One-step Selection Research Articles

Contractor selection: fuzzy-control approach

This paper presents a fuzzy-logic-based system for selecting contractors. This tool, which was based on the fuzzy control technique, was created for the private sector client in traditional design–bid–build projects with one-step selection processes, but its philosophy can also serve other types of clients, industries, contracts, and selection processes. The system develops an assessment of different qualitative and quantitative issues that influence a contractor’s suitability for constructing a specific design in a specific environment (client’s needs and objectives, objectives prioritization, etc.), taking into account the risk of not achieving the client’s objectives. A computer application was developed and validated, including a Delphi analysis with professionals who are experts in contractor selection. The application covers the possibility of using different selection policies, when the essential project objective is cost, time or quality. As part of the validation process, a neural network was developed to prove that the fuzzy-control tool has a behavior that can be recognized by a neural network.

Trypanosoma cruzi as a model system to study the expression of exogenous genes coding for polyamine biosynthetic enzymes. Induction of DFMO resistance in transgenic parasites

Trypanosoma cruzi, the etiologic agent of Chagas' disease, is a polyamine auxotroph organism because its genome contains neither ornithine decarboxylase (ODC) nor arginine decarboxylase (ADC) genes, presumably lost during evolution. After transformation with a recombinant plasmid bearing the complete coding region of Crithidia fasciculata ODC gene, the transgenic parasites were able to synthesize putrescine and simultaneously became susceptible to α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. We have studied the emergence of DFMO-resistant T. cruzi after one-step selection of ODC-transformed parasites cultivated in the presence of high levels of the drug (5 mM). Our results have indicated a duplication of the ODC gene copy number in the drug-resistant cell line. The ODC transcripts and the corresponding translation products showed very significant increases (about 7- and 25-fold, respectively) in DFMO-resistant parasites, while the ODC enzymatic activity was 5 times higher than in drug-sensitive T. cruzi. The unequal increases of ODC protein and enzymatic activity in DFMO-resistant protozoa strongly suggest that in addition to gene amplification and enhanced transcription and translation, the assembly of ODC polypeptide chains into dimeric active enzyme molecules might also contribute to regulate the development of DFMO resistance.

One step engineering of T7-expression strains for protein production: Increasing the host-range of the T7-expression system

The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E. coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6Kγ ori plasmid, pBT20-Δ bla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm R-marker. We showed the engineering of E. coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-β- d-thiogalactopyranoside (IPTG).

TRANSFORMATION OF CHERRY: PRUNUS CERASUS L. 'MONTMORENCY' AND PRUNUS CERASUS × P. CANESCEN 'GISELA 6' MEDIATED BY AGROBACTERIUM TUMEFACIENS AND A TWO-STEP SELECTION SYSTEM

A two-step selection method was established to improve Agrobacterium tumefaciens-mediated stable transformation of sour cherry Prunus cerasus L. 'Montmorency' and rootstock P. cerasus x P. canescens 'Gisela 6'. Whole leaf explants, each with four cuts perpendicular through the midrib, were co-cultivated with EHA105:pBISN1 carrying the neomycin phosphotransferase gene (nptII) and an intron interrupted B-glucuronidase (GUS) reporter gene (gusA). Selection and regeneration of transformed cells and shoots of both cultivars was carried out on selection media starting with 25 mg L -1 kanamycin (Km) for 4 weeks followed by 50 mg L -1 Km for 8 weeks. Compared with a one-step selection using 50 mg L -1 Km, the two-step selection method minimized necrosis of inoculated leaf explants, and the transformation frequency was increased from 3.1% to 7.6% for 'Montmorency' and from 3.3% to 9.7% for 'Gisela 6', respectively. Southern analyses of the GUS- and PCR-positive transformants, 3 for 'Montmorency' and 2 for 'Gisela 6', confirmed stable integration of the transgenes.

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

BackgroundAs a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures.ResultsThe selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.ConclusionThe MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Characterization of clinical isolates of Pseudomonas aeruginosa heterogeneously resistant to carbapenems

Fourteen apparently carbapenem-susceptible Pseudomonas aeruginosa clinical isolates that exhibited colonies within the inhibition zone around carbapenem discs were analysed. MICs of carbapenems were determined and the isolates were genotyped by PFGE. Population analysis, one-step selection of carbapenem-resistant mutants and growth curves of progenitors and carbapenem-resistant subpopulations were performed. Agar dilution MICs of imipenem and meropenem ranged from 0.5 to 4 mg l(-1) and from 0.25 to 2 mg l(-1), respectively. Population analysis confirmed subpopulations that grew in concentrations of up to 18 mg l(-1) and 12 mg l(-1) of imipenem and meropenem, respectively, at frequencies ranging from 6.9 x 10(-5) to 1.1 x 10(-7), suggesting that they might not be detected by standard agar dilution MIC testing. The minority subpopulations exhibited MICs for imipenem ranging from 10 to 20 mg l(-1) and for meropenem from 4 to 14 mg l(-1). The one-step 8 mg l(-1) selection of imipenem-resistant mutants test showed growth in all isolates at frequencies ranging from 3.8 x 10(-4) to 5.1 x 10(-7). Growth curves revealed a prolonged lag phase and a short exponential phase for the heterogeneous subpopulations compared with their respective native subpopulations. These findings may be indicative that the use of carbapenems can lead to selection of P. aeruginosa resistant subpopulations that subsequently cause infections and result in treatment failure.

Production of monoclonal antibodies.

This unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one-step selection and cloning of hybridomas utilizing a semi-solid methylcellulose-based medium (ClonaCell-HY).

Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures: A Universal Tool for Development of Aptamers

Aptamers are DNA (or RNA) ligands selected from large libraries of random DNA sequences and capable of binding different classes of targets with high affinity and selectivity. Both the chances for the aptamer to be selected and the quality of the selected aptamer are largely dependent on the method of selection. Here we introduce selection of aptamers by nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM). The new method has a number of advantages over conventional approaches. First, NECEEM-based selection has exceptionally high efficiency, which allows aptamer development with fewer rounds of selection. Second, NECEEM can be equally used for selecting aptamers and finding their binding parameters. Finally, due to its comprehensive kinetic capabilities, the new method can potentially facilitate selection of aptamers with predefined K(d), k(off), and k(on) of the aptamer-target interaction. In this proof-of-principle work, we describe the theoretical bases of the method and demonstrate its application to a one-step selection of DNA aptamers with nanomolar affinity for protein farnesyltransferase.

Graft engineering for allogeneic haploidentical stem cell transplantation

Haploidentical stem cell transplantation has became a clinical reality in the last 10 years as it provides the chance of transplant for about 50% of patients with hematological malignancies who do not have a matched related or unrelated donor. Proper graft preparation for this type of transplant is crucial and this paper analyses our work over the past decade in the search for the optimal graft processing procedure moving from E-rosetting and soybean agglutination, through a combination of negative or positive selection of hematopoietic stem cells to the current method of one-step positive selection. In preparing a graft for haploidentical transplant, three essential requisites must be met. It must contain (1) a megadose (>10 × 10 6 × kg recipient b.w.) of hematopoietic stem cells to overcome the HLA histocompatibility barrier; (2) very few T-lymphocytes (CD3 + cells <3 × 10 4/kg recipient b.w.) to prevent severe acute and chronic graft-versus-host disease (GvHD); (3) very few B-lymphocytes to prevent Epstein–Barr virus-related lymphoproliferative disorders. With current graft processing technologies based on positive selection of hematopoietic stem cells, these requirements can be met. A 70–80% hematopoietic stem cell recovery ensures the target megadose is achieved in over 70% of cases with a T-cell depletion of more than 4 logs and a B-cell depletion of over 3 logs. Progress in graft processing has ensured primary, sustained engraftment rates of over 90% and has significantly reduced the incidence of severe acute GvHD and EBV-related lymphoproliferative disorders. Modern time-saving automated graft processing devices ensure reproducibility, reliability, and biological safety, which make widespread application of the haploidentical transplant currently feasible.

A pleiotropic defect reducing drug accumulation in cisplatin-resistant cells

The resistance of tumors to cisplatin remains a major cause of treatment failure in cancer patients. Multiple, simultaneous alterations are frequently encountered in cancer cells selected for cisplatin resistance. To determine whether the complex phenotype results from many different cellular alterations, single-step variants were isolated based on one-step selection in cisplatin. Reduced drug accumulation is a common feature of cisplatin-resistant (CP-r) cancer cells, which is probably caused by one or more dominant genes. Pulse-chase labeling and pulse-chase biotinylation of cell surface proteins suggest that membrane protein mislocalization occurs in CP-r cells, caused mainly by a defect in plasma membrane protein recycling, manifested also as a defect in acidification of lysosomes. This membrane protein mislocalization is presumed to reduce cell surface expression of a putative cisplatin carrier or carriers. In cells selected in several steps, decreased expression of folate-binding protein and arsenic-binding proteins, and reduced endocytosis were detected in CP-r cells, contributing to the reduced uptake of cisplatin, methotrexate and other related compounds. Multiple mechanisms in CP-r cells keep cytotoxic platinum compounds out of cells through defective expression of cell surface proteins such as transporters and carriers, and decreased expression of proteins involved in endocytosis.

Combining labelled and unlabelled data in the design of pattern classification systems

There has been much interest in applying techniques that incorporate knowledge from unlabelled data into a supervised learning system but less effort has been made to compare the effectiveness of different approaches and to analyse the behaviour of the learning system when using different ratios of labelled to unlabelled data. In this paper various methods for learning from labelled and unlabelled data are first discussed and categorised into one of three major groups: pre-labelling, post-labelling and semi-supervised approaches. Their generalised formal description and extensive experimental analysis is then provided. The experimental results show that when supported by unlabelled samples much less labelled data is generally required to build a classifier without compromising the classification performance. If only a very limited amount of labelled data is available the results based on random selection of labelled samples show high variability and the performance of the final classifier is more dependent on how reliable the labelled data samples are rather than use of additional unlabelled data. In response to this finding three types of static (one-step) selection methods guided by a clustering information and various options of allocating a number of samples within clusters and their distributions have been proposed and analysed. A significant improvement compared to the random selection of the labelled samples have been observed when using these selective sampling techniques.

Cell Wall Thickening Is a Common Feature of Vancomycin Resistance in Staphylococcus aureus

We have previously shown that a thickened cell wall is responsible for the vancomycin resistance of vancomycin-resistant Staphylococcus aureus (VRSA) (equivalent to vancomycin-intermediate S. aureus and glycopeptide-intermediate S. aureus) strain Mu50 (L. Cui, H. Murakami, K. Kuwahara-Arai, H. Hanaki, and K. Hiramatsu, Antimicrob. Agents Chemother. 44:2276-2285, 2000). However, the mechanism of vancomycin resistance in other VRSA strains remained unclear. In this study, 16 clinical VRSA strains from seven countries were subjected to serial daily passage in drug-free medium. After 10 to 84 days of passage in the nonselective medium, passage-derived strains with decreased MICs of vancomycin (MIC, <4 mg/liter) were obtained. However, all of the passage-derived strains except one (15 of 16) still possessed subpopulations that were resistant to vancomycin as judged by population analysis, and vancomycin-resistant mutant strains were selected from the passage-derived strains by one-step vancomycin selection with a frequency of 4.25 x 10(-6) to 1.64 x 10(-3). The data indicated that vancomycin-resistant cells are frequently generated from the passage-derived strains even after vancomycin selective pressure is lifted. Cell wall thicknesses and MICs of glycopeptides (vancomycin and teicoplanin) and beta-lactams (imipenem and oxacillin) were determined for a total of 48 strains, including 15 sets of three strains: the clinical VRSA strain, the passage-derived strain, and the vancomycin-resistant mutant strain obtained from the passage-derived strain. No simple correlation between glycopeptide and beta-lactam MICs was seen, while significant correlations between MICs of vancomycin and teicoplanin (r = 0.679; P < 0.001) and between MICs of imipenem and oxacillin (r = 0.787; P < 0.001) were recognized. Moreover, all of the VRSA strains had significantly thickened cell walls, which became thinner with the loss of vancomycin resistance during drug-free passages and again became thick in the resistant mutant strains. The data showed that cell wall thickness had high correlation with the MICs of the two glycopeptides (correlation coefficients, 0.908 for vancomycin and 0.655 for teicoplanin) but not with those of the beta-lactam antibiotics tested. These results together with coupled changes of cell wall thickness and vancomycin MICs in 16 isogenic sets of strains indicate that thickening of the cell wall is a common phenotype of clinical VRSA strains and may be a phenotypic determinant for vancomycin resistance in S. aureus.

A more pluralist typology of selection processes

Instead of using only one notion of selection I argue for a broader typology of different types of selection. Three such types are differentiated, namely simple one-step selection, iterated one-step selection, and multi-step selection. It is argued that this more general and more inclusive typology might face more effectively the possible challenges of a general account of selection.

A two-step initial mass function:

If stars originate in transient bound clusters of moderate size, these clusters will decay due to dynamic interactions in which a hard binary forms and ejects most or all the other stars. When the cluster members are chosen at random from a reasonable initial mass function (IMF), the resulting binary characteristics do not match current observations. We nd a signicant improvement in the trends of binary properties from this scenario when an additional constraint is taken into account, namely that there is a distribution of total cluster masses set by the masses of the cloud cores from which the clusters form. Two distinct steps then determine nal stellar masses { the choice of a cluster mass and the formation of the individual stars. We refer to this as a \two-step IMF. Simple statistical arguments are used in this paper to show that a two-step IMF, combined with typical results from dynamic few-body system decay, tends to give better agreement between computed binary characteristics and observations than a one-step mass selection process.

A Genome-Wide Screening in Saccharomyces cerevisiae for Genes That Confer Resistance to the Anticancer Agent Cisplatin

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.

Planungsrechnungen zur Optimierung von Zuchtplänen der Rasse Merinofleischschaf

Abstract. Title of the paper: Alternative selection strategies for the Mutton Merino breed to optimize breedine Systems This paper deals with the investigation of alternative selection strategies for the Mutton Merino breed to improve lamb meat production and fertility. A main topic is the application of ultrasonic scanning to evaluate the meat content on live animals. The aim of model calculations was the optimisation and comparison of five different selection strategies within three different schemes of gene dissimation. First a basic scheme was defined and optimised (selection on field records, one-step selection, no scanning). It was regarded as the reference scheme to which all other breeding Systems were related. The following schemes considered both, selection with and without scanning. A second scheme, also based on field records, includes two-step selection of rams (called improved field test scheme). In a third and fourth scheme a level of uniform environment was assumed for ram progeny testing. These are a central testing Station on the one hand with slaughtering and carcass evaluation on progeny, and one or more contract farms on the other (without slaughtering). For a fifth selection scheme an open nucleus was assumed with ram progeny testing in associated test herds. In a dcterministic approach using the ZPLAN Computer program, the monetary genetic gain for the breeding objeetive (traits lambs weaned, litter weight at weaning, postweaning daily gain and lean meat content) and the profit were calculated for each scheme after optimisation of various biological-technical coefficients. The highest profit was achieved with a nucleus scheme (DM 9,16). Due to low recording costs the basic scheme was second (DM 7,19) and, because of high costs, the Station scheme was last (DM 4,22). The other two schemes were intermediate (DM 6,98 for the scheme with contract farms and DM 6,58 for the improved field test scheme). On an average over all schemes, scanning resulted in a 0,24 DM (i.e. almost 30 per cent) increase of the monetary genetic gain and a threefold higher genetic gain for lean meat percentage. In all schemes scanning lead also to a higher profit because the higher retum of selection based on scanning exceeded the higher costs.

Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.

Characterization of Mycoplasma hominis mutations involved in resistance to fluoroquinolones.

Fluoroquinolone-resistant mutants of Mycoplasma hominis were selected in vitro from the PG21 susceptible reference strain either by multistep selection on increasing concentrations of various fluoroquinolones or by one-step selection on agar medium with ofloxacin. The quinolone resistance-determining regions (QRDR) of the structural genes encoding the A and b subunits of DNA gyrase were amplified by PCR, and the nucleotide sequences of eight multistep-selected resistant strains were compared to those of susceptible strain PG21. Four high-level resistant mutants that were selected on norfloxacin or ofloxacin contained a C-to-T transition in the gyrA QRDR, leading to substitution of Ser-83 by Leu in the GyrA protein. Analysis of the sequence of the gyrB QRDR of the eight multistep-selected mutants did not reveal any difference compared to that of the gyrB QRDR of the reference strain M. hominis PG21. Similar analyses of eight one-step-selected mutants did not reveal any base change in the gyrA and gyrB QRDRs. These results suggest that in M. hominis, like in other bacterial species, a gyrA mutation at Ser-83 is associated with fluoroquinolone resistance.

Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors.

The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene-replacement events into Mycobacterium smegmatis. A sucrose counter-selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrF::K(m)) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one-step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic-exchange mutants. Two-step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.