Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of less than or equal to200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.

Pyrazole or tris(pyrazolyl)ethanol oxo-vanadium(IV) complexes as homogeneous or supported catalysts for oxidation of cyclohexane under mild conditions

The oxovanadium(IV) complexes [VO(acac)(2)(Hpz)].HC(pz)(3) 1.HC(pz)(3) (acac= acetylacetonate, Hpz = pyrazole, pz = pyrazoly1) and [VOCl2{HOCH2C(pz)(3)}] 2 were obtained from reaction of [VO(acac)(2)] with hydrotris(1-pyrazolyl)methane or of VCl(3)with 2,2,2-tris(1-pyrazolyl)ethanol. The compounds were characterized by elemental analysis, IR, Far-IR and EPR spectroscopies, FAB or ESI mass-spectrometry and, for 1, by single crystal X-ray diffraction analysis. 1 and 2 exhibit catalytic activity for the oxidation of cyclohexane to the cyclohexanol and cyclohexanone mixture in homogeneous system (TONS up to 1100) under mild conditions (NCMe, 24h, room temperature) using benzoyl peroxide (BPO), tert-butyl hydroperoxide (TBHP), m-chloroperoxybenzoic acid (mCPBA), hydrogen peroxide or the urea-hydrogen peroxide adduct (UHP) as oxidants. 1 and 2 were also immobilized on a polydimethylsiloxane membrane (1-PDMS or 2-PDMS) and the systems acted as supported catalysts for the cyclohexane oxidation using the above oxidants (TONs up to 620). The best results were obtained with mCPBA or BP0 as oxidant. The effects of various parameters, such as the amount of catalyst, nitric acid, reaction time, type of oxidant and oxidant-to-catalyst molar ratio, were investigated, for both homogeneous and supported systems. (C) 2012 Elsevier B.V. All rights reserved.

Determination of the radiochemical purity of 99mTc-Tetrofosmin: comparision between five methods

GOAL: The manufacturing and distribution of strips of instant thin - layer chromatography with silica gel (ITLC - SG) (reference method) is currently discontinued so there is a need for an alternative method f or the determination of radiochemical purity (RCP) of 99m Tc - tetrofosmin. This study aims to compare five alternative methods proposed by the producer to determine the RCP of 99m Tc - tetrofosmin. METHODS: Nineteen vials of tetrofosmin were radiolabelled with 99m Tc and the percentages of the RCP were determined. Five different methods were compared with the standard RCP testing method (ITLC - SG, 2x20 cm): Whatman 3MM (1x10 cm) with acetone and dichloro - methane (method 1); Whatman 3MM (1x1 0 cm) with ethyl acetate (method 2); aluminum oxide - coated plastic thin - layer chromatography (TLC) plate (1x10 cm) and ethanol (method 3); Whatman 3MM (2x20 cm) with acetone and dichloro - methane (method 4); solid - phase extraction method C18 cartridge (meth od 5). RESULTS: The average values of RCP were 95,30% ± 1,28% (method 1), 93,95 ± 0,61% (method 2), 96,85% ± 0,93% (method 3), 92,94% ± 0,99% (method 4) and 96,25% ± 2,57% (method 5) (n=12 each), and 93,15% ± 1,13% for the standard method (n=19). There we re statistical significant differences in the values obtained for methods 1 (P=0,001), 3 (P=0,000) and 5 (P=0,004), and there were no statistical significant differences in the values obtained for methods 2 (P=0,113) and 4 (P=0,327). CONCLUSION: From the results obtained, methods 2 and 4 showed a higher correlation with the standard method. Unlike method 4, method 2 is less time - consuming than the reference method and can overcome the problems associated with the solvent toxicity. The remaining methods (1, 3 and 5) tended to overestimate RCP value compared to the standard method.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

Materiais e dispositivos sensores para a Norfloxacina

O trabalho consistiu no desenvolvimento e caracterização de sensores potenciométricos com base em polímeros de impressão molecular para a determinação de um antibiótico, a norfloxacina, em aquacultura. A simplicidade, o baixo custo e a interação rápida e reversível dos sensores potenciométricos com os analitos fizeram com que este fosse o tipo de sensor escolhido. O material sensor foi obtido por tecnologia de impressão molecular, baseada em polimerização em bulk, em que a NOR foi a molécula molde e foram utilizados como monómeros para autoconstrução dos sensores o pirrol, isoladamente, ou em conjunto com partículas de sílica gel funcionalizadas com 3-aminopropil. Também foi obtido material sensor, para controlo, em que a molécula molde NOR não estava presente (NIP). As características dos materiais sensores foram sujeitas a análise de microscopia eletrónica SEM e análise por espectrómetro de infravermelhos com transformada de Fourier. Os materiais sensores foram incluídos em membranas poliméricas, que seriam incorporadas em elétrodos. A avaliação do desempenho dos elétrodos foi feita através de curvas de calibração em diferentes meios (PBS, MES e HEPES). Também foi efetuada com sucesso a análise da sensibilidade dos elétrodos em água dopada. As diversas avaliações e análises efetuadas levaram a concluir que o MIP de pirrol com aditivo aniónico, foi o material sensor testado que permitiu obter melhores propriedades de resposta.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

Analysis and treatment of a nineteenth century portrait, study of Artsorb® and a proposal for a microclimate frame

The present work is divided in two parts: Part 1 is focused on the analysis and treatment of a 19th century portrait of Domingos Affonso, which belongs to the Ecomuseu Municipal do Seixal; and Part 2, which is entitled “The Microclimate Frame Project” is focused on the study of Artsorb® and on the planning of a microclimate frame for the painting. In Part 1, a study of the painting’s materials was performed using complementary analytical techniques and the painting’s condition was carefully evaluated. The painting exhibited signs of mould growth, and a more detailed investigation was made of this topic to understand if the fungal community was active and if it represented a real danger to the painting. A treatment was proposed, appropriate to the painting’s condition. A description of the treatment carried out, comprising the treatment options, is also present in this section. Within the study of the microclimate frame, in Part 2, the study of the potential corrosiveness of Artsorb® was a central subject. Artsorb® sheets are one of the most widely used materials for buffering relative humidity fluctuations in microclimate frames and its reported excellent performance is enhanced by its availability in lightweight sheets that can be easily placed inside microclimate frames. However, concerns have arisen regarding the presence of the corrosive salt lithium chloride in the composition of this buffer. Consequently, the present work also aimed to understand the potential risks of using Artsorb® and the possibility of avoiding exposure of lithium chloride to the artworks through the use of Tyvek®. Results from the preliminary tests seem to indicate that Artsorb® releases lithium chloride into air. This study also showed that a Tyvek® cover over Artsorb® reduces but does not eliminate evidence of chlorine contamination, and it significantly reduces the effectiveness of the buffering material. Considering that Artsorb® appears to be unsuitable due to the release of the corrosive salt, that Tyvek® was not efficient as a barrier for lithium chloride or as a permeable material to enable the proper functioning of Artsorb®, the buffering material proposed for the use in the microclimate frames is silica gel without indicator. Based on the choice of buffering material, as a result of this study, a microclimate frame is proposed.

EFEITO DA VELOCIDADE DE SECAGEM SOBRE A EMERGÊNCIA E VIGOR DE SEMENTES DE PUPUNHA (Bactris gasipaes Kunth)

As sementes de pupunha perdem rapidamente a viabilidade quando sua umidade é reduzida, dificultando o seu transporte e armazenamento. Para determinar o efeito da velocidade de secagem, as sementes de pupunha foram submetidas às seguintes condições: a) meio ambiente de laboratório; b) dessecador com sílical gel na proporção de 1:1 (por peso); c) dessecador com silica gel na proporção de 2:1. A cada dois dias, uma amostra de cada ambiente foi utilizada para testes de emergência e determinação da umidade. Conclui-se que: a) a viabilidade e o vigor das sementes de pupunha são dependentes da interação de sua umidade com a velocidade de secagem; b) a emergência e o vigor das sementes são afetados por pequenas reduções na sua umidade, além da velocidade com que esta é rebaixada; c) a secagem mais lenta favorece a emergência e o vigor das sementes de pupunha.

Antimicrobial activity of bergenin from Endopleura uchi (Huber) Cuatrec

Endopleura uchi (Huber) Cuatrec. is an Amazon species traditionally used as treatment for inflammations and female disorders. Bergenin was isolated from ethyl acetate fraction of bark of E. uchi by using column chromatography over sephadex LH-20 and then silica gel 60 flash. Its structure was identified on the basis of its NMR spectra. The antimicrobial activity of bergenin and fractions of methanol extract of E. uchi were evaluated against ATCC microorganisms (Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Candida albicans, C. guilliermondii, Aspergillus flavus, A. nidulans). Clinically isolated strains of all of these microorganisms, along with C. tropicalis, A. niger, Shigella sonnei, Serratia marcenses and Klebsiella pneumoniae were also evaluated. The growth inhibition caused by bergenin, extracts and fractions of E. uchi against ATCC microorganisms were similar to the inhibition to microorganisms clinically isolated. The ethyl acetate fraction and the isolate bergenin inhibit the growth of the yeasts C. albicans, C. tropicalis, and C. guilliermondii, but present lower activity against filamentous fungi Aspergillus flavus, A. nidulans, A. niger, and did not inhibit the Gram positive and Gram negative bacteria. The activity of the ethyl acetate fraction and bergenin are in agreement wit its high concentration found in bark extract of E. uchi. Moreover, the selective activity against three Candida species helps to understand its traditional use against infections that affect women.

Pigmentos de Triatomideos brasileiros

Os extratos em metanolºHCL e amônia-mercaptoetanol de conexivos de Triatomídeos examinados apresentaram espectros de absorção no visível com máximo único e nítido em 400 nm. A cromatografia em camada fina de silica-gel e em papel revelou a pesença de pterinas fluorescentes à luz ultravioleta filtrada em 365 nm a julgar pelos Rf comparados com os de pterinas padrões.

Preparació, estudi estructural i aplicabilitat de derivats de Poli-(4R)-hidroxi-L-prolina a la separació d’enantiòmers per tècniques cromatogràfiques o relacionades

La separació d’enantiòmers (isòmers òptics) és molt important en molts diversos camps, com les síntesis quirals, biologia, i en el camp de la farmacologia especialment. És per això, que es fa necessari de disposar de tècniques i mètodes analítics ràpids, fiables i sensibles per a la separació d’enantiòmers. La present tesi s’emmarca en el camp de la separació d’enantiòmers, concretament en la preparació de fases estacionàries quirals per al seu ús en cromatografia liquida. En aquest sentit, s’ha desenvolupat la síntesi i caracterització de molècules polimèriques quirals derivades de l’aminoàcid L-prolina que incorporades en matrius de gel de sílice poden constituïr columnes quirals per a la separació d’enantiòmers per cromatografia liquida. S’han estudiat les característiques enantioselectives d’aquests nous materials en la separació de molècules quirals, trobant-se ésser satisfactòriament enantioselectius. L’interès que suscita l’obtenció d’enantiòmers a gran escala fa que la recerca s’orienti a la recerca de materials amb elevada capacitat de càrrega, és a dir, que puguin donar lloc a la separació d’elevades quantitats d’enantiòmers. Amb aquesta finalitat s’han dut a terme assaigs de capacitat de càrrega, que han posat de manifest la possible aplicació d’aquests materials a la separació preparativa d’enantiòmers. També s’ha parat especial atenció a l’estudi de les característiques de la matriu de gel de sílice, assajant-se altres materials de sílice més porosos i que permeten així treballar amb fluxos més elevats tot reduint-ne el temps d’anàlisi i els costos associats a la separació preparativa d’enantiòmers. L'estudi conformacional d'aquests nous selectors també ha estat contemplat per tal d'explicar l'enantioselectivitat específica que s'observa en certs dissolvents orgànics en els qual es duu a terme la separació dels enantiòmers.

Methodology in structural determination and synthesis of insect pheromone

By means of ethereal washing of insect pheromone glands of female moths, GC-MS detection along with microchemical reactions and electroantennogram (EAG) survey, six economically important insect species were targeted for pheromone identification. The discovery of a natural pheromone inhibitor, chemo-selectivity and species isolation by pheromone will be described. The modified triple bond migration and triethylamine liganded vinyl cuprate were applied for achiral pheromone synthesis in double bond formation. Some optically active pheromones and their stereoisomers were synthesized through chiral pool or asymmetric synthesis. Some examples of chiral recognition of insects towards their chiral pheromones will be discussed. A CaH2 and silica gel catalyzed Sharpless Expoxidation Reaction was found in shortening the reaction time.

Cardiac glycosides isolated from the Indian-snuff, Maquira sclerophylla Ducke

The hydroalcoholic extract of the powdered bark of the Indian-snuff Maquira sclerophylla Ducke was purified by column chromatography in silica-gel and the major cardenolide isolated from preparative TLC was identified by 1H-NMR, 1 2 C-NMR and IR analyses. The spectra showed that the active substance has strophanthidin as aglicone.

In vitro antibacterial activity of a 7-O-beta-D-glucopyranosyl-nutanocoumarin from Chaptalia nutans (Asteraceae)

Ethanolic crude extracts from the roots of Chaptalia nutans, traditionally used in Brazilian folk medicine, were screened against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by using the disk diffusion test technique. S. aureus with 14 mm inhibition zone was considered susceptible. E. coli and P. aeruginosa without such a zone were considered resistant. As a result of this finding, the ethanolic crude extract was fractionated on silica gel column chromatography into five fractions. The ethyl acetate fraction was active against S. aureus and Bacillus subtilis. Further column chromatography separation of the ethyl acetate fraction afforded 30 fractions, which were assayed against S. aureus. Fractions 16 and 17 showed inhibition zones with S. aureus, indicating the presence of active compounds, and were subjected to purification by repeated preparative thin layer chromatography. The pure compound 7-O-beta-D-glucopyranosyl-nutanocoumarin inhibited B. subtilis and S. aureus at concentrations of 62.5 µg/ml and 125 µg/ml, respectively. The antibacterial property of C. nutans appears to have justified its use for the treatment of wounds, which are contaminated through bacterial infections.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.