 85% sensitive, it exhibited low specificity (80%; Kappa index: 0.75) and was more susceptible to cross-reactivity. We believe that immunological assays should be used in conjunction with Kato-Katz analysis as a supplementary tool for the diagnosis of schistosomiasis for patients with low infection burdens, which are usually hard to detect.

Introduction: The diagnosis of schistosomiasis mansoni on early stages of infection is important to prevent late morbidity. A simple, cheap, sensitive and specific assay for routine diagnosis of schistosome infection based on the detection of specific IgG for schistosomula tegument antigens (ELISA-SmTeg) was developed by our group., Methods: We describe here an acute outbreak involving a travel group of 80 individuals from a non-endemic area of the State of Minas Gerais, Brazil. These individuals were in contact with a freshwater pool where Biomphalaria glabrata was found. Results obtained from our new methodology were compared to IgG antibody titers against soluble worm antigenic preparation (SWAP) by ELISA and, also to parasitological examination, nuclear magnetic resonance and clinical findings., Results: ELISA-SmTeg was capable of detecting 64 positive cases among the 80 individuals participating at the survey with a positivity ratio of 80% and a higher sensitivity than ELISA-SWAP that was only sensitive for 56% of positive cases. Besides, a significant correlation was found for the severity of the infection and the specific IgG titers against SmTeg., Conclusions: Our data showed that ELISA-SmTeg might serve as the initial diagnostic tool for acute stages of the infection in community-based helminth control programs or for the surveillance of individuals from non-endemic areas.

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and other cyclic nitroxides have been shown to inhibit the chlorinating activity of myeloperoxidase (MPO) in vitro and in cells. To examine whether nitroxides inhibit MPO activity in vivo we selected acute carrageenan-induced inflammation on the rat paw as a model. Tempol and three more hydrophobic 4-substituted derivatives (4-azido, 4-benzenesulfonyl, and 4-(4-phenyl-1H-1,2,3-triazol-1-yl)) were synthesized, and their ability to inhibit the in vitro chlorinating activity of MPO and carrageenan-induced inflammation in rat paws was evaluated. All of the tested nitroxides inhibited the chlorinating activity of MPO in vitro with similar IC(50) values (between 1.5 and 1.8 μM). In vivo, the attenuation of carrageenan-induced inflammation showed some correlation with the lipophilicity of the nitroxide at early time points but the differences in the effects were small (<2-fold) compared with the differences in lipophilicity (>200-fold). No inhibition of MPO activity in vivo was evident because the levels of MPO activity in rat paws correlated with the levels of MPO protein. Likewise, paw edema, levels of nitrated and oxidized proteins, and levels of plasma exudation correlated with the levels of MPO protein in the paws of the animals that were untreated or treated with the nitroxides. The effects of the nitroxides in vivo were compared with those of 4-aminobenzoic hydrazide and of colchicine. Taken together, the results indicate that nitroxides attenuate carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting MPO-mediated damage. Accordingly, tempol was shown to inhibit rat neutrophil migration in vitro., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: The correlation between the immunological assay and the antibody titer can offer a tool for the experimental analysis of different phases of the disease., Methods: Two simple immunological assays for Schistosoma mansoni in mice sera samples based on specific IgG detection for worms soluble antigens and eggs soluble antigens were standardized and evaluated in our laboratory. Fifty mice were used in negative and positive groups and the results obtained by enzyme-linked immunosorbent assays (ELISA) assays were compared with the number of worms counted and the IgG titers at different times of infection., Results: Data showed that ELISA using adult worm antigens (ELISA-SWAP) presented a satisfactory correlation between the absorbance value of IgG titers and the individual number of worms counted after perfusion technique (R²=0.62). In addition, ELISA-SWAP differentially detected positive samples with 30 and 60 days post infection (p=0.011 and 0.003, respectively), whereas ELISA using egg antigens (ELISA-SEA) detected samples after 140 days (p=0.03)., Conclusions: These data show that the use of different antigens in immunological methods can be used as potential tools for the analysis of the chronological evolution of S. mansoni infection in murine schistosomiasis. Correlations with human schistosomiasis are discussed.

Worldwide Schistosomiasis mansoni continues to be a serious public health problem. Over the past decades, control programmes have made remarkable progress in reducing S. mansoni infections to a relatively low level in Brazil and African countries. Endemic regions are currently circumscribed in certain core areas where reinfection and repeated chemotherapy are frequent and, consequently, are related to residents with low parasite load. At present, diagnosis is predominately a key step for final disease control although low endemicity area residents are hardly detected by most of the available assays. In this paper, we review the current status and efforts made aiming at the improvement of diagnostic tools for S. mansoni in low endemicity infections. The establishment of diagnostic assays--simple, affordable, sensitive, and specific for field diagnosis of S. mansoni--is essential and should be given high priority.

The nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) reduces tissue injury in animal models of inflammation by mechanisms that are not completely understood. MPO (myeloperoxidase), which plays a fundamental role in oxidant production by neutrophils, is an important target for anti-inflammatory action. By amplifying the oxidative potential of H2O2, MPO produces hypochlorous acid and radicals through the oxidizing intermediates MPO-I [MPO-porphyrin•+-Fe(IV)=O] and MPO-II [MPO-porphyrin-Fe(IV)=O]. Previously, we reported that tempol reacts with MPO-I and MPO-II with second-order rate constants similar to those of tyrosine. However, we noticed that tempol inhibits the chlorinating activity of MPO, in contrast with tyrosine. Thus we studied the inhibition of MPO-mediated taurine chlorination by tempol at pH 7.4 and re-determined the kinetic constants of the reactions of tempol with MPO-I (k=3.5×105 M-1·s-1) and MPO-II, the kinetics of which indicated a binding interaction (K=2.0×10-5 M; k=3.6×10-2 s-1). Also, we showed that tempol reacts extremely slowly with hypochlorous acid (k=0.29 and 0.054 M-1·s-1 at pH 5.4 and 7.4 respectively). The results demonstrated that tempol acts mostly as a reversible inhibitor of MPO by trapping it as MPO-II and the MPO-II-tempol complex, which are not within the chlorinating cycle. After turnover, a minor fraction of MPO is irreversibly inactivated, probably due to its reaction with the oxammonium cation resulting from tempol oxidation. Kinetic modelling indicated that taurine reacts with enzyme-bound hypochlorous acid. Our investigation complements a comprehensive study reported while the present study was underway

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC50 values ranging from 1.0 to 2.8 µM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)