A rapid assay for the coupled cell free generation of oxylipins

We developed a rapid and simple assay for the coupled in vitro synthesis of oxylipins using free unsaturated fatty acids as substrates. Reactions were catalysed with extracts expressed from living plant tissues. Preliminary experiments involving the cell free transformation of fatty acid hydroperoxides revealed that storage or pretreatment of the plant extract rapidly altered its capacity to catalyse the generation of oxidised fatty acid derivatives. This could reflect changes in oxylipin generation that might take place in situ in damaged plant cells during herbivory. All subsequent experiments were performed without dilution, titration or any other modification of the plant extract prior to its addition to the assay system. The assays were used to study, for the first time, tissue-specific differences in fatty acid transformation to divinyl ethers. Root tissues from tomato efficiently catalysed the formation of corneleic and colnelenic acids from linoleic acid and linolenic acids, respectively, whereas leaf, hypocotyl and cotyledon extracts did not promote the formation of these compounds. We observed the efficient generation of 9-oxo-nonanoic acid from the substrate linolenic acid and speculate that this aldehyde could arise either from the action of hydroperoxide lyase on 9-hydroperoxylinolenic acid or by a novel route involving cleavage of colnelenic acid which was also present among the products of the reaction. A potential role of divinyl ethers as substrates for the generation of toxic aldehydes is discussed

Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic–scid/scid mice

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential, but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic–scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34−CD19+ (B-lymphoid) and CD34+ (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains ≈5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38− and CD38+ subsets of CD34+ CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34+CD38− human CB cells in serum-free medium containing flt-3 ligand, Steel factor, interleukin 3, interleukin 6, and granulocyte colony-stimulating factor for 5–8 days resulted in a 100-fold expansion of colony-forming cells, a 4-fold expansion of LTC-IC, and a 2-fold (but significant, P < 0.02) increase in CRU. The culture-derived CRU, like the original CB CRU, generated pluripotent, erythroid, granulopoietic, megakaryopoietic, and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition, their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal.

Sodium nitrite downregulates vascular NADPH oxidase and exerts antihypertensive effects in hypertension

Dietary nitrite and nitrate are important sources of nitric oxide (NO). However, the use of nitrite as an antihypertensive drug may be limited by increased oxidative stress associated with hypertension. We evaluated the antihypertensive effects of sodium nitrite given in drinking water for 4 weeks in two-kidney one-clip (21(1 C) hypertensive rats and the effects induced by nitrite on NO bioavailability and oxidative stress. We found that, even under the increased oxidative stress conditions present in 2K1C hypertension, nitrite reduced systolic blood pressure in a dose-dependent manner. Whereas treatment with nitrite did not significantly change plasma nitrite concentrations in 2K1C rats, it increased plasma nitrate levels significantly. Surprisingly, nitrite treatment exerted antioxidant effects in both hypertensive and sham-normotensive control rats. A series of in vitro experiments was carried out to show that the antioxidant effects induced by nitrite do not involve direct antioxidant effects or xanthine oxidase activity inhibition. Conversely, nitrite decreased vascular NADPH oxidase activity. Taken together, our results show for the first time that nitrite has antihypertensive effects in 2K1C hypertensive rats, which may be due to its antioxidant properties resulting from vascular NADPH oxidase activity inhibition. (C) 2011 Elsevier Inc. All rights reserved.

"In vitro" reconstitution of the fragmentation of vacuoles

Résumé La fragmentation des membranes est un processus commun à beaucoup d'organelles dans une cellule. Les mitochondries, le noyau, le réticulum endoplasmique, les phagosomes, les peroxisomes, l'appareil de Golgi et les lysosomes (vacuoles chez la levure) se fragmentent en plusieurs copies en réponse à des sitmulis environnementaux, tels que des stresses, ou dans une situtation normale durant le cycle cellulaire, afin d' être transférer dans les cellules filles. La fragmentation des membranes est également observée pendant le processus d'endocytose, lors de la formation de vésicules endocytiques, mais également dans tout le traffic intracellulaire, lors de la genèse d'une vésicule de transport. Le processus de fragmentation est donc généralement important. La découverte en 1991 d'une dynamin-like GTPase comme protéine impliquée dans la fragmentation de la membrane plasmique durant l'endocytose a ouvert ce domaine de recherche. Dès lors des dynamines ont été découvertes sur la pluspart des organelles, ce qui suggère un processus de fragmentation des membranes commun à l'ensemble de la cellule. Cependant, l'ensemble des protéines impliquées ainsi que le mécanisme de la fragmentation reste encore à élucider. Mon projet de thèse était d'établir un test in vitro de fragmentation des vacuoles utile à la compréhension du mécanisme de ce processus. Le choix de ce système est judicieux pour plusieurs raisons; premièrement les vacuoles fragmentent naturellement durant le cycle cellulaire, deuxièment leur taille permet de visualiser facilement leur morphologie par simple microscopie optique, finalement elles peuvent être isolées en quantité intéressante avec un haut degré de pureté. In vivo, les vacuoles peuvent être facilement fragmentées par un stress osmotique. Un tel test permet d'identifier des protéines impliquées dans le mécanisme comme dans le criblage que j'ai effectué sur l'ensemble de la collection de délétions des gènes non-essentiels chez la levure. Cependant un test in vitro est ensuite indispensable pour jouer avec les protéines découvertes afin d'en élucider le mécanisme. Avec mon test in vitro, j'ai confirmé l'implication des protéines SNAREs dans la fragmentation et j'ai permis de comprendre la régulation de la quantité de vacuoles et de leur taille par le complexe TORC1 dans une situation de stress. 7 Résumé large public Les cellules de chaque organisme sont composées de différents compartiments appelés organelles. Chacun possède une fonction bien définie afin de permettre la vie et la croissance de la cellule. Ils sont entourés de membrane, qui joue le role de barrière spécifiquement perméable, afin de garder l'intégrité de chacun. Dans des conditions de croissance normale, les cellules prolifèrent. Durant la division cellulaire amenant à la formation d'une nouvelle cellule, chaque organelle doit se diviser afin de fournir l'ensemble des organelles à la cellule fille. La division de chaque organelle nécessite la fragmentation de la membrane les entourant. Des protéines dynamine-like GTPase ont été découvertes sur presque l'ensemble des organelles d'une cellule. Elles sont impliquées dans les processus de fragmentation des membranes. Dès lors l'idée d'un mécanisme commun est apparu. Cependant cette réaction, par sa complexité, ne peut pas impliquer une protéine unique. La découverte d'autres facteurs et la compréhension du mécanisme reste à faire. La première étape peut se faire par étude in vivo, c'est-à-dire avec des cellules entières, la deuxième étape, quant à elle, nécessite d'isoler les protéines impliquées et de jouer avec les différents paramètres, ce qui signifie donc un travail in vitro, séparé des cellules. Mon travail a constisté à établir un procédé expérimental in vitro pour étudier la fragmentation des membranes. Je travaille avec des vacuoles de levures pour étudier les réactions membranaires. Les vacuoles sont les plus grandes organelles présentes dans les levures. Elles sont impliquées principalement dans la digestion. Comme toute organelle, elles se fragmentent durant la division cellulaire. Le procédé expérimental comporte une première étape, l'isolation des vacuoles et, deuxièmement, l'incubation de celles-ci avec des composés essentiels à la réaction. En parallèle, j'ai mis en évidence, par un travail in vivo, de nouvelles protéines impliquées dans le processus de fragmentation des membranes. Ceci a été fait en réalisant un criblage par microscopie d'une collection de mutants. Parmi ces mutants, j'ai cherché ceux qui présentaient un défaut dans la fragmentation des vacuoles. Ces deux procédés expérimentaux, in vitro et in vivo, m'ont permis de découvrir de nouvelles protéines impliquées dans cette réaction, ainsi que de mettre en évidence un mécanisme utlilisé par la cellule pour réguler la fragmentation des vacuoles. 8 Summary Fragmentation of membranes is common for many organelles in a cell. Mitochondria, nucleus, endoplasmic reticulum, phagosomes, peroxisomes, Golgi and lysosomes (vacuoles in yeast) fragment into multiple copies in response to environmental stimuli, such as stresses, or in a normal situation during the cell cycle in order to be transferred into the daughter cell. Fragmentation of membrane occurs during endocytosis, at the latest step in endocytic vesicle formation, and also in intracellular trafficking, when traffic vesicles bud. This field of research was opened in 1991 when a dynamin-like GTPase was found to be involved in fragmentation of the plasma membrane during endocytosis. Since dynamin-like GTPases have been found on most organelles, similarities in their mechanisms of fragmentation might exist. However, many proteins involved in the mechanism of fragmentation remain unknown. My thesis project was to establish an in vitro assay for membrane fragmentation in order to create a tool to study the mechanism of this process. I chose vacuoles as a model organelle for several reasons: first of all, vacuoles fragment under physiological conditions during cell cycle, secondly their size makes their morphology easily visible under the light microscope, and finally vacuoles can be isolated in good amounts with relatively high degrees of purity. In vivo, vacuole fragmentation can be induced with an osmotic shock. Such a simple assay facilitates the identification of new proteins involved in the process. I used this tool to screen of the entire knockout collection of non-essential genes in Saccharomyces cerevisiae for mutants defective in vacuole fragmentation. The in vitro system will be useful to characterize the mutants and to study the mechanism of fragmentation in detail. I used my in vitro assay to confirm the involvement of vacuolar SNARE proteins in fragmentation of the organelle and to uncover that number and size of vacuoles in the cell is regulated by the TORC1 complex via selective stimulation of fragmentation activity.

Two-photon excitation fluorometry in detection of infectious diseases

Because of the heavily overlapping symptoms, pathogen-specific diagnosis and treatment of infectious diseases is difficult based on clinical symptoms alone. Therefore, patients are often treated empirically. More efficient treatment and management of infectious diseases would require rapid point-of-care compatible in vitro diagnostic methods. However, current point-of-care methods are unsatisfactory in performance and in cost structure. The lack of pointof- care methods results in unnecessary use of antibiotics, suboptimal use of virus-specific drugs, and compromised patient care. In this thesis, the applicability of a two-photon excitation fluorometry is evaluated as a tool for rapid detection of infectious diseases. New separation-free immunoassay methodologies were developed and validated for the following application areas: general inflammation markers, pathogen-specific antibodies, pathogen-specific antigens, and antimicrobial susceptibility testing. In addition, dry-reagent methodology and nanoparticulate tracers are introduced in context to the technique. The results show that the new assay technique is a versatile tool for rapid detection of infectious diseases in many different application areas. One particularly attractive area is rapid multianalyte testing of respiratory infections, where the technique was shown to allow simple assay protocols and comparable performance to the state-of-the-art laboratory methods. If implemented in clinical diagnostic use, the new methods could improve diagnostic testing routines, especially in rapid testing of respiratory tract infections.

Accelerating food safety and quality control: The use of antibody engineering techniques in the detec-tion of low-molecular weight food contaminants

The increased awareness and evolved consumer habits have set more demanding standards for the quality and safety control of food products. The production of foodstuffs which fulfill these standards can be hampered by different low-molecular weight contaminants. Such compounds can consist of, for example residues of antibiotics in animal use or mycotoxins. The extremely small size of the compounds has hindered the development of analytical methods suitable for routine use, and the methods currently in use require expensive instrumentation and qualified personnel to operate them. There is a need for new, cost-efficient and simple assay concepts which can be used for field testing and are capable of processing large sample quantities rapidly. Immunoassays have been considered as the golden standard for such rapid on-site screening methods. The introduction of directed antibody engineering and in vitro display technologies has facilitated the development of novel antibody based methods for the detection of low-molecular weight food contaminants. The primary aim of this study was to generate and engineer antibodies against low-molecular weight compounds found in various foodstuffs. The three antigen groups selected as targets of antibody development cause food safety and quality defects in wide range of products: 1) fluoroquinolones: a family of synthetic broad-spectrum antibacterial drugs used to treat wide range of human and animal infections, 2) deoxynivalenol: type B trichothecene mycotoxin, a widely recognized problem for crops and animal feeds globally, and 3) skatole, or 3-methyindole is one of the two compounds responsible for boar taint, found in the meat of monogastric animals. This study describes the generation and engineering of antibodies with versatile binding properties against low-molecular weight food contaminants, and the consecutive development of immunoassays for the detection of the respective compounds.

Enzyme reactions as index of freshness of Fish and Shellfish

An attempt has been made in this study to screen some fish muscle enzymes to assess their potential worth in testing the degree of freshness of fish. A problem with routine enzyme activity determinations is the complexity of the method of enzyme assay. Hence, in the present study as far as possible simple assay techniques were adopted. Several species were screened to assess the possibility of employing this procedure on a large scale. It is hoped that findings of this study will lead to the development of meaningful criteria in testing the freshness of fish. This thesis has been divided into five chapters

Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alternative host model to evaluate Aeromonas virulence

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

Active genetic elements present in the locus of enterocyte effacement in Escherichia coli O26 and their role in mobility

The locus of enterocyte effacement (LEE) is a large multigene chromosomal segment encoding gene products responsible for the generation of attaching and effacing lesions in many diarrheagenic Escherichia coli strains. A recently sequenced LEE harboring a pathogenicity island (PAI) from a Shiga toxin E. coli serotype 026 strain revealed a LEE PAI (designated LEE 026) almost identical to that obtained from a rabbit-specific enteropathogenic 015:H- strain. LEE 026 comprises 59,540 bp and is inserted at 94 min within the mature pheU tRNA locus. The LEE 026 PAI is flanked by two direct repeats of 137 and 136 bp (DR1 and DR2), as well as a gene encoding an integrase belonging to the P4 integrase family. We examined LEE 026 for horizontal gene transfer. By generating mini-LEE plasmids harboring only DR1 or DR2 with or without the integrase-like gene, we devised a simple assay to examine recombination processes between these sequences. Recombination was shown to be integrase dependent in a Delta recA E. coli K-12 strain background. Recombinant plasmids harboring a single direct repeat cloned either with or without the LEE 026 integrase gene were found to insert within the chromosomal pheU locus of E. coli K-12 strains with equal efficiency, suggesting that an endogenous P4-like integrase can substitute for this activity. An integrase with strong homology to the LEE 026 integrase was detected on the K-12 chromosome associated with the leuX tRNA locus at 97 min. Strains deleted for this integrase demonstrated a reduction in the insertion frequency of plasmids harboring only the DR into the pheU locus. These results provide strong evidence that LEE-harboring elements are indeed mobile and suggest that closely related integrases present on the chromosome of E. coli strains contribute to the dynamics of PAI mobility.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.

Comparative evaluation of a simple and sensitive assay for detection of orthomyxo and paramyxoviruses

Studies were done to evaluate comparatively the traditional HA assay and a more recently introduced lectin-neuraminidase (LN) methodologyin search of a simple and sensitive assay for virus detection during laboratorial diagnosis. The results proved the value of LN assay as a sensitive methodologyfor detection of virus particles, presenting results at least equal to those obtained by HA (hemagglutination) assay, with significant values of accumulated frequencies for LN/HA factors (ratios between LN and HA titers) higher than two. The accumulated values of frequencies for LN/HA factors as high as four were very significant, 72.7 (per cent) for influenzavirus and 60.7 (per cent) for Newcastle disease virus (NDV), moreover accumulated frequencies for LN/HA factors even as high as 32 were due to influenzavirus (45.4 per cent) and NDV (7.2 per cent) samples. After the storage period, most of those concentraded samples that even did not present HA titers could be detected through LN assay, demonstrating a lower threshold for virus detection.

Dermatophyte identification in skin and hair samples using a simple and reliable nested polymerase chain reaction assay.

BACKGROUND: Dermatophyte identification in tinea capitis is essential for choosing the appropriate treatment and in tinea infections to identify the possible source. The failure of fungi to grow in cultures frequently occurs, especially in cases of previous antifungal therapy. OBJECTIVES: To develop a rapid polymerase chain reaction (PCR) sequencing assay for dermatophyte identification in tinea capitis and tinea corporis. MATERIAL AND METHODS: Fungal DNA was extracted from hair and skin samples that were confirmed to be positive by direct mycological examination. Dermatophytes were identified by the sequence of a 28S ribosomal DNA subunit amplicon generated by nested PCR. RESULTS: Nested PCR was found to be necessary to obtain amplicons in substantial amounts for dermatophyte identification by sequencing. The results agreed with those of classical mycological identification in 14 of 23, 6 of 10, and 20 of 23 cases of tinea capitis, tinea corporis and tinea pedis, respectively, from which a dermatophyte was obtained in culture. In seven of the 56 cases, another dermatophyte was identified, revealing previous misidentification. A dermatophyte was identified in 12 of 18, three of five, and four of nine cases of tinea capitis, tinea corporis and tinea pedis, respectively, in cases in which no dermatophyte grew in culture. CONCLUSIONS: Although the gold standard dermatophyte identification from clinical samples remains fungal cultures, the assay developed in the present study is especially suitable for tinea capitis. Improved sensitivity for the identification of dermatophyte species was obtained as it is possible to identify the dermatophyte when the fungus fails to grow in cultures.

Validation of a simple and rapid UV spectrophotometric method for dexamethasone assay in tablets

This work reports the validation of an analytical UV spectrophotometric method to assay dexamethasone in tablets (assay and dissolution studies). The method was linear in the range between 1 and 30 µg mL-1 presenting a good correlation coefficient (r = 0.9998, n = 7). Precision and accuracy analysis showed low relative standard deviation (< 2.00%) and good percentual recoveries (95-105%). The procedure was linear, accurate, precise, and robust. The method is simple, and it has low cost. It does not use polluting reagents and can be applied in dissolution studies, being an adequate alternative to assay dexamethasone in tablets.