Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ∼2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ∼60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.

Environmental isolation of black yeast-like fungi involved in human infection

The present study focuses on potential agents of chromoblastomycosis and other endemic diseases in the state of Parana, Southern Brazil. Using a highly selective protocol for chaetothyrialean black yeasts and relatives, environmental samples from the living area of symptomatic patients were analysed. Additional strains were isolated from creosote-treated wood and hydrocarbon-polluted environments, as such polluted sites have been supposed to enhance black yeast prevalence. Isolates showed morphologies compatible with the traditional etiological agents of chromoblastomycosis, e.g. Fonsecaea pedrosoi and Phialophora verrucosa, and of agents of subcutaneous or systemic infections like Cladophialophora bantiana and Exophiala jeanselmei. Some agents of mild disease were indeed encountered. However, molecular analysis proved that most environmental strains differed from known etiologic agents of pronounced disease syndromes: they belonged to the same order, but mostly were undescribed species. Agents of chromoblastomycosis and systemic disease thus far are prevalent on the human host. The hydrocarbon-polluted environments yielded yet another spectrum of chaetothyrialean fungi. These observations are of great relevance because they allow us to distinguish between categories of opportunists, indicating possible differences in pathogenicity and virulence.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae, The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented, A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria, Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group, Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species, A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

Oral disease in animals: The Australian perspective. Isolation and characterisation of black-pigmented bacteria from the oral cavity of marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes; or swabs. Plaque samples were plated onto. non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens, Agar. Plates were incubated in an anaerobic atmosphere and examined after 7-14 days for the presence of black-brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis-like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria. (C) 2002 Elsevier Science Ltd. All rights reserved.

ISOLATION AND MOLECULAR IDENTIFICATION OF POTENTIALLY PATHOGENIC Escherichia coli AND Campylobacter jejuni IN FERAL PIGEONS FROM AN URBAN AREA IN THE CITY OF LIMA, PERU

SUMMARY Feral pigeons (Columbia livia) live in close contact with humans and other animals. They can transmit potentially pathogenic and zoonotic agents. The objective of this study was to isolate and detect strains of diarrheagenic Escherichia coli and Campylobacter jejuniof urban feral pigeons from an area of Lima, Peru. Fresh dropping samples from urban parks were collected for microbiological isolation of E. coli strains in selective agar, and Campylobacterby filtration method. Molecular identification of diarrheagenic pathotypes of E.coliand Campylobacter jejuni was performed by PCR. Twenty-two parks were sampled and 16 colonies of Campylobacter spp. were isolated. The 100% of isolates were identified as Campylobacter jejuni. Furthermore, 102 colonies of E. coli were isolated and the 5.88% resulted as Enteropathogenic (EPEC) type and 0.98% as Shiga toxin-producing E. coli (STEC). The urban feral pigeons of Lima in Peru can act as a reservoir or carriers of zoonotic potentially pathogenic enteric agents.

The comparison of the Butzler medium, filtration technique and their association with isolation of Campylobacter ssp

Campylobacter was isolated in 178 out of 622 stool samples (200 porcine, 220 bovine, and 202 canine). From these 178 samples, the microorganism was identified in 64 samples (36%) isolated only in Butzler selective medium (BSM), 34 samples (19%) using filtration technique (FT), and in 80 samples (45%) using both BSM and FT. Comparison of the proportion of positivity using both techniques showed a significant value (c2 = 9,184; p > 0.001); BSM (36%) being more efficient than FT (19%). The use of both techniques yielded the highest isolation positivity (45%).

A RT-PCR method for selective amplification and phenotypic characterization of all three serotypes of Sabin-related polioviruses from viral mixtures

Outbreaks caused by vaccine-derived polioviruses are challenging the final eradication of paralytic poliomyelitis. Therefore, the surveillance of the acute flaccid paralysis cases based on poliovirus isolation and characterization remains an essential activity. Due to the use of trivalent oral poliovirus vaccine (OPV), mixtures containing more than one serotype of Sabin-related polioviruses are frequently isolated from clinical samples. Because each poliovirus isolate needs to be individually analyzed, we designed polymerase chain reaction primers that can selectively distinguish and amplify a genomic segment of the three Sabin-related poliovirus serotypes present in mixtures, thus, optimizing the diagnosis and providing prompt information to support epidemiologic actions.

Selective tumor localization of radiolabeled anti-human melanoma monoclonal antibody fragment demonstrated in the nude mouse model.

Monoclonal antibodies (Mab) directed against distinct epitopes of the human 240 kD melanoma-associated antigen have been evaluated for their capacity to localize in human melanoma grafted into nude mice. A favorable tumor to normal tissue ratio of 13 was obtained with intact 131I-labeled MAb Me1-14. This ratio was further increased to 43 and 23 by the use of F(ab')2 and Fab fragments, respectively. The specificity of tumor localization was demonstrated by the simultaneous injection of F(ab')2 fragments from MAb Me1-14 and anti-CEA MAb 35, each labeled with a different iodine isotope, into nude mice grafted with a melanoma and colon carcinoma. The fragments from both MAb localized with perfect selectivity in their relevant tumor as shown by differential whole body scanning and by direct measurement of the two isotopes in tumors and normal tissues. These in vivo experimental results suggest that the F(ab')2 fragment from MAb Me1-14 is suitable for melanoma detection by immunoscintigraphy in patients.

Why human islets die? analysis of death signaling pathways during isolation and following cytokine treatments

RESUME Le diabète de type 1 se définit comme un désordre métabolique d'origine auto-immune qui aboutit à la destruction progressive et sélective de la cellule ß-pancréatique sécrétrice d'insuline. Cette maladie représente 10 % des cas de diabète enregistrés dans la population mondiale, et touche les jeunes de moins de 20 ans. Le traitement médical par insulinothérapie corrige le manque d'hormone mais ne prévient pas les nombreuses complications telles que les atteintes cardiaques, neurologiques, rénales, rétiniennes, et les amputations que la maladie provoque. Le remplacement de la cellule ß par transplantation d'îlots de Langerhans est une alternative prometteuse au traitement médical du diabète de type 1. Cependant la greffe d'îlots est encore un traitement expérimental et ne permet pas un contrôle efficace de la glycémie au long terme chez les patients transplantés, et les raisons de cet échec restent mal comprises. L'obstacle immédiat qui se pose est la purification d'un nombre suffisant d'îlots viables et la perte massive de ces îlots dans les premières heures suite à la greffe. Cette tendance presque systématique de la perte fonctionnelle du greffon immédiatement après la transplantation est connue sous le terme de « primary graft non-function » (PNF). En effet, la procédure d'isolement des îlots provoque la destruction des composantes cellulaires et non cellulaires du tissu pancréatique qui jouent un rôle déterminant dans le processus de survie de l'îlot. De plus, la transplantation elle-même expose les cellules à différents stress, notamment le stress par les cytokines inflammatoires qui encourage la mort cellulaire par apoptose et provoque par la suite le rejet de la greffe. L'ensemble de ces mécanismes aboutit a une perte de la masse d'îlot estimée a plus de 60%. Dans ce contexte, nous nous sommes intéressés à définir les voies majeures de stress qui régissent cette perte massive d'îlot par apoptose lors du processus d'isolement et suite à l'exposition immédiate aux cytokines. L'ensemble des résultats obtenus indique que plusieurs voies de signalisation intracellulaire sont recrutées qui s'activent de manière maximale très tôt lors des premières phases de l'isolement. La mise en culture des îlots deux jours permet aux voies activées de revenir aux taux de base. De ce fait nous proposons une stratégie dite de protection qui doit être 1) initiée aussitôt que possible lors de l'isolement des îlots pancréatiques, 2) devrait probablement bloquer l'activation de ces différentes voies de stress mis en évidence lors de notre étude et 3) devrait inclure la mise en culture des îlots purifiés deux jours après l'isolement et avant la transplantation. RESUME LARGE PUBLIC Le diabète est une maladie qui entraîne un taux anormalement élevé de sucre (glucose) dans le sang du à une insuffisance du pancréas endocrine à produire de l'insuline, une hormone qui régule la glycémie (taux de glucose dans le sang). On distingue deux types majeurs de diabètes; le diabète de type 1 ou juvénile ou encore appelé diabète maigre qui se manifeste souvent pendant l'enfance et qui se traduit par une déficience absolue en insuline. Le diabète de type 2 ou diabète gras est le plus fréquent, et touche les sujets de plus de 40 ans qui souffrent d'obésité et qui se traduit par une dysfonction de la cellule ß avec une incapacité à réguler la glycémie malgré la production d'insuline. Dans le diabète de type 1, la destruction de la cellule ß est programmée (apoptose) et est majoritairement provoquée par des médiateurs inflammatoires appelés cytokines qui sont produites localement par des cellules inflammatoires du système immunitaire qui envahissent la cellule ß-pancréatiques. Les cytokines activent différentes voies de signalisation parmi lesquelles on distingue celles des Mitogen-Activated Protein Kinase (MAPKs) composées de trois familles de MAPKs: ERK1/2, p38, et JNK, et la voie NF-κB. Le traitement médical par injections quotidiennes d'insuline permet de contrôler la glycémie mais ne prévient pas les nombreuses complications secondaires liées à cette maladie. La greffe d'îlots de Langerhans est une alternative possible au traitement médical, considérée avantageuse comparée a la greffe du pancréas entier. En effet l'embolisation d'îlots dans le foie par injection intraportale constitue une intervention simple sans complications majeures. Néanmoins la technique de préparation d'îlots altère la fonction endocrine et cause la perte massive d'îlots pancréatiques. De plus, la transplantation elle-même expose la cellule ß à différents stress, notamment le stress par les cytokines inflammatoires qui provoque le rejet de greffon cellulaire. Dans la perspective d'augmenter les rendements des îlots purifiés, nous nous sommes intéressés à définir les voies majeures de stress qui régissent cette perte massive d'îlot lors du processus d'isolement et suite à l'exposition immédiate aux cytokines après transplantation. L'ensemble de ces résultats indique que le stress induit lors de l'isolement des îlots et celui des cytokines recrute différentes voies de signalisation intracellulaire (JNK, p38 et NF-κB) qui s'additionnent entre-elles pour altérer la fonction et la viabilité de l'îlot. De ce fait une stratégie doit être mise en place pour bloquer toute action synergique entre ces différentes voies activées pour améliorer la viabilité et la fonction de la cellule ß lors du greffon cellulaire. SUMMARY Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the progressive and selective destruction of the pancreatic ß-cells that secrete insulin, leading to absolute insulin deficiency. T1DM accounts for about 10% of all diabetes cases, affecting persons younger than 20 years of age. Medical treatment using daily exogenous insulin injection corrects hormone deficiency but does not prevent devastating complications such as heart attack, neuropathy, kidney failure, blindness, and amputation caused by the disease. Pancreatic islet transplantation (PIT) is one strategy that holds promise to cure patients with T1DM, but purified pancreatic islet grafts have failed to maintain long-term glucose homeostasis in human recipients, the reasons for this failure being still poorly understood. There is however a more immediate problem with islet grafting that is dependent upon poor islet recovery from donors and early islet loss following the first hours of grafting. This tendency of islet grafts to fail to function within a short period after transplantation is termed primary graft non-function (PNF). Indeed, the islet isolation procedure itself destroys cellular and non-cellular components of the pancreas that may play a role in supporting islet survival. Further, islet transplantation exposes cells to a variety of stressful stimuli, notably pro-inflammatory cytokines that encourage ß-cell death by apoptosis and lead to early graft failure. Altogether these mechanisms lead to an estimated loss of 60% of the total islet mass. Here, we have mapped the major intracellular stress signaling pathways that may mediate human islet loss by apoptosis during isolation and following cytokine attack. We found that several stress pathways are maximally activated from the earliest stages of the isolation procedure. Culturing islet for two days allow for the activated pathways to return to basal levels. We propose that protective strategies should 1) be initiated as early as possible during isolation of the islets, 2) should probably target the activated stress pathways that we uncovered during our studies and 3) should include culturing islets for two days post-isolation and prior transplantation.

Novel acid phosphatase in Candida glabrata suggests selective pressure and niche specialization in the phosphate signal transduction pathway.

Evolution through natural selection suggests unnecessary genes are lost. We observed that the yeast Candida glabrata lost the gene encoding a phosphate-repressible acid phosphatase (PHO5) present in many yeasts including Saccharomyces cerevisiae. However, C. glabrata still had phosphate starvation-inducible phosphatase activity. Screening a C. glabrata genomic library, we identified CgPMU2, a member of a three-gene family that contains a phosphomutase-like domain. This small-scale gene duplication event could allow for sub- or neofunctionalization. On the basis of phylogenetic and biochemical characterizations, CgPMU2 has neofunctionalized to become a broad range, phosphate starvation-regulated acid phosphatase, which functionally replaces PHO5 in this pathogenic yeast. We determined that CgPmu2, unlike ScPho5, is not able to hydrolyze phytic acid (inositol hexakisphosphate). Phytic acid is present in fruits and seeds where S. cerevisiae grows, but is not abundant in mammalian tissues where C. glabrata grows. We demonstrated that C. glabrata is limited from an environment where phytic acid is the only source of phosphate. Our work suggests that during evolutionary time, the selection for the ancestral PHO5 was lost and that C. glabrata neofunctionalized a weak phosphatase to replace PHO5. Convergent evolution of a phosphate starvation-inducible acid phosphatase in C. glabrata relative to most yeast species provides an example of how small changes in signal transduction pathways can mediate genetic isolation and uncovers a potential speciation gene.

Selective ablation of photovoltaic materials with UV laser sources for monolithic interconnection of devices based on a-Si:H

Lasers are essential tools for cell isolation and monolithic interconnection in thin-film-silicon photovoltaic technologies. Laser ablation of transparent conductive oxides (TCOs), amorphous silicon structures and back contact removal are standard processes in industry for monolithic device interconnection. However, material ablation with minimum debris and small heat affected zone is one of the main difficulty is to achieve, to reduce costs and to improve device efficiency. In this paper we present recent results in laser ablation of photovoltaic materials using excimer and UV wavelengths of diode-pumped solid-state (DPSS) laser sources. We discuss results concerning UV ablation of different TCO and thin-film silicon (a-Si:H and nc-Si:H), focussing our study on ablation threshold measurements and process-quality assessment using advanced optical microscopy techniques. In that way we show the advantages of using UV wavelengths for minimizing the characteristic material thermal affection of laser irradiation in the ns regime at higher wavelengths. Additionally we include preliminary results of selective ablation of film on film structures irradiating from the film side (direct writing configuration) including the problem of selective ablation of ZnO films on a-Si:H layers. In that way we demonstrate the potential use of UV wavelengths of fully commercial laser sources as an alternative to standard backscribing process in device fabrication.

Semi-selective medium for Fusarium graminearum detection in seed samples

Fungi of the genus Fusarium cause a variety of difficult to control diseases in different crops, including winter cereals and maize. Among the species of this genus Fusarium graminearum deserves attention. The aim of this work was to develop a semi-selective medium to study this fungus. In several experiments, substrates for fungal growth were tested, including fungicides and antibiotics such as iprodiona, nystatin and triadimenol, and the antibacterial agents streptomycin and neomycin sulfate. Five seed samples of wheat, barley, oat, black beans and soybeans for F. graminearum detection by using the media Nash and Snyder agar (NSA), Segalin & Reis agar (SRA) and one-quarter dextrose agar (1/4PDA; potato 50g; dextrose 5g and agar 20g), either unsupplemented or supplemented with various concentrations of the antimicrobial agents cited above. The selected components and concentrations (g.L-1) of the proposed medium, Segalin & Reis agar (SRA-FG), were: iprodiona 0.05; nystatin 0,025; triadimenol 0.015; neomycin sulfate 0.05; and streptomycin sulfate, 0.3 added of ¼ potato sucrose agar. In the isolation from seeds of cited plant species, the sensitivity of this medium was similar to that of NSA but with de advantage of maintaining the colony morphological aspects similar to those observed in potato-dextrose-agar medium.

PRESERVATION OF PRIMARY FOREST CHARACTERISTICS DESPITE FRAGMENTATION AND ISOLATION IN A FOREST REMNANT FROM VIÇOSA, MG, BRAZIL1

According to its owners, the Forest of Seu Nico (FSN) from the Viçosa municipality, Minas Gerais, Brazil, never has been logged and is therefore considered a primary forest. Nevertheless, the forest patch suffered impacts due to selective wood and non-timber extraction, fragmentation and isolation. Aim of this study was to test if the FSN, despite impacts, preserved characteristics of primary forests, which are elevated percentages of non-pioneer (>90%), animal-dispersed (>80 %), understory (>50%) and endemic species (~40%). For that, all trees with diameter at breast height equal or major than 3.2 cm within a plot of 100 x 100 m were identified. With 218 tree species found within this hectare, the FSN's species richness is outstanding for the region. The percentages of non-pioneer (92 %), animal-dispersed (85 %), understory (55 %) and endemic species (39.2 %) from the FSN fulfill the criteria proposed for primary forest. Therefore, we conclude that the FSN maintained its characteristics as a primary forest which highlights its importance for the conservation of biotic resources in the region, where similar fragments are lacking or not described yet.

Isolation of DNA sequences with homology to a degenerate FaRP oligonucleotide from the crayfish Procambarus clarkii

The neuropeptide Th1RFamide with the sequence Phe-Met-Arg-Phe-amide was originally isolated in the clam Macrocallista nimbosa (price and Greenberg, 1977). Since its discovery, a large family ofFl\1RFamide-related peptides termed FaRPs have been found to be present in all major animal phyla with functions ranging from modulation of neuronal activity to alteration of muscular contractions. However, little is known about the genetics encoding these peptides, especially in invertebrates. As FaRP-encoding genes have yet to be investigated in the invertebrate Malacostracean subphylum, the isolation and characterization ofFaRP-encoding DNA and mRNA was pursued in this project. The immediate aims of this thesis were: (1) to amplify mRNA sequences of Procambarus clarkii using a degenerate oligonucleotide primer deduced from the common amino acid sequence ofisolated Procambarus FaRPS, (2) to determine if these amplification products encode FaRP gene sequences, and (3) to create a selective cDNA library of sequences recognized by the degenerate oligonucleotide primer. The polymerase chain reaction - rapid amplification of cDNA ends (PCR-RACE) is a procedure in which a single gene-specific primer is used in conjunction with a generalized 3' or 5' primer to amplify copies ofthe region between a single point in the transcript and the 3' or 5' end of cDNA of interest (Frohman et aI., 1988). PCRRACE reactions were optimized with respect to primers used, buffer composition, cycle number, nature ofgenetic substrate to be amplified, annealing, extension and denaturation temperatures and times, and use of reamplification procedures. Amplification products were cloned into plasmid vectors and recombinant products were isolated, as were the recombinant plaques formed in the selective cDNA library. Labeled amplification products were hybridized to recombinant bacteriophage to determine ligated amplification product presence. When sequenced, the five isolated PCR-RACE amplification products were determined not to possess FaRP-encoding sequences. The 200bp, 450bp, and 1500bp sequences showed homology to the Caenorhabditis elegans cosmid K09A11, which encodes for cytochrome P450; transfer-RNA; transposase; and tRNA-Tyr, while the 500bp and 750bp sequences showed homology with the complete genome of the Vaccinia virus. Under the employed amplification conditions the degenerate oligonucleotide primer was observed to bind to and to amplify sequences with either 9 or 10bp of 17bp identity. The selective cDNA library was obselVed to be of extremely low titre. When library titre was increased, white. plaques were isolated. Amplification analysis of eight isolated Agt11 sequences from these plaques indicated an absence of an insertion sequence. The degenerate 17 base oligonucleotide primer synthesized from the common amino acid sequence ofisolated Procambarus FaRPs was thus determined to be non-specific in its binding under the conditions required for its use, and to be insufficient for the isolation and identification ofFaRP-encoding sequences. A more specific primer oflonger sequence, lower degeneracy, and higher melting temperature (TJ is recommended for further investigation into the FaRP-encoding genes of Procambarlls clarkii.

What is the right balance for performance and isolation with virtualization in HPC?

The use of virtualization in high-performance computing (HPC) has been suggested as a means to provide tailored services and added functionality that many users expect from full-featured Linux cluster environments. The use of virtual machines in HPC can offer several benefits, but maintaining performance is a crucial factor. In some instances the performance criteria are placed above the isolation properties. This selective relaxation of isolation for performance is an important characteristic when considering resilience for HPC environments that employ virtualization. In this paper we consider some of the factors associated with balancing performance and isolation in configurations that employ virtual machines. In this context, we propose a classification of errors based on the concept of “error zones”, as well as a detailed analysis of the trade-offs between resilience and performance based on the level of isolation provided by virtualization solutions. Finally, a set of experiments are performed using different virtualization solutions to elucidate the discussion.