In vitro study of the effect of wound dressings on planktonic and biofilms from diabetic foot isolates

The management of wound bioburden has previously been evaluated using various antimicrobial wound dressings on bacterial pathogens isolated from various wounds. In this present study, the antimicrobial effect of silver-impregnated dressings (Acticoat and Silvercel) and honey-impregnated dressing (Medihoney™ Apinate) on both planktonic bacteria and quasi-biofilms by Staphylococcus aureus and Proteus mirabilis were assessed using a 6-well plate and standard agar technique. In the 6-well plate assay, a bacterial suspension of 108 colony forming unit (CFU)/mL was inoculated on each dressing in excess Luria-Bertani broth and incubated at 35 – 37°C for 30 and 60 minutes and 24 hours. After each incubation time, bacteria were recovered in sodium thioglycolate solution (STS) and the CFU/mL determined on LB agar. Dressings were cut into circular shapes (2cm diameter and placed on Mueller Hinton agar plates pre-inoculated with bacterial suspensions to determine their zones of inhibition (ZOI) after 24 hours incubation. None of the dressings was effective to significantly inhibit bacterial growth or biofilm formation at all the times tested. Acticoat and Medihoney™ Apinate produced ZOIs between 1.5 – 15 mm against both Staphylococcus aureus and Proteus mirabilis. It is possible that, dressings augmented with antibiotics can significantly reduce quasi-biofilms on standard agar.

Deteção eletroquímica da norfloxacina e trimetoprim em amostras de urina utilizando polímero molecularmente impresso modificado e grafeno

A norfloxacina e o trimetoprim são dois antibióticos antibacterianos usados para o tratamento de infeções urinárias, intestinais e respiratórias. A maioria dos fármacos exige uma dosagem que garanta os níveis de segurança e eficácia de atuação. A necessidade de dosear os medicamentos e os seus metabolitos é assim um controlo imperioso e em muitos casos regular no tratamento de um paciente. Neste trabalho desenvolveram-se dois sensores eletroquímicos para a deteção da norfloxacina (NFX) e do trimetoprim (TMP), usando como superfície de suporte o carbono vítreo. A busca de novos materiais que conferiram maior seletividade e sensibilidade aos sistemas de deteção e por outro lado apresentem menores riscos para o paciente quando usados em dispositivos que permitam uma análise point-of-care, é especialmente importante e pode ser uma parte crucial do processo de decisão clínica. Assim, os polímeros molecularmente impresos enquadram-se nesse perfil e o seu uso tem vindo a ser cada vez mais avaliado. A impressão molecular é uma tecnologia capaz de produzir polímeros que incorporam as moléculas do analito e que após remoção por solventes específicos, permitem dotá-los de locais específicos de reconhecimento estereoquímico. A seleção do pirrol como polímero molecularmente impresso (MIP) permitiu construir com sucesso os sensores para doseamento dos antibióticos. A fim de aumentar a sensibilidade do método incorporou-se grafeno na superfície do elétrodo. Este material tem vindo a ser largamente utilizado devido às suas propriedades: estrutura molecular, condutividade elétrica e aumento da superfície são algumas das características que mais despertam o interesse para a sua aplicação neste projeto. Os sensores desenvolvidos foram incorporados em sistemas eletroquímicos. Os métodos voltamétricos aplicados foram a voltametria cíclica, a voltametria de onda quadrada e ainda a impedância. As condições de análise foram otimizadas no que respeita à polimerização do pirrol (concentração do polímero, número de ciclos de eletropolimerização e respetivos potenciais aplicados, tempo de incubação, solvente de remoção do analito), ao pH da solução do fármaco, à gama de concentrações dos antibióticos e aos parâmetros voltamétricos dos métodos de análise. Para cada um dos antibióticos um elétrodo não-impresso foi também preparado, usando o procedimento de polimerização mas sem a presença da molécula do analito, e foi usado como controlo. O sensor desenvolvido para o trimetoprim foi usado no doseamento do fármaco em amostras de urina. As amostras foram analisadas sem qualquer diluição, apenas foram centrifugadas para remoção de proteínas e algum interferente. Os sensores construídos apresentaram comportamento linear na gama de concentrações entre 102 e 107 mol/L. Os resultados mostram boa precisão (desvios padrão inferiores a 11%) e os limites de deteção foram de 8,317 e 1,307 mol/L para a norfloxacina e o trimetoprim, respetivamente. Para validação do método foram ainda efetuados ensaios de recuperação tendo obtido valores superiores a 94%.

The use of mannan antigen and anti-mannan antibodies in the diagnosis of invasive candidiasis: recommendations from the Third European Conference on Infections in Leukemia.

INTRODUCTION: Timely diagnosis of invasive candidiasis (IC) remains difficult as the clinical presentation is not specific and blood cultures lack sensitivity and need a long incubation time. Thus, non-culture-based methods for diagnosing IC have been developed. Mannan antigen (Mn) and anti-mannan antibodies (A-Mn) are present in patients with IC. On behalf of the Third European Conference on Infections in Leukemia, the performance of these tests was analysed and reviewed. METHODS: The literature was searched for studies using the commercially available sandwich enzyme-linked immunosorbent assays (Platelia™, Bio-Rad Laboratories, Marnes-la-Coquette, France) for detecting Mn and A-Mn in serum. The target condition of this review was IC defined according to 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Sensitivity, specificity and diagnostic odds ratios (DOR) were calculated for Mn, A-Mn and combined Mn/A-Mn testing. RESULTS: Overall, 14 studies that comprised 453 patients and 767 controls were reviewed. The patient populations included in the studies were mainly haematological and cancer cases in seven studies and mainly intensive care unit and surgery cases in the other seven studies. All studies but one were retrospective in design. Mn sensitivity was 58% (95% confidence interval [CI], 53-62); specificity, 93% (95% CI, 91-94) and DOR, 18 (95% CI 12-28). A-Mn sensitivity was 59% (95% CI, 54-65); specificity, 83% (95% CI, 79-97) and DOR, 12 (95% CI 7-21). Combined Mn/A-Mn sensitivity was 83% (95% CI, 79-87); specificity, 86% (95% CI, 82-90) and DOR, 58 (95% CI 27-122). Significant heterogeneity of the studies was detected. The sensitivity of both Mn and A-Mn varied for different Candida species, and it was the highest for C. albicans, followed by C. glabrata and C. tropicalis. In 73% of 45 patients with candidemia, at least one of the serological tests was positive before the culture results, with mean time advantage being 6 days for Mn and 7 days for A-Mn. In 21 patients with hepatosplenic IC, 18 (86%) had Mn or A-Mn positive test results at a median of 16 days before radiological detection of liver or spleen lesions. CONCLUSIONS: Mn and A-Mn are useful for diagnosis of IC. The performance of combined Mn/A-Mn testing is superior to either Mn or A-Mn testing.

Spezifität der quantitativen Resistenz von Blättern und Früchten der Tomate (Lycopersicon ssp. L.) gegenüber Phytophthora infestans (Mont.) de Bary.

Ziel der vorliegenden Arbeit war es, einen Beitrag zur Resistenzforschung bei Tomaten gegenüber P. infestans zu leisten, um erste Grundlagen für eine mögliche Züchtungsstrategie auf Basis unterschiedlicher quantitativer Resistenzen zu erarbeiten. Hierzu wurde untersucht, inwieweit unterschiedliche qualitative und quantitative Resistenzen bei Tomatenblättern und -früchten vorliegen, und ob hierfür verantwortliche Mechanismen identifiziert werden können. Zudem wurde untersucht, ob isolatspezifische quantitative Resistenzen identifiziert werden können. Zu diesem Zweck wurde mit einer erweiterten Clusteranalyse, basierend auf einer modifizierten Sanghvi-T2 Distanz, ein statistisches Verfahren entwickelt, welches die Identifikation von quantitativen, isolatspezifischen Resistenzen unter der Berücksichtigung der Variabilität ermöglicht. Des weiteren wurde geprüft, inwieweit zwischen den Resistenzausprägungen auf dem Blatt und den Resistenzausprägungen auf der Frucht ein Zusammenhang besteht und inwieweit die im Labor beobachteten Resistenzen unter Freilandbedingungen eine Rolle spielen. Im Labortest wurde die qualitative und quantitative Blattresistenz von 109 Akzessionen aus elf Lycopersicon und Solanum Arten gegenüber zwölf unterschiedlich aggressiven und teilweise auch unterschiedlich virulenten P. infestans Isolaten untersucht (Kap. 3). Die Früchte von 38 Tomatensorten wurden auf ihre Resistenz gegenüber drei P. infestans Isolaten geprüft. Zusätzlich wurde der Einfluss der Fruchtnachreife auf die Resistenzeigenschaften der Tomatenfrüchte gegenüber P. infestans analysiert (Kap. 4). Insgesamt 40 Sorten wurden auch unter Feldbedingungen auf Blatt- und Fruchtbefall untersucht (Kap. 5). Die frühen Stadien der Infektion von Tomatenblättern mit P. infestans Sporangien wurden mikroskopisch bei acht Tomatensorten mit unterschiedlichen quantitativen Reaktionsprofilen und drei Isolaten untersucht (Kap. 6). Hierzu wurden die Entwicklungsstadien von P. infestans Sporangien nach 24h, 48h und 60h nach der Inokulation auf und im Blatt mit der Calcofluor und der KOH - Anilin Blau Färbung sichtbar gemacht. Das Auftreten und die Lokalisation von H2O2 im Blatt nach 48h und 60h nach der Inokulation in Reaktion auf die Infektion wurde mithilfe einer DAB (3,3′ - Diaminobenzidine) Färbung untersucht. Es wurden einige, z.T. auch wahrscheinlich neue, qualitative Blattresistenzen gegenüber P. infestans gefunden, jedoch war keine der 109 Akzessionen vollständig resistent gegenüber allen Isolaten. Für die quantitative Resistenz von Blättern lagen in vielen Fällen isolatspezifische Unterschiede vor. Die Sorte x Isolat Interaktionen konnten mit Hilfe der erweiterten Clusteranalyse erfolgreich analysiert werden und die Akzessionen in Gruppen mit unterschiedlichen quantitativen Resistenzprofilen bzgl. der Interaktion mit den Isolaten und des Resistenzniveaus eingeteilt werden. Für die Fruchtresistenz konnten keine qualitativen Resistenzen gegenüber den drei getesteten Isolaten gefunden werden. Im Gegensatz dazu unterschieden sich die Tomatensorten in ihrer quantitativen Resistenz und Sorten und Isolate interagierten signifikant. Auch für die Fruchtresistenz konnten Gruppen mit unterschiedlichen quantitativen Reaktionsprofilen gebildet werden. Insgesamt nimmt die Anfälligkeit von Tomatenfrüchten mit zunehmender Reife kontinuierlich und signifikant ab. Unter Laborbedingungen korrelierten nur die Sporulationskapazität der Früchte und der prozentuale Blattbefall. Im Feldversuch über zwei Jahre und mit bis zu 40 Tomatensorten war der Zusammenhang hoch signifikant, jedoch asymptotisch, d.h. bereits bei sehr geringem Blattbefall war der Fruchtbefall sehr hoch. Bei den Tomatenherkünften, die sowohl im Labor als auch im Feld auf ihre Anfälligkeit getestet wurden, erschienen die Blattanfälligkeiten ähnlich, während kein klarer Zusammenhang zwischen der Fruchtanfälligkeit im Feld und im Labor bestand. Die Entwicklung von P. infestans auf der Blattoberfläche war unabhängig von der Sorte. Sowohl beim Eindringen und der Etablierung von P. infestans ins Blatt als auch bei der damit verbunden H2O2 Aktivität im Wirt wurden deutliche isolat- und sortenspezifische Effekte gefunden, die aber nur zum Teil mit den quantitativen Unterschieden der Blattresistenz korrespondierten. Sorten, die bei hoher Resistenz unterschiedliche Reaktionsprofile aufweisen, sind grundsätzlich interessante Kreuzungspartner, um die quantitative Resistenz gegenüber P. infestans zu verbessern. Hier sind vor allem Sorten, die sich auch in ihrer H2O2 Aktivität unterscheiden von Interesse.

Avaluació de la toxicitat de metalls pesants i arsènic en diferents models biològics

En aquest estudi, la toxicitat de diversos metalls pesants i l'arsènic va ser analitzada utilitzant diferents models biològics. En la primera part d'aquest treball, el bioassaig de toxicitat Microtox, el qual està basat en la variació de l'emissió lumínica del bacteri luminiscent Vibrio fischeri, va ser utilitzat per establir les corbes dosi-resposta de diferents elements tòxics com el Zn(II), Pb(II), Cu(II), Hg(II), Ag(I), Co(II), Cd(II), Cr(VI), As(V) i As(III) en solucions aquoses. Els experiments es varen portar a terme a pH 6.0 i 7.0 per tal de mostrar que el pH pot influir en la toxicitat final mesurada d'alguns metalls degut als canvis relacionats amb la seva especiació química. Es varen trobar diferents tipus de corbes dosi-resposta depenent del metall analitzat i el pH del medi. En el cas de l'arsènic, l'efecte del pH en la toxicitat de l'arsenat i l'arsenit es va investigar utilitzant l'assaig Microtox en un rang de pHs comprès entre pH 5.0 i 9.0. Els valors d'EC50 determinats per l'As(V) disminueixen, reflectint un augment de la toxicitat, a mesura que el pH de la solució augmenta mentre que, en el cas de l'As(III), els valors d'EC50 quasi bé no varien entre pH 6.0 i 8.0 i només disminueixen a pH 9.0. HAsO42- i H2AsO3- es varen definir com les espècies més tòxiques. Així mateix, una anàlisi estadística va revelar un efecte antagònic entre les espècies químiques d'arsenat que es troben conjuntament a pH 6.0 i 7.0. D'altra banda, els resultats de dos mètodes estadístics per predir la toxicitat i les possibles interaccions entre el Co(II), Cd(II), Cu(II), Zn(II) i Pb(II) en mescles binàries equitòxiques es varen comparar amb la toxicitat observada sobre el bacteri Vibrio fischeri. L'efecte combinat d'aquests metalls va resultar ser antagònic per les mescles de Co(II)-Cd(II), Cd(II)-Zn(II), Cd(II)-Pb(II) i Cu(II)-Pb(II), sinèrgic per Co(II)-Cu(II) i Zn(II)-Pb(II) i additiu en els altres casos, revelant un patró complex de possibles interaccions. L'efecte sinèrgic de la combinació Co(II)-Cu(II) i la forta disminució de la toxicitat del Pb(II) quan es troba en presència de Cd(II) hauria de merèixer més atenció quan s'estableixen les normatives de seguretat ambiental. La sensibilitat de l'assaig Microtox també va ser determinada. Els valors d'EC20, els quals representen la toxicitat llindar mesurable, varen ser determinats per cada element individualment i es va veure que augmenten de la següent manera: Pb(II) < Ag(I) < Hg(II)  Cu(II) < Zn(II) < As(V) < Cd(II)  Co(II) < As(III) < Cr(VI). Aquests valors es varen comparar amb les concentracions permeses en aigues residuals industrials establertes per la normativa oficial de Catalunya (Espanya). L'assaig Microtox va resultar ser suficientment sensible per detectar els elements assajats respecte a les normes oficials referents al control de la contaminació, excepte en el cas del cadmi, mercuri, arsenat, arsenit i cromat. En la segona part d'aquest treball, com a resultats complementaris dels resultats previs obtinguts utilitzant l'assaig de toxicitat aguda Microtox, els efectes crònics del Cd(II), Cr(VI) i As(V) es varen analitzar sobre la taxa de creixement i la viabilitat en el mateix model biològic. Sorprenentment, aquests productes químics nocius varen resultar ser poc tòxics per aquest bacteri quan es mesura el seu efecte després de temps d'exposició llargs. Tot i això, en el cas del Cr(VI), l'assaig d'inhibició de la viabilitat va resultar ser més sensible que l'assaig de toxicitat aguda Microtox. Així mateix, també va ser possible observar un clar fenomen d'hormesis, especialment en el cas del Cd(II), quan s'utilitza l'assaig d'inhibició de la viabilitat. A més a més, diversos experiments es varen portar a terme per intentar explicar la manca de toxicitat de Cr(VI) mostrada pel bacteri Vibrio fischeri. La resistència mostrada per aquest bacteri podria ser atribuïda a la capacitat d'aquest bacteri de convertir el Cr(VI) a la forma menys tòxica de Cr(III). Es va trobar que aquesta capacitat de reducció depèn de la composició del medi de cultiu, de la concentració inicial de Cr(VI), del temps d'incubació i de la presència d'una font de carboni. En la tercera part d'aquest treball, la línia cel·lular humana HT29 i cultius primaris de cèl·lules sanguínies de Sparus sarba es varen utilitzar in vitro per detectar la toxicitat llindar de metalls mesurant la sobreexpressió de proteines d'estrès. Extractes de fangs precedents de diverses plantes de tractament d'aigues residuals i diferents metalls, individualment o en combinació, es varen analitzar sobre cultius cel·lulars humans per avaluar el seu efecte sobre la taxa de creixement i la capacitat d'induir la síntesi de les proteïnes Hsp72 relacionades amb l'estrès cel·lular. No es varen trobar efectes adversos significatius quan els components s'analitzen individualment. Nogensmenys, quan es troben conjuntament, es produeix un afecte advers sobre tan la taxa de creixement com en l'expressió de proteins d'estrès. D'altra banda, cèl·lules sanguínies procedents de Sparus sarba es varen exposar in vitro a diferents concentracions de cadmi, plom i crom. La proteïna d'estrès HSP70 es va sobreexpressar significativament després de l'exposició a concentracions tan febles com 0.1 M. Sota les nostres condicions de treball, no es va evidenciar una sobreexpressió de metal·lotioneïnes. Nogensmenys, les cèl·lules sanguínies de peix varen resultar ser un model biològic interessant per a ser utilitzat en anàlisis de toxicitat. Ambdós models biològics varen resultar ser molt adequats per a detectar acuradament la toxicitat produïda per metalls. En general, l'avaluació de la toxicitat basada en l'anàlisi de la sobreexpressió de proteïnes d'estrès és més sensible que l'avaluació de la toxicitat realitzada a nivell d'organisme. A partir dels resultats obtinguts, podem concloure que una bateria de bioassaigs és realment necessària per avaluar acuradament la toxicitat de metalls ja que existeixen grans variacions entre els valors de toxicitat obtinguts emprant diferents organismes i molts factors ambientals poden influir i modificar els resultats obtinguts.

Comparative effects of fatty acids on endothelial inflammatory gene expression

Background: Endothelial dysfunction may be related to adverse effects of some dietary fatty acids (FAs). Although in vitro studies have failed to show consistent findings, this may reflect the diverse experimental protocols employed and the limited range of FAs and end points studied. Aims: To investigate the effect of dietary FA type (saturated, monounsaturated, n-6 and n-3 polyunsaturated fatty acids), concentration, incubation time and cell stimulation state, on a broad spectrum of endothelial inflammatory gene expression. Methods: Using human umbilical vein endothelial cells, with and without stimulation (+/- 10 ng/ml TNF alpha), the effects of arachidonic (AA), docosahexaenoic (DHA), eicosapentaenoic (EPA), linoleic (LA), oleic (OA) and palmitic acids (PA) (10, 25 and 100 mu M), on the expression of genes encoding a number of inflammatory proteins and transcription factors were assessed by quantitative real time RT-PCR. Results: Individual FAs differentially affect endothelial inflammatory gene expression in a gene-specific manner. EPA, LA and OA significantly up-regulated MCP-1 gene expression compared to AA (p = 0.001, 0.013, 0.008, respectively) and DHA (p < 0.0005, = 0.004, 0.002, respectively). Furthermore, cell stimulation state and FA incubation time significantly influenced reported FA effects on gene expression. Conclusions: The comparative effects of saturated, monounsaturated, n-6 and n-3 polyunsaturated FAs on endothelial gene expression depend on the specific FA investigated, its length of incubation, cell stimulation state and the gene investigated. These findings may explain existing disparity in the literature. This work was funded by the EC, Framework Programme 6 via the LIPGENE project (FOOD-CT-2003-505944).

Enzyme assisted extraction of chitin from shrimp shells (Litopenaeus vannamei)

BACKGROUND: Chemical chitin extraction generates large amounts of wastes and increases partial deacetylation of the product. Therefore, the use of biological methods for chitin extraction is an interesting alternative. The effects of process conditions on enzyme assisted extraction of chitin from the shrimp shells in a systematic way were the focal points of this study. RESULTS: Demineralisation conditions of 25C, 20 min, shells-lactic acid ratio of 1:1.1 w/w; and shells-acetic acid ratio of 1:1.2 w/w, the maximum demineralisation values were 98.64 and 97.57% for lactic and acetic acids, respectively. A total protein removal efficiency of 91.10% by protease from Streptomyces griseus with enzyme-substrate ratio 55 U/g, pH 7.0 and incubation time 3 h is obtained when the particle size range is 50-25 μm, which was identified as the most critical factor. The X-ray diffraction and 13C NMR spectroscopy analysis showed that the lower percent crystallinity and higher degree of acetylation of chitin from enzyme assisted extraction may exhibit better solubility properties and less depolymerisation in comparison with chitin from the chemical extraction. CONCLUSION: The present work investigates the effects of individual factors on process yields, and it has shown that, if the particle size is properly controlled a reaction time of 3 h is more than enough for deproteination by protease. Physicochemical analysis indicated that the enzyme assisted production of chitin seems appropriate to extract chitin, possibly retaining its native structure.

Edible oil from Tiger nut (Cyperus esculentus L.): mechanical pressing and aqueous enzymatic extraction methods

The tiger nut tuber of the Cyperus esculentus L. plant is an unusual storage system with similar amounts of starch and lipid. The extraction of its oil employing both mechanical pressing and aqueous enzymatic extraction (AEE) methods was investigated and an examination of the resulting products was carried out. The effects of particle size and moisture content of the tuber on the yield of tiger nut oil with pressing were initially studied. Smaller particles were found to enhance oil yields while a range of moisture content was observed to favour higher oil yields. When samples were first subjected to high pressures up to 700 MPa before pressing at 38 MPa there was no increase in the oil yields. Ground samples incubated with a mixture of α- Amylase, Alcalase, and Viscozyme (a mixture of cell wall degrading enzyme) as a pre-treatment, increased oil yield by pressing and 90% of oil was recovered as a result. When aqueous enzymatic extraction was carried out on ground samples, the use of α- Amylase, Alcalase, and Celluclast independently improved extraction oil yields compared to oil extraction without enzymes by 34.5, 23.4 and 14.7% respectively. A mixture of the three enzymes further augmented the oil yield and different operational factors were individually studied for their effects on the process. These include time, total mixed enzyme concentration, linear agitation speed, and solid-liquid ratio. The largest oil yields were obtained with a solid-liquid ratio of 1:6, mixed enzyme concentration of 1% (w/w) and 6 h incubation time although the longer time allowed for the formation of an emulsion. Using stationary samples during incubation surprisingly gave the highest oil yields, and this was observed to be as a result of gravity separation occurring during agitation. Furthermore, the use of high pressure processing up to 300 MPa as a pre-treatment enhanced oil yields but additional pressure increments had a detrimental effect. The quality of oils recovered from both mechanical and aqueous enzymatic extraction based on the percentage free fatty acid (% FFA) and peroxide values (PV) all reflected the good stabilities of the oils with the highest % FFA of 1.8 and PV of 1.7. The fatty acid profiles of all oils also remained unchanged. The level of tocopherols in oils were enhanced with both enzyme aided pressing (EAP) and high pressure processing before AEE. Analysis on the residual meals revealed DP 3 and DP 4 oligosaccharides present in EAP samples but these would require further assessment on their identity and quality.

Singlet oxygen generation in the reaction centers of Rhodobacter sphaeroides

Singlet oxygen ((1)O(2)) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. (1)O(2) emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H(2)O: D(2)O mixtures. (1)O(2) was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.

Selective photoinactivation of C. albicans and C. dubliniensis with hypericin

The genus Candida includes different species that have the potential to invade and colonize the human body and C. albicans is the most common cause of skin, nail and mucous infections. The increasing resistance against antifungal drugs has renewed the search for new treatment procedures and antimicrobial photodynamic inactivation (PDI) is a propitious candidate. Hypericin (HY) has several wanted properties to be used as a photosensitizer in this technique including a high quantum yield of singlet oxygen generation, a high extinction coefficient near 600 nm, and a relatively low dark toxicity. Although the phototoxicity of HY on several tumor cells has been reported, the data concerning its photoactivity on microorganisms are scarce. The aim of this study was to obtain the experimental parameters to achieve an acceptable selective hypericinphotoinactivation of two species of Candida comparing with fibroblasts and epithelial cells which are the constituents of some potential host tissues, such mucosas, skin and cavities. Microorganisms and cells were incubated with the same HY concentrations and short incubation time followed by irradiation with equal dose of light. The best conditions to kill just Candida were very low HY concentration (0.1-0.4 mu g ml(-1)) incubated by 10 min and irradiated with LED 590 nm with 6 J cm(-2).

Anti-amnesic effect of enzyme extracted stevioside from Stevia rebaudiana

The increasing consumption of sucrose has resulted in several nutritional and medicinal problems, including obesity. There is an alarming rise in the prevalence of obesity, type 2 diabetes mellitus, and metabolic syndrome in children and adults around the world, partly related to increasing availability of energy-dense, high-calorie foods, and perhaps to increased consumption of sugar and particularly fructose sweetened beverages. Therefore, low calorie sweeteners are urgently required to substitute table sugar.

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase using various parameters such as concentration of enzyme, incubation time and temperature was optimized. The extraction conditions were further optimized using response surface methodology (RSM). Under the optimized conditions, the experimental values were in close agreement with predicted model and resulted in a three times yield enhancement of stevioside.

Various studies have revealed that in addition to sweetening nature of stevisoide, it exerts beneficial effects including antihypertensive, anti-hyperglycemic, anti-human rotavirus, antioxidant, anti-inflammatory and antitumor actions. Its anti-amnesic potential remains to be explored, therefore the present study has been undertaken to investigate the beneficial effect of stevioside in memory deficit of rats employing scopolamine induced amnesia as an animal model.

Significance: Stevia is gaining significance in different parts of the world and is expected to develop into a major source of high potency sweetener for the growing natural food market. There is a strong possibility that Stevia sweeteners could replace aspartame in some diet variants. In addition, Stevia is expected to be used as a part substitute for sugar and also used in combination with other artificial sweeteners in the emerging phase of life cycle.

Effect of silicon and prior deformation of austenite on isothermal transformation in low carbon steels

Isothermal transformation (TTT) behavior of the low carbon steels with two Si contents (0.50 wt pct and 1.35 wt pct) was investigated with and without the prior deformation. The results show that Si and the prior deformation of the austenite have significant effiects on the transformation of the ferrite and bainite. The addition of Si refines the ferrite grains, accelerates the polygonal ferrite transformation and the formation of M/A constituents, leading to the improvement of the strength. The ferrite grains formed under the prior deformation of the austenite become more homogeneous and refined. However, the influence of deformation on the tensile strength of both steels is dependent on the isothermal temperatures. Thermodynamic calculation indicates that Si and prior deformation reduce the incubation time of both ferrite and bainite transformation, but the effiect is weakened by the decrease of the isothermal temperatures.

Synthesis of ethyl acetate employing celite-immobilized lipase of Bacillus cereus MTCC 8372

A wide range of fatty acid esters can be synthesized by esterification and transesterification reactions catalyzed by lipases in non-aqueous systems. In the present study, immobilization of a purified alkaline extra-cellular lipase of Bacillus cereus MTCC 8372 by adsorption on diatomaceous earth (celite) for synthesis of ethyl acetate via transesterification route was investigated. B. cereus lipase was deposited on celite (77% protein binding efficiency) by direct binding from aqueous solution. Immobilized lipase was used to synthesis of ethyl acetate from vinyl acetate and ethanol in n -nonane. Various reaction conditions, such as biocatalyst concentration, substrates concentration, choices of solvents ( n -alkanes), incubation time, temperature, molecular sieves (3Å &times; 1.5 mm), and water activity(a _w ), were optimized. The immobilized lipase (25 mg/ml) was used to perform transesterification in n -alkane(s) that resulted in approximately 73.7 mM of ethyl acetate at 55 °C in n -nonane under shaking (160 rpm) after 15 h, when vinyl acetate and ethanol were used in a equimolar ratio (100 mM each). Addition of molecular sieves (3Å &times; 1.5 mm) as well as effect of water activity of saturated salt solutions (KI, KCl and KNO ₃ ) to the transesterification efficiency has inhibitory effect. Batch operational stability tests indicated that immobilized lipase had retained 50% of its original catalytic activity after four consecutive batches of 15 h each.

Synthesis of ethyl oleate employing synthetic hydrogel-immobilized lipase of Bacillus coagulans MTCC-6375

Ten polymeric hydrogels were chemically synthesized by varying the concentrations of copolymer (DMA) and cross-linker (MBAm) molecules. An alkaline lipase of Bacillus coagulans MTCC-6375 was immobilized onto a poly (MAc-co-DMA-cl-MBAm)-hydrogel support at pH 8.5 and temperature 55ºC in 16 h. The bound lipase possessed 7.6 U.g⁻¹ (matrix) lipase activity with a specific activity of 18 U.mg⁻¹ protein. Hydrogel bound-lipase catalyzed esterification of oleic acid and ethanol to synthesize ethyl oleate in n-nonane. Various kinetic parameters were optimized to produce ethyl oleate using immobilized lipase. The optimal parameters were bound enzyme/substrate (E/S) ratio 0.62 mg/mM, ethanol/oleic acid 100 mM:75 mM or 100 mM:100 mM, incubation time 18 h and reaction temperature 55ºC that resulted in approximately 53% conversion of reactants into ethyl oleate in n-nonane. However, addition of a molecular sieve to the reaction mixture promoted the conversion to 58% in 18 h in n-nonane, which was equivalent to 55 mM of ethyl oleate produced.