Highly Sensitive Neurotransmitters Analysis at Platinum-Ultramicroelectrodes Arrays

An easy way to determine norepinephrine (NE) in biological fluid using a platinum ultramicroelectrode array (Pt-UMEAs) is described. Issues related to UME electrode surface treatment and characterizations are also addressed. At optimized experimental conditions the dynamic concentration range was 1.0 to 10.0 mu mol?L-1 with a detection limit of 40.5 nmol?L-1. The repeatability of current responses for injections of 5 mu mol?L-1 NE was evaluated to be 4.0?% (n=10). This approach obtained excellent sensitivity, a reliable calibration profile and stable electrochemical response for norepinephrine detection. The content of NE in urine samples without any preconcentration, purification, or pretreatment step, was successfully analyzed by the standard addition method using the Pt-UMEAs.

Placentation in the anteaters Myrmecophaga tridactyla and Tamandua tetradactyla (Eutheria, Xenarthra)

Background: Since Xenarthra are serious candidates for being basal to Eutheria, their characteristics, e.g. the placental system, influence perceptions of evolution. However, in the subgroup containing the anteaters, data are very limited. The present study aims to elucidate the nature of the feto-maternal interface in the anteater placenta and to interpret these data within an evolutionary context. Methods: Placentas of two species were investigated with histology, immunohistochemistry and transmission electron microscopy. Results: Remnants of the maternal vessel endothelium were absent, resulting in a fully haemochorial barrier throughout the placenta. Two structurally different parts, the villous and trabecular areas were complex and intermingled. In particular, the trabeculae which consisted of cellular, proliferative trophoblast, associated with connective tissue, were attached to the decidua. The villi contained fetal capillaries and hypertrophied mesenchymal cells that occured near the surface near the end of gestation. The surface of the villi consisted of flat, syncytial trophoblast, interspersed with proliferative trophoblast cells. Conclusions: Based on fundamental differences between anteaters and armadillos, we inferred that placental evolution was more complex than previously thought. The haemochorial pattern of anteaters was likely an ancient condition of xenarthrans. Consequently, villous placentation may be attributed, at least in part, by convergent evolution, but was also characterized by some features that were widespread among xenarthrans.

Efficient RNAi-induced Protein Knockdown in Somatic Cells Using Diced or Chemically Produced Small Interfering RNAs (siRNA)

Background: RNA interference (RNAi) is a post-transcriptional gene silencing process in which double-stranded RNA (dsRNA) directs the degradation of a specific corresponding target mRNA. The mediators of this process are small dsRNAs of approximately 21 to 23 bp in length, called small interfering RNAs (siRNAs), which can be prepared in vitro and used to direct the degradation of specific mRNAs inside cells. Hence, siRNAs represent a powerful tool to study and control gene and cell function. Rapid progress has been made in the use of siRNA as a means to attenuate the expression of any protein for which the cDNA sequence is known. Individual siRNAs can be chemically synthesized, in vitro-transcribed, or expressed in cells from siRNA expression vectors. However, screening for the most efficient siRNAs for post-transcriptional gene silencing in cells in culture is a laborious and expensive process. In this study, the effectiveness of two siRNA production strategies for the attenuation of abundant proteins for DNA repair were compared in human cells: (a) the in vitro production of siRNA mixtures by the Dicer enzyme (Diced siRNAs); and (b) the chemical synthesis of very specific and unique siRNA sequences (Stealth RNai (TM)). Materials, Methods & Results: For in vitro-produced siRNAs, two segments of the human Ku70 (167 bp in exon 5; and 249 bp in exon 13; NM001469) and Xrcc4 (172 bp in exon 2; and 108 bp in exon 6; NM003401) genes were chosen to generate dsRNA for subsequent "Dicing" to create mixtures of siRNAs. The Diced fragments of siRNA for each gene sequence were pooled and stored at -80 degrees C. Alternatively, chemically synthesized Stealth siRNAs were designed and generated to match two very specific gene sequence regions for each target gene of interest (Ku70 and Xrcc4). HCT116 cells were plated at 30% confluence in 24- or 6-well culture plates. The next day, cells were transfected by lipofection with either Diced or Stealth siRNAs for Ku70 or Xrcc4, in duplicate, at various doses, with blank and sham transfections used as controls. Cells were harvested at 0, 24, 48, 72 and 96 h post-transfection for protein determination. The knockdown of specific targeted gene products was quantified by Western blot using GAPDH as control. Transfection of gene-specific siRNA to either Ku70 or Xrcc4 with both Diced and Stealth siRNAs resulted in a down regulation of the targeted proteins to approximately 10 to 20% of control levels 48 h after transfection, with recovery to pre-treatment levels by 96 h. Discussion: By transfecting cells with Diced or chemically synthesized Stealth siRNAs, Ku70 and Xrcc4, two highly expressed proteins in cells, were effectively attenuated, demonstrating the great potential for the use of both siRNA production strategies as tools to perform loss of function experiments in mammalian cells. In fact, down-regulation of Ku70 and Xrcc4 has been shown to reduce the activity of the non-homologous end joining DNA pathway, a very desirable approach for the use of homologous recombination technology for gene targeting or knockout studies. Stealth RNAi (TM) was developed to achieve high specificity and greater stability when compared with mixtures of enzymatically-produced (Diced) siRNA fragments. In this study, both siRNA approaches inhibited the expression of Ku70 and Xrcc4 gene products, with no detectable toxic effects to the cells in culture. However, similar knockdown effects using Diced siRNAs were only attained at concentrations 10-fold higher than with Stealth siRNAs. The application of RNAi technology will expand and continue to provide new insights into gene regulation and as potential applications for new therapies, transgenic animal production and basic research.

Effects of Positive End-expiratory Pressure Titration and Recruitment Maneuver on Lung Inflammation and Hyperinflation in Experimental Acid Aspiration-induced Lung Injury

Background: In acute lung injury positive end-expiratory pressure (PEEP) and recruitment maneuver are proposed to optimize arterial oxygenation. The aim of the study was to evaluate the impact of such a strategy on lung histological inflammation and hyperinflation in pigs with acid aspiration-induced lung injury. Methods: Forty-seven pigs were randomly allocated in seven groups: (1) controls spontaneously breathing; (2) without lung injury, PEEP 5 cm H2O; (3) without lung injury, PEEP titration; (4) without lung injury, PEEP titration + recruitment maneuver; (5) with lung injury, PEEP 5 cm H2O; (6) with lung injury, PEEP titration; and (7) with lung injury, PEEP titration + recruitment maneuver. Acute lung injury was induced by intratracheal instillation of hydrochloric acid. PEEP titration was performed by incremental and decremental PEEP from 5 to 20 cm H2O for optimizing arterial oxygenation. Three recruitment maneuvers (pressure of 40 cm H2O maintained for 20 s) were applied to the assigned groups at each PEEP level. Proportion of lung inflammation, hemorrhage, edema, and alveolar wall disruption were recorded on each histological field. Mean alveolar area was measured in the aerated lung regions. Results: Acid aspiration increased mean alveolar area and produced alveolar wall disruption, lung edema, alveolar hemorrhage, and lung inflammation. PEEP titration significantly improved arterial oxygenation but simultaneously increased lung inflammation in juxta-diaphragmatic lung regions. Recruitment maneuver during PEEP titration did not induce additional increase in lung inflammation and alveolar hyperinflation. Conclusion: In a porcine model of acid aspiration-induced lung injury, PEEP titration aimed at optimizing arterial oxygenation, substantially increased lung inflammation. Recruitment maneuvers further improved arterial oxygenation without additional effects on inflammation and hyperinflation.

Electro-oxidation of ethanol on Pt/C, Rh/C, and Pt/Rh/C-based electrocatalysts investigated by on-line DEMS

The ethanol electro-oxidation reaction was studied on carbon-supported Pt, Rh, and on Pt overlayers deposited on Rh nanoparticles. The synthesized electrocatalysts were characterized by TEM and XRD. The reaction products were monitored by on-line DEMS experiments. Potentiodynamic curves showed higher overall reaction rate for Pt/C when compared to that for Rh/C. However, on-line DEMS measurements revealed higher average current efficiencies for complete ethanol electro-oxidation to CO2 on Rh/C. The average current efficiencies for CO2 formation increased with temperature and with the decrease in the ethanol concentration. The total amount of CO2, on the other hand, was slightly affected by the temperature and ethanol concentration. Additionally, the CO2 signal was observed only in the positive-going scan, none being observed in the negative-going scan, evidencing that the C-C bond breaking occurs only at lower potentials. Thus, the formation of CO2 mainly resulted from oxidative removal of adsorbed CO and CHx,ad species generated at the lower potentials, instead of the electrochemical oxidation of bulk ethanol molecules. The acetaldehyde mass signal, however, was greatly favored after increasing the ethanol concentration from 0.01 to 0.1 mol L-1, on both electrocatalysts, indicating that it is the major reaction product. For the Pt/Rh/C-based electrocatalysts, the Faradaic current and the conversion efficiency for CO2 formation was increased by adjusting the amount of Pt on the surface of the Rh/C nanoparticles. The higher conversion efficiency for CO2 formation on the Pt1Rh/C material was ascribed to its faster and more extensive ethanol deprotonation on the Pt-Rh sites, producing adsorbed intermediates in which the C-C bond cleavage is facilitated. (C) 2012 Elsevier B.V. All rights reserved.

Real-time electrochemical determination of phenolic compounds after benzene oxidation

A sensitive electrochemical sensor was successfully developed on multi-walled carbon nanotubes (MWCNT) and cobalt phthalocyanine (CoPc) modified glassy carbon electrode (GC), and used to detect byproducts formed after the electrolysis of benzene. The GC/MWCNT/CoPc electrode was applied in the detection of phenolic compounds using square wave voltammetry (SWV). The proposed sensor exhibited a sequence in the sensitivity of the tested phenols: catechol > hydroquinone > resorcinol > phenol and 1,4-benzoquinone. The detection limits for individual phenols were also calculated: catechol (15.62 mu g L-1), hydroquinone (17.91 mu g L-1), resorcinol (46.12 mu g L-1), phenol (58.83 mu g L-1) and 1,4-benzoquinone (13.75 mu g L-1). The proposed sensor was successfully applied in the determination of the total amount of phenols formed after the benzene oxidation, and the obtained results were in full agreement with those from the HPLC procedure. (C) 2012 Elsevier B.V. All rights reserved.

Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Use of Hg-Electroplated-Pt Ultramicroelectrode for Determining Elemental Sulphur in Naphtha Samples

This paper describes the applicability of a Hg-electroplated-Pt ultramicroelectrode in the quantification of elemental sulphur in naphtha samples by square-wave voltammetry. A reproducible deposition methodology was studied and is reported in this paper. This methodology is innovative and relies on the quality of the mercury stock solution to obtain reproducible surfaces required for the analytical methodology. All analyses were performed using a Hg-electroplated-Pt ultramicroelectrode (Hg-Pt UME) due to the low sensibility of such devices to ohmic drops in resistive solutions. The responses of the peak areas in voltammetric experiments were linear in all of the range studied. The method developed here is accurate and reproducible, with a detection limit of 0.010 mg L-1 and a good recovery range for both standard solutions of elemental sulphur (85 to 99%) and real naphtha sample (79%). These results attest to the potential for the application of this electroanalytical methodology in determining elemental sulphur in naphtha samples containing mercaptans and disulphides.

Description of atmospheric conditions at the Pierre Auger Observatory using the Global Data Assimilation System (GDAS)

Atmospheric conditions at the site of a cosmic ray observatory must be known for reconstructing observed extensive air showers. The Global Data Assimilation System (GDAS) is a global atmospheric model predicated on meteorological measurements and numerical weather predictions. GDAS provides altitude-dependent profiles of the main state variables of the atmosphere like temperature, pressure, and humidity. The original data and their application to the air shower reconstruction of the Pierre Auger Observatory are described. By comparisons with radiosonde and weather station measurements obtained on-site in Malargue and averaged monthly models, the utility of the GDAS data is shown. (C) 2012 Elsevier B.V. All rights reserved.

Importance of anterior segment tomography for the diagnosis of corneal ectasia

Purpose: The methodology currently used for interpretation of the cornea and anterior segment tomography for the diagnosis of corneal ectasia and its susceptibility. Methods: Description of the methodology and clinical interpretation of corneal and anterior segment tomography indexes; report of three cases demonstrating the importance of this new diagnostic tool (Pentacam HR (R)) in ophthalmological practice. Conclusion: The use of corneal and anterior segment tomography seems to be an effective method to increase the sensitivity and specificity for the diagnosis and early detection of corneal ectasia.

Estudo morfofuncional dos rins de cães da raça Golden Retriever afetados pela distrofia muscular (GRMD)

A Distrofia Muscular de Duchenne (DMD) é uma miopatia severa de caráter recessivo ligada ao cromossomo X e o modelo animal de estudo mais relevante é o Golden Retriever Muscular Dystrophy (GRMD). Além das severas alterações que ocorrem na musculatura estriada, muitos estudos mostram que outras estruturas, inclusive viscerais, podem se mostrar alteradas nesta patologia. Desta forma, este trabalho objetivou análisar e comparar possíveis alterações estruturais e funcionais do rim em cães GRMD. Neste modelo de estudo, foi possível observar a presença das faces convexa e côncava, do hilo renal e dos pólos craniais e caudais dos rins. O órgão mostrou-se envolto por uma cápsula fibrosa. Em um corte sagital do órgão, notou-se a presença das regiões cortical e medular e da pelve renal. Na análise microscópica foi possível identificar a zona medular e cortical com suas estruturas: os corpúsculos renais formados pelo glomérulo e pela cápsula de Bowman, os túbulos contorcidos proximais e distais, os ductos coletores, vasos sanguíneos e os segmentos das Alças de Henle. As dosagens séricas de creatinina e uréia encontram-se dentro dos limites de normalidade. Desta forma, de acordo com os nossos resultados, podemos concluir que os animais afetados estudados, não apresentaram alterações estruturais ou funcionais dos rins, o que nos permitir sugerir que apesar da ingestão hídrica comprometida, a estrutura renal, mantem- se preservada nos animais GRMD.

Antennas for the detection of radio emission pulses from cosmic-ray induced air showers at the Pierre Auger Observatory

The Pierre Auger Observatory is exploring the potential of the radio detection technique to study extensive air showers induced by ultra-high energy cosmic rays. The Auger Engineering Radio Array (AERA) addresses both technological and scientific aspects of the radio technique. A first phase of AERA has been operating since September 2010 with detector stations observing radio signals at frequencies between 30 and 80 MHz. In this paper we present comparative studies to identify and optimize the antenna design for the final configuration of AERA consisting of 160 individual radio detector stations. The transient nature of the air shower signal requires a detailed description of the antenna sensor. As the ultra-wideband reception of pulses is not widely discussed in antenna literature, we review the relevant antenna characteristics and enhance theoretical considerations towards the impulse response of antennas including polarization effects and multiple signal reflections. On the basis of the vector effective length we study the transient response characteristics of three candidate antennas in the time domain. Observing the variation of the continuous galactic background intensity we rank the antennas with respect to the noise level added to the galactic signal.

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells. Microsc. Res. Tech. 75:766770, 2012. (C) 2011 Wiley Periodicals, Inc.

Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice

Vinolo MA, Rodrigues HG, Festuccia WT, Crisma AR, Alves VS, Martins AR, Amaral CL, Fiamoncini J, Hirabara SM, Sato FT, Fock RA, Malheiros G, dos Santos MF, Curi R. Tributyrin attenuates obesity-associated inflammation and insulin resistance in high-fat-fed mice. Am J Physiol Endocrinol Metab 303: E272-E282, 2012. First published May 22, 2012; doi:10.1152/ajpendo.00053.2012.-The aim of this study was to investigate whether treatment with tributyrin (Tb; a butyrate prodrug) results in protection against diet-induced obesity and associated insulin resistance. C57BL/6 male mice fed a standard chow or high-fat diet were treated with Tb (2 g/kg body wt, 10 wk) and evaluated for glucose homeostasis, plasma lipid profile, and inflammatory status. Tb protected mice against obesity and obesity-associated insulin resistance and dyslipidemia without food consumption being affected. Tb attenuated the production of TNF alpha and IL-1 beta by peritoneal macrophages and their expression in adipose tissue. Furthermore, in the adipose tissue, Tb reduced the expression of MCP-1 and infiltration by leukocytes and restored the production of adiponectin. These effects were associated with a partial reversion of hepatic steatosis, reduction in liver and skeletal muscle content of phosphorylated JNK, and an improvement in muscle insulin-stimulated glucose uptake and Akt signaling. Although part of the beneficial effects of Tb are likely to be secondary to the reduction in body weight, we also found direct protective actions of butyrate reducing TNF alpha production after LPS injection and in vitro by LPS- or palmitic acid-stimulated macrophages and attenuating lipolysis in vitro and in vivo. The results, reported herein, suggest that Tb may be useful for the treatment and prevention of obesity-related metabolic disorders.

Descrição morfológica do diafragma do sagui-de-tufo-branco (Callithrix jacchus)

O músculo diafragma, encontrado apenas nos mamíferos, é o principal músculo no processo respiratório, servindo de fronteira entre as cavidades torácica e abdominal. Sua importância também ganha destaque em pesquisas realizadas no âmbito dos enxertos, empregando-se diversos tipos de membranas biológicas para o reparo de defeitos diafragmáticos, os quais podem gerar hérnias diafragmáticas. Apesar de muitos estudos já conduzidos para com os primatas não humanos, especialmente no que tange a espécie do novo mundo Callithrix jacchus (Sagui-de-tufo-branco), oriundo do nordeste brasileiro, as pesquisas envolvendo o uso do diafragma em tal espécie é inexistente. Deste modo objetivou-se caracterizar a morfologia e a biometria do diafragma na espécie Callithrix jacchus de ambos os sexos, analisando possíveis divergências estruturais entre machos e fêmeas. Para tal foram utilizados quatros animais, 2 machos e 2 fêmeas, adultos, que vieram a óbito por causas naturais, provenientes de um criadouro comercial. Após fixação em solução de formaldeído 10% os animais foram devidamente dissecados para fotodocumentação e em seguida o diafragma coletado para efetuação da biometria (comprimento e largura) com o uso de um paquímetro e para o processamento histológico por meio da coloração de hematoxilina-eosina e tricrômio de masson, da porção muscular. As mensurações feitas permitiram concluir que não houve diferenças signifcativas entre machos e femeas. A topografia e a presença de três aberturas (forame da veia cava caudal, hiato aórtico e esofágico) na extensão do diafragma corroboram com descrições na literatura classica para outros mamíferos. A presença de um centro tendíneo em "V" difere do encontrado para animais como o peixe-boi e porquinho-da-india, mas é similar ao encontrado para o gambá-de-orelhas-brancas e rato albino. No que diz respeito aos achados histológicos conclui-se que as fibras musculares estão dispostas de forma organizada, apresentam diâmetro grande e núcleos basais, tendo, portanto, características similares do músculo estriado esquelético tanto nos animais machos como nas fêmeas.