Interplay between nitric oxide and gonadotrophin-releasing hormone in the neuromodulation of the corpus luteum during late pregnancy in the rat.

BACKGROUND: Nitric oxide and GnRH are biological factors that participate in the regulation of reproductive functions. To our knowledge, there are no studies that link NO and GnRH in the sympathetic ganglia. Thus, the aim of the present work was to investigate the influence of NO on GnRH release from the coeliac ganglion and its effect on luteal regression at the end of pregnancy in the rat. METHODS: The ex vivo system composed by the coeliac ganglion, the superior ovarian nerve, and the ovary of rats on day 21 of pregnancy was incubated for 180 min with the addition, into the ganglionic compartment, of L-NG-nitro arginine methyl ester (L-NAME), a non-selective NO synthase inhibitor. The control group consisted in untreated organ systems. RESULTS: The addition of L-NAME in the coeliac ganglion compartment decreased NO as well as GnRH release from the coeliac ganglion. In the ovarian compartment, and with respect to the control group, we observed a reduced release of GnRH, NO, and noradrenaline, but an increased production of progesterone, estradiol, and expression of their limiting biosynthetic enzymes, 3ß-HSD and P450 aromatase, respectively. The inhibition of NO production by L-NAME in the coeliac ganglion compartment also reduced luteal apoptosis, lipid peroxidation, and nitrotyrosine, whereas it increased the total antioxidant capacity within the corpora lutea. CONCLUSION: Collectively, the results indicate that NO production by the coeliac ganglion modulates the physiology of the ovary and luteal regression during late pregnancy in rats.

The production of nitric oxide in the coeliac ganglion modulates the effect of cholinergic neurotransmission on the rat ovary during the preovulatory period.

The aim of the present work was to investigate whether the nitric oxide produced by the nitric oxide/nitric oxide synthase (NO/NOS) system present in the coeliac ganglion modulates the effects of cholinergic innervation on oxidative status, steroidogenesis and apoptotic mechanisms that take place in the rat ovary during the first proestrous. An ex vivo Coeliac Ganglion- Superior Ovarian Nerve- Ovary (CG-SON-O) system was used. Cholinergic stimulation of the CG was achieved by 10-6 M Acetylcholine (Ach). Furthermore, 400 µM Aminoguanidine (AG) - an inhibitor of inducible-NOS was added in the CG compartment in absence and presence of Ach. It was found that Ach in the CG compartment promotes apoptosis in ovarian tissue, probably due to the oxidative stress generated. AG in the CG compartment decreases the release of NO and progesterone, and increases the release of estradiol from the ovary. The CG co-treatment with Ach and AG counteracts the effects of the ganglionic cholinergic agonist on ovarian oxidative stress, increases hormone production and decreases Fas mRNA expression. These results suggest that NO is an endogenous modulator of cholinergic neurotransmission in CG, with implication in ovarian steroidogenesis and the apoptotic mechanisms that take place in the ovary during the preovulatory period in rats.

Cesarean section rates in Ecuador: a 13-year comparative analysis between public and private health systems.

OBJECTIVE: To demonstrate the prevalence of cesearean sections (C-sections) in Ecuador and their distribution between private and public health centers. METHODS: An observational population-based study was conducted of patients discharged from public and private hospitals in Ecuador after a C-section or vaginal delivery. Data were collected by the Ecuadorian National Institute of Statistics and Census (INEC) between 2001 and 2013. RESULTS: The overall national C-section rate in the private health care system is double the rate in the public health care system. Over the 13 years of the study, C-sections accounted for 57.5% of births in the private sector, while the public sector proportion did not exceed 22.3%. Countrywide, less than 36% of C-sections were found to be clinically justified by parallel analysis of absolute or relative indications. Acute fetal distress (AFD) was more frequently reported in private centers compared to public ones (446 per 10 000 live births versus 274 per 10 000). Since 2001, the number of births by cesarean section increased by more than 50% (R2 = 0.7306, P < 0.05), with an annual growth rate of 4.03%. In Guayaquil, the largest city in Ecuador, up to 74% of live births occurred by C-section. CONCLUSION: National data show that C-sections are performed more frequently in Ecuador than the rate recommended by the World Health Organization, especially in the private health care system. Private centers also report higher rates of AFD, which implies that this diagnosis is either overused in private centers or underrecognized in public centers. Although several factors might be influencing these trends, no data are available to determine the relative importance of economics, practicality, and medical or personal concerns of mothers and physicians in deciding which method of delivery should be used.

OBJETIVO: Demostrar la prevalencia de las cesáreas en Ecuador y su distribución entre centros privados y públicos de salud. MÉTODOS: Se realizó un estudio de observación basado en la población de pacientes dadas de alta de hospitales públicos y privados en Ecuador después de una cesárea o un parto vaginal. Los datos fueron recopilados por el Instituto Nacional de Estadísticas y Censo (INEC) de Ecuador entre el 2001 y el 2013. RESULTADOS: La tasa nacional de cesáreas en el sistema privado de atención de salud es el doble de la tasa que se observa en el sistema público de salud. Durante los 13 años que duró el estudio, las cesáreas representaron 57,5% de los nacimientos en el sector privado, mientras que la proporción en el sector público no superó 22,3%. A nivel de todo el país, se observó que menos de 36% de las cesáreas estaban clínicamente justificadas con un análisis paralelo de indicaciones absolutas o relativas. Se notificaron casos de sufrimiento fetal agudo con mayor frecuencia en los centros privados comparados con los públicos (446 por 10 000 nacidos vivos frente a 274 por 10 000 n.v.). Desde el 2001, el número de nacimientos por cesárea aumentó más de 50% (R2 = 0,7306, P < 0,05), con una tasa de crecimiento anual de 4,03%. En Guayaquil, la ciudad más grande de Ecuador, hasta 74% de los nacidos vivos nacieron por cesárea. CONCLUSIONES: Los datos nacionales muestran que las cesáreas se realizan en Ecuador con una frecuencia mayor a la tasa recomendada por la Organización Mundial de la Salud, especialmente en el sistema privado de atención de salud. Los centros privados también notifican tasas más altas de sufrimiento fetal agudo, lo que implica que este diagnóstico se utiliza excesivamente en los centros privados o no se lo reconoce sufi- cientemente en los centros públicos. Aunque varios factores podrían estar influyendo sobre estas tendencias, no se tienen datos para determinar la importancia relativa de los factores económicos, la practicidad y las inquietudes médicas o personales de las madres y los médicos al decidir el método de parto.

Oxidative stress and altered steroidogenesis in the ovary by cholinergic stimulation of coeliac ganglion in the first proestrous in rats. Implication of nitric oxide.

An ex-vivo Coeliac Ganglion-Superior Ovarian Nerve-Ovary (CG-SON-O) system from virgin rats in the first proestrous was used to test whether cholinergic stimulation of CG affects oxidative status and steroidogenesis in the ovary. The CG and the O were placed in separate buffered-compartments, connected by the SON, and the CG was stimulated by acetylcholine (Ach). To test a possible role of nitric oxide (NO) in the ovarian response to cholinergic stimulation of CG, aminoguanidine (AG) - an inhibitor of inducible-NO synthase was added to the O compartment. After 180 min incubation, the oxidative status was assessed in O whereas nitrite and steroidogenesis were assessed at 30, 120 and 180 min. Ach in CG decreased the total antioxidant capacity, but increased NO production and protein carbonization in O. Ach stimulation of CG increased estradiol, but decreased progesterone release in O by reducing the mRNAs related to their synthesis and degradation. The addition of AG to the O compartment caused an opposite effect, which was more pronounced in the presence of Ach in the CG compartment than in its absence. These results show that the stimulation of the extrinsic-cholinergic innervation of the O increases the concentration of NO, causes oxidative stress and modulates steroidogenesis in the first rat proestrous.

[Creutzfeldt-Jakob disease: Report of one case]. / Desafíos en el diagnóstico de enfermedad de Creutzfeldt-Jakob: Caso clínico.

Creutzfeldt-Jakob disease has a higher incidence in Chile than in other countries. The post mortem pathological characterization of brain tissue is necessary to reach a definitive diagnosis. We report a 73 years old man with a history compatible with of a rapidly progressive dementia, in which the first electroencephalographic study showed a pattern consistent with non-convulsive status epilepticus. Besides discarding this diagnosis, it was necessary to rule out other causes of rapidly progressive dementia such as Hashimoto encephalopathy. Finally, the sustained clinical deterioration with no response to anticonvulsants and corticosteroids, the imaging studies, a serial electroencephalographic monitoring study and the detection of 14-3-3 protein in cerebrospinal fluid were the keys to achieve the diagnosis of the disease.

Immuno-spin trapping from biochemistry to medicine: advances, challenges, and pitfalls. Focus on protein-centered radicals.

BACKGROUND: Immuno-spin trapping (IST) is based on the reaction of a spin trap with a free radical to form a stable nitrone adduct, followed by the use of antibodies, rather than traditional electron paramagnetic resonance spectroscopy, to detect the nitrone adduct. IST has been successfully applied to mechanistic in vitro studies, and recently, macromolecule-centered radicals have been detected in models of drug-induced agranulocytosis, hepatotoxicity, cardiotoxicity, and ischemia/reperfusion, as well as in models of neurological, metabolic and immunological diseases. SCOPE OF THE REVIEW: To critically evaluate advances, challenges, and pitfalls as well as the scientific opportunities of IST as applied to the study of protein-centered free radicals generated in stressed organelles, cells, tissues and animal models of disease and exposure. MAJOR CONCLUSIONS: Because the spin trap has to be present at high enough concentrations in the microenvironment where the radical is formed, the possible effects of the spin trap on gene expression, metabolism and cell physiology have to be considered in the use of IST and in the interpretation of results. These factors have not yet been thoroughly dealt with in the literature. GENERAL SIGNIFICANCE: The identification of radicalized proteins during cell/tissue response to stressors will help define their role in the complex cellular response to stressors and pathogenesis; however, the fidelity of spin trapping/immuno-detection and the effects of the spin trap on the biological system should be considered. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Combined molecular MRI and immuno-spin-trapping for in vivo detection of free radicals in orthotopic mouse GL261 gliomas.

Free radicals play a major role in gliomas. By combining immuno-spin-trapping (IST) and molecular magnetic resonance imaging (mMRI), in vivo levels of free radicals were detected within mice bearing orthotopic GL261 gliomas. The nitrone spin trap DMPO (5,5-dimethyl pyrroline N-oxide) was administered prior to injection of an anti-DMPO probe (anti-DMPO antibody covalently bound to a bovine serum albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent) to trap tumor-associated free radicals. mMRI detected the presence of anti-DMPO adducts by either a significant sustained increase (p<0.001) in MR signal intensity or a significant decrease (p<0.001) in T1 relaxation, measured as %T1 change. In vitro assessment of the anti-DMPO probe indicated a significant decrease (p<0.0001) in T1 relaxation in GL261 cells that were oxidatively stressed with hydrogen peroxide, compared to controls. The biotin moiety of the anti-DMPO probe was targeted with fluorescently-labeled streptavidin to locate the anti-DMPO probe in excised brain tissues. As a negative control a non-specific IgG antibody covalently bound to the albumin-Gd-DTPA-biotin construct was used. DMPO adducts were also confirmed in tumor tissue from animals administered DMPO, compared to non-tumor brain tissue. GL261 gliomas were found to have significantly increased malondialdehyde (MDA) protein adducts (p<0.001) and 3-nitrotyrosine (3-NT) (p<0.05) compared to normal mouse brain tissue, indicating increased oxidized lipids and proteins, respectively. Co-localization of the anti-DMPO probe with either 3-NT or 4-hydroxynonenal was also observed. This is the first report regarding the detection of in vivo levels of free radicals from a glioma model.

[Adverse socio-economic conditions associated with the presence of obesity and its metabolic comorbidities in adults in San Luis, Argentina ]. / Bajo nivel de instrucción asociado a la presencia de obesidad y sus comorbilidades metabólicas en adultos de Argentina.

The prevalence of obesity depends on biopsychosocial and environmental factors and represents a risk factor for communicable and non-communicable diseases. Objectives: To determine the association between demographic, socioeconomic and lifestyle characteristics and the presence of obesity and its metabolic comorbidities (MC) in adults in San Luis City, Argentina. Observational population-based cross-sectional study of 306 individuals aged 18-85 years from San Luis, Argentina, selected by multistage random sampling, with an overweight prevalence of 35% and a 0.05 margin of error. Socioeconomic, demographic, and lifestyle variables were assessed, and multiple logistic regression models were fitted with the presence of obesity and MC as outcomes and sociodemographic and lifestyle characteristics as covariates. Obesity was found in 17.3% of participants, diabetes in 3%, high blood pressure (HBP) in 11%, dyslipidemia in 3.3% and coronary ischemic complications (CIC) in 13%. The proportion of residents with at least one of these conditions was 26.8%. Low Educational level (EL) was positively associated with the presence of obesity (OR 3.58; IC95% 1.04-12.24; p=0,04), and its MC (OR 5.25; IC95% 1.05-26.23; p=0.04) with respect to high EL. Similarly, the possibility of presenting CIC was increased in people with medium EL (OR 5.8; IC95% 1.12-30.19; p=0.03). On the other hand, the possibility of presenting diabetes increased by 17% with increasing body mass index (BMI) (OR 1.17; IC95% 1.03-1.34; p=0.01). Finally, women were more likely to present HBP (OR 3.71; IC95% 1.01-13.72; p=0.04) and CIC (OR 3,43; IC95% 1,06-11,10; p=0,03). Conclusion: the increase in age, female sex and medium and low NI are factors and conditions of vulnerability that predispose an increase in the prevalence of MC in adults from San Luis, Argentina.

La prevalencia de obesidad depende de factores biopsicosociales y ambientales, y es un factor de riesgo para enfermedades transmisibles y no transmisibles. Objetivo: Determinar la asociación entre las características demográficas, socioeconómicas y del estilo de vida, y la presencia de obesidad y sus comorbilidades metabólicas (CM) en adultos de la ciudad de San Luis, Argentina. Estudio observacional-poblacional-transversal en 306 individuos entre 18 y 85 años de San Luis, Argentina, seleccionados mediante un muestreo aleatorio multietápico, considerando una prevalencia de sobrepeso del 35% y un error de 0,05. Se indagaron variables socioeconómicas, demográficas, del estilo de vida, y se ajustaron modelos de regresión logística múltiple incluyendo presencia de obesidad y CM como variable de respuesta, y características sociodemográficas y del estilo de vida como covariables. El 17,3% de los participantes presentó obesidad, un 3% diabetes, el 11% hipertensión arterial (HTA), el 3,3% dislipidemia y un 13% complicaciones isquémicas coronarias (CIC). La proporción de habitantes con al menos una de esas patologías fue del 26,8%. Un bajo Nivel de instrucción (NI) se asoció positivamente con la presencia de obesidad (OR 3,58; IC95% 1,04-12,24; p=0,04), y sus CM (OR 5,25; IC95% 1,05-26,23; p=0,04) respecto al NI alto. Asimismo, la posibilidad de presentar CIC se vio aumentada en personas con NI medio (OR 5,8; IC95% 1,12-30,19; p=0,03). Por otro lado, la posibilidad de presentar diabetes aumenta un 17% a medida que aumenta el índice de masa corporal (IMC) (OR 1,17; IC95% 1,03-1,34; p=0,01). Finalmente, las mujeres fueron más propensas a presentar HTA (OR 3,71; IC95% 1,01-13,72; p=0,04) y CIC (OR 3,43; IC95% 1,06-11,10; p=0,03). Conclusión: el aumento en la edad, el sexo femenino y el NI medio y bajo son factores y condiciones de vulnerabilidad que predisponen un aumento en la prevalencia de CM en adultos de San Luis, Argentina.

An intracerebroventricular injection of AΒ (1-42) modifies temporal profiles of spatial memory performance and oxidative status in the temporal cortex rat.

Alzheimer's dementia (AD) is a neurodegenerative disorder that causes memory loss and dementia in older adults. Intracellular accumulation of Aß causes an imbalance in the oxidative status and cognitive dysfunctions. Besides oxidative stress and loss of memory, Alzheimer's patients show dysfunction of the circadian rhythms. The objective of this work was to evaluate the consequences of an intracerebroventricular injection of Aß (1-42) on temporal patterns of cognitive performance, as well as on lipid peroxidation, protein oxidation and total antioxidant capacity levels, in the rat temporal cortex. Holtzman male rats from control and Aß-injected groups were used in this study. We found that MDA, protein carbonyls and total antioxidant capacity levels displayed day-night oscillations in the rat temporal cortex and spatial memory performance also varied rhythmically. An intracerebroventricular injection of Aß (1-42) modified temporal patterns of cognitive performance as well as daily profiles of parameters of oxidative stress. Thus, elevated levels of Aß aggregates induces alterations in daily rhythmicity of parameters of oxidative stress and, consequently, would affect cellular clock activity, affecting the spatial memory performance in the AD.

Hypothyroidism modifies lipid composition of polymorphonuclear leukocytes.

Thyroid hormones are important regulators of lipid metabolism. Polymorphonuclear leukocytes (PMN) are essential components of innate immune response. Our goal was to determine whether hypothyroidism affects lipid metabolism in PMN cells. Wistar rats were made hypothyroid by administrating 0.1 g/L 6-propyl-2-thiouracil (PTU) in drinking water during 30 days. Triacylglycerides (TG), cholesterol and phospholipids were determined in PMN and serum by conventional methods. The mRNA expression of LDL receptor (LDL-R), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoAR), sterol regulatory element binding protein 2 (SREBP-2), and diacylglycerol acyltransferase 2 (DGAT-2) were quantified by Real-Time PCR. Cellular neutral lipids were identified by Nile red staining. We found hypothyroidism decreases serum TG whereas it increases them in PMN. This result agrees with those observed in Nile red preparations, however DAGT-2 expression was not modified. Cholesterol synthesizing enzyme HMGCoAR mRNA and protein was reduced in PMN of hypothyroid rats. As expected, cholesterol content decreased in the cells although it increased in serum. Hypothyroidism also reduced relative contents of palmitic, stearic, and arachidonic acids, whereas increased the myristic, linoleic acids, and the unsaturation index in PMN. Thus, hypothyroidism modifies PMN lipid composition. These findings would emphasize the importance of new research to elucidate lipid-induced alterations in specific function(s) of PMN.

Transnasal Humidified Rapid Insufflation Ventilatory Exchange in Laryngotracheal Resection and Reconstruction: A Report of Two Pregnant Cases.

Introduction: The incidence of tracheal stenosis is progressively increasing. A risk factor for developing this clinical condition is a history of prolonged endotracheal intubation. A transnasal humidified rapid insufflation ventilatory exchange, known as THRIVE, has gained importance in tracheal resection surgeries. Case Presentation: Herein, we describe the anesthetic management of two obstetric patients, a 19-year-old and 29-year-old, with a history of prolonged endotracheal intubation and a diagnosis of tracheal stenosis. The patients required the resection of the tracheal segment and end-to-end anastomosis. The anesthetic management focused on THRIVE using a high-flow nasal cannula. Conclusions: This system proved to be a safe anesthetic technique for pregnant women and the fetus. Furthermore, it allowed surgeons to better visualize the surgical field without the risk of accidental injury to the endotracheal tube.

Protein Radical Formation Resulting from Eosinophil Peroxidase-catalyzed Oxidation of Sulfite.

Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of bisulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical ((.)SO(3)(-)). This free radical further reacts with oxygen to form peroxymonosulfate anion radical ((-)O(3)SOO(.)) and the very reactive sulfate anion radical (SO(4)()), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H(2)O(2) is able to oxidize bisulfite, ultimately forming the sulfate anion radical (SO(4)()), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H(2)O(2) in the presence of bisulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H(2)O(2) induced the formation of radical-derived DMPO spin-trapped human serum albumin and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in bisulfite-exacerbated eosinophilic inflammatory disorders.

Myeloperoxidase-induced genomic DNA-centered radicals.

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.

Detection of Vibrio cholerae aDNA in human burials from the fifth cholera pandemic in Argentina (1886-1887 AD).

OBJECTIVE: Detecting traces of ancient DNA of Vibrio cholerae to provide genetic information associated with the fifth cholera pandemic. MATERIALS: Sediment samples from the sacral foramina of four individuals were analyzed, recovered from a mass grave near an institution dedicated exclusively to the isolation and treatment of citizens infected with cholera in the late 19th century in the city of Cordoba, Argentina. METHODS: Paleogenetic techniques (ancient DNA extraction, PCR amplification, and Sanger sequencing) were applied. Specific primers for Vibrio cholerae (VCR, ctxA, ctxB, and tcpA) were designed. RESULTS: By amplifying and sequencing the Vibrio cholerae repeats fragment, the infection in at least one individual was confirmed. CONCLUSIONS: The synthesis of the paleogenetic results with the archaeological and historical evidence strongly supports that at least one individual from the mass grave in Cordoba, Argentina, was a victim of the fifth cholera pandemic. SIGNIFICANCE: Confirming the presence of the disease through multiple lines of evidence, including genetic, archaeological, and historical analyses, strengthens and affirms our understanding of the presence, effects, and potential evolutionary paths of the disease in the past. LIMITATIONS: Vibrio cholerae repeats were sequenced in one individual, while the remaining genes could not be amplified, which is likely related to gene copy number. SUGGESTIONS FOR FUTURE RESEARCH: Paleogenetic examination of ancient samples from different locations will broaden our understanding of the origin, evolution, and past dissemination of Vibrio cholerae epidemic strains.

An intracerebroventricular injection of amyloid-beta peptide (1-42) aggregates modifies daily temporal organization of clock factors expression, protein carbonyls and antioxidant enzymes in the rat hippocampus.

Alzheimer disease (AD) is the most frequent form of dementia in the elderly. It is characterized by the deterioration of memory and learning. The histopathological hallmarks of AD include the presence of extracellular deposits of amyloid beta peptide, intracellular neurofibrillary tangles, neuron and synapse loss, in the brain, including the hippocampus. Accumulation of Aß peptide causes an increase in intracellular reactive oxygen species (ROS) and free radicals associated to a deficient antioxidant defense system. Besides oxidative stress and cognitive deficit, AD patients show alterations in their circadian rhythms. The objective of this work was to investigate the effects of an intracerebroventricular injection of amyloid beta peptide Aß(1-42) aggregates on temporal patterns of protein oxidation, antioxidant enzymes and clock factors in the rat hippocampus. Four-month-old male Holtzman rats divided into the groups control (CO) and Aß-injected (Aß), were maintained under 12 h-light12h-dark conditions and received water and food ad-libitum. Hippocampus samples were isolated every 6 h during a 24 h period. Our results showed daily patterns of protein carbonyls, catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as Rorα and Rev-erbß mRNA, in the rat hippocampus. Interestingly, an intracerebroventricular injection of Aß aggregates modified daily oscillation of protein carbonyls levels, phase-shifted daily rhythms of clock genes and had a differential effect on the daily expression and activity of CAT and GPx. Thus, Aß aggregates might affect clock-mediated transcriptional regulation of antioxidant enzymes, by affecting the formation of BMAL1:CLOCK heterodimer, probably, as a consequence of the alteration of the redox state observed in rats injected with Aß.

Effect of an Intracerebroventricular Injection of Aggregated Beta-amyloid (1-42) on Daily Rhythms of Oxidative Stress Parameters in the Prefrontal Cortex.

Accumulation of amyloid peptides in the brain plays a key role in the pathogenesis of Alzheimer's disease (AD). Aggregated beta-amyloid (Aß) peptide increases intracellular reactive oxygen species associated to a deficient antioxidant defense system. Prefrontal cortex plays a key role in memory and learning and is especially susceptible to oxidative stress. The objective of this work was to investigate the effects of an intracerebroventricular (i.c.v.) injection of Aß (1-42) on 24 h patterns of oxidative stress parameters and antioxidant defenses in the rat prefrontal cortex. Four-month-old male Holtzman rats were divided into two groups defined as: control (CO) and Aß-injected (Aß). Rats were maintained under12 h-light:12 h-dark conditions and received water and food ad libitum. Tissues samples were isolated every 6 h during a 24 h period. Interestingly, we found that an i.c.v. injection of Aß(1-42) increased lipid peroxidation, reduced total antioxidant capacity level, phase-shifted the daily peak of reduced glutathione, and had a differential effect on the oscillating catalase and glutathione peroxidase specific activity. Thus, elevated levels of Aß aggregates-a pathogenic hallmark of AD, caused altered temporal patterns of the cellular redox state in prefrontal cortex rat. These findings might contribute, at least in part, to the understanding of the molecular and biochemical basis of redox changes caused by circadian rhythms alterations observed in AD patients.

New Records of Anopheles benarrochi B (Diptera: Culicidae) in Malaria Hotspots in the Amazon Regions of Ecuador and Peru.

The increase in malaria transmission in the Amazon region motivated vector control units of the Ministry of Health of Ecuador and Peru to investigate Anopheles (Diptera: Culicidae) species present in transmission hotspots. Mosquitoes were collected using prokopack aspirators and CDC light traps (Ecuador) and human landing catch in Peru. In Ecuador, 84 Anopheles were captured from Pastaza, Morona Santiago, and Orellana provinces and identified morphologically [An. (An.) apicimacula Dyar and Knab, An. (Nys.) near benarrochi, An. (Nys.) near oswaldoi, An. (Nys.) near strodei, An. (An.) nimbus (Theobald, 1902), and An. (Nyssorhynchus) sp.]. In Peru, 1,150 Anopheles were collected in Andoas District. A subsample of 166 specimens was stored under silica and identified as An. near oswaldoi, An. darlingi, and An. (An.) mattogrossensis Lutz and Neiva. COI barcode region sequences were obtained for 137 adults (107 from Peru, 30 from Ecuador) identified by ITS2 PCR-RFLP as An. benarrochi Gabaldon, Cova Garcia, and Lopez and retained in the final analysis. Haplotypes from the present study plus An. benarrochi B GenBank sequences grouped separately from Brazilian An. benarrochi GenBank sequences by 44 mutation steps, indicating that the present study specimens were An. benarrochi B. Our findings confirm the presence of An. benarrochi B in Ecuador and reported here for the first time from the Amazonian provinces of Orellana and Morona Santiago. Furthermore, we confirm that the species collected in Andoas District in the Datem del Maranon Province, Peru, is An. benarrochi B, and we observed that it is highly anthropophilic. Overall, the known distribution of An. benarrochi B has been extended and includes southern Colombia, much of Peru and eastern Ecuador.

Cu,Zn-superoxide dismutase-driven free radical modifications: copper- and carbonate radical anion-initiated protein radical chemistry.

The understanding of the mechanism, oxidant(s) involved and how and what protein radicals are produced during the reaction of wild-type SOD1 (Cu,Zn-superoxide dismutase) with H2O2 and their fate is incomplete, but a better understanding of the role of this reaction is needed. We have used immuno-spin trapping and MS analysis to study the protein oxidations driven by human (h) and bovine (b) SOD1 when reacting with H2O2 using HSA (human serum albumin) and mBH (mouse brain homogenate) as target models. In order to gain mechanistic information about this reaction, we considered both copper- and CO3(*-) (carbonate radical anion)-initiated protein oxidation. We chose experimental conditions that clearly separated SOD1-driven oxidation via CO(*-) from that initiated by copper released from the SOD1 active site. In the absence of (bi)carbonate, site-specific radical-mediated fragmentation is produced by SOD1 active-site copper. In the presence of (bi)carbonate and DTPA (diethylenetriaminepenta-acetic acid) (to suppress copper chemistry), CO(*-) produced distinct radical sites in both SOD1 and HSA, which caused protein aggregation without causing protein fragmentation. The CO(*-) produced by the reaction of hSOD1 with H2O2 also produced distinctive DMPO (5,5-dimethylpyrroline-N-oxide) nitrone adduct-positive protein bands in the mBH. Finally, we propose a biochemical mechanism to explain CO(*-) production from CO2, enhanced protein radical formation and protection by (bi)carbonate against H2O2-induced fragmentation of the SOD1 active site. Our present study is important for establishing experimental conditions for studying the molecular mechanism and targets of oxidation during the reverse reaction of SOD1 with H2O2; these results are the first step in analysing the critical targets of SOD1-driven oxidation during pathological processes such as neuroinflammation.

Pulmonary Neutrophilic Inflammation and Noncommunicable Diseases: Pathophysiology, Redox Mechanisms, Biomarkers, and Therapeutics.

Significance: Pulmonary neurophilic inflammation (PNI) is the homing and activation of neutrophil with damage to the microvasculature. This process is involved in pulmonary damage in patients exposed to airborne pollutants (exogenous stressors) and also to systemic inflammation/oxidative stress (endogenous stressors) associated with noncommunicable diseases (NCDs). Recent Advances: PNI is an important trigger of the early onset and progression of NCD in susceptible patients exposed to airborne pollutants. Irritation of the lung microvasculature by exogenous and endogenous stressors causes PNI. Circulating endogenous stressors in NCD can cause PNI. Critical Issues: Air pollution-triggered PNI causes increased circulating endogenous stressors that can trigger NCD in susceptible patients. Systemic inflammation/oxidative stress associated with NCD can cause PNI. Inflammation/end-oxidation products of macromolecules are also potential biomarkers and therapeutic targets for NCD-triggered PNI- and PNI-triggered NCD. Future Directions: Understanding the molecular mechanism of PNI triggered by exogenous or endogenous stressors will help explain the early onset of NCD in susceptible patients exposed to air pollution. It can also help undercover biomarkers and mechanism-based therapeutic targets in air pollutant-triggered PNI, PNI-triggered NCD, and NCD-triggered PNI.

Polyclonal antibody production anti Pc_312-324 peptide. Its potential use in electrochemical immunosensors for transgenic soybean detection.

A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean.