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Sample records for yeast identification system

  1. Evaluation of Automated Yeast Identification System

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.

    1996-01-01

    One hundred and nine teleomorphic and anamorphic yeast isolates representing approximately 30 taxa were used to evaluate the accuracy of the Biolog yeast identification system. Isolates derived from nomenclatural types, environmental, and clinica isolates of known identity were tested in the Biolog system. Of the isolates tested, 81 were in the Biolog database. The system correctly identified 40, incorrectly identified 29, and was unable to identify 12. Of the 28 isolates not in the database, 18 were given names, whereas 10 were not. The Biolog yeast identification system is inadequate for the identification of yeasts originating from the environment during space program activities.

  2. Species level identification and antifungal susceptibility of yeasts isolated from various clinical specimens and evaluation of Integral System Yeasts Plus.

    PubMed

    Bicmen, Can; Doluca, Mine; Gulat, Sinem; Gunduz, Ayriz T; Tuksavul, Fevziye

    2012-07-01

    It is essential to use easy, standard, cost-effective and accurate methods for identification and susceptibility testing of yeasts in routine practice. This study aimed to establish the species distribution and antifungal susceptibility of yeast isolates and also to evaluate the performance of the colorimetric and commercially available Integral System Yeasts Plus (ISYP). Yeast isolates (n=116) were identified by conventional methods and ISYP. Antifungal susceptibility testing was performed by the microdilution method according to the standards of CLSI M27-A3 and ISYP. Candida albicans (50%) was the most common species isolated, followed by C. parapsilosis (25%) (mostly in blood samples). According to the CLSI M27-S3 criteria, resistance rates for amphotericin B, flucytosine, fluconazole, itraconazole, and voriconazole were 0%, 0%, 4.6%, 4.5% and 1.8%, respectively. Resistance for miconazole (MIC >1 mg/L) was found as 17.9%. Sixty-two (53.4%) of the isolates which were analyzed by ISYP showed disagreement with those identified by the conventional methods and API ID 32C identification kit or a specific identification code could not be assigned by ISYP. The performance of ISYP could be indicated as low for all antifungal drugs tested according to the ROC analysis (AUC: 0.28-0.56). As the current version of ISYP displays a poor performance, it is recommended to use the other commercial systems whose results are approved as reliable and in agreement with those of the reference methods in identification and susceptibility testing of yeasts. PMID:22842602

  3. [Rapid identification and susceptibility to killer toxins of yeasts isolated from non-systemic mycoses].

    PubMed

    Sangorrín, M P; Lopes, C A; Rivero, A; Caballero, A C

    2007-01-01

    Rapid identification and susceptibility to killer toxins of yeasts isolated from non-systemic mycoses. The use of quick and reliable yeast identification methods, as well as the development of new antifungal agents with more specific targets, will enable a more efficient treatment of mycoses. In the present work, a total of 53 clinical isolates obtained from non-systemic infections in Neuquén Hospitals and an ophthalmologic clinic in Buenos Aires during 2005, were identified by means of a rapid molecular method (ITS1-5.8S ADNr-ITS2 PCR-RFLP). Additionally, the killer susceptibility of the isolates was tested against reference and indigenous killer yeasts on plate tests. Eight yeast species were identified among the clinical isolates: Candida albicans (52%), Candida parapsilosis (17%), Candida tropicalis (10%), Candida krusei (5%), Candida glabrata (4%), Candida guilliermondii (4%), Kluyveromyces lactis (4%) and Saccharomyces cerevisiae (4%). Sixty-nine percent of the isolates corresponding to the predominant species (C. albicans) were related to vaginal infections. On the other hand, 61% of the yeasts associated with ocular infections were identified as C. parapsilosis. Two indigenous killer isolates DVMais5 and HCMeiss5, belonging to Pichia anomala and P. kluyveri respectively, exhibited the broadest killer spectrum against clinical isolates.

  4. Multicenter Study Evaluating the Vitek MS System for Identification of Medically Important Yeasts

    PubMed Central

    Westblade, Lars F.; Jennemann, Rebecca; Branda, John A.; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B.; Ginocchio, Christine C.; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Rychert, Jenna A.; Sercia, Linda

    2013-01-01

    The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories. PMID:23658267

  5. Multicenter study evaluating the Vitek MS system for identification of medically important yeasts.

    PubMed

    Westblade, Lars F; Jennemann, Rebecca; Branda, John A; Bythrow, Maureen; Ferraro, Mary Jane; Garner, Omai B; Ginocchio, Christine C; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Rychert, Jenna A; Sercia, Linda; Burnham, Carey-Ann D

    2013-07-01

    The optimal management of fungal infections is correlated with timely organism identification. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is revolutionizing the identification of yeasts isolated from clinical specimens. We present a multicenter study assessing the performance of the Vitek MS system (bioMérieux) in identifying medically important yeasts. A collection of 852 isolates was tested, including 20 Candida species (626 isolates, including 58 C. albicans, 62 C. glabrata, and 53 C. krusei isolates), 35 Cryptococcus neoformans isolates, and 191 other clinically relevant yeast isolates; in total, 31 different species were evaluated. Isolates were directly applied to a target plate, followed by a formic acid overlay. Mass spectra were acquired using the Vitek MS system and were analyzed using the Vitek MS v2.0 database. The gold standard for identification was sequence analysis of the D2 region of the 26S rRNA gene. In total, 823 isolates (96.6%) were identified to the genus level and 819 isolates (96.1%) were identified to the species level. Twenty-four isolates (2.8%) were not identified, and five isolates (0.6%) were misidentified. Misidentified isolates included one isolate of C. albicans (n = 58) identified as Candida dubliniensis, one isolate of Candida parapsilosis (n = 73) identified as Candida pelliculosa, and three isolates of Geotrichum klebahnii (n = 6) identified as Geotrichum candidum. The identification of clinically relevant yeasts using MS is superior to the phenotypic identification systems currently employed in clinical microbiology laboratories.

  6. Evaluation of the Biolog MicroStation system for yeast identification

    NASA Technical Reports Server (NTRS)

    McGinnis, M. R.; Molina, T. C.; Pierson, D. L.; Mishra, S. K.

    1996-01-01

    One hundred and fifty-nine isolates representing 16 genera and 53 species of yeasts were processed with the Biolog MicroStation System for yeast identification. Thirteen genera and 38 species were included in the Biolog database. For these 129 isolates, correct identifications to the species level were 13.2, 39.5 and 48.8% after 24, 48 and 72 hours incubation at 30 degrees C, respectively. Three genera and 15 species which were not included in the Biolog database were also tested. Of the 30 isolates studied, 16.7, 53.3 and 56.7% of the isolates were given incorrect names from the system's database after 24,48 and 72 h incubation at 30 degrees C, respectively. The remaining isolates of this group were not identified.

  7. Evaluation of the API 20C yeast identification system for the differentiation of some dematiaceous fungi.

    PubMed Central

    Espinel-Ingroff, A; McGinnis, M R; Pincus, D H; Goldson, P R; Kerkering, T M

    1989-01-01

    Ninety-seven isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii, Phaeoannellomyces werneckii, Phialophora spp., Wangiella dermatitidis, and Xylohypha bantiana were used to evaluate the API 20C Yeast Identification System for the differentiation of dematiaceous fungi. Using the API 20C system, we were able to distinguish most species of Phialophora and Cladosporium and to separate L. hoffmannii from the species of Phialophora tested; X. bantiana from C. carrionii, C. resinae, and C. sphaerospermum; and W. dermatitidis from Exophiala jeanselmei and Exophiala spinifera. Ninety-two (60.1%) of 153 possible species-pair combinations were separated. PMID:2808678

  8. Comparative Evaluation of BD Phoenix and Vitek 2 Systems for Species Identification of Common and Uncommon Pathogenic Yeasts

    PubMed Central

    Posteraro, Brunella; Ruggeri, Alberto; De Carolis, Elena; Torelli, Riccardo; Vella, Antonietta; De Maio, Flavio; Ricciardi, Walter; Posteraro, Patrizia

    2013-01-01

    The BD Phoenix system was evaluated for species-level identification of yeasts (250 clinical isolates) and compared with the Vitek 2 system, using ribosomal internal transcribed spacer (ITS) sequence analysis as the gold standard. Considering only the species included in each system's database, 96.3% (236/245) and 91.4% (224/245) of the isolates were correctly identified by BD Phoenix and Vitek 2, respectively. PMID:23966500

  9. [Evaluation of common commercial systems for the identification of yeast isolates in microbiology laboratories: a multicenter study].

    PubMed

    Karabıçak, Nilgün; Uludağ Altun, Hatice; Karatuna, Onur; Hazırolan, Gülşen; Aksu, Neriman; Adiloğlu, Ali; Akyar, Işın

    2015-04-01

    Accurate and rapid identification of yeast isolates have become important in recent years for not only antifungal susceptibility testing due to the species-specific clinical resistance breakpoints but also early initiation of appropriate antifungal therapy. In clinical microbiology laboratories species identification of yeasts is often performed with several commercial systems based on biochemical properties and rarely according to the physiological and morphological characteristics. The aim of this study was to compare the two common commercial systems, VITEK 2 YST ID Card (Vitek; bioMérieux, France) and API 20C AUX (API; bioMérieux, France) with conventional mycological methods. A total of 473 clinical yeast strains isolated from clinical specimens in different university and training/research hospitals and identified by Vitek system were included in the study. The isolates were re-identified with API and conventional methods including morphological identification in the Mycology Reference Laboratory of the Public Health Institute of Turkey. Candida dubliniensis MYA 583, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019, Candida albicans ATCC 10231 and Cryptococcus neoformans ATCC 32268 were used as quality control strains and those standard strains were studied consecutively 10 days with both of the methods. The results of identification by Vitek and API were compared with the results of conventional methods for those 473 yeast isolates [6 genus (Candida, Cryptococcus, Blastoshizomyces, Rhodotorula, Saccharomyces, Trichosporon), 17 species (5 common and 12 rarely isolated)]. The performances of the systems were better (Vitek: 95%; API: 96%) for the commonly detected species (C.albicans, C.parapsilosis, C.glabrata, C.tropicalis and C.krusei) than those for rarely detected species (Vitek: 78.4%; API: 71.6%) (p= 0.155). Misidentification or unidentification were mostly detected for C.parapsilosis (Vitek: 6/87; API: 7/87) and C.glabrata (Vitek: 9/104; API

  10. Yeast one-hybrid gγ recruitment system for identification of protein lipidation motifs.

    PubMed

    Fukuda, Nobuo; Doi, Motomichi; Honda, Shinya

    2013-01-01

    Fatty acids and isoprenoids can be covalently attached to a variety of proteins. These lipid modifications regulate protein structure, localization and function. Here, we describe a yeast one-hybrid approach based on the Gγ recruitment system that is useful for identifying sequence motifs those influence lipid modification to recruit proteins to the plasma membrane. Our approach facilitates the isolation of yeast cells expressing lipid-modified proteins via a simple and easy growth selection assay utilizing G-protein signaling that induces diploid formation. In the current study, we selected the N-terminal sequence of Gα subunits as a model case to investigate dual lipid modification, i.e., myristoylation and palmitoylation, a modification that is widely conserved from yeast to higher eukaryotes. Our results suggest that both lipid modifications are required for restoration of G-protein signaling. Although we could not differentiate between myristoylation and palmitoylation, N-terminal position 7 and 8 play some critical role. Moreover, we tested the preference for specific amino-acid residues at position 7 and 8 using library-based screening. This new approach will be useful to explore protein-lipid associations and to determine the corresponding sequence motifs.

  11. [Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples].

    PubMed

    Gayibova, Ülkü; Dalyan Cılo, Burcu; Ağca, Harun; Ener, Beyza

    2014-07-01

    Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C

  12. [Comparison of Phoenix™ Yeast ID Panel and API® ID 32C commercial systems for the identification of Candida species isolated from clinical samples].

    PubMed

    Gayibova, Ülkü; Dalyan Cılo, Burcu; Ağca, Harun; Ener, Beyza

    2014-07-01

    Opportunistic fungal pathogens are one of the important causes of nosocomial infections, and several different types of yeasts, especially Candida species are increasingly recovered from immunocompromised patients. Since many of the yeasts are resistant to the commonly used antifungal agents, the introduction of appropriate therapy depends on rapid and accurate identification. The aims of this study were to compare the commercial identification systems namely API® ID 32C (bioMerieux, France) and Phoenix™ Yeast ID Panel (Becton Dickinson Diagnostics, USA) for the identification of Candida species and to evaluate the effect of morphological findings in the identification process. A total of 211 yeast strains isolated from different clinical samples (111 urine, 34 blood/vascular catheter, 27 upper/lower respiratory tract, 16 abscess/pus, 13 throat/vagina swabs and 10 sterile body fluids) of 137 patients hospitalized in Uludag University Health and Research Center between October 2013 to January 2014, were included in the study. Samples were cultured on blood agar, chromogenic agar (CHROMagar Candida, BD, USA) and Saboraud's dextrose agar (SDA), and isolated yeast colonies were evaluated with germ tube test and morphological examination by microscopy on cornmeal/Tween-80 agar. The isolates were identified as well by two commercial systems according to the manufacturers' recommendations. Discrepant results between the systems were tried to be resolved by using morphological characteristics of the yeasts. Of the isolates 159 were identified identical by both of the systems, and the concordance between those systems were estimated as 75.4%. According to the concordant identification, the most frequently isolated species was C.albicans (44.1%) followed by C.tropicalis (9.9%), C.glabrata (9.5%), C.parapsilosis (8.5%) and C.kefyr (8.1%). The concordance rate was 81.7% in identification of frequently isolated species (C.albicans, C.tropicalis, C.parapsilosis, C.glabrata, C

  13. Yeast killer systems.

    PubMed Central

    Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

    1997-01-01

    The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

  14. Rapid methods for identification of yeasts.

    PubMed Central

    Huppert, M; Harper, G; Sun, S H; Delanerolle, V

    1975-01-01

    Opportunistic infections by yeasts have been implicated as one of the major causes of complications in the compromised patient. Rapid recognition and identification of these yeasts is essential for patient management, but conventional liquid medium methods for completing identification tests are cumbersome and time consuming. Rapid tests have been devised based on modifications of methods commonly used in bacteriology. These rapid methods included tests for carbohydrate and nitrate assimilation, fermentation, and urease production. These were compared with several current methods for accuracy of results, for time to final identification, and for economy of time and reagents. In addition, the usual tests for pseudogerm tube formation, for production of hyphae or pseudohyphae, and for growth temperatures were included. The rapid tests achieved 96% or better accuracy compared with expected results, and 46 species of yeasts were identified in 1 to 2 days compared with the 10 to 14 days required by conventional liquid culture methods. Images PMID:1241586

  15. [Construction and Identification of the Bait Vector Containing Duck Circovirus Cap Gene for the Yeast Two-hybrid System].

    PubMed

    Xu, Yu; Zhang, Zhilong; Lu, Yanyan; Zhang, Lei; Li, Pengfei; Jia, Renyong

    2015-05-01

    To construct a bait expression vector containing the duck circovirus Cap gene for use in the yeast two-hybrid system, the whole cap codon-optimized gene was inserted into pGBKT7 vector and confirmed by PCR, restriction enzyme digestion, and sequence analysis. After transformation into a Y2HGold yeast strain, the expression of Cap protein was analyzed by Western blotting. Toxicity and self-activation of the bait protein were detected using different dropout minimal base. PCR reaction, restriction enzyme digestion, and sequencing analyses indicated that the duck circovirus Cap gene was correctly inserted into pG- BKT7. Western blotting showed that the whole Cap protein was expressed. The recombinant bait protein had no toxicity and self-activation. Therefore, the bait vector with the Cap gene was constructed successfully, providing a foundation for future screening for interacting proteins in the yeast two-hybrid system.

  16. Development of a membrane-anchored ligand and receptor yeast two-hybrid system for ligand-receptor interaction identification

    PubMed Central

    Li, Jingjing; Gao, Jin; Han, Lei; Zhang, Yinjie; Guan, Wen; Zhou, Liang; Yu, Yan; Han, Wei

    2016-01-01

    Identifying interactions between ligands and transmembrane receptors is crucial for understanding the endocrine system. However, the present approaches for this purpose are still not capable of high-throughput screening. In this report, a membrane-anchored ligand and receptor yeast two-hybrid (MALAR-Y2H) system was established. In the method, an extracellular ligand is linked with an intracellular split-ubiquitin reporter system via a chimeric transmembrane structure. Meanwhile, the prey proteins of transmembrane receptors are fused to the other half of the split-ubiquitin reporter system. The extracellular interaction of ligands and receptors can lead to the functional recovery of the ubiquitin reporter system in yeast, and eventually lead to the expression of report genes. Consequently, the system can be used to detect the interactions between extracellular ligands and their transmembrane receptors. To test the efficiency and universality of the method, interactions between several pairs of ligands and receptors of mouse were analyzed. The detecting results were shown to be thoroughly consistent with the present knowledge, indicating MALAR-Y2H can be utilized for such purpose with high precision, high efficiency and strong universality. The characteristics of the simple procedure and high-throughput potential make MALAR-Y2H a powerful platform to study protein-protein interaction networks between secreted proteins and transmembrane proteins. PMID:27762338

  17. Improved identification of yeast species directly from positive blood culture media by combining Sepsityper specimen processing and Microflex analysis with the matrix-assisted laser desorption ionization Biotyper system.

    PubMed

    Yan, Yingjun; He, Ying; Maier, Thomas; Quinn, Criziel; Shi, Gongyi; Li, Haijing; Stratton, Charles W; Kostrzewa, Markus; Tang, Yi-Wei

    2011-07-01

    Current methods for identification of yeast from blood cultures may take several days after these microorganisms have been observed by Gram stain smears from positive blood cultures. We explored the use of a matrix-assisted laser desorption ionization (MALDI) Biotyper system in combination with Sepsityper specimen processing and Microflex analysis for improved detection and identification of yeast species directly from positive blood culture specimens demonstrating yeast-like organisms by Gram stain. The limit of detection of yeast species in blood culture medium was determined to be 5.9 × 10(5) CFU, with intra- and interstrain coefficients of variation of 1.8 to 3.6% and 2.9%, respectively. A total of 42 yeast-containing positive blood culture specimens were processed, and the identification results were compared to those obtained by routinely used phenotypic methods. Specimens with discrepant results between the Biotyper and phenotypic methods were identified on the basis of internal transcribed spacer region sequencing. The MALDI Biotyper system correctly identified the 42 specimens to species level, including 28 (66.7%) Candida albicans, 8 (19.0%) Candida parapsilosis, and 5 (11.9%) Candida tropicalis isolates and 1 (2.4%) Cryptococcus neoformans isolate. The entire procedure, from specimen extraction to final result reporting, can be completed within 1 h. Our data indicated that the Sepsityper specimen processing and Microflex analysis by the MALDI Biotyper system provide a rapid and reliable tool for yeast species identification directly from positive blood culture media.

  18. Yeast metabolic state identification using micro-fiber optics spectroscopy

    NASA Astrophysics Data System (ADS)

    Silva, J. S.; Castro, C. C.; Vicente, A. A.; Tafulo, P.; Jorge, P. A. S.; Martins, R. C.

    2011-05-01

    Saccharomyces cerevisiae morphology is known to be dependent on the cell physiological state and environmental conditions. On their environment, wild yeasts tend to form complex colonies architectures, such as stress response and pseudohyphal filaments morphologies, far away from the ones found inside bioreactors, where the regular cell cycle is observed under controlled conditions (e.g. budding and flocculating colonies). In this work we explore the feasibility of using micro-fiber optics spectroscopy to classify Saccharomyces cerevisiae S288C colony structures in YPD media, under different growth conditions, such as: i) no alcohol; ii) 1 % (v/v) Ethanol; iii) 1 % (v/v) 1-butanol; iv) 1 % (v/v) Isopropanol; v) 1 % (v/v) Tert-Amyl alcohol (2 Methyl-2-butanol); vi) 0,2 % (v/v) 2-Furaldehyde; vii) 5 % (w/v) 5 (Hydroxymethyl)-furfural; and viii) 1 % (w/v) (-)-Adenosine3', 5'cyclic monophosphate. The microscopy system includes a hyperspectral camera apparatus and a micro fiber (sustained by micro manipulator) optics system for spectroscopy. Results show that micro fiber optics system spectroscopy has the potential for yeasts metabolic state identification once the spectral signatures of colonies differs from each others. This technique associated with others physico-chemical information can benefit the creation of an information system capable of providing extremely detailed information about yeast metabolic state that will aid both scientists and engineers to study and develop new biotechnological products.

  19. Species identification of invasive yeasts including Candida in Pakistan: limitations of phenotypic methods

    PubMed Central

    Farooqi, Joveria; Jabeen, Kauser; Saeed, Noureen; Zafar, Afia; Brandt, Mary Eleanor; Hasan, Rumina

    2015-01-01

    Objective To compare phenotypic and genotypic methods of yeast identification. Methods The in-vitro cross-sectional study was conducted from January 2006 to May 2009. Invasive yeasts isolated at the clinical microbiology laboratory at the Aga Khan University (AKU), Karachi, Pakistan, were identified. Speciation by phenotypic and molecular methods was compared. All yeasts isolated during the study period from blood and other invasive sites were identified using standard methods. Isolates were shipped to Mycotic Diseases Branch, Centres for Disease Control and Prevention, Atlanta, Georgia, USA, for identification by Luminex flow cytometric multianalyte profiling (xMAP) system. Ribosomal ITS2 DNA sequencing was performed on isolates not identified by Luminex. Result Of the 214 invasive yeasts evaluated, Candida species were 209 (97.7%) while the frequency of non-Candida species was 5 (2.3%). Overall agreement between phenotypic and molecular identification was 81.3%, 90.3% amongst the more common Candida species, and only 38.8% amongst the uncommon yeasts. Conclusion Phenotypic methods of identification proved adequate for common Candida species, but were deficient in recognising rare Candida and non-Candida yeasts, highlighting the importance of molecular methods for identification. PMID:23866432

  20. Microfermentation Test For Identification Of Yeast

    NASA Technical Reports Server (NTRS)

    Pierson, D. L.; Mishra, S. K.; Molina, Thomas C.

    1995-01-01

    Microfermentation test developed as supplementary method for use in identifying yeasts, especially in clinical and environmental studies. In comparison with traditional fermentation tests, simpler and easier, and requiries less equipment, material, and laboratory space. Results obtained in days instead of weeks.

  1. [Evaluation of Vitek 2 for the identification of Candida yeasts].

    PubMed

    Ochiuzzi, María E; Cataldi, Silvana; Guelfand, Liliana; Maldonado, Ivana; Arechavala, Alicia

    2014-01-01

    The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24%) o API ID 32 C (76%) kits (bioMérieux, Marcy L'Etoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3%. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species.

  2. [Evaluation of Vitek 2 for the identification of Candida yeasts].

    PubMed

    Ochiuzzi, María E; Cataldi, Silvana; Guelfand, Liliana; Maldonado, Ivana; Arechavala, Alicia

    2014-01-01

    The aim of this investigation was to evaluate the performance of Vitek 2 YST cards (bioMérieux, Inc., Hazelwood, MO, USA) for the identification of yeasts of the genus Candida. A total of 168 isolates were analyzed and the results were compared to those of the API 20 C AUX (24%) o API ID 32 C (76%) kits (bioMérieux, Marcy L'Etoile, France). Each isolate was grown in chromogenic agar and in corn meal agar (Oxoid, UK) to observe its micromorphology. C. albicans and C. dublininesis were identified by additional biochemical and molecular tests. The agreement observed was 98.3%. Only three isolates were incorrectly identified by Vitek 2: one strain of C .tropicalis and one strain of C. krusei were identified as C. parapsilosis by YST while one strain of C. krusei was identified with low discrimination. The average time for obtaining results was 18.25 h. Vitek 2 is a simple, safe and useful system for the identification of significant Candida species. PMID:25011593

  3. Identification of Critical Amino Acids Conferring Lethality in VopK, a Type III Effector Protein of Vibrio cholerae: Lessons from Yeast Model System

    PubMed Central

    Bankapalli, Leela Krishna; Mishra, Rahul Chandra; Singh, Balvinder; Raychaudhuri, Saumya

    2015-01-01

    VopK, a type III effector protein, has been implicated in the pathogenesis of Vibrio cholerae strains belonging to diverse serogroups. Ectopic expression of this protein exhibits strong toxicity in yeast model system. In order to map critical residues in VopK, we scanned the primary sequence guided by available data on various toxins and effector proteins. Our in silico analysis of VopK indicated the presence of predicted MCF1-SHE (SHxxxE) serine peptidase domain at the C-terminus region of the protein. Substitution of each of the predicted catalytic triad residues namely Ser314, His353 and Glu357 with alanine resulted in recombinant VopK proteins varying in lethality as evaluated in yeast model system. We observed that replacement of glutamate357 to alanine causes complete loss in toxicity while substitutions of serine314 and histidine353 with alanine exhibited partial loss in toxicity without affecting the stability of variants. In addition, replacement of another conserved serine residue at position 354 (S354) within predicted S314H353E357 did not affect toxicity of VopK. In essence, combined in silico and site directed mutagenesis, we have identified critical amino acids contributing to the lethal activity of VopK in yeast model system. PMID:26488395

  4. Differential identification of Candida species and other yeasts by analysis of (/sup 35/S)methionine-labeled polypeptide profiles

    SciTech Connect

    Shen, H.D.; Choo, K.B.; Tsai, W.C.; Jen, T.M.; Yeh, J.Y.; Han, S.H.

    1988-12-01

    This paper describes a scheme for differential identification of Candida species and other yeasts based on autoradiographic analysis of protein profiles of (/sup 35/S)methionine-labeled cellular proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using ATCC strains as references, protein profile analysis showed that different Candida and other yeast species produced distinctively different patterns. Good agreement in results obtained with this approach and with other conventional systems was observed. Being accurate and reproducible, this approach provides a basis for the development of an alternative method for the identification of yeasts isolated from clinical specimens.

  5. Yeast identification in floral nectar of Mimulus aurantiacus (Invited)

    NASA Astrophysics Data System (ADS)

    Kyauk, C.; Belisle, M.; Fukami, T.

    2009-12-01

    Nectar is such a sugar-rich resource that serves as a natural habitat in which microbes thrive. As a result, yeasts arrive to nectar on the bodies of pollinators such as hummingbirds and bees. Yeasts use the sugar in nectar for their own needs when introduced. This research focuses on the identification of different types of yeast that are found in the nectar of Mimulus aurantiacus (commonly known as sticky monkey-flower). Unopened Mimulus aurantiacus flower buds were tagged at Jasper Ridge and bagged three days later. Floral nectar was then extracted and plated on potato dextrose agar. Colonies on the plates were isolated and DNA was extracted from each sample using QIAGEN DNeasy Plant Mini Kit. The DNA was amplified through PCR and ran through gel electrophoresis. The PCR product was used to clone the nectar samples into an E.coli vector. Finally, a phylogenetic tree was created by BLAST searching sequences in GenBank using the Internal Transcribed Space (ITS) locus. It was found that 18 of the 50 identified species were Candida magnifica, 14 was Candida rancensis, 6 were Crytococcus albidus and there were 3 or less of the following: Starmella bombicola, Candida floricola, Aureobasidium pullulans, Pichia kluyvera, Metschnikowa cibodaserisis, Rhodotorua colostri, and Malassezia globosa. The low diversity of the yeast could have been due to several factors: time of collection, demographics of Jasper Ridge, low variety of pollinators, and sugar concentration of the nectar. The results of this study serve as a necessary first step for a recently started research project on ecological interactions between plants, pollinators, and nectar-living yeast. More generally, this research studies the use of the nectar-living yeast community as a natural microcosm for addressing basic questions about the role of dispersal and competitive and facilitative interactions in ecological succession.

  6. Production of serpins using yeast expression systems.

    PubMed

    Pemberton, Philip A; Bird, Phillip I

    2004-02-01

    Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered. PMID:14698631

  7. Human heart cell proteins interacting with a C-terminally truncated 2A protein of coxsackie B3 virus: identification by the yeast two-hybrid system.

    PubMed

    Zhao, Tiansheng; Huang, Xiaotian; Xia, Yanhua

    2016-04-01

    Protein 2A is a non-structural protein of coxsackievirus B3 (CVB3), an important human pathogen that can cause a variety of human diseases. Protein 2A not only participates in viral life cycle, but also regulates host cell functions; however, the underlying mechanisms remain poorly understood. In order to better understand the molecular mechanisms of CVB3 2A's function, the yeast two-hybrid (Y2H) system was adopted to screen for CVB3 2A interactive proteins in the human heart cDNA library. Full-length 2A shows strong transcriptional activity in yeast cells, which interferes with the application of Y2H system; therefore, a series of 2A deletion mutants were constructed. Analysis of transcriptional self-activation revealed that 2A lost its transcriptional activity after truncation of 60 amino acids (aa) at the N-terminus or deletion of 17 aa at the C-terminus. Choosing the 2A mutant with 17 aa deletion at the C-terminus as the bait protein, four interactive cellular proteins were identified, including TIMP4, MYL2, COX7C, and ENO1. These proteins are mostly related to protein degradation and metabolism. Although the interactions detected by the Y2H system should be considered as preliminary results, the finding of proteins translated from a human heart cDNA library that interacts with the CVB3 2A will stimulate experiments testing the reactivity of a translational mixture derived from that library with full-length 2A protein, followed by co-immunoprecipitation studies.

  8. Performance of CHROMAGAR candida and BIGGY agar for identification of yeast species

    PubMed Central

    Yücesoy, Mine; Marol, Serhat

    2003-01-01

    Background The importance of identifying the pathogenic fungi rapidly has encouraged the development of differential media for the presumptive identification of yeasts. In this study two differential media, CHROMagar Candida and bismuth sulphite glucose glycine yeast agar, were evaluated for the presumptive identification of yeast species. Methods A total number of 270 yeast strains including 169 Candida albicans, 33 C. tropicalis, 24 C. glabrata, 18 C. parapsilosis, 12 C. krusei, 5 Trichosporon spp., 4 C. kefyr, 2 C. lusitaniae, 1 Saccharomyces cerevisiae and 1 Geotrichum candidum were included. The strains were first identified by germ tube test, morphological characteristics on cornmeal tween 80 agar and Vitek 32 and API 20 C AUX systems. In parallel, they were also streaked onto CHROMagar Candida and bismuth sulphite glucose glycine yeast agar plates. The results were read according to the color, morphology of the colonies and the existance of halo around them after 48 hours of incubation at 37°C. Results The sensitivity and specificity values for C. albicans strains were found to be 99.4, 100% for CHROMagar Candida and 87.0, 75.2% for BiGGY agar, respectively. The sensitivity of CHROMagar Candida to identify C. tropicalis, C. glabrata and C. krusei ranged between 90.9 and 100% while the specificity was 100%. The sensitivity rates for BiGGY agar were 66.6 and 100% while the specificity values were found to be 95.4 and 100% for C. tropicalis and C. krusei, respectively. Conclusions It can be concluded that the use of CHROMagar Candida is an easy and reliable method for the presumptive identification of most commonly isolated Candida species especially C. albicans, C. tropicalis and C. krusei. The lower sensitivity and specificity of BiGGY agar to identify commonly isolated Candida species potentially limits the clinical usefulness of this agar. PMID:14613587

  9. Prospective evaluation of a matrix-assisted laser desorption ionization-time of flight mass spectrometry system in a hospital clinical microbiology laboratory for identification of bacteria and yeasts: a bench-by-bench study for assessing the impact on time to identification and cost-effectiveness.

    PubMed

    Tan, K E; Ellis, B C; Lee, R; Stamper, P D; Zhang, S X; Carroll, K C

    2012-10-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.

  10. Identification and assessment of kefir yeast potential for sugar/ethanol-resistance

    PubMed Central

    Miguel, M.G.C.P.; Cardoso, P.G.; Magalhães-Guedes, K.T.; Schwan, R.F.

    2013-01-01

    Biochemical and molecular analysis was used for identification of different kefir yeasts species from Brazil, Canada and the United States of America. The sugar/ethanol-resistant activity of the yeasts was evaluated. Saccharomyces cerevisiae and Kluyveromyces marxianus had the highest growth rates, suggesting biotechnological applications possible for these strains. PMID:24159292

  11. Identification of propulsion systems

    NASA Technical Reports Server (NTRS)

    Merrill, Walter; Guo, Ten-Huei; Duyar, Ahmet

    1991-01-01

    This paper presents a tutorial on the use of model identification techniques for the identification of propulsion system models. These models are important for control design, simulation, parameter estimation, and fault detection. Propulsion system identification is defined in the context of the classical description of identification as a four step process that is unique because of special considerations of data and error sources. Propulsion system models are described along with the dependence of system operation on the environment. Propulsion system simulation approaches are discussed as well as approaches to propulsion system identification with examples for both air breathing and rocket systems.

  12. Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process.

    PubMed

    Basílio, A C M; de Araújo, P R L; de Morais, J O F; da Silva Filho, E A; de Morais, M A; Simões, D A

    2008-04-01

    Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.

  13. Comparative Evaluation of the Bruker Biotyper and Vitek MS Matrix-Assisted Laser Desorption Ionization–Time Of Flight (MALDI-TOF) Mass Spectrometry Systems for Identification of Yeasts of Medical Importance

    PubMed Central

    De Carolis, Elena; Infurnari, Laura; Vella, Antonietta; Clementi, Nicola; Vaccaro, Luisa; Ruggeri, Alberto; Posteraro, Brunella; Burioni, Roberto; Clementi, Massimo; Sanguinetti, Maurizio

    2013-01-01

    We report the first comparative evaluation between the Bruker Biotyper MS (BMS) and the Vitek MS (VMS) for the identification of yeasts. The rate of correct identifications at the species level was comparable using the commercial databases (89.8% versus 84.3%; P = 0.712), but higher for BMS using an in-house-extended database (100% versus 84.3%; P = 0.245). Importantly, the rate of misidentification was significantly higher for VMS (1% versus 12.1%; P < 0.0001), including the rate of major errors (0% versus 4.5%; P = 0.0036). PMID:23678071

  14. [Identification of Host Factors Interacting with the Movement Protein of Apple Chlorotic Leaf Spot Virus by Yeast Two-Hybrid System].

    PubMed

    He, Yikun; Zhong, Min; Zhang, Yu; Wang, Yanan; Cao, Keqiang

    2015-03-01

    In order to identify host factors which interact with the movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV), ACLSV MP was cloned into the bait vector pGBKT7 and used to screen a cDNA library of Malus sylvestris cv. R12740-7A, which had previously been constructed by yeast two-hybrid sequencing transformation. The protein functions of the identified host factors were determined according to their gene annotations in GenBank. The result showed that the bait plasmid pGBKT7-MP showed no virulence or self-activating effect on yeast strain Y2H Gold. Sixty-nine interactor proteins were identified, which were divided into the following 10 classes according to their described functions: hydrolases; pathogenesis-related proteins; DNA binding proteins; phosphatases; ligases; proteins with catalytic activity; phenylalanine ammonialyases; peroxidases; NAD binding proteins; and proteins of unknown function. Bioinformatic analysis of gene homology suggested that phosphatases, pathogenesis-related proteins and glyceraldehyde-3-phosphate dehydrogenase A may play an important role in the interaction between virus and host. This study may provide a theoretical basis for the further study of viral pathogenesis and virus-host interaction mechanisms.

  15. Evaluation of PNA FISH® Yeast Traffic Light in identification of Candida species from blood and non-blood culture specimens.

    PubMed

    Radic, Marina; Goic-Barisic, Ivana; Novak, Anita; Rubic, Zana; Tonkic, Marija

    2016-08-01

    PNA FISH(®) (peptide nucleic acid fluorescent in situ hybridization) Yeast Traffic Light (PNA FISH(®) YTL) assay is a commercially avaliable method for rapid identification of Candida spp. directly from positive blood cultures. This report provides a one-year experience in identification of yeasts from 25 specimens (15 positive blood cultures and 10 other clinically significant specimens) using PNA FISH(®) YTL and comparing it to VITEK 2 System. Overall, assay identification compatibility with VITEK 2 System was found among 21/25 (84%) isolates tested. Only 3/25 (12%) of the isolates were not identified, and one isolate was misidentified by the PNA FISH(®) YTL assay. Our results show that the assay is a reliable method in identification of Candida spp. not only from blood cultures, but even from other clinically significant specimens (urine cultures, catheter tip cultures, peritoneal fluid cultures) when compared to automated method like VITEK 2 System. This novel application of the PNA FISH(®) YTL assay could therefore contribute to cost savings and significant benefit to patients, as rapid information about isolated yeast species is provided. PMID:27067303

  16. Evaluation of PNA FISH® Yeast Traffic Light in identification of Candida species from blood and non-blood culture specimens.

    PubMed

    Radic, Marina; Goic-Barisic, Ivana; Novak, Anita; Rubic, Zana; Tonkic, Marija

    2016-08-01

    PNA FISH(®) (peptide nucleic acid fluorescent in situ hybridization) Yeast Traffic Light (PNA FISH(®) YTL) assay is a commercially avaliable method for rapid identification of Candida spp. directly from positive blood cultures. This report provides a one-year experience in identification of yeasts from 25 specimens (15 positive blood cultures and 10 other clinically significant specimens) using PNA FISH(®) YTL and comparing it to VITEK 2 System. Overall, assay identification compatibility with VITEK 2 System was found among 21/25 (84%) isolates tested. Only 3/25 (12%) of the isolates were not identified, and one isolate was misidentified by the PNA FISH(®) YTL assay. Our results show that the assay is a reliable method in identification of Candida spp. not only from blood cultures, but even from other clinically significant specimens (urine cultures, catheter tip cultures, peritoneal fluid cultures) when compared to automated method like VITEK 2 System. This novel application of the PNA FISH(®) YTL assay could therefore contribute to cost savings and significant benefit to patients, as rapid information about isolated yeast species is provided.

  17. Identification of superior lipid producing Lipomyces and Myxozyma yeasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oleaginous yeasts are of interest for production of single cell oils from sugars. Here 17 members of the Lipomyces and Myxozyma clade were screened for lipid production when cultured on glucose. The highest ranking yeasts included L. tetrasporus (21 g/l), L. kononenkoae (19.6 g/l), and L. lipofer (1...

  18. Identification of Pathogenic Rare Yeast Species in Clinical Samples: Comparison between Phenotypical and Molecular Methods▿

    PubMed Central

    Cendejas-Bueno, Emilio; Gomez-Lopez, Alicia; Mellado, Emilia; Rodriguez-Tudela, Juan L.; Cuenca-Estrella, Manuel

    2010-01-01

    Species identification using both phenotypic and molecular methods and antifungal susceptibility tests was carried out with 60 uncommon clinical yeasts. Our data show that phenotypic methods were insufficient for correct identification (only 25%) and that most of the wrongly identified strains showed a resistant antifungal profile. PMID:20237094

  19. Prospective evaluation of the VITEK MS for the routine identification of bacteria and yeast in the clinical microbiology laboratory: assessment of accuracy of identification and turnaround time.

    PubMed

    Charnot-Katsikas, Angella; Tesic, Vera; Boonlayangoor, Sue; Bethel, Cindy; Frank, Karen M

    2014-02-01

    This study assessed the accuracy of bacterial and yeast identification using the VITEK MS, and the time to reporting of isolates before and after its implementation in routine clinical practice. Three hundred and sixty-two isolates of bacteria and yeast, consisting of a variety of clinical isolates and American Type Culture Collection strains, were tested. Results were compared with reference identifications from the VITEK 2 system and with 16S rRNA sequence analysis. The VITEK MS provided an acceptable identification to species level for 283 (78 %) isolates. Considering organisms for which genus-level identification is acceptable for routine clinical care, 315 isolates (87 %) had an acceptable identification. Six isolates (2 %) were identified incorrectly, five of which were Shigella species. Finally, the time for reporting the identifications was decreased significantly after implementation of the VITEK MS for a total mean reduction in time of 10.52 h (P<0.0001). Overall, accuracy of the VITEK MS was comparable or superior to that from the VITEK 2. The findings were also comparable to other studies examining the accuracy of the VITEK MS, although differences exist, depending on the diversity of species represented as well as on the versions of the databases used. The VITEK MS can be incorporated effectively into routine use in a clinical microbiology laboratory and future expansion of the database should provide improved accuracy for the identification of micro-organisms.

  20. Advantages of Using Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry as a Rapid Diagnostic Tool for Identification of Yeasts and Mycobacteria in the Clinical Microbiological Laboratory

    PubMed Central

    Chen, Jonathan H. K.; Yam, Wing-Cheong; Ngan, Antonio H. Y.; Fung, Ami M. Y.; Woo, Wai-Lan; Yan, Mei-Kum; Choi, Garnet K. Y.; Ho, Pak-Leung; Cheng, Vincent C. C.

    2013-01-01

    Yeast and mycobacteria can cause infections in immunocompromised patients and normal hosts. The rapid identification of these organisms can significantly improve patient care. There has been an increasing number of studies on using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for rapid yeast and mycobacterial identifications. However, studies on direct comparisons between the Bruker Biotyper and bioMérieux Vitek MS systems for the identification of yeast and mycobacteria have been limited. This study compared the performance of the two systems in their identification of 98 yeast and 102 mycobacteria isolates. Among the 98 yeast isolates, both systems generated species-level identifications in >70% of the specimens, of which Candida albicans was the most commonly cultured species. At a genus-level identification, the Biotyper system identified more isolates than the Vitek MS system for Candida (75/78 [96.2%]versus 68/78 [87.2%], respectively; P = 0.0426) and non-Candida yeasts (18/20 [90.0%]versus 7/20 [35.0%], respectively; P = 0.0008). For mycobacterial identification, the Biotyper system generated reliable identifications for 89 (87.3%) and 64 (62.8%) clinical isolates at the genus and species levels, respectively, from solid culture media, whereas the Vitek MS system did not generate any reliable identification. The MS method differentiated 12/21 clinical species, despite the fact that no differentiation between Mycobacterium abscessus and Mycobacterium chelonae was found by using 16S rRNA gene sequencing. In summary, the MALDI-TOF MS method provides short turnaround times and a standardized working protocol for the identification of yeast and mycobacteria. Our study demonstrates that MALDI-TOF MS is suitable as a first-line test for the identification of yeast and mycobacteria in clinical laboratories. PMID:24048537

  1. Identification of Medically Important Candida and Non-Candida Yeast Species by an Oligonucleotide Array▿

    PubMed Central

    Leaw, Shiang Ning; Chang, Hsien Chang; Barton, Richard; Bouchara, Jean-Philippe; Chang, Tsung Chain

    2007-01-01

    The incidence of yeast infections has increased in the recent decades, with Candida albicans still being the most common cause of infections. However, infections caused by less common yeasts have been widely reported in recent years. Based on the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 77 species of clinically relevant yeasts belonging to 16 genera. The ITS regions were amplified by PCR with a pair of fungus-specific primers, followed by hybridization of the digoxigenin-labeled PCR product to a panel of oligonucleotide probes immobilized on a nylon membrane for species identification. A collection of 452 yeast strains (419 target and 33 nontarget strains) was tested, and a sensitivity of 100% and a specificity of 97% were obtained by the array. The detection limit of the array was 10 pg of yeast genomic DNA per assay. In conclusion, yeast identification by the present method is highly reliable and can be used as an alternative to the conventional identification methods. The whole procedure can be finished within 24 h, starting from isolated colonies. PMID:17507521

  2. Comparative analysis of Gram's stain, PNA-FISH and Sepsityper with MALDI-TOF MS for the identification of yeast direct from positive blood cultures.

    PubMed

    Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C

    2014-10-01

    Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting.

  3. [Evaluation of mass spectrometry: MALDI-TOF MS for fast and reliable yeast identification].

    PubMed

    Relloso, María S; Nievas, Jimena; Fares Taie, Santiago; Farquharson, Victoria; Mujica, María T; Romano, Vanesa; Zarate, Mariela S; Smayevsky, Jorgelina

    2015-01-01

    The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification.

  4. Identification of putative regulatory region of insulin-like androgenic gland hormone gene (IAG) in the prawn Macrobrachium nipponense and proteins that interact with IAG by using yeast two-hybrid system.

    PubMed

    Ma, Ke-Yi; Li, Jia-Le; Qiu, Gao-Feng

    2016-04-01

    Insulin-like androgenic gland hormone gene (IAG) is a sex regulator specifically expressed in male crustaceans, controlling the male sexual differentiation, spermatogenesis and reproductive strategy. Our previous study reported the cloning and characterization of the prawn Macrobrachium nipponense IAG (MnIAG). In this study, we further identified a 2214-bp MnIAG 5'-flanking region, and analyzed its transcription factor binding sites and transcriptional activity. The results showed that there were two potential promoter core sequences, three TATA boxes and one CAAT box existing in the MnIAG 5'-flanking region as well as many potential transcription factor binding sites, such as SRY, Sox-5, GATA-1, etc. Notably, the transcriptional activity was weak in this region, and a negative regulatory region was found in -604 to -231bp. In addition, we constructed M. nipponense yeast libraries and identified proteins interacting with the MnIAG protein by yeast two hybridization assay. The yeast two-hybrid screening yielded ten positive clones, of which five were annotated by NCBI database, namely heat shock protein 21, NADH dehydrogenase, zinc finger protein, beta-N-acetylglucosaminidase and a hypothetical protein. The identification of MnIAG putative regulatory region and proteins that interact with IAG will facilitate our understanding of the regulatory role of MnIAG and provide a foundation for deep insight into the prawn sex differentiation mechanism and signaling transduction pathways. PMID:26979275

  5. Author Identification Systems

    ERIC Educational Resources Information Center

    Wagner, A. Ben

    2009-01-01

    Many efforts are currently underway to disambiguate author names and assign unique identification numbers so that publications by a given scholar can be reliably grouped together. This paper reviews a number of operational and in-development services. Some systems like ResearcherId.Com depend on self-registration and self-identification of a…

  6. Identification of yeasts from clinical specimens by oxidase test.

    PubMed

    Kumar, S; Arora, B S; Mathur, M D

    2000-10-01

    A total of 100 yeasts and yeast like fungi isolates from clinical specimens were negative for oxidase production on Sabouraud dextrose agar. When grown on Columbia agar, chocolate agar, tryptose agar, Mueller-Hinton agar, brain heart infusion and a medium resembling Sabouraud's dextrose agar but with starch instead of dextrose, all the isolate of Candida albicans (55), C. guilliermondii (6), C. parapsilosis (14), C. tropicalis (6), C. pseudotropicalis (6) and Crytococcus neoformans (2) were positive for oxidase producation. Torulopsis glabrata (2), Saccharomyces cervisiae (2) and two out of seven isolates of C. krusei were negative for oxidase test. PMID:11344606

  7. Yeast systems for the commercial production of heterologous proteins.

    PubMed

    Buckholz, R G; Gleeson, M A

    1991-11-01

    Yeasts are attractive hosts for the production of heterologous proteins. Unlike prokaryotic systems, their eukaryotic subcellular organization enables them to carry out many of the post-translational folding, processing and modification events required to produce "authentic" and bioactive mammalian proteins. In addition, they retain the advantages of a unicellular microorganism, with respect to rapid growth and ease of genetic manipulation. The vast majority of yeast expression work has focused on the well-characterized baker's yeast Saccharomyces cerevisiae. However, with the development of DNA transformation technologies, a growing number of non-Saccharomyces yeasts are becoming available as hosts for recombinant polypeptide production. These include Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomyces pombe, Schwanniomyces occidentalis and Yarrowia lipolytica. The performance of these alternative yeast expression systems is reviewed here relative to S. cerevisiae, and the advantages and limitations of these systems are discussed.

  8. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  9. Quantum system identification.

    PubMed

    Burgarth, Daniel; Yuasa, Kazuya

    2012-02-24

    The aim of quantum system identification is to estimate the ingredients inside a black box, in which some quantum-mechanical unitary process takes place, by just looking at its input-output behavior. Here we establish a basic and general framework for quantum system identification, that allows us to classify how much knowledge about the quantum system is attainable, in principle, from a given experimental setup. We show that controllable closed quantum systems can be estimated up to unitary conjugation. Prior knowledge on some elements of the black box helps the system identification. We present an example in which a Bell measurement is more efficient to identify the system. When the topology of the system is known, the framework enables us to establish a general criterion for the estimability of the coupling constants in its Hamiltonian.

  10. Identification of food and beverage spoilage yeasts from DNA sequence analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

  11. Modification of potassium nitrate assimilation test for identification of clinically important yeasts.

    PubMed Central

    Pincus, D H; Salkin, I F; Hurd, N J; Levy, I L; Kemna, M A

    1988-01-01

    The modification of an auxanographic method used in yeast species identification to determine potassium nitrate (KNO3) assimilation resulted in a simple and inexpensive KNO3 assimilation test medium. The medium provided accurate and reliable results in less than 24 h. PMID:3343330

  12. Identification of Medically Important Yeast Species by Sequence Analysis of the Internal Transcribed Spacer Regions

    PubMed Central

    Leaw, Shiang Ning; Chang, Hsien Chang; Sun, Hsiao Fang; Barton, Richard; Bouchara, Jean-Philippe; Chang, Tsung Chain

    2006-01-01

    Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods. PMID:16517841

  13. Optimized System Identification

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Longman, Richard W.

    1999-01-01

    In system identification, one usually cares most about finding a model whose outputs are as close as possible to the true system outputs when the same input is applied to both. However, most system identification algorithms do not minimize this output error. Often they minimize model equation error instead, as in typical least-squares fits using a finite-difference model, and it is seen here that this distinction is significant. Here, we develop a set of system identification algorithms that minimize output error for multi-input/multi-output and multi-input/single-output systems. This is done with sequential quadratic programming iterations on the nonlinear least-squares problems, with an eigendecomposition to handle indefinite second partials. This optimization minimizes a nonlinear function of many variables, and hence can converge to local minima. To handle this problem, we start the iterations from the OKID (Observer/Kalman Identification) algorithm result. Not only has OKID proved very effective in practice, it minimizes an output error of an observer which has the property that as the data set gets large, it converges to minimizing the criterion of interest here. Hence, it is a particularly good starting point for the nonlinear iterations here. Examples show that the methods developed here eliminate the bias that is often observed using any system identification methods of either over-estimating or under-estimating the damping of vibration modes in lightly damped structures.

  14. Identification of protein/target molecule interactions using yeast surface-displayed cDNA libraries

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    We describe a novel expression cloning method based on screening yeast surface-displayed human cDNA libraries by direct affinity interaction to identify cellular proteins binding to a broad spectrum of target molecules. Being a eukaryote, yeast protein expression pathways are similar to those found in mammalian cells, and therefore mammalian protein fragments displayed on the yeast cell wall are more likely to be properly folded and functional than proteins displayed using prokaryotic systems. Yeast surface displayed human cDNA libraries have been successfully used to screen for proteins that bind to post-translationally modified phosphorylated peptides, small signaling molecule phosphatidylinositides, and monoclonal antibodies. In this article we describe protocols for using yeast surface-displayed cDNA libraries, coupled with fluorescence-activated cell sorting (FACS), to select protein fragments with affinity for various target molecules including post-translationally modified peptides, small signaling molecules, monoclonal phage antibodies, and monoclonal IgG molecules. PMID:21365493

  15. [The gas-liquid cromatography with mass spectrometry for the identification of yeasts].

    PubMed

    Paredes-Salido, F; Mira-Gutiérrez, J; Sasián-Macías, P; García-Martos, P

    2001-03-01

    Methods based on gas chromatography, have been used for identification of the yeasts. In order to know the value of the patterns obtained by this method, we have used this technique and mass spectrometry on 44 strains belonging to 16 genus and 21 species of collection yeasts, identifying the corresponding peaks to 22 fatty acids methyl esters by means of the reaction times of the corresponding standards and the confirmation of molecular weigh by mass spectrometry. The correlation coefficient was of 0.848965. The chromatographic technique seems of great utility for the determination of lipidotypes.

  16. Global identification of yeast chromosome interactions using Genome conformation capture.

    PubMed

    Rodley, C D M; Bertels, F; Jones, B; O'Sullivan, J M

    2009-11-01

    The association of chromosomes with each other and other nuclear components plays a critical role in nuclear organization and Genome function. Here, using a novel and generally applicable methodology (Genome conformation capture [GCC]), we reveal the network of chromosome interactions for the yeast Saccharomyces cerevisiae. Inter- and intra-chromosomal interactions are non-random and the number of interactions per open reading frame depends upon the dispensability of the gene product. Chromosomal interfaces are organized and provide evidence of folding within chromosomes. Interestingly, the genomic connections also involve the 2 microm plasmid and the mitochondrial genome. Mitochondrial interaction partners include genes of alpha-proteobacterial origin and the ribosomal DNA. Organization of the 2 microm plasmid aligns two inverted repeats (IR1 and IR2) and displays the stability locus on a prominent loop thus making it available for plasmid clustering. Our results form the first global map of chromosomal interactions in a eukaryotic nucleus and demonstrate the highly connected nature of the yeast genome. These results have significant implications for understanding eukaryotic genome organization.

  17. Performance of mass spectrometric identification of bacteria and yeasts routinely isolated in a clinical microbiology laboratory using MALDI-TOF MS

    PubMed Central

    Wang, Weiping; Xi, Haiyan; Huang, Mei; Wang, Jie; Fan, Ming; Chen, Yong; Shao, Haifeng

    2014-01-01

    Background Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to identifying bacterial and yeast strains. The aim of this study was to evaluate the clinical performance of the VITEK® MS system in the identification of bacteria and yeast strains routinely isolated from clinical samples. Methods We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria and yeasts regardless of phylum or source of isolation. Discordant results were resolved with 16S rDNA or internal transcribed spacer (ITS) gene sequencing. Colonies (a single deposit on a MALDI disposable target without any prior extraction step) were analyzed using the VITEK® MS system. Peptide spectra acquired by the system were compared with the VITEK® MS IVD database Version 2.0, and the identification scores were recorded. Results Of the 1,181 isolates (1,061 bacterial isolates and 120 yeast isolates) analyzed, 99.5% were correctly identified by MALDI-TOF mass spectrometry; 95.7% identified to the species level, 3.6% identified to the genus level, and 0.3% identified within a range of species belonging to different genera. Conversely, 0.1% of isolates were misidentified and 0.4% were unidentified, partly because the species were not included in the database. Re-testing using a second deposit provided a successful identification for 0.5% of isolates unidentified with the first deposit. Our results show that the VITEK® MS system has exceptional performance in identifying bacteria and yeast by comparing acquired peptide spectra to those contained in its database. Conclusions MALDI-TOF mass spectrometry is a rapid, accurate, and relatively inexpensive method for bacterial and yeast identification. Our results demonstrate that the VITEK® MS system is a fast and reliable technique, and has the potential to replace conventional phenotypic

  18. [Presumptive identification of Candida spp. and other clinically important yeasts: usefulness of Brilliance Candida Agar].

    PubMed

    Alfonso, Claudia; López, Mónica; Arechavala, Alicia; Perrone, María Del Carmen; Guelfand, Liliana; Bianchi, Mario

    2010-06-30

    Fungal infections caused by yeasts have increased during the last decades and invasive forms represent a serious problem for human health. Candida albicans is the species most frequently isolated from clinical samples. However, other emerging yeast pathogens are increasingly responsible for mycotic infections, and some of them are resistant to some antifungal drugs. Consequently, it is necessary to have methods that can provide a rapid presumptive identification at species level. Numerous chromogenic agar media have been shown to be of value as diagnostic tools. We have compared a chromogenic medium, Brilliance Candida Agar, with CHROMagar Candida, the chromogenic medium most used in our country. A multicentre study was conducted in 16 Hospitals belonging to the Mycology Net of Buenos Aires City Government. A total of 240 yeast isolates were included in this research. The new chromogenic agar showed results very similar to those obtained with CHROMagar Candida.

  19. Molecular-based identification of yeasts isolated from bovine clinical mastitis in Japan.

    PubMed

    Hayashi, Tomohito; Sugita, Takashi; Hata, Eiji; Katsuda, Ken; Zhang, Enshi; Kiku, Yoshio; Sugawara, Kazue; Ozawa, Tomomi; Matsubara, Tomoko; Ando, Takaaki; Obayashi, Tetsu; Ito, Takaaki; Yabusaki, Takahiro; Kudo, Katsunori; Yamamoto, Hiroshi; Koiwa, Masateru; Oshida, Toshio; Tagawa, Yuichi; Kawai, Kazuhiro

    2013-01-01

    This study analyzed molecular-based identification of yeasts that associated with bovine clinical mastitis in Japan. Over 3,200 quarter milk samples from Holstein dairy cows collected in 2011 on Hokkaido and Honshu islands were examined. Yeast isolates were characterized by polymerase chain reaction amplification and sequencing of the D1/D2 region of the 26S rDNA. Molecular characterization confirmed that Candida spp. and Pichia spp. were most frequently isolated species. Our molecular analysis of mastitic milk samples demonstrated the prevalence of Pichia kudriavzevii(22/58) and Candida tropicalis(14/58). In addition, we demonstrated that molecular analysis of the D1/D2 region of the 26S rDNA is a rapid and reliable method for identifying clinically significant yeasts in dairy hygiene, including potentially new or emerging pathogenic species. PMID:23100116

  20. Identification of yeast and human 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) transporters.

    PubMed

    Ceschin, Johanna; Saint-Marc, Christelle; Laporte, Jean; Labriet, Adrien; Philippe, Chloé; Moenner, Michel; Daignan-Fornier, Bertrand; Pinson, Benoît

    2014-06-13

    5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation. PMID:24778186

  1. Identification of Yeast and Human 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters*

    PubMed Central

    Ceschin, Johanna; Saint-Marc, Christelle; Laporte, Jean; Labriet, Adrien; Philippe, Chloé; Moenner, Michel; Daignan-Fornier, Bertrand; Pinson, Benoît

    2014-01-01

    5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) is the precursor of the active monophosphate form (AICAR), a small molecule with potent anti-proliferative and low energy mimetic properties. The molecular bases for AICAR toxicity at the cellular level are poorly understood. Here, we report the isolation and characterization of several yeast AICAr-hypersensitive mutants. Identification of the cognate genes allowed us to establish that thiamine transporters Thi7 and Thi72 can efficiently take up AICAr under conditions where they are overexpressed. We establish that, under standard growth conditions, Nrt1, the nicotinamide riboside carrier, is the major AICAr transporter in yeast. A study of AICAR accumulation in human cells revealed substantial disparities among cell lines and confirmed that AICAr enters cells via purine nucleoside transporters. Together, our results point to significant differences between yeast and human cells for both AICAr uptake and AICAR accumulation. PMID:24778186

  2. Isolation of plant transcription factors using a modified yeast one-hybrid system

    PubMed Central

    Lopato, Sergiy; Bazanova, Natalia; Morran, Sarah; Milligan, Andrew S; Shirley, Neil; Langridge, Peter

    2006-01-01

    Background The preparation of expressional cDNA libraries for use in the yeast two-hybrid system is quick and efficient when using the dedicated Clontech™ product, the MATCHMAKER Library Construction and Screening Kit 3. This kit employs SMART technology for the amplification of full-length cDNAs, in combination with cloning using homologous recombination. Unfortunately, such cDNA libraries prepared directly in yeast can not be used for the efficient recovery of purified plasmids and thus are incompatible with existing yeast one-hybrid systems, which use yeast transformation for the library screen. Results Here we propose an adaptation of the yeast one-hybrid system for identification and cloning of transcription factors using a MATCHMAKER cDNA library. The procedure is demonstrated using a cDNA library prepared from the liquid part of the multinucleate coenocyte of wheat endosperm. The method is a modification of a standard one-hybrid screening protocol, utilising a mating step to introduce the library construct and reporter construct into the same cell. Several novel full length transcription factors from the homeodomain, AP2 domain and E2F families of transcription factors were identified and isolated. Conclusion In this paper we propose a method to extend the compatibility of MATCHMAKER cDNA libraries from yeast two-hybrid screens to one-hybrid screens. The utility of the new yeast one-hybrid technology is demonstrated by the successful cloning from wheat of full-length cDNAs encoding several transcription factors from three different families. PMID:16504065

  3. Identification of food and beverage spoilage yeasts from DNA sequence analyses.

    PubMed

    Kurtzman, Cletus P

    2015-11-20

    Detection, identification and classification of yeasts have undergone major changes in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences has resulted in a major revision of yeast systematics resulting in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed, especially in the context of food and beverage spoilage yeasts.

  4. Accurate identification of centromere locations in yeast genomes using Hi-C.

    PubMed

    Varoquaux, Nelle; Liachko, Ivan; Ay, Ferhat; Burton, Joshua N; Shendure, Jay; Dunham, Maitreya J; Vert, Jean-Philippe; Noble, William S

    2015-06-23

    Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations. We describe a method, Centurion, that jointly infers the locations of all centromeres in a single genome from Hi-C data by exploiting the centromeres' tendency to cluster in three-dimensional space. We first demonstrate the accuracy of Centurion in identifying known centromere locations from high coverage Hi-C data of budding yeast and a human malaria parasite. We then use Centurion to infer centromere locations in 14 yeast species. Across all microbes that we consider, Centurion predicts 89% of centromeres within 5 kb of their known locations. We also demonstrate the robustness of the approach in datasets with low sequencing depth. Finally, we predict centromere coordinates for six yeast species that currently lack centromere annotations. These results show that Centurion can be used for centromere identification for diverse species of yeast and possibly other microorganisms.

  5. Accurate identification of centromere locations in yeast genomes using Hi-C

    PubMed Central

    Varoquaux, Nelle; Liachko, Ivan; Ay, Ferhat; Burton, Joshua N.; Shendure, Jay; Dunham, Maitreya J.; Vert, Jean-Philippe; Noble, William S.

    2015-01-01

    Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations. We describe a method, Centurion, that jointly infers the locations of all centromeres in a single genome from Hi-C data by exploiting the centromeres’ tendency to cluster in three-dimensional space. We first demonstrate the accuracy of Centurion in identifying known centromere locations from high coverage Hi-C data of budding yeast and a human malaria parasite. We then use Centurion to infer centromere locations in 14 yeast species. Across all microbes that we consider, Centurion predicts 89% of centromeres within 5 kb of their known locations. We also demonstrate the robustness of the approach in datasets with low sequencing depth. Finally, we predict centromere coordinates for six yeast species that currently lack centromere annotations. These results show that Centurion can be used for centromere identification for diverse species of yeast and possibly other microorganisms. PMID:25940625

  6. Rapid identification of moulds and arthroconidial yeasts from positive blood cultures by MALDI-TOF mass spectrometry.

    PubMed

    de Almeida, João N; Sztajnbok, Jaques; da Silva, Afonso Rafael; Vieira, Vinicius Adriano; Galastri, Anne Layze; Bissoli, Leandro; Litvinov, Nadia; Del Negro, Gilda Maria Barbaro; Motta, Adriana Lopes; Rossi, Flávia; Benard, Gil

    2016-11-01

    Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS™ system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS™ [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy. PMID:27317582

  7. Polyphasic identification of yeasts isolated from bark of cork oak during the manufacturing process of cork stoppers.

    PubMed

    Villa-Carvajal, Mercedes; Coque, Juan José R; Alvarez-Rodríguez, María Luísa; Uruburu, Federico; Belloch, Carmela

    2004-05-01

    A two-step protocol was used for the identification of 52 yeasts isolated from bark of cork oak at initial stages of the manufacturing process of cork stoppers. The first step in the identification was the separation of the isolates into groups by their physiological properties and RFLPs of the ITS-5.8S rRNA gene. The second step was the sequencing of the D1/D2 domains of the 26S rRNA gene of selected isolates representing the different groups. The results revealed a predominance of basidiomycetous yeasts (11 species), while only two species represented the ascomycetous yeasts. Among the basidiomycetous yeasts, members representing the species Rhodosporidium kratochvilovae and Rhodotorula nothofagi, that have been previously isolated from plant material, were the most abundant. Yeasts pertaining to the species Debaryomyces hansenii var. fabryii, Rhodotorula mucilaginosa and Trichosporon mucoides were isolated in small numbers.

  8. Identification of Chemical-Genetic Interactions via Parallel Analysis of Barcoded Yeast Strains.

    PubMed

    Suresh, Sundari; Schlecht, Ulrich; Xu, Weihong; Miranda, Molly; Davis, Ronald W; Nislow, Corey; Giaever, Guri; St Onge, Robert P

    2016-01-01

    The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here. PMID:27587778

  9. Identification of Chemical-Genetic Interactions via Parallel Analysis of Barcoded Yeast Strains.

    PubMed

    Suresh, Sundari; Schlecht, Ulrich; Xu, Weihong; Miranda, Molly; Davis, Ronald W; Nislow, Corey; Giaever, Guri; St Onge, Robert P

    2016-09-01

    The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here.

  10. Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast.

    PubMed

    Vandebroek, Tom; Vanhelmont, Thomas; Terwel, Dick; Borghgraef, Peter; Lemaire, Katleen; Snauwaert, Johan; Wera, Stefaan; Van Leuven, Fred; Winderickx, Joris

    2005-08-30

    Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.

  11. Yeast prions: structure, biology, and prion-handling systems.

    PubMed

    Wickner, Reed B; Shewmaker, Frank P; Bateman, David A; Edskes, Herman K; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E

    2015-03-01

    A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.

  12. Yeast Prions: Structure, Biology, and Prion-Handling Systems

    PubMed Central

    Shewmaker, Frank P.; Bateman, David A.; Edskes, Herman K.; Gorkovskiy, Anton; Dayani, Yaron; Bezsonov, Evgeny E.

    2015-01-01

    SUMMARY A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered β-sheet-rich protein aggregates with β-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants. PMID:25631286

  13. Potential of yeast secretory vesicles in biodelivery systems.

    PubMed

    Kutralam-Muniasamy, Gurusamy; Flores-Cotera, Luis B; Perez-Guevara, Fermin

    2015-06-01

    Membranous vesicular organelles (MVOs), such as secretory vesicles and exosomes, perform a variety of biological functions ranging from secretion to cellular communication in eukaryotic cells. Exosomes, particularly those of mammalian cells, have been widely studied as potential carriers in human therapeutic applications. However, no study has yet demonstrated the use of yeast secretory vesicles for such applications. Therefore, we explore here the current state of knowledge on yeast secretory vesicles and their potential use in therapeutic delivery systems. We focus on the characteristics shared by exosomes and yeast secretory vesicles to provide insights into the use of the latter as delivery vehicles. From this perspective, we speculate on the potential application of post-Golgi vesicles (PGVs) in the biomedical field. PMID:25843637

  14. Are the Conventional Commercial Yeast Identification Methods Still Helpful in the Era of New Clinical Microbiology Diagnostics? A Meta-Analysis of Their Accuracy

    PubMed Central

    Efremov, Ljupcho; Leoncini, Emanuele; Amore, Rosarita; Posteraro, Patrizia; Ricciardi, Walter

    2015-01-01

    Accurate identification of pathogenic species is important for early appropriate patient management, but growing diversity of infectious species/strains makes the identification of clinical yeasts increasingly difficult. Among conventional methods that are commercially available, the API ID32C, AuxaColor, and Vitek 2 systems are currently the most used systems in routine clinical microbiology. We performed a systematic review and meta-analysis to estimate and to compare the accuracy of the three systems, in order to assess whether they are still of value for the species-level identification of medically relevant yeasts. After adopting rigorous selection criteria, we included 26 published studies involving Candida and non-Candida yeasts that were tested with the API ID32C (674 isolates), AuxaColor (1,740 isolates), and Vitek 2 (2,853 isolates) systems. The random-effects pooled identification ratios at the species level were 0.89 (95% confidence interval [CI], 0.80 to 0.95) for the API ID32C system, 0.89 (95% CI, 0.83 to 0.93) for the AuxaColor system, and 0.93 (95% CI, 0.89 to 0.96) for the Vitek 2 system (P for heterogeneity, 0.255). Overall, the accuracy of studies using phenotypic analysis-based comparison methods was comparable to that of studies using molecular analysis-based comparison methods. Subanalysis of studies conducted on Candida yeasts showed that the Vitek 2 system was significantly more accurate (pooled ratio, 0.94 [95% CI, 0.85 to 0.99]) than the API ID32C system (pooled ratio, 0.84 [95% CI, 0.61 to 0.99]) and the AuxaColor system (pooled ratio, 0.76 [95% CI, 0.67 to 0.84]) with respect to uncommon species (P for heterogeneity, <0.05). Subanalysis of studies conducted on non-Candida yeasts (i.e., Cryptococcus, Rhodotorula, Saccharomyces, and Trichosporon) revealed pooled identification accuracies of ≥98% for the Vitek 2, API ID32C (excluding Cryptococcus), and AuxaColor (only Rhodotorula) systems, with significant low or null levels of

  15. Are the Conventional Commercial Yeast Identification Methods Still Helpful in the Era of New Clinical Microbiology Diagnostics? A Meta-Analysis of Their Accuracy.

    PubMed

    Posteraro, Brunella; Efremov, Ljupcho; Leoncini, Emanuele; Amore, Rosarita; Posteraro, Patrizia; Ricciardi, Walter; Sanguinetti, Maurizio

    2015-08-01

    Accurate identification of pathogenic species is important for early appropriate patient management, but growing diversity of infectious species/strains makes the identification of clinical yeasts increasingly difficult. Among conventional methods that are commercially available, the API ID32C, AuxaColor, and Vitek 2 systems are currently the most used systems in routine clinical microbiology. We performed a systematic review and meta-analysis to estimate and to compare the accuracy of the three systems, in order to assess whether they are still of value for the species-level identification of medically relevant yeasts. After adopting rigorous selection criteria, we included 26 published studies involving Candida and non-Candida yeasts that were tested with the API ID32C (674 isolates), AuxaColor (1,740 isolates), and Vitek 2 (2,853 isolates) systems. The random-effects pooled identification ratios at the species level were 0.89 (95% confidence interval [CI], 0.80 to 0.95) for the API ID32C system, 0.89 (95% CI, 0.83 to 0.93) for the AuxaColor system, and 0.93 (95% CI, 0.89 to 0.96) for the Vitek 2 system (P for heterogeneity, 0.255). Overall, the accuracy of studies using phenotypic analysis-based comparison methods was comparable to that of studies using molecular analysis-based comparison methods. Subanalysis of studies conducted on Candida yeasts showed that the Vitek 2 system was significantly more accurate (pooled ratio, 0.94 [95% CI, 0.85 to 0.99]) than the API ID32C system (pooled ratio, 0.84 [95% CI, 0.61 to 0.99]) and the AuxaColor system (pooled ratio, 0.76 [95% CI, 0.67 to 0.84]) with respect to uncommon species (P for heterogeneity, <0.05). Subanalysis of studies conducted on non-Candida yeasts (i.e., Cryptococcus, Rhodotorula, Saccharomyces, and Trichosporon) revealed pooled identification accuracies of ≥98% for the Vitek 2, API ID32C (excluding Cryptococcus), and AuxaColor (only Rhodotorula) systems, with significant low or null levels of

  16. High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories ▿

    PubMed Central

    van Veen, S. Q.; Claas, E. C. J.; Kuijper, Ed J.

    2010-01-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory. PMID:20053859

  17. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories.

    PubMed

    van Veen, S Q; Claas, E C J; Kuijper, Ed J

    2010-03-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.

  18. Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

    PubMed

    Arana-Sánchez, A; Segura-García, L E; Kirchmayr, M; Orozco-Ávila, I; Lugo-Cervantes, E; Gschaedler-Mathis, A

    2015-02-01

    The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.

  19. Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

    PubMed

    Arana-Sánchez, A; Segura-García, L E; Kirchmayr, M; Orozco-Ávila, I; Lugo-Cervantes, E; Gschaedler-Mathis, A

    2015-02-01

    The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected. PMID:25566818

  20. Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

    PubMed Central

    Chao, Qiao-Ting; Lee, Tai-Fen; Teng, Shih-Hua; Peng, Li-Yun; Chen, Ping-Hung; Teng, Lee-Jene; Hsueh, Po-Ren

    2014-01-01

    We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex

  1. Enrichment by organomercurial agarose and identification of cys-containing peptides from yeast cell lysates.

    PubMed

    Raftery, Mark J

    2008-05-01

    Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.

  2. Clinical evaluation of the FilmArray blood culture identification panel in identification of bacteria and yeasts from positive blood culture bottles.

    PubMed

    Altun, Osman; Almuhayawi, Mohammed; Ullberg, Måns; Ozenci, Volkan

    2013-12-01

    The FilmArray platform (FA; BioFire, Salt Lake City, UT) is a closed diagnostic system allowing high-order multiplex PCR analysis with automated readout of results directly from positive blood cultures in 1 h. In the present study, we evaluated the clinical performance of the FilmArray blood culture identification (BCID) panel, which includes 19 bacteria, five yeasts, and three antibiotic resistance genes. In total, 206 blood culture bottles were included in the study. The FilmArray could identify microorganisms in 153/167 (91.6%) samples with monomicrobial growth. Thirteen of the 167 (7.8%) microorganisms were not covered by the FilmArray BCID panel. In 6/167 (3.6%) samples, the FilmArray detected an additional microorganism compared to blood culture. When polymicrobial growth was analyzed, the FilmArray could detect all target microorganisms in 17/24 (71%) samples. Twelve blood culture bottles that yielded a positive signal but showed no growth were also negative by FilmArray. In 3/206 (1.5%) bottles, the FilmArray results were invalid. The results of the FilmArray were reproducible, as demonstrated by the testing and retesting of five bottles in the same day and a longitudinal follow-up of five other blood cultures up to 4 weeks. The present study shows that the FilmArray is a rapid identification method with high performance in direct identification of bacteria and yeasts from positive blood culture bottles.

  3. Systems-level engineering of nonfermentative metabolism in yeast.

    PubMed

    Kennedy, Caleb J; Boyle, Patrick M; Waks, Zeev; Silver, Pamela A

    2009-09-01

    We designed and experimentally validated an in silico gene deletion strategy for engineering endogenous one-carbon (C1) metabolism in yeast. We used constraint-based metabolic modeling and computer-aided gene knockout simulations to identify five genes (ALT2, FDH1, FDH2, FUM1, and ZWF1), which, when deleted in combination, predicted formic acid secretion in Saccharomyces cerevisiae under aerobic growth conditions. Once constructed, the quintuple mutant strain showed the predicted increase in formic acid secretion relative to a formate dehydrogenase mutant (fdh1 fdh2), while formic acid secretion in wild-type yeast was undetectable. Gene expression and physiological data generated post hoc identified a retrograde response to mitochondrial deficiency, which was confirmed by showing Rtg1-dependent NADH accumulation in the engineered yeast strain. Formal pathway analysis combined with gene expression data suggested specific modes of regulation that govern C1 metabolic flux in yeast. Specifically, we identified coordinated transcriptional regulation of C1 pathway enzymes and a positive flux control coefficient for the branch point enzyme 3-phosphoglycerate dehydrogenase (PGDH). Together, these results demonstrate that constraint-based models can identify seemingly unrelated mutations, which interact at a systems level across subcellular compartments to modulate flux through nonfermentative metabolic pathways. PMID:19564482

  4. Mapping the Interactions of Dengue Virus NS1 Protein with Human Liver Proteins Using a Yeast Two-Hybrid System: Identification of C1q as an Interacting Partner

    PubMed Central

    Allonso, Diego; Nogueira, Mauricio L.; Mohana-Borges, Ronaldo

    2013-01-01

    Dengue constitutes a global health concern. The clinical manifestation of this disease varies from mild febrile illness to severe hemorrhage and/or fatal hypovolemic shock. Flavivirus nonstructural protein 1 (NS1) is a secreted glycoprotein that is displayed on the surface of infected cells but is absent in viral particles. NS1 accumulates at high levels in the plasma of dengue virus (DENV)-infected patients, and previous reports highlight its involvement in immune evasion, dengue severity, liver dysfunction and pathogenesis. In the present study, we performed a yeast two-hybrid screen to search for DENV2 NS1-interacting partners using a human liver cDNA library. We identified fifty genes, including human complement component 1 (C1q), which was confirmed by coimmunoprecipitation, ELISA and immunofluorescence assays, revealing for the first time the direct binding of this protein to NS1. Furthermore, the majority of the identified genes encode proteins that are secreted into the plasma of patients, and most of these proteins are classified as acute-phase proteins (APPs), such as plasminogen, haptoglobin, hemopexin, α-2-HS-glycoprotein, retinol binding protein 4, transferrin, and C4. The results presented here confirm the direct interaction of DENV NS1 with a key protein of the complement system and suggest a role for this complement protein in the pathogenesis of DENV infection. PMID:23516407

  5. Detection and identification of wild yeasts in Champús, a fermented Colombian maize beverage.

    PubMed

    Osorio-Cadavid, Esteban; Chaves-López, Clemencia; Tofalo, Rosanna; Paparella, Antonello; Suzzi, Giovanna

    2008-09-01

    The aim of this study was to identify and characterise the predominant yeasts in Champús, a traditional Colombian cereal-based beverage with a low alcoholic content. Samples of Champús from 20 production sites in the Cauca Valley region were analysed. A total of 235 yeast isolates were identified by conventional microbiological analyses and by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of ITS1-5.8S rDNA-ITS2. The dominant species were: Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia fermentans, Pichia kluyveri var. kluyveri, Zygosaccharomyces fermentati, Torulospora delbruekii, Galactomyces geotrichum and Hanseniaspora spp. Model Champús systems were inoculated with single strains of some isolated sporogenus species and the aromatic profiles were analysed by SPME. Analysis of data showed that Champús strains produced high amounts of esters. The aromatic compounds produced by Saccharomyces and non-Saccharomyces yeasts from Champús can exert a relevant influence on the sensory characteristics of the fermented beverage. The Champús strains could thus represent an important source for new yeast biotypes with potential industrial applications.

  6. Killer systems of the yeast Saccharomyces cerevisiae

    SciTech Connect

    Nesterova, G.F.

    1989-01-01

    The killer systems of Saccharomyces cerevisiae are an unusual class of cytoplasmic symbionts of primitive eukaryotes. The genetic material of these symbionts is double-stranded RNA. They are characterized by the linearity of the genome, its fragmentation into a major and a minor fraction, which replicate separately, and their ability to control the synthesis of secretory mycocin proteins possessing a toxic action on closely related strains. The secretion of mycocins at the same time ensures acquiring of resistance to them. Strains containing killer symbionts are toxigenic and resistant to the action of their own toxin, but strains that are free of killer double-stranded RNAs are sensitive to the action of mycocins. The killer systems of S. cerevisiae have retained features relating them to viruses and are apparently the result of evolution of infectious viruses. The occurrences of such systems among monocellular eukaryotic organisms is an example of complication of the genome by means of its assembly from virus-like components. We discuss the unusual features of replication and the expression of killer systems and their utilization in the construction of vector molecules.

  7. Evaluation of the Yeast Traffic Light PNA FISH Probes for Identification of Candida Species from Positive Blood Cultures

    PubMed Central

    Hall, Leslie; Le Febre, Kara M.; Deml, Sharon M.; Wohlfiel, Sherri L.

    2012-01-01

    The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures. PMID:22238445

  8. Evaluation of the Yeast Traffic Light PNA FISH probes for identification of Candida species from positive blood cultures.

    PubMed

    Hall, Leslie; Le Febre, Kara M; Deml, Sharon M; Wohlfiel, Sherri L; Wengenack, Nancy L

    2012-04-01

    The Yeast Traffic Light PNA FISH kit (YTL) correctly identified Candida spp. in 207/216 (96%) positive blood cultures. Discordant results were seen with known cross-reacting species and cultures containing Candida lambica and Rhodotorula mucilaginosa. The YTL provides rapid, reliable identification of the five common Candida species found in blood cultures.

  9. Studying protein complexes by the yeast two-hybrid system.

    PubMed

    Rajagopala, Seesandra V; Sikorski, Patricia; Caufield, J Harry; Tovchigrechko, Andrey; Uetz, Peter

    2012-12-01

    Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

  10. NADP Regulates the Yeast GAL Induction System

    SciTech Connect

    Kumar,P.; Yao, Y.; Sternglanz, R.; Johnston, S.; Joshua-Tor, L.

    2008-01-01

    Transcriptional regulation of the galactose-metabolizing genes in Saccharomyces cerevisiae depends on three core proteins: Gal4p, the transcriptional activator that binds to upstream activating DNA sequences (UASGAL); Gal80p, a repressor that binds to the carboxyl terminus of Gal4p and inhibits transcription; and Gal3p, a cytoplasmic transducer that, upon binding galactose and adenosine 5'-triphosphate, relieves Gal80p repression. The current model of induction relies on Gal3p sequestering Gal80p in the cytoplasm. However, the rapid induction of this system implies that there is a missing factor. Our structure of Gal80p in complex with a peptide from the carboxyl-terminal activation domain of Gal4p reveals the existence of a dinucleotide that mediates the interaction between the two. Biochemical and in vivo experiments suggests that nicotinamide adenine dinucleotide phosphate (NADP) plays a key role in the initial induction event.

  11. Multisensor fusion for system identification

    NASA Astrophysics Data System (ADS)

    Sim, Sung-Han; Cho, Soojin; Park, Jong-Woong; Kim, Hyunjun

    2014-04-01

    System identification is a fundamental process for developing a numerical model of a physical structure. The system identification process typically involves in data acquisition; particularly in civil engineering applications accelerometers are preferred due to its cost-effectiveness, low noise, and installation convenience. Because the measured acceleration responses result in translational degrees of freedom (DOF) in the numerical model, moment-resisting structures such as beam and plate are not appropriately represented by the models. This study suggests a system identification process that considers both translational and rotational DOFs by using accelerometers and gyroscopes. The proposed approach suggests a systematic way of obtaining dynamic characteristics as well as flexibility matrix from two different measurements of acceleration and angular velocity. Numerical simulation and laboratory experiment are conducted to validate the efficacy of the proposed system identification process.

  12. Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast

    PubMed Central

    Sun, Xiaoying; Hirai, Go; Ueki, Masashi; Hirota, Hiroshi; Wang, Qianqian; Hongo, Yayoi; Nakamura, Takemichi; Hitora, Yuki; Takahashi, Hidekazu; Sodeoka, Mikiko; Osada, Hiroyuki; Hamamoto, Makiko; Yoshida, Minoru; Yashiroda, Yoko

    2016-01-01

    Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4+ and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast. PMID:26892493

  13. Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast.

    PubMed

    Sun, Xiaoying; Hirai, Go; Ueki, Masashi; Hirota, Hiroshi; Wang, Qianqian; Hongo, Yayoi; Nakamura, Takemichi; Hitora, Yuki; Takahashi, Hidekazu; Sodeoka, Mikiko; Osada, Hiroyuki; Hamamoto, Makiko; Yoshida, Minoru; Yashiroda, Yoko

    2016-02-19

    Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4(+) and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

  14. Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method.

    PubMed

    Kappachery, Sajeesh; Yu, Jae Woong; Baniekal-Hiremath, Gangadhar; Park, Se Won

    2013-01-01

    Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato. PMID:24296077

  15. Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory.

    PubMed

    Ninghui, Guo; Bing, Wang; Wei, Ren; Mengmeng, Liu; Meiling, Chu; Dongya, Meng; Liqiong, Yao; Wencheng, Xue

    2015-01-01

    This study aimed to develop a method for rapid and accurate identification of yeasts obtained in the clinic, especially from immunocompromised patients, in order to provide a timely and appropriate antifungal therapy. A total of 112 Candida isolates were analyzed in this study; 28 of them were used to validate the PCR-HRM method in species identification in a blinded manner. Strains were identified by conventional techniques that use VITEK 2 YST cards and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). These methods were compared to the newly developed technique based on real-time polymerase-chain reaction high-resolution melting (PCR-HRM). Discordant results were resolved with internal transcribed spacer (ITS) gene sequencing, the "golden standard" used to evaluate the reliability of all methods in identifying yeasts at the species level. PCR-HRM sensitivity was assessed with the isolated strains. VITEK 2, MALDI-TOF-MS, and PCR-HRM accurately identified 89.2% (74/83), 97.6% (81/83), 100% (83/83) of the isolates, respectively. PCR-HRM detection limit was 1fg/μl of yeasts. In validation assays, a 100% accuracy rate was achieved by the use of PCR-HRM. Therefore, the PCR-HRM method is a rapid, sensitive, and specific diagnostic approach, which provides a cost-effective and more suitable alternative for yeast identification in a clinical laboratory. Future research is needed for automation of data acquisition.

  16. Influence of the farming system on the epiphytic yeasts and yeast-like fungi colonizing grape berries during the ripening process.

    PubMed

    Martins, Guilherme; Vallance, Jessica; Mercier, Anne; Albertin, Warren; Stamatopoulos, Panagiotis; Rey, Patrice; Lonvaud, Aline; Masneuf-Pomarède, Isabelle

    2014-05-01

    Grape berries are colonized by a wide array of epiphytic microorganisms such as yeast and filamentous fungi. This microbiota plays a major role in crop health and also interferes with the winemaking process. In this study, culture-dependent and -independent methods were used to investigate the dynamics and diversity of the yeast and yeast-like microorganisms on the grape berry surface during maturation and the influence of cropping systems in this microflora. The results showed a significant impact of both the farming system and the maturity stage on the epiphytic yeast and yeast-like community. A quantitative approach based on counting cultivable populations indicated an increase in the yeast and yeast-like population during the grape ripening process, reaching a maximum when the berries became overripe. The cultivable yeast and yeast-like population also varied significantly depending on the farming system. Microorganism counts were significantly higher for organically- than conventionally-farmed grapes. The yeast and yeast-like community structures were analysed by culture independent methods, using CE-SSCP. The results revealed changes in the genetic structure of the yeast and yeast-like community throughout the ripening process, as well as the impact of the farming system. Copper-based fungicide treatments were revealed as the main factor responsible for the differences in microbial population densities between samples of different farming systems. The results showed a negative correlation between copper levels and yeast and yeast-like populations, providing evidence that copper inhibited this epiphytic community. Taken together, our results showed that shifts in the microbial community were related to changes in the composition of the grape-berry surface, particularly sugar exudation and the occurrence of copper residues from pesticide treatments. PMID:24603471

  17. Influence of the farming system on the epiphytic yeasts and yeast-like fungi colonizing grape berries during the ripening process.

    PubMed

    Martins, Guilherme; Vallance, Jessica; Mercier, Anne; Albertin, Warren; Stamatopoulos, Panagiotis; Rey, Patrice; Lonvaud, Aline; Masneuf-Pomarède, Isabelle

    2014-05-01

    Grape berries are colonized by a wide array of epiphytic microorganisms such as yeast and filamentous fungi. This microbiota plays a major role in crop health and also interferes with the winemaking process. In this study, culture-dependent and -independent methods were used to investigate the dynamics and diversity of the yeast and yeast-like microorganisms on the grape berry surface during maturation and the influence of cropping systems in this microflora. The results showed a significant impact of both the farming system and the maturity stage on the epiphytic yeast and yeast-like community. A quantitative approach based on counting cultivable populations indicated an increase in the yeast and yeast-like population during the grape ripening process, reaching a maximum when the berries became overripe. The cultivable yeast and yeast-like population also varied significantly depending on the farming system. Microorganism counts were significantly higher for organically- than conventionally-farmed grapes. The yeast and yeast-like community structures were analysed by culture independent methods, using CE-SSCP. The results revealed changes in the genetic structure of the yeast and yeast-like community throughout the ripening process, as well as the impact of the farming system. Copper-based fungicide treatments were revealed as the main factor responsible for the differences in microbial population densities between samples of different farming systems. The results showed a negative correlation between copper levels and yeast and yeast-like populations, providing evidence that copper inhibited this epiphytic community. Taken together, our results showed that shifts in the microbial community were related to changes in the composition of the grape-berry surface, particularly sugar exudation and the occurrence of copper residues from pesticide treatments.

  18. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

    PubMed Central

    de Godoy, Lyris MF; Olsen, Jesper V; de Souza, Gustavo A; Li, Guoqing; Mortensen, Peter; Mann, Matthias

    2006-01-01

    Background Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage. Results To probe the yeast proteome in depth and determine factors currently preventing complete analysis, we grew yeast cells, extracted proteins and separated them by one-dimensional gel electrophoresis. Peptides resulting from trypsin digestion were analyzed by liquid chromatography mass spectrometry on a linear ion trap-Fourier transform mass spectrometer with very high mass accuracy and sequencing speed. We achieved unambiguous identification of more than 2,000 proteins, including very low abundant ones. Effective dynamic range was limited to about 1,000 and effective sensitivity to about 500 femtomoles, far from the subfemtomole sensitivity possible with single proteins. We used SILAC (stable isotope labeling by amino acids in cell culture) to generate one-to-one pairs of true peptide signals and investigated if sensitivity, sequencing speed or dynamic range were limiting the analysis. Conclusion Advanced mass spectrometry methods can unambiguously identify more than 2,000 proteins in a single proteome. Complex mixture analysis is not limited by sensitivity but by a combination of dynamic range (high abundance peptides preventing sequencing of low abundance ones) and by effective sequencing speed. Substantially increased coverage of the yeast proteome appears feasible with further development in software and instrumentation. PMID:16784548

  19. Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

    PubMed

    Ojini, Irene; Gammie, Alison

    2015-07-21

    Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers.

  20. Assessment of Reproducibility of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Bacterial and Yeast Identification

    PubMed Central

    Westblade, Lars F.; Garner, Omai B.; MacDonald, Karen; Bradford, Constance; Pincus, David H.; Mochon, A. Brian; Jennemann, Rebecca; Manji, Ryhana; Bythrow, Maureen; Lewinski, Michael A.; Burnham, Carey-Ann D.

    2015-01-01

    Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the identification of clinical bacterial and yeast isolates. However, data describing the reproducibility of MALDI-TOF MS for microbial identification are scarce. In this study, we show that MALDI-TOF MS-based microbial identification is highly reproducible and can tolerate numerous variables, including differences in testing environments, instruments, operators, reagent lots, and sample positioning patterns. Finally, we reveal that samples of bacterial and yeast isolates prepared for MALDI-TOF MS identification can be repeatedly analyzed without compromising organism identification. PMID:25926486

  1. Yeast Systems Biology: Our Best Shot at Modeling a Cell

    PubMed Central

    Boone, Charles

    2014-01-01

    THE Genetics Society of America’s Edward Novitski Prize recognizes an extraordinary level of creativity and intellectual ingenuity in the solution of significant problems in genetics research. The 2014 recipient, Charles Boone, has risen to the top of the emergent discipline of postgenome systems biology by focusing on the global mapping of genetic interaction networks. Boone invented the synthetic genetic array (SGA) technology, which provides an automated method to cross thousands of strains carrying precise mutations and map large-scale yeast genetic interactions. These network maps offer researchers a functional wiring diagram of the cell, which clusters genes into specific pathways and reveals functional connections. PMID:25316779

  2. Identification of benzoquinones in pretreated lignocellulosic feedstocks and inhibitory effects on yeast.

    PubMed

    Stagge, Stefan; Cavka, Adnan; Jönsson, Leif J

    2015-12-01

    Pretreatment of lignocellulosic biomass under acidic conditions gives rise to by-products that inhibit fermenting microorganisms. An analytical procedure for identification of p-benzoquinone (BQ) and 2,6-dimethoxybenzoquinone (DMBQ) in pretreated biomass was developed, and the inhibitory effects of BQ and DMBQ on the yeast Saccharomyces cerevisiae were assessed. The benzoquinones were analyzed using ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. Pretreatment liquids examined with regard to the presence of BQ and DMBQ originated from six different lignocellulosic feedstocks covering agricultural residues, hardwood, and softwood, and were produced through impregnation with sulfuric acid or sulfur dioxide at varying pretreatment temperature (165-204 °C) and residence time (6-20 min). BQ was detected in all six pretreatment liquids in concentrations ranging up to 6 mg/l, while DMBQ was detected in four pretreatment liquids in concentrations ranging up to 0.5 mg/l. The result indicates that benzoquinones are ubiquitous as by-products of acid pretreatment of lignocellulose, regardless of feedstock and pretreatment conditions. Fermentation experiments with BQ and DMBQ covered the concentration ranges 2 mg/l to 1 g/l and 20 mg/l to 1 g/l, respectively. Even the lowest BQ concentration tested (2 mg/l) was strongly inhibitory to yeast, while 20 mg/l DMBQ gave a slight negative effect on ethanol formation. This work shows that benzoquinones should be regarded as potent and widespread inhibitors in lignocellulosic hydrolysates, and that they warrant attention besides more well-studied inhibitory substances, such as aliphatic carboxylic acids, phenols, and furan aldehydes. PMID:26384342

  3. Systems identification - reprise and projections

    NASA Technical Reports Server (NTRS)

    Taylor, L. W., Jr.

    1974-01-01

    A state-of-the-arts review is given for the field of system identification. Progress in the field is traced from the early models of dynamic systems by Sir Isaac Newton up to the present day use of advanced techniques for numerous applications.

  4. Advances in rotorcraft system identification

    NASA Astrophysics Data System (ADS)

    Hamel, Peter G.; Kaletka, Jürgen

    1997-03-01

    System identification can best be described as the extraction of system characteristics from measured flight test data. Therefore it provides an excellent tool for determining and improving mathematical models for a wide range of applications. The increasing need for accurate models for the design of high bandwidth control systems for rotorcraft has initiated a high interest in and a more intensive use of system identification. This development was supported by the AGARD FVP Working Group 18 on ‘Rotorcraft System Identification’, which brought together specialists from research organisations and industry, tasked with exploring the potential of this tool. In the Group, the full range of identification approaches was applied to dedicated helicopter flight-test-data including data quality checking and the determination and verification of flight mechanical models. It was mainly concentrated on the identification of six degrees of freedom rigid body models, which provide a realistic description of the rotorcraft dynamics for the lower and medium frequency range. The accomplishment of the Working Group has increased the demand for applying these techniques more routinely and, in addition, for extending the model order by including explicit rotor degrees of freedom. Such models also accurately characterize the higher frequency range needed for high bandwidth control system designs. In the specific case of the DLR In-Flight Simulator BO 105 ATTHeS, the application of the identified higher order models for the model-following control system was a major prerequisite for the obtained high simulation quality.

  5. Receptor-mediated mitophagy in yeast and mammalian systems

    PubMed Central

    Liu, Lei; Sakakibara, Kaori; Chen, Quan; Okamoto, Koji

    2014-01-01

    Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease. PMID:24903109

  6. Advances in Identification of Clinical Yeast Isolates by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

    PubMed Central

    Buchan, Blake W.

    2013-01-01

    Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification is being adopted by clinical laboratories for routine identification of microorganisms. To date, the majority of studies have focused on the performance and optimization of MALDI-TOF MS for the identification of bacterial isolates. We review recent literature describing the use of MALDI-TOF MS for the routine identification of a variety of yeasts and yeast-like isolates. Specific topics include the effect of optimized or streamlined extraction methods, modified scoring thresholds, expanded reference libraries, and the possibility of conducting antifungal susceptibility testing using MALDI-TOF MS. PMID:23426924

  7. Modified yeast-two-hybrid system to identify proteins interacting with the growth factor progranulin.

    PubMed

    Tian, Qing-Yun; Zhao, Yun-Peng; Liu, Chuan-ju

    2012-01-17

    Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing, inflammation, and host defense. PGRN also functions as a neurotrophic factor, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel, extracellular matrix protein, and growth factor. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor.

  8. Development of a transformation system for the flavinogenic yeast Candida famata.

    PubMed

    Voronovsky, Andriy A; Abbas, Charles A; Fayura, Lyubov R; Kshanovska, Barbara V; Dmytruk, Kostyantyn V; Sybirna, Kateryna A; Sibirny, Andriy A

    2002-08-01

    Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned. PMID:12702288

  9. Identification and Characterization of Ixodes scapularis Antigens That Elicit Tick Immunity Using Yeast Surface Display

    PubMed Central

    Daffre, Sirlei; DePonte, Kathleen; Hovius, Joppe W. R.; Veer, Cornelis van't; van der Poll, Tom; Bakhtiari, Kamran; Meijers, Joost C. M.; Boder, Eric T.; van Dam, Alje P.; Fikrig, Erol

    2011-01-01

    Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding. PMID:21246036

  10. Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

    PubMed

    Schuijt, Tim J; Narasimhan, Sukanya; Daffre, Sirlei; DePonte, Kathleen; Hovius, Joppe W R; Van't Veer, Cornelis; van der Poll, Tom; Bakhtiari, Kamran; Meijers, Joost C M; Boder, Eric T; van Dam, Alje P; Fikrig, Erol

    2011-01-05

    Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r) P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

  11. Automated drug identification system

    NASA Technical Reports Server (NTRS)

    Campen, C. F., Jr.

    1974-01-01

    System speeds up analysis of blood and urine and is capable of identifying 100 commonly abused drugs. System includes computer that controls entire analytical process by ordering various steps in specific sequences. Computer processes data output and has readout of identified drugs.

  12. New and emerging yeast pathogens.

    PubMed Central

    Hazen, K C

    1995-01-01

    The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed. PMID:8665465

  13. [Identification of C(2)M interacting proteins by yeast two-hybrid screening].

    PubMed

    Shanshan, Yue; Laixin, Xia

    2015-11-01

    The synaptonemal complex (SC) is a huge structure which assembles between the homologous chromosomes during meiotic prophase I. Drosophila germ cell-specific nucleoprotein C(2)M clustering at chromosomes can induce SC formation. To further study the molecular function and mechanism of C(2)M in meiosis, we constructed a bait vector for C(2)M and used the yeast two-hybrid system to identify C(2)M interacting proteins. Forty interacting proteins were obtained, including many DNA and histone binding proteins, ATP synthases and transcription factors. Gene silencing assays in Drosophila showed that two genes, wech and Psf1, may delay the disappearance of SC. These results indicate that Wech and Psf1 may form a complex with C(2)M to participate in the formation or stabilization of the SC complex.

  14. Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica.

    PubMed

    Barahona, Salvador; Yuivar, Yassef; Socias, Gabriel; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

    2016-07-01

    The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities.

  15. Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica.

    PubMed

    Barahona, Salvador; Yuivar, Yassef; Socias, Gabriel; Alcaíno, Jennifer; Cifuentes, Víctor; Baeza, Marcelo

    2016-07-01

    The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities. PMID:27215207

  16. A bimodal biometric identification system

    NASA Astrophysics Data System (ADS)

    Laghari, Mohammad S.; Khuwaja, Gulzar A.

    2013-03-01

    Biometrics consists of methods for uniquely recognizing humans based upon one or more intrinsic physical or behavioral traits. Physicals are related to the shape of the body. Behavioral are related to the behavior of a person. However, biometric authentication systems suffer from imprecision and difficulty in person recognition due to a number of reasons and no single biometrics is expected to effectively satisfy the requirements of all verification and/or identification applications. Bimodal biometric systems are expected to be more reliable due to the presence of two pieces of evidence and also be able to meet the severe performance requirements imposed by various applications. This paper presents a neural network based bimodal biometric identification system by using human face and handwritten signature features.

  17. Structural Aspects of System Identification

    NASA Technical Reports Server (NTRS)

    Glover, Keith

    1973-01-01

    The problem of identifying linear dynamical systems is studied by considering structural and deterministic properties of linear systems that have an impact on stochastic identification algorithms. In particular considered is parametrization of linear systems so that there is a unique solution and all systems in appropriate class can be represented. It is assumed that a parametrization of system matrices has been established from a priori knowledge of the system, and the question is considered of when the unknown parameters of this system can be identified from input/output observations. It is assumed that the transfer function can be asymptotically identified, and the conditions are derived for the local, global and partial identifiability of the parametrization. Then it is shown that, with the right formulation, identifiability in the presence of feedback can be treated in the same way. Similarly the identifiability of parametrizations of systems driven by unobserved white noise is considered using the results from the theory of spectral factorization.

  18. On-Orbit System Identification

    NASA Technical Reports Server (NTRS)

    Mettler, E.; Milman, M. H.; Bayard, D.; Eldred, D. B.

    1987-01-01

    Information derived from accelerometer readings benefits important engineering and control functions. Report discusses methodology for detection, identification, and analysis of motions within space station. Techniques of vibration and rotation analyses, control theory, statistics, filter theory, and transform methods integrated to form system for generating models and model parameters that characterize total motion of complicated space station, with respect to both control-induced and random mechanical disturbances.

  19. Nanogold labeling of the yeast endosomal system for ultrastructural analyses.

    PubMed

    Mari, Muriel; Griffith, Janice; Reggiori, Fulvio

    2014-07-14

    Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belonging to this network of organelles interconnected by vesicular transport, cause severe pathologies including cancer and neurobiological disorders. It is therefore of prime relevance to understand the mechanisms underlying the biogenesis and organization of the endosomal system. The yeast Saccharomyces cerevisiae has been pivotal in this task. To specifically label and analyze at the ultrastructural level the endosomal system of this model organism, we present here a detailed protocol for the positively charged nanogold uptake by spheroplasts followed by the visualization of these particles through a silver enhancement reaction. This method is also a valuable tool for the morphological examination of mutants with defects in endosomal trafficking. Moreover, it is not only applicable for ultrastructural examinations but it can also be combined with immunogold labelings for protein localization investigations.

  20. Identification of yeasts during alcoholic fermentation of tchapalo, a traditional sorghum beer from Côte d'Ivoire.

    PubMed

    N'guessan, Kouadio Florent; Brou, Kouakou; Jacques, Noémie; Casaregola, Serge; Dje, Koffi Marcellin

    2011-05-01

    This study investigated the diversity and dynamics of yeasts involved in alcoholic fermentation of a traditional sorghum beer from Côte d'Ivoire, tchapalo. A total of 240 yeast strains were isolated from fermenting sorghum wort inoculated with dry yeast from two geographic regions of Côte d'Ivoire (Abidjan and Bondoukou). Initial molecular identification to the species level was carried out using RFLP of PCR-amplified internal transcribed spacers of rDNA (ITS1-5.8S-ITS2). Ten different profiles were obtained from the restriction of PCR products with the three endonucleases HaeIII, CfoI and HinfI. Sequence analysis of the D1/D2 domain of the 26S rDNA and the ACT1 gene allowed us to assign these groups to six different species: Saccharomyces cerevisiae-like, Candida tropicalis, Pichia kudriavzevii, Pichia kluyveri, Kodamaea ohmeri and Meyerozyma caribbica. The most frequent species associated with tchapalo fermentation was S. cerevisiae-like (87.36%), followed by C. tropicalis (5.45%) and M. caribbica (2.71%). S. cerevisiae-like strains were diploid heterozygotes and exhibited three to four nucleotides divergence from the type strain in the D1/D2 domain and several indels in the more discriminant sequence of the intron of the ACT1 gene. During the process, the yeast species isolated and their frequencies varied according to the geographic origin of the dry yeast. The occurrence of some species was sporadic and only two non-Saccharomyces species were found in the final product. PMID:21318423

  1. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    PubMed

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B

    2014-05-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  2. Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

    PubMed Central

    Duina, Andrea A.; Miller, Mary E.; Keeney, Jill B.

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans. PMID:24807111

  3. Automated systems for identification of microorganisms.

    PubMed Central

    Stager, C E; Davis, J R

    1992-01-01

    Automated instruments for the identification of microorganisms were introduced into clinical microbiology laboratories in the 1970s. During the past two decades, the capabilities and performance characteristics of automated identification systems have steadily progressed and improved. This article explores the development of the various automated identification systems available in the United States and reviews their performance for identification of microorganisms. Observations regarding deficiencies and suggested improvements for these systems are provided. PMID:1498768

  4. Triclabendazole protects yeast and mammalian cells from oxidative stress: identification of a potential neuroprotective compound.

    PubMed

    Lee, Yong Joo; Burlet, Elodie; Wang, Shaoxiao; Xu, Baoshan; Huang, Shile; Galiano, Floyd J; Witt, Stephan N

    2011-10-14

    The Prestwick and NIH chemical libraries were screened for drugs that protect baker's yeast from sugar-induced cell death (SICD). SICD is triggered when stationary-phase yeast cells are transferred from spent rich medium into water with 2% glucose and no other nutrients. The rapid, apoptotic cell death occurs because reactive oxygen species (ROS) accumulate. We found that triclabendazole, which is used to treat liver flukes in cattle and man, partially protects against SICD. Characterization of triclabendazole revealed that it also protects yeast cells from death induced by the Parkinson's disease-related protein alpha-synuclein (α-syn), which is known to induce the accumulation of ROS. PMID:21946065

  5. The MICE Particle Identification System

    NASA Astrophysics Data System (ADS)

    Bogomilov, M.; MICE Collaboration

    2011-06-01

    The Muon Ionization Cooling Experiment (MICE) at the ISIS accelerator located at the Rutherford Appleton Laboratory, UK, will be the first experiment to study muon cooling at high precision. Demonstration of muon ionization cooling is a major technological step towards the construction of a neutrino factory or a muon collider. A muon beam is produced via pion decay in the MICE beam line within a range of emittances and momenta. Muon purity is assured by a system of detectors for particle identification (PID). We describe briefly the PID system here.

  6. Yeast as a Heterologous Model System to Uncover Type III Effector Function

    PubMed Central

    Popa, Crina; Coll, Núria S.; Valls, Marc; Sessa, Guido

    2016-01-01

    Type III effectors (T3E) are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. “Favourite” targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK) signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure–function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations of S

  7. On-orbit system parameter identification

    NASA Technical Reports Server (NTRS)

    Simonian, Stepan S.

    1988-01-01

    Viewgraphs and discussion on on-orbit system parameter identification are included. Topics covered include: dynamic programming filter (DPF); cost function and estimator; frequency domain formulation structrual dynamic identification; and attributes of DPF.

  8. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit.

    PubMed

    Garner, Cherilyn D; Starr, Jennifer K; McDonough, Patrick L; Altier, Craig

    2010-06-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

  9. Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit▿

    PubMed Central

    Garner, Cherilyn D.; Starr, Jennifer K.; McDonough, Patrick L.; Altier, Craig

    2010-01-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods—traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit—showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population. PMID:20392917

  10. Modular coherence of protein dynamics in yeast cell polarity system

    PubMed Central

    Gao, Juntao Tony; Guimerà, Roger; Li, Hua; Pinto, Inês Mendes; Sales-Pardo, Marta; Wai, Stephanie C.; Rubinstein, Boris; Li, Rong

    2011-01-01

    In this study, we investigated on a systems level how complex protein interactions underlying cell polarity in yeast determine the dynamic association of proteins with the polar cortical domain (PCD) where they localize and perform morphogenetic functions. We constructed a network of physical interactions among >100 proteins localized to the PCD. This network was further divided into five robust modules correlating with distinct subprocesses associated with cell polarity. Based on this reconstructed network, we proposed a simple model that approximates a PCD protein's molecular residence time as the sum of the characteristic time constants of the functional modules with which it interacts, weighted by the number of edges forming these interactions. Regression analyses showed excellent fitting of the model with experimentally measured residence times for a large subset of the PCD proteins. The model is able to predict residence times using small training sets. Our analysis also revealed a scaffold protein that imposes a local constraint of dynamics for certain interacting proteins. PMID:21502521

  11. Identification of human proteins functionally conserved with the yeast putative adaptors ADA2 and GCN5.

    PubMed Central

    Candau, R; Moore, P A; Wang, L; Barlev, N; Ying, C Y; Rosen, C A; Berger, S L

    1996-01-01

    Transcriptional adaptor proteins are required for full function of higher eukaryotic acidic activators in the yeast Saccharomyces cerevisiae, suggesting that this pathway of activation is evolutionarily conserved. Consistent with this view, we have identified possible human homologs of yeast ADA2 (yADA2) and yeast GCN5 (yGCN5), components of a putative adaptor complex. While there is overall sequence similarity between the yeast and human proteins, perhaps more significant is conservation of key sequence features with other known adaptors. We show several functional similarities between the human and yeast adaptors. First, as shown for yADA2 and yGCN5, human ADA2 (hADA2) and human GCN5 (hGCN5) interacted in vivo in a yeast two-hybrid assay. Moreover, hGCN5 interacted with yADA2 in this assay, suggesting that the human proteins form similar complexes. Second, both yADA2 and hADA2 contain cryptic activation domains. Third, hGCN5 and yGCN5 had similar stabilizing effects on yADA2 in vivo. Furthermore, the region of yADA2 that interacted with yGCN5 mapped to the amino terminus of yADA2, which is highly conserved in hADA2. Most striking, is the behavior of the human proteins in human cells. First, GAL4-hADA2 activated transcription in HeLa cells, and second, either hADA2 or hGCN5 augmented GAL4-VP16 activation. These data indicated that the human proteins correspond to functional homologs of the yeast adaptors, suggesting that these cofactors play a key role in transcriptional activation. PMID:8552087

  12. Direct identification and recognition of yeast species from clinical material by using albicans ID and CHROMagar Candida plates.

    PubMed Central

    Baumgartner, C; Freydiere, A M; Gille, Y

    1996-01-01

    Two chromogenic media, Albicans ID and CHROMagar Candida agar plates, were compared with a reference medium, Sabouraud-chloramphenicol agar, and standard methods for the identification of yeast species. This study involved 951 clinical specimens. The detection rates for the two chromogenic media for polymicrobial specimens were 20% higher than that for the Sabouraud-chloramphenicol agar plates. The rates of identification of Candida albicans for Albicans ID and CHROMagar Candida agar plates were, respectively, 37.0 and 6.0% after 24 h of incubation and 93.6 and 92.2% after 72 h of incubation, with specificities of 99.8 and 100%. Furthermore, CHROMagar Candida plates identified 13 of 14 Candida tropicalis and 9 of 12 Candida krusei strains after 48 h of incubation. PMID:8789038

  13. Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.

    PubMed

    Wagemans, Jeroen; Lavigne, Rob

    2015-01-01

    Yeast two-hybrid (Y2H) screening permits identification of completely new protein interaction partners for a protein of interest, in addition to confirming binary protein-protein interactions. After discussing the general advantages and drawbacks of Y2H and existing alternatives, this chapter provides a detailed protocol for traditional Gal4p-based Y2H library screens in Saccharomyces cerevisiae AH109. This includes bait transformation, bait auto-activation testing, prey library transformation, Y2H evaluation, and subsequent identification of the prey plasmids. Moreover, a one-on-one mating protocol to confirm interactions between suspected partners is given. Finally, a quantitative α-galactosidase assay protocol to compare interaction strengths is provided.

  14. Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control.

    PubMed Central

    Ben Yehuda, S; Dix, I; Russell, C S; Levy, S; Beggs, J D; Kupiec, M

    1998-01-01

    The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction. It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene. The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development. In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S. cerevisiae. The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats. We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant. Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated. These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes. PMID:9769104

  15. Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India.

    PubMed

    Jeyaram, K; Singh, W Mohendro; Capece, Angela; Romano, Patrizia

    2008-05-31

    In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from

  16. Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae.

    PubMed

    Park, Yang-Nim; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

    2011-09-01

    The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.

  17. System/observer/controller identification toolbox

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Horta, Lucas G.; Phan, Minh

    1992-01-01

    System Identification is the process of constructing a mathematical model from input and output data for a system under testing, and characterizing the system uncertainties and measurement noises. The mathematical model structure can take various forms depending upon the intended use. The SYSTEM/OBSERVER/CONTROLLER IDENTIFICATION TOOLBOX (SOCIT) is a collection of functions, written in MATLAB language and expressed in M-files, that implements a variety of modern system identification techniques. For an open loop system, the central features of the SOCIT are functions for identification of a system model and its corresponding forward and backward observers directly from input and output data. The system and observers are represented by a discrete model. The identified model and observers may be used for controller design of linear systems as well as identification of modal parameters such as dampings, frequencies, and mode shapes. For a closed-loop system, an observer and its corresponding controller gain directly from input and output data.

  18. Parameter identification for nonlinear aerodynamic systems

    NASA Technical Reports Server (NTRS)

    Pearson, Allan E.

    1991-01-01

    Work continues on frequency analysis for transfer function identification, both with respect to the continued development of the underlying algorithms and in the identification study of two physical systems. Some new results of a theoretical nature were recently obtained that lend further insight into the frequency domain interpretation of the research. Progress in each of those areas is summarized. Although not related to the system identification problem, some new results were obtained on the feedback stabilization of linear time lag systems.

  19. Analyzing fission yeast multidrug resistance mechanisms to develop a genetically tractable model system for chemical biology.

    PubMed

    Kawashima, Shigehiro A; Takemoto, Ai; Nurse, Paul; Kapoor, Tarun M

    2012-07-27

    Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a "closed" mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

  20. Identification of Aspergillus Brla Response Elements (Bres) by Genetic Selection in Yeast

    PubMed Central

    Chang, Y. C.; Timberlake, W. E.

    1993-01-01

    The brlA gene of Aspergillus nidulans plays a central role in controlling conidiophore development. To test the hypothesis that brlA encodes a transcriptional regulator and to identify sites of interaction for the BrlA polypeptide, we expressed brlA in Saccharomyces cerevisiae (yeast) strains containing Aspergillus DNA sequences inserted upstream of a minimal yeast promoter fused to the Escherichia coli lacZ gene. Initially, a DNA fragment from the promoter region of the developmentally regulated rodA gene was tested and shown to mediate brlA-dependent transcriptional activation. Two additional DNA fragments were selected from an Aspergillus genomic library by their ability to respond to brlA in yeast. These fragments contained multiple copies of a sequence motif present in the rodA fragment, which we propose to be sites for BrlA interaction and designate brlA response elements (BREs). DNA fragments containing BREs upstream of a minimal Aspergillus promoter were capable of conferring developmental regulation in Aspergillus. Deletion of BREs from the upstream region of rodA greatly decreased its developmental induction. Multiple copies of a synthetic oligonucleotide with the consensus sequence identified among the BREs mediated brlA-dependent transcriptional activation in yeast. The results show that a primary activity of brlA is transcriptional activation and tentatively identify sites of interaction for the BrlA polypeptide. PMID:8417986

  1. Identification of novel noncoding transcripts in telomerase-negative yeast using RNA-seq

    PubMed Central

    Niederer, Rachel O.; Papadopoulos, Nickolas; Zappulla, David C.

    2016-01-01

    Telomerase is a ribonucleoprotein that maintains the ends of linear chromosomes in most eukaryotes. Loss of telomerase activity results in shortening of telomeric DNA and eventually a specific G2/M cell-cycle arrest known as senescence. In humans, telomere shortening occurs during aging, while inappropriate activation of telomerase is associated with approximately 90% of cancers. Previous studies have identified several classes of noncoding RNAs (ncRNA) also associated with aging-related senescence and cancer, but whether ncRNAs are also involved in short-telomere-induced senescence in yeast is unknown. Here, we report 112 putative novel lncRNAs in the yeast Saccharomyces cerevisiae, 41 of which are only expressed in telomerase-negative yeast. Expression of approximately half of the lncRNAs is strongly correlated with that of adjacent genes, suggesting this subset may influence transcription of neighboring genes. Our results reveal a new potential mechanism governing adaptive changes in senescing and post-senescent survivor yeast cells. PMID:26786024

  2. Identification and characterisation of xylanolytic yeasts isolated from decaying wood and sugarcane bagasse in Brazil.

    PubMed

    Lara, Carla A; Santos, Renata O; Cadete, Raquel M; Ferreira, Carla; Marques, Susana; Gírio, Francisco; Oliveira, Evelyn S; Rosa, Carlos A; Fonseca, César

    2014-06-01

    In this study, yeasts associated with lignocellulosic materials in Brazil, including decaying wood and sugarcane bagasse, were isolated, and their ability to produce xylanolytic enzymes was investigated. A total of 358 yeast isolates were obtained, with 198 strains isolated from decaying wood and 160 strains isolated from decaying sugarcane bagasse samples. Seventy-five isolates possessed xylanase activity in solid medium and were identified as belonging to nine species: Candida intermedia, C. tropicalis, Meyerozyma guilliermondii, Scheffersomyces shehatae, Sugiyamaella smithiae, Cryptococcus diffluens, Cr. heveanensis, Cr. laurentii and Trichosporon mycotoxinivorans. Twenty-one isolates were further screened for total xylanase activity in liquid medium with xylan, and five xylanolytic yeasts were selected for further characterization, which included quantitative analysis of growth in xylan and xylose and xylanase and β-D-xylosidase activities. The yeasts showing the highest growth rate and cell density in xylan, Cr. laurentii UFMG-HB-48, Su. smithiae UFMG-HM-80.1 and Sc. shehatae UFMG-HM-9.1a, were, simultaneously, those exhibiting higher xylanase activity. Xylan induced the highest level of (extracellular) xylanase activity in Cr. laurentii UFMG-HB-48 and the highest level of (intracellular, extracellular and membrane-associated) β-D-xylosidase activity in Su. smithiae UFMG-HM-80.1. Also, significant β-D-xylosidase levels were detected in xylan-induced cultures of Cr. laurentii UFMG-HB-48 and Sc. shehatae UFMG-HM-9.1a, mainly in extracellular and intracellular spaces, respectively. Under xylose induction, Cr. laurentii UFMG-HB-48 showed the highest intracellular β-D-xylosidase activity among all the yeast tested. C. tropicalis UFMG-HB 93a showed its higher (intracellular) β-D-xylosidase activity under xylose induction and higher at 30 °C than at 50 °C. This study revealed different xylanolytic abilities and strategies in yeasts to metabolise xylan and

  3. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the rapid identification of yeasts causing bloodstream infections.

    PubMed

    Ghosh, A K; Paul, S; Sood, P; Rudramurthy, S M; Rajbanshi, A; Jillwin, T J; Chakrabarti, A

    2015-04-01

    Few studies have systematically standardised and evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identification of yeasts from bloodstream infections. This is rapidly becoming pertinent for early identification of yeasts and appropriate antifungal therapy. We used 354 yeast strains identified by polymerase chain reaction (PCR) sequencing for standardisation and 367 blind clinical strains for validation of our MALDI-TOF MS protocols. We also evaluated different sample preparation methods and found the on-plate formic acid extraction method as most cost- and time-efficient. The MALDI-TOF assay correctly identified 98.9% of PCR-sequenced yeasts. Novel main spectrum projections (MSP) were developed for Candida auris, C. viswanathii and Kodamaea ohmeri, which were missing from the Bruker MALDI-TOF MS database. Spectral cut-offs computed by receiver operating characteristics (ROC) analysis showed 99.4% to 100% accuracy at a log score of ≥ 1.70 for C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis. The differences in the species-specific scores of our standardisation and blind validation strains were not statistically significant, implying the optimal performance of our test protocol. The MSPs of the three new species also were validated. We conclude that MALDI-TOF MS is a rapid, accurate and reliable tool for identification of bloodstream yeasts. With proper standardisation, validation and regular database expansion, its efficiency can be further enhanced.

  4. Structural system identification of a composite shell

    SciTech Connect

    Red-Horse, J.R.; Carne, T.G.; James, G.H.; Witkowski, W.R.

    1991-12-31

    Structural system identification is undergoing a period of renewed interest. Probabilistic approaches to physical parameter identification in analysis finite element models make uncertainty in test results an important issue. In this paper, we investigate this issue with a simple, though in many ways representative, structural system. The results of two modal parameter identification techniques are compared and uncertainty estimates, both through bias and random errors, are quantified. The importance of the interaction between test and analysis is also highlighted. 25 refs.

  5. Structural system identification of a composite shell

    SciTech Connect

    Red-Horse, J.R.; Carne, T.G.; James, G.H.; Witkowski, W.R.

    1991-01-01

    Structural system identification is undergoing a period of renewed interest. Probabilistic approaches to physical parameter identification in analysis finite element models make uncertainty in test results an important issue. In this paper, we investigate this issue with a simple, though in many ways representative, structural system. The results of two modal parameter identification techniques are compared and uncertainty estimates, both through bias and random errors, are quantified. The importance of the interaction between test and analysis is also highlighted. 25 refs.

  6. Identification of mass disaster victims: the Swiss identification system.

    PubMed

    Mühlemann, H R; Steiner, E; Brandestini, M

    1979-01-01

    A new, simple, and reliable forensic identification system has been described. It permits the rapid and positive identification of victims of catastrophies such as airplane accidents, battles, floods, and fires. An electronic microprocessing unit directs a mechanical engraver to encode up to 13 alphanumeric characters on a small gold disk 0.25 mm thick and 2.0 mm in diameter. The identification chip is sealed in a 0.8-mm deep cavity prepared with a specially designed diamond burr in the lingual enamel of a molar tooth. The sealant is a stained composite material shown experimentally to be leakage proof, fire resistant, and readily detectable in teeth exposed to high temperatures. At the identification center the gold disk can easily be recovered and the victim positively identified without recourse to time-consuming comparison of dental records. Minimal training is required for the forensic personnel. PMID:512601

  7. Comparative Study of the New Colorimetric VITEK 2 Yeast Identification Card versus the Older Fluorometric Card and of CHROMagar Candida as a Source Medium with the New Card

    PubMed Central

    Aubertine, C. L.; Rivera, M.; Rohan, S. M.; Larone, D. H.

    2006-01-01

    The new VITEK 2 colorimetric card was compared to the previous fluorometric card for identification of yeast. API 20C was considered the “gold standard.” The new card consistently performed better than the older card. Isolates from CHROMagar Candida plates were identified equally as well as those from Sabouraud dextrose agar. PMID:16390976

  8. Identification of Candida species and susceptibility testing with Sensititre YeastOne microdilution panel to 9 antifungal agents

    PubMed Central

    Kucukates, Emine; Gultekin, Nuh N.; Alisan, Zeynep; Hondur, Nur; Ozturk, Recep

    2016-01-01

    Objectives: To determine the species incidence and susceptibility pattern to 9 antifungal agents of yeasts isolated from various clinical specimens of colonized or infected patients treated in the coronary and surgical intensive care units (ICU). Methods: A total of 421 ICU patients were treated at the Cardiology Institute, Istanbul University, Istanbul, Turkey between June 2013 and May 2014, and 44 Candida species were isolated from blood, urine, endotracheal aspiration fluid, sputum, and wounds of 16 ICU patients. Identification of Candida was performed using CHROMagar. Antifungal susceptibility was determined by a Sensititre YeastOne colorimetric microdilution panel. Results: Candida albicans (C. albicans) was the most commonly observed microorganism 23 (54%); the other microorganisms isolated were Candida tropicalis 12 (27%), Candida glabrata 5 (11%), Candida parapsilosis 1 (2%), Candida lusitaniae 1 (2%), Candida sake 1 (2%), and Geotrichum capitatum 1 (2%). All isolates were susceptible to amphotericin B and 5-flucytosine. Geotrichum capitatum excepted, the other isolates were also susceptible to anidulafungin, micafungin, and caspofungin. Candida parapsilosis was found to be susceptible to all the studied antifungals. High MIC rates for azole group of antifungal drugs were found for C. albicans, C. tropicalis, and C. glabrata. The rate of colonisation was 3.8% (16/421). Only 0.7% (3/421) patients out of a total of 421 developed candidemia. Conclusion: We found that the yeast colonization and infection rates of patients in our ICUs are very low. Candida albicans is still the most common species. We detected a decreasing susceptibility to azole compounds. PMID:27381534

  9. Aniline blue-containing buffered charcoal-yeast extract medium for presumptive identification of Legionella species

    SciTech Connect

    Holmes, R.L.

    1982-04-01

    By utilizing buffered charcoal-yeast extract medium containing 0.01% aniline blue in conjunction with a long-wave UV light, the differentiation of five species of Legionella was facilitated. L. pneumophila, when grown on this medium, did not absorb the aniline blue dye; however, L. micdadei, L. dumoffii, L. bozemanii, and L. gormanii absorbed the dye in varying amounts and produced colonies of various shades of blue.

  10. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system

    PubMed Central

    Enkler, Ludovic; Richer, Delphine; Marchand, Anthony L.; Ferrandon, Dominique; Jossinet, Fabrice

    2016-01-01

    Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism. PMID:27767081

  11. Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast.

    PubMed

    Burchett, S A; Scott, A; Errede, B; Dohlman, H G

    2001-07-13

    Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome. Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo. As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety. Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12. Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7. These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK. PMID:11337509

  12. PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

    PubMed

    Colabella, Fernando; Libkind, Diego

    2016-01-01

    It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts.

  13. PCR-based method for the rapid identification of astaxanthin-accumulating yeasts (Phaffia spp.).

    PubMed

    Colabella, Fernando; Libkind, Diego

    2016-01-01

    It has been recently found that the natural distribution, habitat, and genetic diversity of astaxanthin-producing yeasts (i.e. Phaffia rhodozyma, synonym Xanthophyllomyces dendrorhous) is much greater than previously thought. P. rhodozyma is biotechnologically exploited due to its ability to produce the carotenoid pigment astaxanthin and thus, it is used as a natural source of this pigment for aquaculture. P. rhodozyma was also capable of synthesizing the potent UVB sunscreen mycosporine-glutaminol-glucoside (MGG). Therefore, further environmental studies are needed to elucidate its ecological aspects and detect new potential strains for the production of astaxanthin and MGG. However, obtaining new isolates of P. rhodozyma and related species is not always easy due to its low abundance and the presence of other sympatric and pigmented yeasts. In this work we report a successful development of a species-specific primer which has the ability to quickly and accurately detecting isolates representing all known lineages of the genus Phaffia (including novel species of the genus) and excluding closely related taxa. For this purpose, a primer of 20 nucleotides (called PhR) was designed to be used in combination with universal primers ITS3 and NL4 in a multiplex amplification. The proposed method has the sensitivity and specificity required for the precise detection of new isolates, and therefore represents an important tool for the environmental search for novel astaxanthin-producing yeasts. PMID:26922472

  14. Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

    SciTech Connect

    Himelblau, E.; Amasino, R.M.; Mira, H.; Penarrubia, L.; Lin, S.J.; Culotta, V.C.

    1998-08-01

    A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and in fluorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO{sub 4}, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

  15. Stochastic system identification in structural dynamics

    USGS Publications Warehouse

    Safak, Erdal

    1988-01-01

    Recently, new identification methods have been developed by using the concept of optimal-recursive filtering and stochastic approximation. These methods, known as stochastic identification, are based on the statistical properties of the signal and noise, and do not require the assumptions of current methods. The criterion for stochastic system identification is that the difference between the recorded output and the output from the identified system (i.e., the residual of the identification) should be equal to white noise. In this paper, first a brief review of the theory is given. Then, an application of the method is presented by using ambient vibration data from a nine-story building.

  16. Dictyostelium discoideum as a Novel Host System to Study the Interaction between Phagocytes and Yeasts

    PubMed Central

    Koller, Barbara; Schramm, Christin; Siebert, Susann; Triebel, János; Deland, Eric; Pfefferkorn, Anna M.; Rickerts, Volker; Thewes, Sascha

    2016-01-01

    The social amoeba Dictyostelium discoideum is a well-established model organism to study the interaction between bacteria and phagocytes. In contrast, research using D. discoideum as a host model for fungi is rare. We describe a comprehensive study, which uses D. discoideum as a host model system to investigate the interaction with apathogenic (Saccharomyces cerevisiae) and pathogenic (Candida sp.) yeast. We show that Dictyostelium can be co-cultivated with yeasts on solid media, offering a convenient test to study the interaction between fungi and phagocytes. We demonstrate that a number of D. discoideum mutants increase (atg1−, kil1−, kil2−) or decrease (atg6−) the ability of the amoebae to predate yeast cells. On the yeast side, growth characteristics, reduced phagocytosis rate, as well as known virulence factors of C. albicans (EFG1, CPH1, HGC1, ICL1) contribute to the resistance of yeast cells against predation by the amoebae. Investigating haploid C. albicans strains, we suggest using the amoebae plate test for screening purposes after random mutagenesis. Finally, we discuss the potential of our adapted amoebae plate test to use D. discoideum for risk assessment of yeast strains.

  17. System identification for robust control design

    SciTech Connect

    Dohner, J.L.

    1995-04-01

    System identification for the purpose of robust control design involves estimating a nominal model of a physical system and the uncertainty bounds of that nominal model via the use of experimentally measured input/output data. Although many algorithms have been developed to identify nominal models, little effort has been directed towards identifying uncertainty bounds. Therefore, in this document, a discussion of both nominal model identification and bounded output multiplicative uncertainty identification will be presented. This document is divided into several sections. Background information relevant to system identification and control design will be presented. A derivation of eigensystem realization type algorithms will be presented. An algorithm will be developed for calculating the maximum singular value of output multiplicative uncertainty from measured data. An application will be given involving the identification of a complex system with aliased dynamics, feedback control, and exogenous noise disturbances. And, finally, a short discussion of results will be presented.

  18. Identification of Telomerase RNAs from Filamentous Fungi Reveals Conservation with Vertebrates and Yeasts

    PubMed Central

    Kuprys, Paulius V.; Davis, Shaun M.; Hauer, Tyler M.; Meltser, Max; Tzfati, Yehuda; Kirk, Karen E.

    2013-01-01

    Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5′ splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi. PMID:23555591

  19. Identification of genes responsible for improved cryoresistance in fermenting yeast cells.

    PubMed

    Tanghe, A; Teunissen, A; Van Dijck, P; Thevelein, J M

    2000-04-10

    Using repetitive freezing and thawing, different mutant industrial Saccharomyces cerevisiae strains with increased freeze resistance have been isolated. To get a better insight in the mechanisms responsible for this elevated resistance and to give us the opportunity to modify other strains so that they become more suitable for use in frozen dough preparations, we applied the microarray technology in order to identify genes that are differentially expressed in a freeze-resistant mutant when compared to a freeze-sensitive industrial yeast strain. PMID:10791754

  20. Identification of Malassezia yeast species isolated from patients with pityriasis versicolor.

    PubMed

    Petry, Vanessa; Tanhausen, Fernanda; Weiss, Luciana; Milan, Thais; Mezzari, Adelina; Weber, Magda Blessmann

    2011-01-01

    Pityriasis versicolor (PV) is a disease with worldwide distribution. Twelve different species of Malassezia yeast have been described. The objective of this study was to determine which species of Malassezia are more prevalent in patients with pityriasis versicolor. Samples were collected by scraping the lesions of 87 patients with a clinical suspicion of pityriasis versicolor. The samples were then submitted to fungal microscopy and culture to identify the species. The species found were: Malassezia sympodialis (30%), Malassezia furfur (25.7%), Malassezia globosa (22.7%), Malassezia restricta (12.1%), Malassezia obtusa (7.6%) and Malassezia slooffiae (1.5%).

  1. Systems Level Analysis of the Yeast Osmo-Stat

    PubMed Central

    Talemi, Soheil Rastgou; Tiger, Carl-Fredrik; Andersson, Mikael; Babazadeh, Roja; Welkenhuysen, Niek; Klipp, Edda; Hohmann, Stefan; Schaber, Jörg

    2016-01-01

    Adaptation is an important property of living organisms enabling them to cope with environmental stress and maintaining homeostasis. Adaptation is mediated by signaling pathways responding to different stimuli. Those signaling pathways might communicate in order to orchestrate the cellular response to multiple simultaneous stimuli, a phenomenon called crosstalk. Here, we investigate possible mechanisms of crosstalk between the High Osmolarity Glycerol (HOG) and the Cell Wall Integrity (CWI) pathways in yeast, which mediate adaptation to hyper- and hypo-osmotic challenges, respectively. We combine ensemble modeling with experimental investigations to test in quantitative terms different hypotheses about the crosstalk of the HOG and the CWI pathways. Our analyses indicate that for the conditions studied i) the CWI pathway activation employs an adaptive mechanism with a variable volume-dependent threshold, in contrast to the HOG pathway, whose activation relies on a fixed volume-dependent threshold, ii) there is no or little direct crosstalk between the HOG and CWI pathways, and iii) its mainly the HOG alone mediating adaptation of cellular osmotic pressure for both hyper- as well as hypo-osmotic stress. Thus, by iteratively combining mathematical modeling with experimentation we achieved a better understanding of regulatory mechanisms of yeast osmo-homeostasis and formulated new hypotheses about osmo-sensing. PMID:27515486

  2. Systems Level Analysis of the Yeast Osmo-Stat.

    PubMed

    Talemi, Soheil Rastgou; Tiger, Carl-Fredrik; Andersson, Mikael; Babazadeh, Roja; Welkenhuysen, Niek; Klipp, Edda; Hohmann, Stefan; Schaber, Jörg

    2016-01-01

    Adaptation is an important property of living organisms enabling them to cope with environmental stress and maintaining homeostasis. Adaptation is mediated by signaling pathways responding to different stimuli. Those signaling pathways might communicate in order to orchestrate the cellular response to multiple simultaneous stimuli, a phenomenon called crosstalk. Here, we investigate possible mechanisms of crosstalk between the High Osmolarity Glycerol (HOG) and the Cell Wall Integrity (CWI) pathways in yeast, which mediate adaptation to hyper- and hypo-osmotic challenges, respectively. We combine ensemble modeling with experimental investigations to test in quantitative terms different hypotheses about the crosstalk of the HOG and the CWI pathways. Our analyses indicate that for the conditions studied i) the CWI pathway activation employs an adaptive mechanism with a variable volume-dependent threshold, in contrast to the HOG pathway, whose activation relies on a fixed volume-dependent threshold, ii) there is no or little direct crosstalk between the HOG and CWI pathways, and iii) its mainly the HOG alone mediating adaptation of cellular osmotic pressure for both hyper- as well as hypo-osmotic stress. Thus, by iteratively combining mathematical modeling with experimentation we achieved a better understanding of regulatory mechanisms of yeast osmo-homeostasis and formulated new hypotheses about osmo-sensing. PMID:27515486

  3. Dental orthopantomogram biometrics system for human identification.

    PubMed

    Singh, Sandeep; Bhargava, Darpan; Deshpande, Ashwini

    2013-07-01

    Fingerprinting is the most widely accepted method of identification of people. But in cases of disfigured, decomposed, burnt or fragmented bodies, it is of limited value. Teeth and dental restorations on the other hand are extremely resistant to destruction by fire. They retain a number of their original characteristics, which are often unique and hence offer a possibility of rather accurate and legally acceptable identification of such remains. This study was undertaken to evaluate the utility of orthopantomography for human identification and propose a coding system for orthopantomogram (OPG), which can be utilized as an identification tool in forensic sciences.

  4. Practical reactor systems for yeast cell immobilization using biomass support particles

    SciTech Connect

    Black, G.M.; Webb, C.; Mattews, T.M.; Atkinson, B.

    1984-01-01

    The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.

  5. Microbe domestication and the identification of the wild genetic stock of lager-brewing yeast

    PubMed Central

    Libkind, Diego; Hittinger, Chris Todd; Valério, Elisabete; Gonçalves, Carla; Dover, Jim; Johnston, Mark; Gonçalves, Paula; Sampaio, José Paulo

    2011-01-01

    Domestication of plants and animals promoted humanity's transition from nomadic to sedentary lifestyles, demographic expansion, and the emergence of civilizations. In contrast to the well-documented successes of crop and livestock breeding, processes of microbe domestication remain obscure, despite the importance of microbes to the production of food, beverages, and biofuels. Lager-beer, first brewed in the 15th century, employs an allotetraploid hybrid yeast, Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis), a domesticated species created by the fusion of a Saccharomyces cerevisiae ale-yeast with an unknown cryotolerant Saccharomyces species. We report the isolation of that species and designate it Saccharomyces eubayanus sp. nov. because of its resemblance to Saccharomyces bayanus (a complex hybrid of S. eubayanus, Saccharomyces uvarum, and S. cerevisiae found only in the brewing environment). Individuals from populations of S. eubayanus and its sister species, S. uvarum, exist in apparent sympatry in Nothofagus (Southern beech) forests in Patagonia, but are isolated genetically through intrinsic postzygotic barriers, and ecologically through host-preference. The draft genome sequence of S. eubayanus is 99.5% identical to the non-S. cerevisiae portion of the S. pastorianus genome sequence and suggests specific changes in sugar and sulfite metabolism that were crucial for domestication in the lager-brewing environment. This study shows that combining microbial ecology with comparative genomics facilitates the discovery and preservation of wild genetic stocks of domesticated microbes to trace their history, identify genetic changes, and suggest paths to further industrial improvement. PMID:21873232

  6. Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.

    PubMed

    Athenstaedt, K; Zweytick, D; Jandrositz, A; Kohlwein, S D; Daum, G

    1999-10-01

    Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.

  7. Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

    PubMed Central

    Athenstaedt, Karin; Zweytick, Dagmar; Jandrositz, Anita; Kohlwein, Sepp Dieter; Daum, Günther

    1999-01-01

    Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism. PMID:10515935

  8. Isolation and Identification of Yeasts from Wild Flowers Collected around Jangseong Lake in Jeollanam-do, Republic of Korea, and Characterization of the Unrecorded Yeast Bullera coprosmaensis.

    PubMed

    Han, Sang-Min; Hyun, Se-Hee; Lee, Hyang Burm; Lee, Hye Won; Kim, Ha-Kun; Lee, Jong-Soo

    2015-09-01

    Several types of yeasts were isolated from wild flowers around Jangseong Lake in Jeollanam-do, Republic of Korea and identified by comparing the nucleotide sequences of the PCR amplicons for the D1/D2 variable domain of the 26S ribosomal DNA using Basic Local Alignment Search Tool (BLAST) analysis. In total, 60 strains from 18 species were isolated, and Pseudozyma spp. (27 strains), which included Pseudozyma rugulosa (7 strains) and Pseudozyma aphidis (6 strains), was dominant species. Among the 60 strains, Bullera coprosmaensis JS00600 represented a newly recorded yeast strain in Korea, and its microbiological characteristics were investigated. The yeast cell has an oval-shaped morphology measuring 1.4 × 1.7 µm in size. Bullera coprosmaensis JS00600 is an asporous yeast that exhibits no pseudomycelium formation. It grew well in vitamin-free medium as well as in yeast extract-malt extract broth and yeast extract-peptone-dextrose (YPD) broth, and it is halotolerant growing in 10% NaCl-containing YPD broth.

  9. Identification and Control of Mechanical Systems

    NASA Astrophysics Data System (ADS)

    Juang, Jer-Nan; Phan, Minh Q.

    2001-08-01

    The control of vibrating systems is a significant issue in the design of aircraft, spacecraft, bridges, and high-rise buildings. This book discusses the control of vibrating systems, integrating structural dynamics, vibration analysis, modern control, and system identification. By integrating these subjects engineers will need only one book, rather than several texts or courses, to solve vibration control problems. The authors cover key developments in aerospace control and identification theory, including virtual passive control, observer and state-space identification, and data-based controller synthesis. They address many practical issues and applications, and show examples of how various methods are applied to real systems. Some methods show the close integration of system identification and control theory from the state-space perspective, rather than from the traditional input-output model perspective of adaptive control. This text will be useful for advanced undergraduate and beginning graduate students in aerospace, mechanical, and civil engineering, as well as for practicing engineers.

  10. Study of amyloids using yeast

    PubMed Central

    Wickner, Reed B.; Kryndushkin, Dmitry; Shewmaker, Frank; McGlinchey, Ryan; Edskes, Herman K.

    2012-01-01

    Summary Saccharomyces cerevisiae has been a useful model organism in such fields as the cell cycle, regulation of transcription, protein trafficking and cell biology, primarily because of its ease of genetic manipulation. This is no less so in the area of amyloid studies. The endogenous yeast amyloids described to date include prions, infectious proteins (Table 1), and some cell wall proteins (1). and amyloids of humans and a fungal prion have also been studied using the yeast system. Accordingly, the emphasis of this chapter will be on genetic, biochemical, cell biological and physical methods particularly useful in the study of yeast prions and other amyloids studied in yeast. We limit our description of these methods to those aspects which have been most useful in studying yeast prions, citing more detailed expositions in the literature. Volumes on yeast genetics methods (2–4), and on amyloids and prions (5, 6) are useful, and Masison has edited a volume of Methods on “Identification, analysis and characterization of fungal prions” which covers some of this territory (7). We also outline some useful physical methods, pointing the reader to more extensive and authoratative descriptions. PMID:22528100

  11. Extracellular enzyme production and phylogenetic distribution of yeasts in wastewater treatment systems.

    PubMed

    Yang, Qingxiang; Zhang, Hao; Li, Xueling; Wang, Zhe; Xu, Ying; Ren, Siwei; Chen, Xuanyu; Xu, Yuanyuan; Hao, Hongxin; Wang, Hailei

    2013-02-01

    The abilities of yeasts to produce different extracellular enzymes and their distribution characteristics were studied in municipal, inosine fermentation, papermaking, antibiotic fermentation, and printing and dyeing wastewater treatment systems. The results indicated that of the 257 yeasts, 16, 14, 55, and 11 produced lipase, protease, manganese dependant peroxidase (MnP), and lignin peroxidase (LiP), respectively. They were distributed in 12 identified and four unidentified genera, in which Candida rugosa (AA-M17) and an unidentified Saccharomycetales (AA-Y5), Pseudozyma sp. (PH-M15), Candida sp. (MO-Y11), and Trichosporon montevideense (MO-M16) were shown to have the highest activity of lipase, protease, Mnp, and LiP, respectively. No yeast had amylase, cellulose, phytase, or laccase activity. Although only 60 isolates produced ligninolytic enzymes, 249 of the 257 yeasts could decolorize different dyes through the mechanism of biodegradation (222 isolates) or bio-sorption. The types of extracellular enzymes that the yeasts produced were significantly shaped by the types of wastewater treated. PMID:23261999

  12. Yeast and Fungal Prions: Amyloid-Handling Systems, Amyloid Structure, and Prion Biology.

    PubMed

    Wickner, R B; Edskes, H K; Gorkovskiy, A; Bezsonov, E E; Stroobant, E E

    2016-01-01

    Yeast prions (infectious proteins) were discovered by their outré genetic properties and have become important models for an array of human prion and amyloid diseases. A single prion protein can become any of many distinct amyloid forms (called prion variants or strains), each of which is self-propagating, but with different biological properties (eg, lethal vs mild). The folded in-register parallel β sheet architecture of the yeast prion amyloids naturally suggests a mechanism by which prion variant information can be faithfully transmitted for many generations. The yeast prions rely on cellular chaperones for their propagation, but can be cured by various chaperone imbalances. The Btn2/Cur1 system normally cures most variants of the [URE3] prion that arise. Although most variants of the [PSI+] and [URE3] prions are toxic or lethal, some are mild in their effects. Even the most mild forms of these prions are rare in the wild, indicating that they too are detrimental to yeast. The beneficial [Het-s] prion of Podospora anserina poses an important contrast in its structure, biology, and evolution to the yeast prions characterized thus far.

  13. Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification

    PubMed Central

    Markov, Dmitriy A; Savkina, Maria; Anikin, Michael; Del Campo, Mark; Ecker, Karen; Lambowitz, Alan M; De Gnore, Jon P; McAllister, William T

    2009-01-01

    The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP–protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP–TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP–mtRNAP fusion, pulled down associated proteins, and identified them by LC–MS–MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity. Copyright © 2009 John Wiley & Sons, Ltd. PMID:19536766

  14. Isolation and Identification of Black Yeasts by Enrichment on Atmospheres of Monoaromatic Hydrocarbons

    PubMed Central

    Zhao, Jingjun; Zeng, Jingsi; de Hoog, G. Sybren; Attili-Angelis, Derlene

    2010-01-01

    Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species. PMID:20333373

  15. A yeast surface display system for the discovery of ligands that trigger cell activation.

    PubMed

    Cho, B K; Kieke, M C; Boder, E T; Wittrup, K D; Kranz, D M

    1998-11-01

    Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface. For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell. In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR. Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

  16. MALDI-TOF MS-based identification of black yeasts of the genus Exophiala.

    PubMed

    Özhak-Baysan, Betil; Öğünç, Dilara; Döğen, Aylin; Ilkit, Macit; de Hoog, G Sybren

    2015-05-01

    In this study, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Exophiala species. The analysis included a total of 110 Exophiala isolates, including 15 CBS strains representing 4 species, Exophiala dermatitidis (61), E. phaeomuriformis (36), E. crusticola (9), and E. heteromorpha (4), that had been previously identified based on internal transcribed spacer (ITS) regions. We also compared the relative efficacies of Sabouraud glucose agar (SGA) and Columbia agar (CA) for use in MALDI-TOF MS. Remarkably, we obtained a log-score value ≥2.0 by using either SGA or CA for all 15 CBS strains, indicating species-level identification. The remaining 95 Exophiala strains were identified to the genus or species levels, with identification rates of 96.8% and 90.5%, using SGA or CA, respectively. Most of the E. dermatitidis (100% and 92.9%), E. phaeomuriformis (80.6% and 83.9%), E. crusticola (50% and 100%), and E. heteromorpha (100% and 100%) isolates were correctly identified using SGA or CA, respectively. Furthermore, 58.9% and 26.3% of the strains had log-score values of ≥2.0 by using SGA and CA, respectively. Our results indicate that MALDI-TOF MS is a rapid and reliable technique with high rates of correct taxonomic identification. PMID:25851261

  17. Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species.

    PubMed

    Melhem, M S C; Bertoletti, A; Lucca, H R L; Silva, R B O; Meneghin, F A; Szeszs, M W

    2013-12-01

    Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods. PMID:24688520

  18. Use of the VITEK 2 system to identify and test the antifungal susceptibility of clinically relevant yeast species

    PubMed Central

    Melhem, MSC; Bertoletti, A; Lucca, HRL; Silva, RBO; Meneghin, FA; Szeszs, MW

    2013-01-01

    Eleven quality control isolates (Candida albicans ATCC 64548, C. tropicalis ATCC 200956, C. glabrata ATCC 90030, C. lusitaniae ATCC 200951, C. parapsilosis ATCC 22019, C. krusei ATCC 6258, C. dubliniensis ATCC 6330, Saccharomyces cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 90012, C. gattii FIOCRUZ-CPF 60, and Trichosporon mucoides ATCC 204094) and 32 bloodstream isolates, including C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, C. krusei, C. guilliermondii, C. pelliculosa (Pichia anomala), C. haemulonii, C. lusitaniae, and C. kefyr were identified at the species level by the VITEK 2 system. A set of clinical isolates (32 total) were used as challenge strains to evaluate the ability of the VITEK 2 system to determine the antifungal susceptibility of yeasts compared with the CLSI and EUCAST BMD reference standards. The VITEK 2 system correctly identified 100% of the challenge strains. The identification of yeast species and the evaluation of their susceptibility profiles were performed in an automated manner by the VITEK 2 system after approximately 15 h of growth for most species of Candida. The VITEK 2 system ensures that each test is performed in a standardized manner and provides quantitative MIC results that are reproducible and accurate when compared with the BMD reference methods. This system was able to determine the MICs of amphotericin B, flucytosine, voriconazole, and fluconazole in 15 h or less for the most common clinically relevant Candida species. In addition, the VITEK 2 system could reliably identify resistance to flucytosine, voriconazole, and fluconazole and exhibits excellent quantitative and qualitative agreement with the CLSI or EUCAST broth microdilution reference methods. PMID:24688520

  19. Identification of medically relevant species of arthroconidial yeasts by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    PubMed

    Kolecka, Anna; Khayhan, Kantarawee; Groenewald, Marizeth; Theelen, Bart; Arabatzis, Michael; Velegraki, Aristea; Kostrzewa, Markus; Mares, Mihai; Taj-Aldeen, Saad J; Boekhout, Teun

    2013-08-01

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used for an extensive identification study of arthroconidial yeasts, using 85 reference strains from the CBS-KNAW yeast collection and 134 clinical isolates collected from medical centers in Qatar, Greece, and Romania. The test set included 72 strains of ascomycetous yeasts (Galactomyces, Geotrichum, Saprochaete, and Magnusiomyces spp.) and 147 strains of basidiomycetous yeasts (Trichosporon and Guehomyces spp.). With minimal preparation time, MALDI-TOF MS proved to be an excellent diagnostic tool that provided reliable identification of most (98%) of the tested strains to the species level, with good discriminatory power. The majority of strains were correctly identified at the species level with good scores (>2.0) and seven of the tested strains with log score values between 1.7 and 2.0. The MALDI-TOF MS results obtained were consistent with validated internal transcribed spacer (ITS) and/or large subunit (LSU) ribosomal DNA sequencing results. Expanding the mass spectrum database by increasing the number of reference strains for closely related species, including those of nonclinical origin, should enhance the usefulness of MALDI-TOF MS-based diagnostic analysis of these arthroconidial fungi in medical and other laboratories.

  20. Choosing Between Yeast and Bacterial Expression Systems: Yield Dependent

    NASA Technical Reports Server (NTRS)

    Miller, Rebecca S.; Malone, Christine C.; Moore, Blake P.; Burk, Melissa; Crawford, Lisa; Karr, Laurel J.; Curreri, Peter A. (Technical Monitor)

    2002-01-01

    Green fluorescent protein (GFP) is a naturally occurring fluorescent protein isolated from the jellyfish Aequorea victoria. The intrinsic fluorescence of the protein is due to a chromophore located in the center of the molecule. Its usefulness has been established as a marker for gene expression and localization of gene products. GFP has recently been utilized as a model protein for crystallization studies at NASA/MSFC, both in earth-based and in microgravity experiments. Because large quantities of purified protein were needed, the cDNA of GFP was cloned into the Pichia pastoris pPICZ(alpha) C strain, with very little protein secreted into the media. Microscopic analysis prior to harvest showed gigantic green fluorescent yeast, but upon harvesting most protein was degraded. Trial fermentations of GFP cloned into pPICZ A for intracellular expression provided unsatisfactory yield. GFP cloned into E, coli was overexpressed at greater than 150 mg/liter, with purification yields at greater than 100mg/liter.

  1. Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

    PubMed Central

    2012-01-01

    Background Chitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS. Results Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS. Conclusions Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides

  2. Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

    PubMed

    Eakle, K A; Bernstein, M; Emr, S D

    1988-10-01

    SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind

  3. Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

    PubMed Central

    Cvijović, Marija; Dalevi, Daniel; Bilsland, Elizabeth; Kemp, Graham JL; Sunnerhagen, Per

    2007-01-01

    Background The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis. Results We have used comparative genomics to identify novel uORFs in yeast with a high likelihood of having a translational regulatory role. We examined uORFs, previously shown to play a role in regulation of translation in Saccharomyces cerevisiae, for evolutionary conservation within seven Saccharomyces species. Inspection of the set of conserved uORFs yielded the following three characteristics useful for discrimination of functional from spurious uORFs: a length between 4 and 6 codons, a distance from the start of the main ORF between 50 and 150 nucleotides, and finally a lack of overlap with, and clear separation from, neighbouring uORFs. These derived rules are inherently associated with uORFs with properties similar to the GCN4 locus, and may not detect most uORFs of other types. uORFs with high scores based on these rules showed a much higher evolutionary conservation than randomly selected uORFs. In a genome-wide scan in S. cerevisiae, we found 34 conserved uORFs from 32 genes that we predict to be functional; subsequent analysis showed the majority of these to be located within transcripts. A total of 252 genes were found containing conserved uORFs with properties indicative of a functional role; all but 7 are novel. Functional content analysis of this set identified an overrepresentation of genes involved in transcriptional control and development. Conclusion Evolutionary conservation of uORFs in yeasts can be traced up to 100 million years of separation. The

  4. Nuclear Materials Identification System Operational Manual

    SciTech Connect

    Chiang, L.G.

    2001-04-10

    This report describes the operation and setup of the Nuclear Materials Identification System (NMIS) with a {sup 252}Cf neutron source at the Oak Ridge Y-12 Plant. The components of the system are described with a description of the setup of the system along with an overview of the NMIS measurements for scanning, calibration, and confirmation of inventory items.

  5. Modeling, system identification, and control of ASTREX

    NASA Technical Reports Server (NTRS)

    Abhyankar, Nandu S.; Ramakrishnan, J.; Byun, K. W.; Das, A.; Cossey, Derek F.; Berg, J.

    1993-01-01

    The modeling, system identification and controller design aspects of the ASTREX precision space structure are presented in this work. Modeling of ASTREX is performed using NASTRAN, TREETOPS and I-DEAS. The models generated range from simple linear time-invariant models to nonlinear models used for large angle simulations. Identification in both the time and frequency domains are presented. The experimental set up and the results from the identification experiments are included. Finally, controller design for ASTREX is presented. Simulation results using this optimal controller demonstrate the controller performance. Finally the future directions and plans for the facility are addressed.

  6. Multi-level RF identification system

    DOEpatents

    Steele, Kerry D.; Anderson, Gordon A.; Gilbert, Ronald W.

    2004-07-20

    A radio frequency identification system having a radio frequency transceiver for generating a continuous wave RF interrogation signal that impinges upon an RF identification tag. An oscillation circuit in the RF identification tag modulates the interrogation signal with a subcarrier of a predetermined frequency and modulates the frequency-modulated signal back to the transmitting interrogator. The interrogator recovers and analyzes the subcarrier signal and determines its frequency. The interrogator generates an output indicative of the frequency of the subcarrier frequency, thereby identifying the responding RFID tag as one of a "class" of RFID tags configured to respond with a subcarrier signal of a predetermined frequency.

  7. Modeling, system identification, and control of ASTREX

    NASA Astrophysics Data System (ADS)

    Abhyankar, Nandu S.; Ramakrishnan, J.; Byun, K. W.; Das, A.; Cossey, Derek F.; Berg, J.

    1993-02-01

    The modeling, system identification and controller design aspects of the ASTREX precision space structure are presented in this work. Modeling of ASTREX is performed using NASTRAN, TREETOPS and I-DEAS. The models generated range from simple linear time-invariant models to nonlinear models used for large angle simulations. Identification in both the time and frequency domains are presented. The experimental set up and the results from the identification experiments are included. Finally, controller design for ASTREX is presented. Simulation results using this optimal controller demonstrate the controller performance. Finally the future directions and plans for the facility are addressed.

  8. On Markov parameters in system identification

    NASA Technical Reports Server (NTRS)

    Phan, Minh; Juang, Jer-Nan; Longman, Richard W.

    1991-01-01

    A detailed discussion of Markov parameters in system identification is given. Different forms of input-output representation of linear discrete-time systems are reviewed and discussed. Interpretation of sampled response data as Markov parameters is presented. Relations between the state-space model and particular linear difference models via the Markov parameters are formulated. A generalization of Markov parameters to observer and Kalman filter Markov parameters for system identification is explained. These extended Markov parameters play an important role in providing not only a state-space realization, but also an observer/Kalman filter for the system of interest.

  9. Identification of Chinese cabbage sentrin as a suppressor of Bax-induced cell death in yeast.

    PubMed

    Sawitri, Widhi Dyah; Slameto, Slameto; Sugiharto, Bambang; Kim, Kyung-Min

    2012-05-01

    Studies into the cell death program termed apoptosis have resulted in new information regarding how cells control and execute their own demise, including insights into the mechanism by which death-preventing factors can inhibit Bax-induced caspase activation. We investigated high temperature stress-induced cell death in Brassica rapa. Using a yeast functional screening from a Brassica rapa cDNA library, the BH5-127 EST clone encoding an apoptotic suppressor peptide was identified. However, a phylogenic tree showed that BH5-127 clusters within a clade containing SUMO-1 (Small Ubiquitin-like Modifier- 1). BH5-127 was confirmed similar to have function to SUMO-1 as Fas suppression. Expression of BH5-127 showed that substantial suppression of cell death survived on SD-galactose-Leu--Ura- medium. The results suggest that BrSE (Brassica rapa Sentrin EST, BH5-127) is one of the important regulatory proteins in programming cell death, especially in the seedling stage of Chinese cabbage.

  10. Identification and transcription control of fission yeast genes repressed by an ammonium starvation growth arrest.

    PubMed

    Bonnet, C; Perret, E; Dumont, X; Picard, A; Caput, D; Lenaers, G

    2000-01-15

    In fission yeast Schizosaccharomyces pombe, ammonium starvation induces a growth arrest, a cell cycle exit in G(1) and a further switch to meiosis. This process is regulated by the cAMP-dependent protein kinase and the Wis1-dependent MAP kinase cascade, and downstream transcription factors. In order to understand how cells adapt their genetic programme to the switch from mitotic cycling to starvation, a differential transcript analysis comparing mRNA from exponentially growing and ammonium-starved cells was performed. Genes repressed by this stimulus mainly concern cell growth, i.e. protein synthesis and global metabolism. Comparison of the expression of two of them, the ribosomal proteins Rps6 and TCTP, in many different growing conditions, evidenced a strong correlation, suggesting that their transcriptions are coordinately regulated. Nevertheless, by repeating the ammonium starvation on strains constitutively activated for the PKA pathway (Deltacgs1), or unable to activate the Wis1-dependent MAP kinase pathway (Deltawis1), or with both characteristics (Deltacgs1+Deltawis1), the transcriptional inhibition was found to be governed either by the PKA pathway, or by the Wis1 pathway, or by both. These results suggest that during the switch from exponential growth to ammonium starvation, cell homeostasis is maintained by downregulating the transcription of the most expressed genes by a PKA and a Wis1-dependent process. Accession Nos for the S30 and L14 ribosomal protein cDNA sequences are AJ2731 and AJ2732, respectively.

  11. Identification of the structural and functional human homolog of the yeast ubiquitin conjugating enzyme UBC9.

    PubMed Central

    Yasugi, T; Howley, P M

    1996-01-01

    Ubiquitin conjugating enzymes (UBCs) are a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. We have identified a human homolog of Scubc9 (termed hubc9) using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S.cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression. PMID:8668529

  12. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

  13. Engineered Saccharomyces cerevisiae strain for improved xylose utilization with a three-plasmid SUMO yeast expression system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A three-plasmid yeast expression system utilizing the portable small ubiquitin-like modifier (SUMO) vector set combined with the efficient endogenous yeast protease Ulp1 was developed for production of large amounts of soluble functional protein in Saccharomyces cerevisiae. Each vector has a differ...

  14. Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies.

    PubMed

    Lasserre, Jean-Paul; Dautant, Alain; Aiyar, Raeka S; Kucharczyk, Roza; Glatigny, Annie; Tribouillard-Tanvier, Déborah; Rytka, Joanna; Blondel, Marc; Skoczen, Natalia; Reynier, Pascal; Pitayu, Laras; Rötig, Agnès; Delahodde, Agnès; Steinmetz, Lars M; Dujardin, Geneviève; Procaccio, Vincent; di Rago, Jean-Paul

    2015-06-01

    Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as 'petite-positivity'), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast.

  15. Yeast as a system for modeling mitochondrial disease mechanisms and discovering therapies

    PubMed Central

    Lasserre, Jean-Paul; Dautant, Alain; Aiyar, Raeka S.; Kucharczyk, Roza; Glatigny, Annie; Tribouillard-Tanvier, Déborah; Rytka, Joanna; Blondel, Marc; Skoczen, Natalia; Reynier, Pascal; Pitayu, Laras; Rötig, Agnès; Delahodde, Agnès; Steinmetz, Lars M.; Dujardin, Geneviève; Procaccio, Vincent; di Rago, Jean-Paul

    2015-01-01

    ABSTRACT Mitochondrial diseases are severe and largely untreatable. Owing to the many essential processes carried out by mitochondria and the complex cellular systems that support these processes, these diseases are diverse, pleiotropic, and challenging to study. Much of our current understanding of mitochondrial function and dysfunction comes from studies in the baker's yeast Saccharomyces cerevisiae. Because of its good fermenting capacity, S. cerevisiae can survive mutations that inactivate oxidative phosphorylation, has the ability to tolerate the complete loss of mitochondrial DNA (a property referred to as ‘petite-positivity’), and is amenable to mitochondrial and nuclear genome manipulation. These attributes make it an excellent model system for studying and resolving the molecular basis of numerous mitochondrial diseases. Here, we review the invaluable insights this model organism has yielded about diseases caused by mitochondrial dysfunction, which ranges from primary defects in oxidative phosphorylation to metabolic disorders, as well as dysfunctions in maintaining the genome or in the dynamics of mitochondria. Owing to the high level of functional conservation between yeast and human mitochondrial genes, several yeast species have been instrumental in revealing the molecular mechanisms of pathogenic human mitochondrial gene mutations. Importantly, such insights have pointed to potential therapeutic targets, as have genetic and chemical screens using yeast. PMID:26035862

  16. Identification of novel transcriptional regulators of Zat12 using comprehensive yeast one-hybrid screens.

    PubMed

    Ben Daniel, Bat-Hen; Cattan, Esther; Wachtel, Chaim; Avrahami, Dorit; Glick, Yair; Malichy, Asaf; Gerber, Doron; Miller, Gad

    2016-08-01

    To appropriately acclimate to environmental stresses, plants have to rapidly activate a specific transcriptional program. Yet, the identity and function of many of the transcriptional regulators that mediate early responses to abiotic stress stimuli is still unknown. In this work we employed the promoter of the multi-stress-responsive zinc-finger protein Zat12 in yeast one-hybrid (Y1H) screens to identify early abiotic stress-responsive transcriptional regulators. Analysis of Zat12 promoter fragments fused to luciferase underlined an approximately 200 bp fragment responsive to NaCl and to reactive oxygen species (ROS). Using these segments and others as baits against Y1H control or stress Arabidopsis prey libraries, we identified 15 potential Zat12 transcriptional regulators. Among the prominent proteins identified were known transcription factors including bZIP29 and ANAC91 as well as unknown function proteins such as a homolog of the human USB1, a U6 small nuclear RNA (snRNA) processing protein, and dormancy/auxin-associated family protein 2 (DRM2). Altered expression of Zat12 during high light stress in the knockout mutants further indicated the involvement of these proteins in the regulation of Zat12. Using a state of the art microfluidic approach we showed that AtUSB1 and DRM2 can specifically bind dsDNA and were able to identify the preferred DNA-binding motif of all four proteins. Overall, the proteins identified in this work provide an important start point for charting the earliest signaling network of Zat12 and of other genes required for acclimation to abiotic stresses. PMID:26923089

  17. Laser/rf personnel identification system

    NASA Astrophysics Data System (ADS)

    Zari, Michael C.; Ward, Reeder N.; Hess, David A.; Anderson, Christopher S.

    1995-05-01

    This paper documents the design of a Laser/RF Personnel Identification System developed for the US Army Communications and Electronics Command (CECOM) for soldier identification. The system has dual use applications, including law enforcement officer protection, and includes a laser interrogation unit with a programmable activation code. The interrogation unit is very directive for low probability of intercept (LPI), which is of interest during covert operations. A responder unit, worn by the law enforcement personnel or soldier, transmits an LPI radio frequency (RF) response only after receiving the proper interrogation code. The basic subsystems for the identification system are a laser interrogation unit, an RF responder unit, and a programming/synchronization unit. In this paper, the operating principles for the subsystems are reviewed and design issues are discussed. In addition to the design performed for CECOM, a breadboard system was developed to validate the concept. Hardware implementation is reviewed and field testing of the breadboard is presented.

  18. [Evaluation of a new chromogenic medium (Candida ID) for the isolation and presumptive identification of Candida albicans and other medically important yeasts].

    PubMed

    Quindós, G; Alonso-Vargas, R; Helou, S; Arechavala, A; Martín-Mazuelos, E; Negroni, R

    2001-03-01

    Candidiasis is a frequent human infection caused mainly by Candida albicans. However, other species are emerging as important pathogens, as Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei or Candida guilliermondii. Rapid identification of clinical isolates could facilitate diagnosis and treatment. Candida ID (bioMerieux, Spain) is a new medium for the isolation and presumptive identification of yeasts: C. albicans grows as blue colonies, and C. tropicalis, C. guilliermondii, Candida kefyr and Candida lusitaniae as pink ones. The utility of Candida ID was evaluated with more than 700 clinical isolates and type culture collection strains from different genera including Candida, Cryptococcus, Saccharomyces, and Rhodotorula. Presumptive identification was confirmed by germ tube test, microscopic morphology and chlamydoconidia production on corn meal agar and carbohydrate assimilation on API-ATB ID 32C or Vitek (bioMerieux). Growth on Candida ID was rapid (18-24 h) for most of the yeast strains tested. Sensitivity and specificity of identification of C. albicans was significantly high (>98%), since a very low number of isolates were found to be false negative or false positive. A better result was obtained for species growing as pink colonies (>99.5%). Detection of different species of medical important yeasts was easy with Candida ID, as perfectly distinct colors and textures of colonies were observed on this medium. Candida ID allowed the discrimination between C. glabrata (creamy and smooth) and C. krusei (rough and white) colonies. Other species showed different colony textures and colours, white being the predominant colour. Candida ID was very useful for the presumptive identification C. albicans isolates.

  19. Detection and identification of yeasts from formalin-fixed, paraffin-embedded tissue by use of PCR-electrospray ionization mass spectrometry.

    PubMed

    Simner, Patricia J; Buckwalter, Seanne P; Uhl, James R; Wengenack, Nancy L; Pritt, Bobbi S

    2013-11-01

    Diagnosis of yeast infection is typically accomplished by fungal smear and culture, histopathologic examination, and/or serologic studies. Newer assays based on mass spectrometry may be useful for yeast identification when histologic examination is inconclusive, fungal cultures are not ordered, or cultures fail to yield a causative agent. The purpose of this study was to evaluate the ability of the PLEX-ID broad fungal assay to accurately detect and identify yeasts in formalin-fixed paraffin-embedded (FFPE) tissues. Tissue samples from 78 FFPE specimens with both histopathology and corresponding culture results for a variety of yeasts were tested using the PLEX-ID broad fungal assay. A 40-μm FFPE tissue section from each specimen was digested with proteinase K followed by nucleic acid extraction and PCR using broad-range fungal primers. Yeast DNA in amplified products was identified using electrospray ionization mass spectrometry. Discordant results were resolved by D2 rRNA gene sequencing. PLEX-ID analysis detected yeast DNA in 78.2% (61/78) of the cases, of which 91.8% (56/61) were concordant with culture results. Of the 5 discordant positive results, 4 PLEX-ID results were considered to result from environmental contaminants, while 1 clinically important discrepancy was observed (Blastomyces dermatitidis by culture and Cryptococcus neoformans by PLEX-ID). Sequencing of the discordant sample was unsuccessful. The majority of histopathology results (89.7% [70/78]) correlated with culture results. The PLEX-ID broad fungal assay identifies fungi directly from FFPE tissues and can be a useful adjunct to traditional culture and histopathology tests.

  20. Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

    PubMed

    Pang, Wei; Coghill, George M

    2015-05-01

    In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed.

  1. Identification of the Molecular Mechanisms for Cell-Fate Selection in Budding Yeast through Mathematical Modeling

    PubMed Central

    Li, Yongkai; Yi, Ming; Zou, Xiufen

    2013-01-01

    The specification and maintenance of cell fates is essential to the development of multicellular organisms. However, the precise molecular mechanisms in cell fate selection are, to our knowledge, poorly understood due to the complexity of multiple interconnected pathways. In this study, model-based quantitative analysis is used to explore how to maintain distinguished cell fates between cell-cycle commitment and mating arrest in budding yeast. We develop a full mathematical model of an interlinked regulatory network based on the available experimental data. By theoretically defining the Start transition point, the model is able to reproduce many experimental observations of the dynamical behaviors in wild-type cells as well as in Ste5-8A and Far1-S87A mutants. Furthermore, we demonstrate that a moderate ratio between Cln1/2→Far1 inhibition and Cln1/2→Ste5 inhibition is required to ensure a successful switch between different cell fates. We also show that the different ratios of the mutual Cln1/2 and Far1 inhibition determine the different cell fates. In addition, based on a new, definition of network entropy, we find that the Start point in wild-type cells coincides with the system’s point of maximum entropy. This result indicates that Start is a transition point in the network entropy. Therefore, we theoretically explain the Start point from a network dynamics standpoint. Moreover, we analyze the biological bistablity of our model through bifurcation analysis. We find that the Cln1/2 and Cln3 production rates and the nonlinearity of SBF regulation on Cln1/2 production are potential determinants for irreversible entry into a new cell fate. Finally, the quantitative computations further reveal that high specificity and fidelity of the cell-cycle and mating pathways can guarantee specific cell-fate selection. These findings show that quantitative analysis and simulations with a mathematical model are useful tools for understanding the molecular mechanisms in

  2. State Identification in Nonlinear Systems

    SciTech Connect

    Holloway, James Paul

    2005-02-06

    A state estimation method based on finding a system state that causes a model to match a set of system measurements is regularized by requiring that sudden changes in system state be avoided. The required optimization is accomplished by a pattern search algorithm. The method does not require derivative information or linearization of the model. Is has been applied to a 10 dimensional model of a fast reactor system.

  3. Robust orthogonal recombination system for versatile genomic elements rearrangement in yeast Saccharomyces Cerevisiae

    PubMed Central

    Lin, Qiuhui; Qi, Hao; Wu, Yi; Yuan, Yingjin

    2015-01-01

    Rearrangement of genomic DNA elements in a dynamic controlled fashion is a fundamental challenge. Site-specific DNA recombinases have been tamed as a powerful tool in genome editing. Here, we reported a DNA element rearrangement on the basis of a pairwise orthogonal recombination system which is comprised of two site-specific recombinases of Vika/vox and Cre/loxp in yeast Saccharomyces Creevisiae. Taking the advantage of the robust pairwise orthogonality, we showed that multi gene elements could be organized in a programmed way, in which rationally designed pattern of loxP and vox determined the final genotype after expressing corresponding recombinases. Finally, it was demonstrated that the pairwise orthogonal recombination system could be utilized to refine synthetic chromosome rearrangement and modification by loxP-mediated evolution, SCRaMbLE, in yeast cell carrying a completely synthesized chromosome III. PMID:26477943

  4. Robust orthogonal recombination system for versatile genomic elements rearrangement in yeast Saccharomyces cerevisiae.

    PubMed

    Lin, Qiuhui; Qi, Hao; Wu, Yi; Yuan, Yingjin

    2015-01-01

    Rearrangement of genomic DNA elements in a dynamic controlled fashion is a fundamental challenge. Site-specific DNA recombinases have been tamed as a powerful tool in genome editing. Here, we reported a DNA element rearrangement on the basis of a pairwise orthogonal recombination system which is comprised of two site-specific recombinases of Vika/vox and Cre/loxp in yeast Saccharomyces Creevisiae. Taking the advantage of the robust pairwise orthogonality, we showed that multi gene elements could be organized in a programmed way, in which rationally designed pattern of loxP and vox determined the final genotype after expressing corresponding recombinases. Finally, it was demonstrated that the pairwise orthogonal recombination system could be utilized to refine synthetic chromosome rearrangement and modification by loxP-mediated evolution, SCRaMbLE, in yeast cell carrying a completely synthesized chromosome III.

  5. Continuous-Time Bilinear System Identification

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan

    2003-01-01

    The objective of this paper is to describe a new method for identification of a continuous-time multi-input and multi-output bilinear system. The approach is to make judicious use of the linear-model properties of the bilinear system when subjected to a constant input. Two steps are required in the identification process. The first step is to use a set of pulse responses resulting from a constant input of one sample period to identify the state matrix, the output matrix, and the direct transmission matrix. The second step is to use another set of pulse responses with the same constant input over multiple sample periods to identify the input matrix and the coefficient matrices associated with the coupling terms between the state and the inputs. Numerical examples are given to illustrate the concept and the computational algorithm for the identification method.

  6. Analysis of the structure and function of EMRE in a yeast expression system.

    PubMed

    Yamamoto, Takenori; Yamagoshi, Ryohei; Harada, Kazuki; Kawano, Mayu; Minami, Naoki; Ido, Yusuke; Kuwahara, Kana; Fujita, Atsushi; Ozono, Mizune; Watanabe, Akira; Yamada, Akiko; Terada, Hiroshi; Shinohara, Yasuo

    2016-06-01

    The mitochondrial calcium uniporter (MCU) complex is a highly-selective calcium channel, and this complex is believed to consist of a pore-forming subunit, MCU, and its regulatory subunits. As yeast cells lack orthologues of the mammalian proteins, the yeast expression system for the mammalian calcium uniporter subunits is useful for investigating their functions. We here established a yeast expression system for the native-form mouse MCU and 4 other subunits. This expression system enabled us to precisely reconstitute the properties of the mammalian MCU complex in yeast mitochondria. Using this expression system, we analyzed the essential MCU regulator (EMRE), which is a key subunit for Ca(2+) uptake but whose functions and structure remain unclear. The topology of EMRE was revealed: its N- and C-termini projected into the matrix and the inter membrane space, respectively. The expression of EMRE alone was insufficient for Ca(2+) uptake; and co-expression of MCU with EMRE was necessary. EMRE was independent of the protein levels of other subunits, indicating that EMRE was not a protein-stabilizing factor. Deletion of acidic amino acids conserved in EMRE did not significantly affect Ca(2+) uptake; thus, EMRE did not have basic properties of ion channels such as ion-selectivity filtration and ion concentration. Meanwhile, EMRE closely interacted with the MCU on both sides of the inner membrane, and this interaction was essential for Ca(2+) uptake. This close interaction suggested that EMRE might be a structural factor for opening of the MCU-forming pore.

  7. Stabilization of the yeast desaturase system by low levels of oxygen

    NASA Technical Reports Server (NTRS)

    Volkmann, C. M.; Klein, H. P.

    1983-01-01

    The stability of particulate palmitoyl-CoA desaturase preparations from anaerobically grown yeast cells was increased by exposure to low levels of oxygen. The stabilizing effect of oxygen may be based upon the increased amounts of palmitoleic acid and ergosterol that become available to the cells. These results suggest the evolutinary appearance of this system at a time when atmospheric oxygen was at a low level.

  8. Molecular comparisons for identification of food spoilage yeasts and prediction of species that may develop in different food products

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Spoilage of foods and beverages by yeasts is often characterized by objectionable odors, appearance, taste, texture or build-up of gas in packaging containers, resulting in loss of the product. Seldom is human health compromised by products spoiled by yeasts even though some spoilage is caused by sp...

  9. In vitro evaluation of atmospheric particulate matter and sedimentation particles using yeast bioassay system.

    PubMed

    Mori, Taiki; Inudo, Makiko; Takao, Yuji; Koga, Minoru; Takemasa, Takehiro; Shinohara, Ryota; Arizono, Koji

    2007-01-01

    Little information on the evaluation of airborne particulate matter (APM) and sedimentation particles from subway stations is available. The thermal metamorphism of train wheels generating toxic particles in subway stations is a possibility. In this study, the toxicity and physiological effects of particles from subway stations were evaluated using a yeast bioassay system. Estrogenic and antiestrogenic activities of APM in APM extracts from subway stations were determined. No estrogenic activity was found in the APM fractions and their S9-activated APM samples. Sedimentation dust samples also showed no estrogen activity. In contrast, extracts from sedimentation dust samples showed antiestrogen activity. Marked yeast toxicity was observed in the samples extracted from sedimentation dust. Potent yeast toxicity was also found in the S9-activated extracts from sedimentation dust. The results suggest that sedimentation dust from a semiclosed area of a subway system has antiestrogen activity, although both the origin and generation system of this activity are uncertain. These pollutants in sedimentation dust may change to a more toxic form in vivo by S9 activation. PMID:17762843

  10. Parametric uncertain identification of a robotic system

    NASA Astrophysics Data System (ADS)

    Angel, L.; Viola, J.; Hernández, C.

    2016-07-01

    This paper presents the parametric uncertainties identification of a robotic system of one degree of freedom. A MSC-ADAMS / MATLAB co-simulation model was built to simulate the uncertainties that affect the robotic system. For a desired trajectory, a set of dynamic models of the system was identified in presence of variations in the mass, length and friction of the system employing least squares method. Using the input-output linearization technique a linearized model plant was defined. Finally, the maximum multiplicative uncertainty of the system was modelled giving the controller desired design conditions to achieve a robust stability and performance of the closed loop system.

  11. Optical disk uses in criminal identification systems

    NASA Astrophysics Data System (ADS)

    Sypherd, Allen D.

    1990-08-01

    A significant advancement in law enforcement tools has been made possible by the rapid and innovative development of electronic imaging for criminal identification systems. In particular, development of optical disks capable of high-capacity and random-access storage has provided a unique marriage of application and technology. Fast random access to any record, non-destructive reading of stored images, electronic sorting and transmission of images and an accepted legal basis for evidence are a few of the advantages derived from optical disk technology. This paper discusses the application of optical disk technology to both Automated Fingerprint Identification Systems (AFIS) and Automated Mugshot Retrieval Systems (AMRS). The following topics are addressed in light of AFIS and AMRS user requirements and system capabilities: Write once vs. rewritable, gray level and storage requirements, multi-volume library systems, data organization and capacity trends.

  12. 78 FR 58785 - Unique Device Identification System

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-24

    ... discussed in section VII. B. ``Compliance Dates.'' FOR FURTHER INFORMATION CONTACT: Jay Crowley, UDI... device identification system, as required by section 519(f) of the FD&C Act (see 77 FR 40736). On July 9...(f) of the FD&C Act (see 77 FR 69393). The preamble to the July 2012 proposal describes...

  13. Identification of Metabolic Pathway Systems.

    PubMed

    Dolatshahi, Sepideh; Voit, Eberhard O

    2016-01-01

    The estimation of parameters in even moderately large biological systems is a significant challenge. This challenge is greatly exacerbated if the mathematical formats of appropriate process descriptions are unknown. To address this challenge, the method of dynamic flux estimation (DFE) was proposed for the analysis of metabolic time series data. Under ideal conditions, the first phase of DFE yields numerical representations of all fluxes within a metabolic pathway system, either as values at each time point or as plots against their substrates and modulators. However, this numerical result does not reveal the mathematical format of each flux. Thus, the second phase of DFE selects functional formats that are consistent with the numerical trends obtained from the first phase. While greatly facilitating metabolic data analysis, DFE is only directly applicable if the pathway system contains as many dependent variables as fluxes. Because most actual systems contain more fluxes than metabolite pools, this requirement is seldom satisfied. Auxiliary methods have been proposed to alleviate this issue, but they are not general. Here we propose strategies that extend DFE toward general, slightly underdetermined pathway systems.

  14. Identification of Metabolic Pathway Systems

    PubMed Central

    Dolatshahi, Sepideh; Voit, Eberhard O.

    2016-01-01

    The estimation of parameters in even moderately large biological systems is a significant challenge. This challenge is greatly exacerbated if the mathematical formats of appropriate process descriptions are unknown. To address this challenge, the method of dynamic flux estimation (DFE) was proposed for the analysis of metabolic time series data. Under ideal conditions, the first phase of DFE yields numerical representations of all fluxes within a metabolic pathway system, either as values at each time point or as plots against their substrates and modulators. However, this numerical result does not reveal the mathematical format of each flux. Thus, the second phase of DFE selects functional formats that are consistent with the numerical trends obtained from the first phase. While greatly facilitating metabolic data analysis, DFE is only directly applicable if the pathway system contains as many dependent variables as fluxes. Because most actual systems contain more fluxes than metabolite pools, this requirement is seldom satisfied. Auxiliary methods have been proposed to alleviate this issue, but they are not general. Here we propose strategies that extend DFE toward general, slightly underdetermined pathway systems. PMID:26904095

  15. Microbial exopolymers link predator and prey in a model yeast biofilm system.

    PubMed

    Joubert, L-M; Wolfaardt, G M; Botha, A

    2006-08-01

    Protistan grazing on biofilms is potentially an important conduit enabling energy flow between microbial trophic levels. Contrary to the widely held assumption that protistan feeding primarily involves ingestion of biofilm cells, with negative consequences for the biofilm, this study demonstrated preferential grazing on the noncellular biofilm matrix by a ciliate, with selective ingestion of yeast and bacterial cells of planktonic origin over attached and biofilm-derived planktonic cells. Introducing a ciliate to two biofilm-forming Cryptococcus species, as well as two bacterial species in a model biofilm system, fluorescent probes were applied to determine ingestion of cellular and noncellular biofilm fractions. Fluoromicroscopy, as well as photometric quantification, confirmed that protistan grazing enhanced yeast biofilm metabolism, and an increase in biofilm biomass and viability. We propose that the extracellular polymeric matrix of biofilms may act as an interface regulating interaction between predator and prey, while serving as source of nutrients and energy for protists.

  16. Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system.

    PubMed

    Qi, Mei; Huang, Meijuan; Chen, Fan

    2002-04-01

    Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis. PMID:18763077

  17. An efficient automatic firearm identification system

    NASA Astrophysics Data System (ADS)

    Chuan, Zun Liang; Liong, Choong-Yeun; Jemain, Abdul Aziz; Ghani, Nor Azura Md.

    2014-06-01

    Automatic firearm identification system (AFIS) is highly demanded in forensic ballistics to replace the traditional approach which uses comparison microscope and is relatively complex and time consuming. Thus, several AFIS have been developed for commercial and testing purposes. However, those AFIS are still unable to overcome some of the drawbacks of the traditional firearm identification approach. The goal of this study is to introduce another efficient and effective AFIS. A total of 747 firing pin impression images captured from five different pistols of same make and model are used to evaluate the proposed AFIS. It was demonstrated that the proposed AFIS is capable of producing firearm identification accuracy rate of over 95.0% with an execution time of less than 0.35 seconds per image.

  18. Characteristics of an immobilized yeast cell system using very high gravity for the fermentation of ethanol.

    PubMed

    Ji, Hairui; Yu, Jianliang; Zhang, Xu; Tan, Tianwei

    2012-09-01

    The characteristics of ethanol production by immobilized yeast cells were investigated for both repeated batch fermentation and continuous fermentation. With an initial sugar concentration of 280 g/L during the repeated batch fermentation, more than 98% of total sugar was consumed in 65 h with an average ethanol concentration and ethanol yield of 130.12 g/L and 0.477 g ethanol/g consumed sugar, respectively. The immobilized yeast cell system was reliable for at least 10 batches and for a period of 28 days without accompanying the regeneration of Saccharomyces cerevisiae inside the carriers. The multistage continuous fermentation was carried out in a five-stage column bioreactor with a total working volume of 3.75 L. The bioreactor was operated for 26 days at a dilution rate of 0.015 h(-1). The ethanol concentration of the effluent reached 130.77 g/L ethanol while an average 8.18 g/L residual sugar remained. Due to the high osmotic pressure and toxic ethanol, considerable yeast cells died without regeneration, especially in the last two stages, which led to the breakdown of the whole system of multistage continuous fermentation.

  19. Scheffersomyces stipitis: a comparative systems biology study with the Crabtree positive yeast Saccharomyces cerevisiae

    PubMed Central

    2012-01-01

    Background Scheffersomyces stipitis is a Crabtree negative yeast, commonly known for its capacity to ferment pentose sugars. Differently from Crabtree positive yeasts such as Saccharomyces cerevisiae, the onset of fermentation in S. stipitis is not dependent on the sugar concentration, but is regulated by a decrease in oxygen levels. Even though S. stipitis has been extensively studied due to its potential application in pentoses fermentation, a limited amount of information is available about its metabolism during aerobic growth on glucose. Here, we provide a systems biology based comparison between the two yeasts, uncovering the metabolism of S. stipitis during aerobic growth on glucose under batch and chemostat cultivations. Results Starting from the analysis of physiological data, we confirmed through 13C-based flux analysis the fully respiratory metabolism of S. stipitis when growing both under glucose limited or glucose excess conditions. The patterns observed showed similarity to the fully respiratory metabolism observed for S. cerevisiae under chemostat cultivations however, intracellular metabolome analysis uncovered the presence of several differences in metabolite patterns. To describe gene expression levels under the two conditions, we performed RNA sequencing and the results were used to quantify transcript abundances of genes from the central carbon metabolism and compared with those obtained with S. cerevisiae. Interestingly, genes involved in central pathways showed different patterns of expression, suggesting different regulatory networks between the two yeasts. Efforts were focused on identifying shared and unique families of transcription factors between the two yeasts through in silico transcription factors analysis, suggesting a different regulation of glycolytic and glucoenogenic pathways. Conclusions The work presented addresses the impact of high-throughput methods in describing and comparing the physiology of Crabtree positive and Crabtree

  20. Identification of Wild Yeast Strains and Analysis of Their β-Glucan and Glutathione Levels for Use in Makgeolli Brewing

    PubMed Central

    Kang, Sun Hee; Kim, Hye Ryun; Kim, Jae Ho; Ahn, Byung Hak; Kim, Tae Wan

    2014-01-01

    Makgeolli, also known as Takju, is a non-filtered traditional Korean alcoholic beverage that contains various floating matter, including yeast cells, which contributes to its high physiological functionality. In the present study, we assessed the levels of β-glucan and glutathione in various yeast strains isolated from traditional Korean Nuruk and selected a β-glucan- and glutathione-rich yeast strain to add value to Makgeolli by enhancing its physiological functionality through increased levels of these compounds. Yeast β-glucan levels ranged from 6.26% to 32.69% (dry basis) and were strongly species-dependent. Dried Saccharomyces cerevisiae isolated from Nuruk contained 25.53 µg/mg glutathione, 0.70 µg/mg oxidized glutathione, and 11.69 µg/g and 47.85 µg/g spermidine and L-ornithine monohydrochloride, respectively. To produce functional Makgeolli, a β-glucan- and glutathione-rich yeast strain was selected in a screening analysis. Makgeolli fermented with the selected yeast strain contained higher β-glucan and glutathione levels than commercial Makgeolli. Using the selected yeast strain to produce Makgeolli with high β-glucan and glutathione content may enable the production of functional Makgeolli. PMID:25606008

  1. 33 CFR 401.20 - Automatic Identification System.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...' maritime Differential Global Positioning System radiobeacon services; or (7) The use of a temporary unit... 33 Navigation and Navigable Waters 3 2012-07-01 2012-07-01 false Automatic Identification System... Identification System. (a) Each of the following vessels must use an Automatic Identification System...

  2. 33 CFR 401.20 - Automatic Identification System.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...' maritime Differential Global Positioning System radiobeacon services; or (7) The use of a temporary unit... 33 Navigation and Navigable Waters 3 2013-07-01 2013-07-01 false Automatic Identification System... Identification System. (a) Each of the following vessels must use an Automatic Identification System...

  3. 33 CFR 401.20 - Automatic Identification System.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...' maritime Differential Global Positioning System radiobeacon services; or (7) The use of a temporary unit... 33 Navigation and Navigable Waters 3 2011-07-01 2011-07-01 false Automatic Identification System... Identification System. (a) Each of the following vessels must use an Automatic Identification System...

  4. 33 CFR 401.20 - Automatic Identification System.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...' maritime Differential Global Positioning System radiobeacon services; or (7) The use of a temporary unit... 33 Navigation and Navigable Waters 3 2014-07-01 2014-07-01 false Automatic Identification System... Identification System. (a) Each of the following vessels must use an Automatic Identification System...

  5. A Heritable Recombination system for synthetic Darwinian evolution in yeast.

    PubMed

    Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa; Cornish, Virginia W

    2012-12-21

    Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology. PMID:23412545

  6. Parameter identification for nonlinear aerodynamic systems

    NASA Technical Reports Server (NTRS)

    Pearson, Allan E.

    1990-01-01

    Parameter identification for nonlinear aerodynamic systems is examined. It is presumed that the underlying model can be arranged into an input/output (I/O) differential operator equation of a generic form. The algorithm estimation is especially efficient since the equation error can be integrated exactly given any I/O pair to obtain an algebraic function of the parameters. The algorithm for parameter identification was extended to the order determination problem for linear differential system. The degeneracy in a least squares estimate caused by feedback was addressed. A method of frequency analysis for determining the transfer function G(j omega) from transient I/O data was formulated using complex valued Fourier based modulating functions in contrast with the trigonometric modulating functions for the parameter estimation problem. A simulation result of applying the algorithm is given under noise-free conditions for a system with a low pass transfer function.

  7. Identification of general linear mechanical systems

    NASA Technical Reports Server (NTRS)

    Sirlin, S. W.; Longman, R. W.; Juang, J. N.

    1983-01-01

    Previous work in identification theory has been concerned with the general first order time derivative form. Linear mechanical systems, a large and important class, naturally have a second order form. This paper utilizes this additional structural information for the purpose of identification. A realization is obtained from input-output data, and then knowledge of the system input, output, and inertia matrices is used to determine a set of linear equations whereby we identify the remaining unknown system matrices. Necessary and sufficient conditions on the number, type and placement of sensors and actuators are given which guarantee identificability, and less stringent conditions are given which guarantee generic identifiability. Both a priori identifiability and a posteriori identifiability are considered, i.e., identifiability being insured prior to obtaining data, and identifiability being assured with a given data set.

  8. Structural system identification: Structural dynamics model validation

    SciTech Connect

    Red-Horse, J.R.

    1997-04-01

    Structural system identification is concerned with the development of systematic procedures and tools for developing predictive analytical models based on a physical structure`s dynamic response characteristics. It is a multidisciplinary process that involves the ability (1) to define high fidelity physics-based analysis models, (2) to acquire accurate test-derived information for physical specimens using diagnostic experiments, (3) to validate the numerical simulation model by reconciling differences that inevitably exist between the analysis model and the experimental data, and (4) to quantify uncertainties in the final system models and subsequent numerical simulations. The goal of this project was to develop structural system identification techniques and software suitable for both research and production applications in code and model validation.

  9. Microbial identification system for Space Station Freedom

    NASA Technical Reports Server (NTRS)

    Brown, Harlan D.; Scarlett, Janie B.; Skweres, Joyce A.; Fortune, Russell L.; Staples, John L.; Pierson, Duane L.

    1989-01-01

    The Environmental Health System (EHS) and Health Maintenance Facility (HMF) on Space Station Freedom will require a comprehensive microbiology capability. This requirement entails the development of an automated system to perform microbial identifications on isolates from a variety of environmental and clinical sources and, when required, to perform antimicrobial sensitivity testing. The unit currently undergoing development and testing is the Automated Microbiology System II (AMS II) built by Vitek Systems, Inc. The AMS II has successfully completed 12 months of laboratory testing and evaluation for compatibility with microgravity operation. The AMS II is a promising technology for use on Space Station Freedom.

  10. A program for identification of linear systems

    NASA Technical Reports Server (NTRS)

    Buell, J.; Kalaba, R.; Ruspini, E.; Yakush, A.

    1971-01-01

    A program has been written for the identification of parameters in certain linear systems. These systems appear in biomedical problems, particularly in compartmental models of pharmacokinetics. The method presented here assumes that some of the state variables are regularly modified by jump conditions. This simulates administration of drugs following some prescribed drug regime. Parameters are identified by a least-square fit of the linear differential system to a set of experimental observations. The method is especially suited when the interval of observation of the system is very long.

  11. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using yeast models of OXPHOS deficiencies.

    PubMed

    Fontanesi, Flavia; Diaz, Francisca; Barrientos, Antoni

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. Several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, this unit describes the creation and study of yeast models of mitochondrial OXPHOS deficiencies.

  12. Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover

    PubMed Central

    Sitepu, Irnayuli R.; Jin, Mingjie; Fernandez, J. Enrique; da Costa Sousa, Leonardo; Balan, Venkatesh; Boundy-Mills, Kyria L.

    2015-01-01

    Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40% of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Pre-culturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals. PMID:25052467

  13. Identification of a novel pathway involving a GATA transcription factor in yeast and possibly in plant Zn uptake and homeostasis.

    PubMed

    Milner, Matthew J; Pence, Nicole S; Liu, Jiping; Kochian, Leon V

    2014-03-01

    To gain a better understanding of the regulation of Zn homeostasis in plants and the degree of conservation of Zn homeostasis between plants and yeast, a cDNA library from the Zn/Cd hyperaccumulating plant species, Noccaea caerulescens, was screened for its ability to restore growth under Zn limiting conditions in the yeast mutant zap1Δ. ZAP1 is a transcription factor that activates the Zn dependent transcription of yeast genes involved in Zn uptake, including ZRT1, the yeast high affinity Zn transporter. From this screen two members of the E2F family of transcription factors were found to activate ZRT1 expression in a Zn independent manner. The activation of ZRT1 by the plant E2F proteins involves E2F-mediated activation of a yeast GATA transcription factor which in turn activates ZRT1 expression. A ZRT1 promoter region necessary for activation by E2F and GATA proteins is upstream of two zinc responsive elements previously shown to bind ZAP1 in ZRT1. This activation may not involve direct binding of E2F to the ZRT1 promoter. The expression of E2F genes in yeast does not replace function of ZAP1; instead it appears to activate a novel GATA regulatory pathway involved in Zn uptake and homeostasis that is not Zn responsive.

  14. Identification of oleaginous yeast strains able to accumulate high intracellular lipids when cultivated in alkaline pretreated corn stover.

    PubMed

    Sitepu, Irnayuli R; Jin, Mingjie; Fernandez, J Enrique; da Costa Sousa, Leonardo; Balan, Venkatesh; Boundy-Mills, Kyria L

    2014-09-01

    Microbial oil is a potential alternative to food/plant-derived biodiesel fuel. Our previous screening studies identified a wide range of oleaginous yeast species, using a defined laboratory medium known to stimulate lipid accumulation. In this study, the ability of these yeasts to grow and accumulate lipids was further investigated in synthetic hydrolysate (SynH) and authentic ammonia fiber expansion (AFEX™)-pretreated corn stover hydrolysate (ACSH). Most yeast strains tested were able to accumulate lipids in SynH, but only a few were able to grow and accumulate lipids in ACSH medium. Cryptococcus humicola UCDFST 10-1004 was able to accumulate as high as 15.5 g/L lipids, out of a total of 36 g/L cellular biomass when grown in ACSH, with a cellular lipid content of 40 % of cell dry weight. This lipid production is among the highest reported values for oleaginous yeasts grown in authentic hydrolysate. Preculturing in SynH media with xylose as sole carbon source enabled yeasts to assimilate both glucose and xylose more efficiently in the subsequent hydrolysate medium. This study demonstrates that ACSH is a suitable medium for certain oleaginous yeasts to convert lignocellullosic sugars to triacylglycerols for production of biodiesel and other valuable oleochemicals.

  15. Capability of yeast derivatives to adhere enteropathogenic bacteria and to modulate cells of the innate immune system.

    PubMed

    Ganner, Anja; Schatzmayr, Gerd

    2012-07-01

    Yeast derivatives including yeast cell wall components are promising alternatives to antibiotics with respect to the promotion of health and performance in livestock, based on their capacity to bind enteropathogenic bacteria and to beneficially modulate the immune system. However, these mode(s) of action both in vitro and in vivo are still not well understood. Furthermore, standardization and reproducibility of in vitro techniques (microbiology, cell culture assays) are critical features for the application of yeast derivatives as well as for the proof of effectiveness. Yeast cell wall products are suggested as anti-adhesive agents and are thus proposed to prevent attachment of certain intestinal bacteria by providing alternative adhesion sites to enterobacteria, which contain mannose-specific type I fimbriae such as Escherichia coli or Salmonella spp. and which is well documented. Various in vitro assay techniques have become of paramount importance for biotechnological research since they allow for determination and quantification of potential mode(s) of action. However, in vitro assays may be criticized by product end users as not accurately reflecting in vivo responses. Pro and cons of different assays and their bias will be discussed specifically regarding yeast cell wall components and adhesion of enteropathogenic bacteria. Immunomodulation is a therapeutic approach intervening in auto-regulating processes of the defense system. Yeast derivatives such as beta-glucans are proposed to interact with cells of the innate immune system by receptor recognition. Controversial data in literature and mode(s) of action are reviewed and discussed here.

  16. Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system

    PubMed Central

    Li, Dan; Wang, Xiao-Zhong; Yu, Jie-Ping; Chen, Zhi-Xin; Huang, Yue-Hong; Tao, Qi-Min

    2004-01-01

    AIM: To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC). METHODS: With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with β-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was peformed to confirm the binding of putative proteins to X protein in the yeast cells. RESULTS: Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through β-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (cox III). Furthermore, mating experiment identified that the binding of cox III to X protein was specific. CONCLUSION: cox III protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein. PMID:15334674

  17. Design and evaluation of a microfluidic system for inhibition studies of yeast cell signaling

    NASA Astrophysics Data System (ADS)

    Hamngren, Charlotte; Dinér, Peter; Grøtli, Morten; Goksör, Mattias; Adiels, Caroline B.

    2012-10-01

    In cell signaling, different perturbations lead to different responses and using traditional biological techniques that result in averaged data may obscure important cell-to-cell variations. The aim of this study was to develop and evaluate a four-inlet microfluidic system that enables single-cell analysis by investigating the effect on Hog1 localization post a selective Hog1 inhibitor treatment during osmotic stress. Optical tweezers was used to position yeast cells in an array of desired size and density inside the microfluidic system. By changing the flow rates through the inlet channels, controlled and rapid introduction of two different perturbations over the cell array was enabled. The placement of the cells was determined by diffusion rates flow simulations. The system was evaluated by monitoring the subcellular localization of a fluorescently tagged kinase of the yeast "High Osmolarity Glycerol" (HOG) pathway, Hog1-GFP. By sequential treatment of the yeast cells with a selective Hog1 kinase inhibitor and sorbitol, the subcellular localization of Hog1-GFP was analysed on a single-cell level. The results showed impaired Hog1-GFP nuclear localization, providing evidence of a congenial design. The setup made it possible to remove and add an agent within 2 seconds, which is valuable for investigating the dynamic signal transduction pathways and cannot be done using traditional methods. We are confident that the features of the four-inlet microfluidic system will be a valuable tool and hence contribute significantly to unravel the mechanisms of the HOG pathway and similar dynamic signal transduction pathways.

  18. Identification of dynamic systems, theory and formulation

    NASA Technical Reports Server (NTRS)

    Maine, R. E.; Iliff, K. W.

    1985-01-01

    The problem of estimating parameters of dynamic systems is addressed in order to present the theoretical basis of system identification and parameter estimation in a manner that is complete and rigorous, yet understandable with minimal prerequisites. Maximum likelihood and related estimators are highlighted. The approach used requires familiarity with calculus, linear algebra, and probability, but does not require knowledge of stochastic processes or functional analysis. The treatment emphasizes unification of the various areas in estimation in dynamic systems is treated as a direct outgrowth of the static system theory. Topics covered include basic concepts and definitions; numerical optimization methods; probability; statistical estimators; estimation in static systems; stochastic processes; state estimation in dynamic systems; output error, filter error, and equation error methods of parameter estimation in dynamic systems, and the accuracy of the estimates.

  19. Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants.

    PubMed

    Zhou, G L; Miyazaki, Y; Nakagawa, T; Tanaka, K; Shishido, K; Matsuda, H; Kawamukai, M

    1998-04-01

    The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function. PMID:9579081

  20. [Respiratory system of Pichia guilliermondii yeasts with different levels of flavinogenesis].

    PubMed

    Zviagil'skaia, R A; Fedorovich, D V; Shavlovskiĭ, G M

    1978-01-01

    The yeast Pichia guilliermondii was grown on media with different content of iron and its respiration system was studied. When the yeast was cultivated on a complete medium, its respiratory chain operated at the maximum rate in the exponential growth phase, i. e. all the three points of phosphorylation were involved; cytochrome oxidase was the only terminal oxidase. When the growth was decelerated and at the stationary phase, the alternative autooxidable cyanide-resistant pathway inhibited with salicyl hydroxamate partly functioned. Iron deficiency in the medium resulted in a two-three-fold decrease in the content of total and non-hemin iron in the cells, changes in the character and rate of growth, a decrease in the biomass yield, a high rate of flavinogenesis, a slight decrease in the respiration activity, though no drastic changes in the respiration system occurred. This system is represented, as in the case of cells grown on a complete medium, by a typical cytochrome system and an alternative autooxidable pathway. The absence of principal differences in the respiration systems of normal and iron-deficient cells, as well as the operation of the first point of coupling in flavinogenic cells, makes it doubtful that Fenh-proteins of the first segment of the respiratory chain are involved in the regulation of flavinogenesis. PMID:745565

  1. System identification of Drosophila olfactory sensory neurons.

    PubMed

    Kim, Anmo J; Lazar, Aurel A; Slutskiy, Yevgeniy B

    2011-02-01

    The lack of a deeper understanding of how olfactory sensory neurons (OSNs) encode odors has hindered the progress in understanding the olfactory signal processing in higher brain centers. Here we employ methods of system identification to investigate the encoding of time-varying odor stimuli and their representation for further processing in the spike domain by Drosophila OSNs. In order to apply system identification techniques, we built a novel low-turbulence odor delivery system that allowed us to deliver airborne stimuli in a precise and reproducible fashion. The system provides a 1% tolerance in stimulus reproducibility and an exact control of odor concentration and concentration gradient on a millisecond time scale. Using this novel setup, we recorded and analyzed the in-vivo response of OSNs to a wide range of time-varying odor waveforms. We report for the first time that across trials the response of OR59b OSNs is very precise and reproducible. Further, we empirically show that the response of an OSN depends not only on the concentration, but also on the rate of change of the odor concentration. Moreover, we demonstrate that a two-dimensional (2D) Encoding Manifold in a concentration-concentration gradient space provides a quantitative description of the neuron's response. We then use the white noise system identification methodology to construct one-dimensional (1D) and two-dimensional (2D) Linear-Nonlinear-Poisson (LNP) cascade models of the sensory neuron for a fixed mean odor concentration and fixed contrast. We show that in terms of predicting the intensity rate of the spike train, the 2D LNP model performs on par with the 1D LNP model, with a root mean-square error (RMSE) increase of about 5 to 10%. Surprisingly, we find that for a fixed contrast of the white noise odor waveforms, the nonlinear block of each of the two models changes with the mean input concentration. The shape of the nonlinearities of both the 1D and the 2D LNP model appears to be

  2. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  3. Power system identification toolbox: Phase two progress

    SciTech Connect

    Trudnowski, D.J.

    1994-08-01

    This report describes current progress on a project funded by the Bonneville Power Administration (BPA) to develop a set of state-of-the-art analysis software (termed the Power System Identification [PSI] Toolbox) for fitting dynamic models to measured data. The project is being conducted as a three-phase effort. The first phase, completed in late 1992, involved investigating the characteristics of the analysis techniques by evaluating existing software and developing guidelines for best use. Phase Two includes extending current software, developing new analysis algorithms and software, and demonstrating and developing applications. The final phase will focus on reorganizing the software into a modular collection of documented computer programs and developing user manuals with instruction and application guidelines. Phase Two is approximately 50% complete; progress to date and a vision for the final product of the PSI Toolbox are described. The needs of the power industry for specialized system identification methods are particularly acute. The industry is currently pushing to operate transmission systems much closer to theoretical limits by using real-time, large-scale control systems to dictate power flows and maintain dynamic stability. Reliably maintaining stability requires extensive system-dynamic modeling and analysis capability, including measurement-based methods. To serve this need, the BPA has developed specialized system-identification computer codes through in-house efforts and university contract research over the last several years. To make full integrated use of the codes, as well as other techniques, the BPA has commissioned Pacific Northwest Laboratory (PNL) to further develop the codes and techniques into the PSI Toolbox.

  4. Intellectual system of identification of Arabic graphics

    NASA Astrophysics Data System (ADS)

    Abdoullayeva, Gulchin G.; Aliyev, Telman A.; Gurbanova, Nazakat G.

    2001-08-01

    The studies made by using the domain of graphic images allowed creating facilities of the artificial intelligence for letters, letter combinations etc. for various graphics and prints. The work proposes a system of recognition and identification of symbols of the Arabic graphics, which has its own specificity as compared to Latin and Cyrillic ones. The starting stage of the recognition and the identification is coding with further entry of information into a computer. Here the problem of entry is one of the essentials. For entry of a large volume of information in the unit of time a scanner is usually employed. Along with the scanner the authors suggest their elaboration of technical facilities for effective input and coding of the information. For refinement of symbols not identified from the scanner mostly for a small bulk of information the developed coding devices are used directly in the process of writing. The functional design of the software is elaborated on the basis of the heuristic model of the creative activity of a researcher and experts in the description and estimation of states of the weakly formalizable systems on the strength of the methods of identification and of selection of geometric features.

  5. Identification of two hydrophilins that contribute to the desiccation and freezing tolerance of yeast (Saccharomyces cerevisiae) cells.

    PubMed

    Dang, Nghiem X; Hincha, Dirk K

    2011-06-01

    Hydrophilins are a group of proteins that are present in all organisms and that have been defined as being highly hydrophilic and rich in glycine. They are assumed to play important roles in cellular dehydration tolerance. There are 12 genes in the yeast Saccharomyces cerevisiae that encode hydrophilins and most of these genes are stress responsive. However, the functional role of yeast hydrophilins, especially in desiccation and freezing tolerance, is largely unknown. Here, we selected six candidate hydrophilins for further analysis. All six proteins were predicted to be intrinsically disordered, i.e. to have no stable structure in solution. The contribution of these proteins to the desiccation and freezing tolerance of yeast was investigated in the respective knock-out strains. Only the disruption of the genes YJL144W and YMR175W (SIP18) resulted in significantly reduced desiccation tolerance, while none of the strains was affected in its freezing tolerance under our experimental conditions. Complementation experiments showed that yeast cells overexpressing these two genes were both more desiccation and freezing tolerant, confirming the role of these two hydrophilins in yeast dehydration stress tolerance.

  6. Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast.

    PubMed

    Duong, C T; Strack, L; Futschik, M; Katou, Y; Nakao, Y; Fujimura, T; Shirahige, K; Kodama, Y; Nevoigt, E

    2011-11-01

    Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc-ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc-ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc-ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.

  7. Biodiversity of yeast mycobiota in "sucuk," a traditional Turkish fermented dry sausage: phenotypic and genotypic identification, functional and technological properties.

    PubMed

    Ozturk, Ismet; Sagdic, Osman

    2014-11-01

    In this study, yeasts from Turkish fermented sucuks were identified and their functional and technological properties were evaluated. Two hundred fifty-five yeast isolates were obtained from 35 different sucuk samples from different regions of Turkey. The yeast isolates were determined as genotypic using 2 different polymerase chain reaction (PCR) methods (rep-PCR and RAPD-PCR). Functional and technological properties of including proteolytic, lipolytic, and catalase activities, tolerance to NaCl and bile, as well as growing rates at different temperature and pH conditions selected yeast strains were also evaluated. Candida zeylanoides and Debaryomyces hansenii were dominant strains in sucuk samples. All C. zeylanoides and D. hansenii tested could grow at the condition of 15% NaCl and 0.3% bile salt. However, none of the strains were able to grow at 37 °C, even though catalase activity, weak proteolytic and lipolytic activities was still observed. D. hansenii were able to grow only at pH 3, while some of C. zeylanoides could grow at lower pH levels (pH 2). Three and 4 strains of C. zeylanoides showed β-hemolysis activity and nitrate reduction ability to nitrite, respectively. D. hansenii did not have properties, which are β-hemolysis, nitrate reduction, or hydrogen sulfide production. Overall, diverse yeast mycobiota present in Turkish fermented sucuk and their functional and technological properties were revealed with this study. PMID:25273925

  8. Identification of interacting proteins with aryl hydrocarbon receptor in scallop Chlamys farreri by yeast two hybrid screening.

    PubMed

    Cai, Yuefeng; Pan, Luqing; Miao, Jingjing; Liu, Tong

    2016-11-01

    The aryl hydrocarbon receptor (AhR) belongs to the basic-helix-loop helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. AhR has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes, as well as the mediation of the toxicity of certain xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the AhR is well-studied as a mediator of the toxicity of certain xenobiotics in marine bivalves, the normal physiological function remains unknown. In order to explore the function of the AhR, the bait protein expression plasmid pGBKT7-CfAhR and the cDNA library of gill from Chlamys farreri were constructed. By yeast two hybrid system, after multiple screening with the high screening rate medium, rotary verification, sequencing and bioinformatics analysis, the interactions of the CfAhR with receptor for activated protein kinase C 1 (RACK1), thyroid peroxidase-like protein (TPO), Toll-like receptor 4(TLR 4), androglobin-like, store-operated Ca(2+) entry (SocE), ADP/ATP carrier protein, cytochrome b, thioesterase, actin, ferritin subunit 1, poly-ubiquitin, short-chain collagen C4-like and one hypothetical protein in gill cells were identified. This study suggests that the CfAhR played fundamental roles in immune system homeostasis, oxidative stress response, and in grow and development of C. farreri. The elucidation of these protein interactions is of much importance both in understanding the normal physiological function of AhR, and as potential targets for further research on protein function in AhR interactions. PMID:27497785

  9. Identification of interacting proteins with aryl hydrocarbon receptor in scallop Chlamys farreri by yeast two hybrid screening.

    PubMed

    Cai, Yuefeng; Pan, Luqing; Miao, Jingjing; Liu, Tong

    2016-11-01

    The aryl hydrocarbon receptor (AhR) belongs to the basic-helix-loop helix (bHLH) Per-Arnt-Sim (PAS) family of transcription factors. AhR has been known primarily for its role in the regulation of several drug and xenobiotic metabolizing enzymes, as well as the mediation of the toxicity of certain xenobiotics, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Although the AhR is well-studied as a mediator of the toxicity of certain xenobiotics in marine bivalves, the normal physiological function remains unknown. In order to explore the function of the AhR, the bait protein expression plasmid pGBKT7-CfAhR and the cDNA library of gill from Chlamys farreri were constructed. By yeast two hybrid system, after multiple screening with the high screening rate medium, rotary verification, sequencing and bioinformatics analysis, the interactions of the CfAhR with receptor for activated protein kinase C 1 (RACK1), thyroid peroxidase-like protein (TPO), Toll-like receptor 4(TLR 4), androglobin-like, store-operated Ca(2+) entry (SocE), ADP/ATP carrier protein, cytochrome b, thioesterase, actin, ferritin subunit 1, poly-ubiquitin, short-chain collagen C4-like and one hypothetical protein in gill cells were identified. This study suggests that the CfAhR played fundamental roles in immune system homeostasis, oxidative stress response, and in grow and development of C. farreri. The elucidation of these protein interactions is of much importance both in understanding the normal physiological function of AhR, and as potential targets for further research on protein function in AhR interactions.

  10. Short communication: Identification and technological characterization of yeast strains isolated from samples of water buffalo Mozzarella cheese.

    PubMed

    Aponte, M; Pepe, O; Blaiotta, G

    2010-06-01

    Sixty yeast cultures were isolated from samples of water buffalo Mozzarella, a popular "pasta filata" cheese, originating on 16 farms located in the provinces of Salerno, Caserta, and Frosinone (Italy). Strains were identified by means of 5.8S internal transcribed spacer rDNA PCR-RFLP combined with 26S rRNA gene partial sequencing and characterized for their ability to exert biochemical properties of technological interest. The recorded dominance of fermenting yeasts such as the lactose-fermenting Kluyveromyces marxianus (38.3% of the total isolates) and the galactose-fermenting Saccharomyces cerevisiae (21.6% of the total isolates) suggests that these yeasts contribute to the organoleptic definition of the water buffalo Mozzarella. The speciographic analysis revealed the presence of 7 other species rarely or never reported in a dairy environment belonging to the genera Pichia and Candida, whose role in Mozzarella cheese organoleptic properties need to be further investigated.

  11. Mammalian ribosomal and chaperone protein RPS3A counteracts α-synuclein aggregation and toxicity in a yeast model system.

    PubMed

    De Graeve, Stijn; Marinelli, Sarah; Stolz, Frank; Hendrix, Jelle; Vandamme, Jurgen; Engelborghs, Yves; Van Dijck, Patrick; Thevelein, Johan M

    2013-11-01

    Accumulation of aggregated forms of αSyn (α-synuclein) into Lewy bodies is a known hallmark associated with neuronal cell death in Parkinson's disease. When expressed in the yeast Saccharomyces cerevisiae, αSyn interacts with the plasma membrane, forms inclusions and causes a concentration-dependent growth defect. We have used a yeast mutant, cog6Δ, which is particularly sensitive to moderate αSyn expression, for screening a mouse brain-specific cDNA library in order to identify mammalian proteins that counteract αSyn toxicity. The mouse ribosomal and chaperone protein RPS3A was identified as a suppressor of αSyn [WT (wild-type) and A53T] toxicity in yeast. We demonstrated that the 50 N-terminal amino acids are essential for this function. The yeast homologues of RPS3A were not effective in suppressing the αSyn-induced growth defect, illustrating the potential of our screening system to identify modifiers that would be missed using yeast gene overexpression as the first screening step. Co-expression of mouse RPS3A delayed the formation of αSyn-GFP inclusions in the yeast cells. The results of the present study suggest that the recently identified extraribosomal chaperonin function of RPS3A also acts on the neurodegeneration-related protein αSyn and reveal a new avenue for identifying promising candidate mammalian proteins involved in αSyn functioning.

  12. Automated Firearms Identification System (AFIDS), phase 1

    NASA Technical Reports Server (NTRS)

    Blackwell, R. J.; Framan, E. P.

    1974-01-01

    Items critical to the future development of an automated firearms identification system (AFIDS) have been examined, with the following specific results: (1) Types of objective data, that can be utilized to help establish a more factual basis for determining identity and nonidentity between pairs of fired bullets, have been identified. (2) A simulation study has indicated that randomly produced lines, similar in nature to the individual striations on a fired bullet, can be modeled and that random sequences, when compared to each other, have predictable relationships. (3) A schematic diagram of the general concept for AFIDS has been developed and individual elements of this system have been briefly tested for feasibility. Future implementation of such a proposed system will depend on such factors as speed, utility, projected total cost and user requirements for growth. The success of the proposed system, when operational, would depend heavily on existing firearms examiners.

  13. Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

    PubMed

    Shin, John J; Aftab, Qurratulain; Austin, Pamela; McQueen, Jennifer A; Poon, Tak; Li, Shu Chen; Young, Barry P; Roskelley, Calvin D; Loewen, Christopher J R

    2016-09-01

    A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain

  14. Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets

    PubMed Central

    Shin, John J.; Aftab, Qurratulain; Austin, Pamela; McQueen, Jennifer A.; Poon, Tak; Li, Shu Chen; Young, Barry P.; Roskelley, Calvin D.

    2016-01-01

    ABSTRACT A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C–COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial

  15. Genomics and the making of yeast biodiversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces ...

  16. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    PubMed

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.

  17. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System.

    PubMed

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

  18. Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

    PubMed Central

    Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

    2015-01-01

    Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

  19. An Efficient Genome-Wide Fusion Partner Screening System for Secretion of Recombinant Proteins in Yeast

    PubMed Central

    Bae, Jung-Hoon; Hyun Sung, Bong; Kim, Hyun-Jin; Park, Soon-Ho; Lim, Kwang-Mook; Kim, Mi-Jin; Lee, Cho-Ryong; Sohn, Jung-Hoon

    2015-01-01

    To produce rarely secreted recombinant proteins in the yeast Saccharomyces cerevisiae, we developed a novel genome-wide optimal translational fusion partner (TFP) screening system that involves recruitment of an optimal secretion signal and fusion partner. A TFP library was constructed from a genomic and truncated cDNA library by using the invertase-based signal sequence trap technique. The efficiency of the system was demonstrated using two rarely secreted proteins, human interleukin (hIL)-2 and hIL-32. Optimal TFPs for secretion of hIL-2 and hIL-32 were easily selected, yielding secretion of these proteins up to hundreds of mg/L. Moreover, numerous uncovered yeast secretion signals and fusion partners were identified, leading to efficient secretion of various recombinant proteins. Selected TFPs were found to be useful for the hypersecretion of other recombinant proteins at yields of up to several g/L. This screening technique could provide new methods for the production of various types of difficult-to-express proteins. PMID:26195161

  20. High-throughput fluorescence screening assay for the identification and comparison of antimicrobial peptides' activity on various yeast species.

    PubMed

    Kodedová, Marie; Sychrová, Hana

    2016-09-10

    New antifungal compounds that circumvent the resistance of the pathogen by directly damaging yeast cell surface structures are promising agents for the treatment of fungal infections, due to their different mechanism of action from current clinically used antifungal drugs. We present here a rapid and cost-effective fluorescence method suitable for identifying new potent drugs that directly target yeast cell surface structures, causing cell permeabilization and thus bypassing the multidrug resistance mechanisms of pathogens. The fluorescence assay enabled us to detect with high sensitivity damage to the Candida plasma membrane (its hyperpolarization and permeabilization) as a result of short-term exposure to the antifungal compounds. Results can be obtained in 1-2h with minimal effort and consumption of the tested compounds, also 96 samples can be analysed simultaneously. We used this method to study antimicrobial peptides isolated from the venom of bees and their synthetic analogs, compare the potency of the peptides and determine their minimal effective concentrations. The antimicrobial peptides were able to kill yeast cells at low concentrations within a 15-min treatment, the LL-III peptide exhibited a broad spectrum of antifungal activity on various Saccharomyces, pathogenic Candida and osmotolerant yeast species. PMID:27369550

  1. Saccharomyces cerivisiae as a model system for kidney disease: what can yeast tell us about renal function?

    PubMed

    Kolb, Alexander R; Buck, Teresa M; Brodsky, Jeffrey L

    2011-07-01

    Ion channels, solute transporters, aquaporins, and factors required for signal transduction are vital for kidney function. Because mutations in these proteins or in associated regulatory factors can lead to disease, an investigation into their biogenesis, activities, and interplay with other proteins is essential. To this end, the yeast, Saccharomyces cerevisiae, represents a powerful experimental system. Proteins expressed in yeast include the following: 1) ion channels, including the epithelial sodium channel, members of the inward rectifying potassium channel family, and cystic fibrosis transmembrane conductance regulator; 2) plasma membrane transporters, such as the Na(+)-K(+)-ATPase, the Na(+)-phosphate cotransporter, and the Na(+)-H(+) ATPase; 3) aquaporins 1-4; and 4) proteins such as serum/glucocorticoid-induced kinase 1, phosphoinositide-dependent kinase 1, Rh glycoprotein kidney, and trehalase. The variety of proteins expressed and studied emphasizes the versatility of yeast, and, because of the many available tools in this organism, results can be obtained rapidly and economically. In most cases, data gathered using yeast have been substantiated in higher cell types. These attributes validate yeast as a model system to explore renal physiology and suggest that research initiated using this system may lead to novel therapeutics.

  2. Identification of a small molecule yeast TORC1 inhibitor with a flow cytometry-based multiplex screen

    PubMed Central

    Chen, Jun; Young, Susan M.; Allen, Chris; Seeber, Andrew; Péli-Gulli, Marie-Pierre; Panchaud, Nicolas; Waller, Anna; Ursu, Oleg; Yao, Tuanli; Golden, Jennifer E.; Strouse, J. Jacob; Carter, Mark B.; Kang, Huining; Bologa, Cristian G.; Foutz, Terry D.; Edwards, Bruce S.; Peterson, Blake R.; Aubé, Jeffrey; Werner-Washburne, Margaret; Loewith, Robbie J.; De Virgilio, Claudio; Sklar, Larry A.

    2012-01-01

    TOR (target of rapamycin) is a serine/threonine kinase, evolutionarily conserved from yeast to human, which functions as a fundamental controller of cell growth. The moderate clinical benefit of rapamycin in mTOR-based therapy of many cancers favors the development of new TOR inhibitors. Here we report a high throughput flow cytometry multiplexed screen using five GFP-tagged yeast clones that represent the readouts of four branches of the TORC1 signaling pathway in budding yeast. Each GFP-tagged clone was differentially color-coded and the GFP signal of each clone was measured simultaneously by flow cytometry, which allows rapid prioritization of compounds that likely act through direct modulation of TORC1 or proximal signaling components. A total of 255 compounds were confirmed in dose-response analysis to alter GFP expression in one or more clones. To validate the concept of the high throughput screen, we have characterized CID 3528206, a small molecule most likely to act on TORC1 as it alters GFP expression in all five GFP clones in an analogous manner to rapamycin. We have shown that CID 3528206 inhibited yeast cell growth, and that CID 3528206 inhibited TORC1 activity both in vitro and in vivo with EC50s of 150 nM and 3.9 μM, respectively. The results of microarray analysis and yeast GFP collection screen further support the notion that CID 3528206 and rapamycin modulate similar cellular pathways. Together, these results indicate that the HTS has identified a potentially useful small molecule for further development of TOR inhibitors. PMID:22260433

  3. Autonomous Frequency-Domain System-Identification Program

    NASA Technical Reports Server (NTRS)

    Yam, Yeung; Mettler, Edward; Bayard, David S.; Hadaegh, Fred Y.; Milman, Mark H.; Scheid, Robert E.

    1993-01-01

    Autonomous Frequency Domain Identification (AU-FREDI) computer program implements system of methods, algorithms, and software developed for identification of parameters of mathematical models of dynamics of flexible structures and characterization, by use of system transfer functions, of such models, dynamics, and structures regarded as systems. Software considered collection of routines modified and reassembled to suit system-identification and control experiments on large flexible structures.

  4. Modulating the potency of an activator in a yeast in vitro transcription system.

    PubMed Central

    Ohashi, Y; Brickman, J M; Furman, E; Middleton, B; Carey, M

    1994-01-01

    The intrinsic stimulatory potential or potency of a eukaryotic gene activator is controlled by the interaction between the activation domain and the transcriptional machinery. To further understand this interaction, we undertook a biochemical study to identify parameters that could be used to modulate activator potency. We considered how varying the number of activation domains, their flexibility, and the number of promoter sites affects potency in a yeast nuclear extract. The effects of GAL4 derivatives bearing either one, two, or four herpes simplex virus VP16 activation domains (amino acids 413 to 454) were measured on DNA templates containing one or two GAL4 sites in a Saccharomyces cerevisiae nuclear extract. We found that multimerized VP16 activation domains acted synergistically to increase the potency of the activators. The spacing between the activation domains was critical, such that the increased flexibility imparted by a protein linker contributed to increased activator potency. With highly potent activators, the levels of transcription stimulated on a single site were saturating, whereas the stimulatory effect of weaker activators increased with the number of sites. We discuss how these biochemical studies relate to the mechanism of gene activation and synergy in a yeast in vitro system. Images PMID:8139572

  5. Industrial systems biology and its impact on synthetic biology of yeast cell factories.

    PubMed

    Fletcher, Eugene; Krivoruchko, Anastasia; Nielsen, Jens

    2016-06-01

    Engineering industrial cell factories to effectively yield a desired product while dealing with industrially relevant stresses is usually the most challenging step in the development of industrial production of chemicals using microbial fermentation processes. Using synthetic biology tools, microbial cell factories such as Saccharomyces cerevisiae can be engineered to express synthetic pathways for the production of fuels, biopharmaceuticals, fragrances, and food flavors. However, directing fluxes through these synthetic pathways towards the desired product can be demanding due to complex regulation or poor gene expression. Systems biology, which applies computational tools and mathematical modeling to understand complex biological networks, can be used to guide synthetic biology design. Here, we present our perspective on how systems biology can impact synthetic biology towards the goal of developing improved yeast cell factories. Biotechnol. Bioeng. 2016;113: 1164-1170. © 2015 Wiley Periodicals, Inc.

  6. Screening of APP interaction proteins by DUALmembrane yeast two-hybrid system

    PubMed Central

    Yu, You; Li, Yinan; Zhang, Yan

    2015-01-01

    Alzheimer’s disease (AD) is one of the most common forms of neurodegenerative disease. There is a growing interest in the amyloid precursor protein (APP) over the years due to its involvement in AD. Besides its role in pathological mechanisms of AD, APP participates in many signaling pathways as well. APP functions through protein-protein interactions, and in this report staufen 1 (STAU1) is demonstrated to have interaction with APP, using yeast two-hybrid screening and co-immunoprecipitation in mammalian system. STAU1 belongs to the double-stranded RNA binding protein family and can mediate mRNA degradation in mammalian system, implicating that APP may be involved in the regulation of mRNA as well. PMID:26045787

  7. System Identification of X-33 Neural Network

    NASA Technical Reports Server (NTRS)

    Aggarwal, Shiv

    2003-01-01

    Modern flight control research has improved spacecraft survivability as its goal. To this end we need to have a failure detection system on board. In case the spacecraft is performing imperfectly, reconfiguration of control is needed. For that purpose we need to have parameter identification of spacecraft dynamics. Parameter identification of a system is called system identification. We treat the system as a black box which receives some inputs that lead to some outputs. The question is: what kind of parameters for a particular black box can correlate the observed inputs and outputs? Can these parameters help us to predict the outputs for a new given set of inputs? This is the basic problem of system identification. The X33 was supposed to have the onboard capability of evaluating the current performance and if needed to take the corrective measures to adapt to desired performance. The X33 is comprised of both rocket and aircraft vehicle design characteristics and requires, in general, analytical methods for evaluating its flight performance. Its flight consists of four phases: ascent, transition, entry and TAEM (Terminal Area Energy Management). It spends about 200 seconds in ascent phase, reaching an altitude of about 180,000 feet and a speed of about 10 to 15 Mach. During the transition phase which lasts only about 30 seconds, its altitude may increase to about 190,000 feet but its speed is reduced to about 9 Mach. At the beginning of this phase, the Main Engine is Cut Off (MECO) and the control is reconfigured with the help of aerosurfaces (four elevons, two flaps and two rudders) and reaction control system (RCS). The entry phase brings down the altitude of X33 to about 90,000 feet and its speed to about Mach 3. It spends about 250 seconds in this phase. Main engine is still cut off and the vehicle is controlled by complex maneuvers of aerosurfaces. The last phase TAEM lasts for about 450 seconds and the altitude and speed, both are reduced to zero. The

  8. Usefulness of CHROMagar Candida Medium, Biochemical Methods--API ID32C and VITEK 2 Compact and Two MALDI-TOF MS Systems for Candida spp. Identification.

    PubMed

    Stefaniuk, Elzbieta; Baraniak, Anna; Fortuna, Monika; Hryniewicz, Waleria

    2016-01-01

    This study was conducted to compare of the yeasts identification results obtained with two new systems using the MALDI-TOF MS technique with the ones obtained using the routine identification methods of Candida spp. in clinical microbiology laboratories. All 124 Candida spp. isolates were recovered from the routine examination of clinical specimens in microbiological laboratories and collected in the Centre of Quality Control in Microbiology in Warsaw (Poland). Our findings confirm the high agreement (98%) of fungal identification using the standard, biochemistry laboratory methods and mass spectrometry technique. PMID:27282002

  9. System Identification of a Vortex Lattice Aerodynamic Model

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Kholodar, Denis; Dowell, Earl H.

    2001-01-01

    The state-space presentation of an aerodynamic vortex model is considered from a classical and system identification perspective. Using an aerodynamic vortex model as a numerical simulator of a wing tunnel experiment, both full state and limited state data or measurements are considered. Two possible approaches for system identification are presented and modal controllability and observability are also considered. The theory then is applied to the system identification of a flow over an aerodynamic delta wing and typical results are presented.

  10. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein.

    PubMed

    Vojtek, A B; Cooper, J A

    1993-07-01

    CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

  11. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    PubMed

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. PMID:27480687

  12. In-silico identification and characterization of organic and inorganic chemical stress responding genes in yeast (Saccharomyces cerevisiae).

    PubMed

    Barozai, Muhammad Younas Khan; Bashir, Farrukh; Muzaffar, Shafia; Afzal, Saba; Behlil, Farida; Khan, Muzaffar

    2014-10-15

    To study the life processes of all eukaryotes, yeast (Saccharomyces cerevisiae) is a significant model organism. It is also one of the best models to study the responses of genes at transcriptional level. In a living organism, gene expression is changed by chemical stresses. The genes that give response to chemical stresses will provide good source for the strategies in engineering and formulating mechanisms which are chemical stress resistant in the eukaryotic organisms. The data available through microarray under the chemical stresses like lithium chloride, lactic acid, weak organic acids and tomatidine were studied by using computational tools. Out of 9335 yeast genes, 388 chemical stress responding genes were identified and characterized under different chemical stresses. Some of these are: Enolases 1 and 2, heat shock protein-82, Yeast Elongation Factor 3, Beta Glucanase Protein, Histone H2A1 and Histone H2A2 Proteins, Benign Prostatic Hyperplasia, ras GTPase activating protein, Establishes Silent Chromatin protein, Mei5 Protein, Nondisjunction Protein and Specific Mitogen Activated Protein Kinase. Characterization of these genes was also made on the basis of their molecular functions, biological processes and cellular components.

  13. Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica.

    PubMed

    Morita, Tomotake; Ito, Emi; Kitamoto, Hiroko K; Takegawa, Kaoru; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

    2010-11-01

    The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surface-active properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA_004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA_004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained ΔPaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed ΔPaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast. PMID:20564650

  14. Identification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein

    PubMed Central

    1988-01-01

    We have produced monoclonal antibodies against purified nuclei from the yeast Saccharomyces cerevisiae and have characterized three different antibodies that recognize a protein with an apparent molecular weight of 38,000, termed p38. Subcellular fractionation shows that virtually all of p38 occurs in the nuclear fraction. High concentrations of salt (1 M) or urea (6 M) effectively solubilize p38 from a nuclear envelope fraction prepared by digestion of nuclei with DNase. Indirect immunofluorescence demonstrates a crescent shaped distribution of p38 at the inner periphery of the nucleus, with p38 extending between dividing pairs of cells during (closed) mitosis. Postembedding immunogold electron microscopy shows decoration of the densely stained "crescent" region of the yeast nucleus, confirming the localization of p38 to the nucleolus. One of the monoclonals, D77, cross reacts on immunoblots with a single protein of molecular weight 37,000 from purified rat liver nuclei. Indirect immunofluorescence localizes this protein to the nucleolus, and shows that it is dispersed throughout the cell during mitosis. The yeast and rat liver nucleolar proteins behave similarly when electrophoresed in two dimensions, and appear to have basic pI values. Analysis of immunological cross-reactivity using D77, and antibodies specific for nucleolar proteins from other sources, suggests that the rat liver protein is fibrillarin, and demonstrates that p38 shares epitopes with fibrillarin, as well as with other vertebrate nucleolar proteins. PMID:3292539

  15. Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

    PubMed

    Röder, Christoph; König, Helmut; Fröhlich, Jürgen

    2007-09-01

    Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples. PMID:17596183

  16. Inactivation of yeast and filamentous fungi by the lactoperoxidase-hydrogen peroxide-thiocyanate-system.

    PubMed

    Popper, L; Knorr, D

    1997-02-01

    The antifungal activity of the lactoperoxidase (LPO) system with glucose oxidase (GOD) as source of hydrogen peroxide was determined in salt solution and in apple juice. The test organisms Rhodutorula rubra and Saccharomyces cerevisiae were cultivated aerobically in apple juice, Mucor rouxii was grown on wort agar adjusted to pH 4.5. Aspergillus niger and Byssochlamys fulva were kept on malt extract agar. Spores of the filamentous fungi were harvested by suspension in salt solution supplemented with Tween 80 and checked microscopically. The antifungal activity of the combined enzyme system was tested with initial counts of approx. 10(5) cfu.ml-1 (yeast cells or spores) suspended in salt solution supplemented with 25 mg.l-1 thiocyanate and 20 g.l-1 glucose or in apple juice supplemented with the same amount of thiocyanate. The tests were performed with 25 ml of the medium in 100 ml Erlenmeyer flasks shaken at 28 degrees C under aerobic conditions. Inactivation was achieved for all test organisms in both media. The yeast strains were found to be least stable while B. fulva was most resistant. A combination of 5 U.ml-1 LPO with 0.5 to 1 U.ml-1 GOD was sufficient for complete inactivation of this mold in salt solution within 2 h. The enzyme system also showed antifungal activity in apple juice at acid pH (3.2), although its effectiveness was reduced. In this medium, B. fulva was inactivated by 20 U.ml-1 LPD and 1 U.ml-1 GOD within 4 h. R. rubra and S. cerevisfiae were unable to survive in apple juice at 5 U.ml-1 LPO combined with 1 U.ml-1 GOD. For inhibition by GOD alone, higher amounts of this enzyme were needed and even then only M. rouxii and R. rubra have been affected within the concentration range tested (maximum 3 U.ml-1).

  17. Nonlinear identification of ionic polymer actuator systems

    NASA Astrophysics Data System (ADS)

    Kothera, Curt S.; Lacy, Seth L.; Erwin, R. Scott; Leo, Donald J.

    2004-07-01

    Ionic polymers are a class of electromechanically coupled materials that can be used as flexible transducers. When set up in the cantilever configuration, the actuators exhibit a large bending deflection when an electric field is applied across their thickness. Being a relatively new research topic, the governing physical and chemical mechanisms are not yet fully understood. Experimental results have demonstrated nonlinear dynamic behavior. The nonlinear dynamics can be seen in the response of current, displacement, and velocity of the actuator. This work presents results for the nonlinear identification of ionic polymer actuator systems driven at a specific frequency. Identification results using a 5th-degree Volterra expansion show that the nonlinear distortion can be accurately modeled. Using such a high power in the series expansion is necessary to capture the most dominant harmonics, as evidenced when examining the power spectral density of the response. An investigation of how nonlinearities enter into the response is also performed. By analyzing both the actuation current and tip velocity, results show that both the voltage to current and current to velocity stages influence the nonlinear response, but the voltage to current stage is more dominantly nonlinear.

  18. Fractional System Identification: An Approach Using Continuous Order-Distributions

    NASA Technical Reports Server (NTRS)

    Hartley, Tom T.; Lorenzo, Carl F.

    1999-01-01

    This paper discusses the identification of fractional- and integer-order systems using the concept of continuous order-distribution. Based on the ability to define systems using continuous order-distributions, it is shown that frequency domain system identification can be performed using least squares techniques after discretizing the order-distribution.

  19. Apoptosis in yeast: a new model system with applications in cell biology and medicine.

    PubMed

    Madeo, Frank; Engelhardt, Silvia; Herker, Eva; Lehmann, Nina; Maldener, Corinna; Proksch, Astrid; Wissing, Silke; Fröhlich, Kai-Uwe

    2002-07-01

    Apoptosis is a highly coordinated cellular suicide program crucial for metazoan health and diseases. Although its increasing importance in cancer, neurodegenerative disorders and AIDS led to intense research and a better understanding of apoptosis, many details of its regulation or the apoptotic phenotypes are poorly understood. The complex regulatory network and the often contradictory results obtained with human cell lines made application of an easier model system desirable. Apoptosis in yeast promises to provide a better understanding of the genetics of apoptosis. During the past 2 years, scientists were successful in identifying new cell-death regulators of humans, plants and fungi using Saccharomyces cerevisiae. The finding of apoptotic phenotypes, even in protists, suggests that apoptosis developed in unicellular organisms long before the evolutionary separation between fungi, plants and metazoan animals occurred.

  20. The yeast model system as a tool towards the understanding of apoptosis regulation by sphingolipids.

    PubMed

    Rego, António; Trindade, Dário; Chaves, Susana R; Manon, Stéphen; Costa, Vítor; Sousa, Maria João; Côrte-Real, Manuela

    2014-02-01

    It has been established that sphingolipids are engaged in the regulation of apoptosis both as direct executors and as signalling molecules. However, the peculiarities of this class of bioactive lipids, namely the interconnectivity of their metabolic pathways, the specific subcellular localization where they are generated and the transport mechanisms involved, introduce a considerably high level of complexity in deciphering their role in the signalling and regulation of programmed cell death. Although yeast is undeniably a simple model, the conservation of the sphingolipid metabolism and of the core machinery engaged in regulated cell death has already provided valuable clues to the understanding of metabolic pathways involved in distinct cellular processes, including apoptosis. It can be anticipated that studies using this model system will further unravel mechanisms underlying the regulation of apoptosis by sphingolipids and contribute to novel therapeutic strategies against serious human diseases associated with dysfunction of sphingolipid-dependent cell death programmes. PMID:24103214

  1. MEMS-based flow cytometry: microfluidics-based cell identification system by fluorescent imaging.

    PubMed

    Wu, W K; Liang, C K; Huang, J Z

    2004-01-01

    This study utilizes MEMS technology to realize a novel low-cost microfluidics-based biochip system for flow-type cell handling. Powered by vacuum pump, the microfluidic driving system enables cells to move in order one by one in the biochip by an effect of sheath flow prefocus. Then, cells are guided to a fluorescent inspection region where two detection tasks such as cell image identification and cell counting are conducted. Currently, the glass-based biochip has been manufactured and all the related devices have been well set up in our laboratory. With this proposed prototype system, typical results about cell separation of yeast cell and PC-3 cell are available and their separated images are also presented, respectively. PMID:17270801

  2. Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system.

    PubMed

    Wolf, Sylvi; Diel, Patrick; Parr, Maria Kristina; Rataj, Felicitas; Schänzer, Willhelm; Vollmer, Günter; Zierau, Oliver

    2011-04-01

    The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0-307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

  3. Thermal Signature Identification System (TheSIS)

    NASA Technical Reports Server (NTRS)

    Merritt, Scott; Bean, Brian

    2015-01-01

    We characterize both nonlinear and high order linear responses of fiber-optic and optoelectronic components using spread spectrum temperature cycling methods. This Thermal Signature Identification System (TheSIS) provides much more detail than conventional narrowband or quasi-static temperature profiling methods. This detail allows us to match components more thoroughly, detect subtle reversible shifts in performance, and investigate the cause of instabilities or irreversible changes. In particular, we create parameterized models of athermal fiber Bragg gratings (FBGs), delay line interferometers (DLIs), and distributed feedback (DFB) lasers, then subject the alternative models to selection via the Akaike Information Criterion (AIC). Detailed pairing of components, e.g. FBGs, is accomplished by means of weighted distance metrics or norms, rather than on the basis of a single parameter, such as center wavelength.

  4. Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non-homologous end joining-mediated integrative transformation with genes from Saccharomyces cerevisiae.

    PubMed

    Yarimizu, Tohru; Nonklang, Sanom; Nakamura, Junpei; Tokuda, Shuya; Nakagawa, Takaaki; Lorreungsil, Sasithorn; Sutthikhumpha, Surasit; Pukahuta, Charida; Kitagawa, Takao; Nakamura, Mikiko; Cha-Aim, Kamonchai; Limtong, Savitree; Hoshida, Hisashi; Akada, Rinji

    2013-12-01

    The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non-homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR-amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour-intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ-mediated integrative transformation with PCR-amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus.

  5. Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

    PubMed

    Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

    2016-04-01

    Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. PMID

  6. Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes

    PubMed Central

    2004-01-01

    Morphological analysis of a conditional yeast mutant in acetyl-CoA carboxylase acc1ts/mtr7, the rate-limiting enzyme of fatty acid synthesis, suggested that the synthesis of C26 VLCFAs (very-long-chain fatty acids) is important for maintaining the structure and function of the nuclear membrane. To characterize this C26-dependent pathway in more detail, we have now examined cells that are blocked in pathways that require C26. In yeast, ceramide synthesis and remodelling of GPI (glycosylphosphatidylinositol)-anchors are two pathways that incorporate C26 into lipids. Conditional mutants blocked in either ceramide synthesis or the synthesis of GPI anchors do not display the characteristic alterations of the nuclear envelope observed in acc1ts, indicating that the synthesis of another C26-containing lipid may be affected in acc1ts mutant cells. Lipid analysis of isolated nuclear membranes revealed the presence of a novel C26-substituted PI (phosphatidylinositol). This C26-PI accounts for approx. 1% of all the PI species, and is present in both the nuclear and the plasma membrane. Remarkably, this C26-PI is the only C26-containing glycerophospholipid that is detectable in wild-type yeast, and the C26-substitution is highly specific for the sn-1 position of the glycerol backbone. To characterize the biophysical properties of this lipid, it was chemically synthesized. In contrast to PIs with normal long-chain fatty acids (C16 or C18), the C26-PI greatly reduced the bilayer to hexagonal phase transition of liposomes composed of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE). The biophysical properties of this lipid are thus consistent with a possible role in stabilizing highly curved membrane domains. PMID:15270698

  7. Lightweight autonomous chemical identification system (LACIS)

    NASA Astrophysics Data System (ADS)

    Lozos, George; Lin, Hai; Burch, Timothy

    2012-06-01

    Smiths Detection and Intelligent Optical Systems have developed prototypes for the Lightweight Autonomous Chemical Identification System (LACIS) for the US Department of Homeland Security. LACIS is to be a handheld detection system for Chemical Warfare Agents (CWAs) and Toxic Industrial Chemicals (TICs). LACIS is designed to have a low limit of detection and rapid response time for use by emergency responders and could allow determination of areas having dangerous concentration levels and if protective garments will be required. Procedures for protection of responders from hazardous materials incidents require the use of protective equipment until such time as the hazard can be assessed. Such accurate analysis can accelerate operations and increase effectiveness. LACIS is to be an improved point detector employing novel CBRNE detection modalities that includes a militaryproven ruggedized ion mobility spectrometer (IMS) with an array of electro-resistive sensors to extend the range of chemical threats detected in a single device. It uses a novel sensor data fusion and threat classification architecture to interpret the independent sensor responses and provide robust detection at low levels in complex backgrounds with minimal false alarms. The performance of LACIS prototypes have been characterized in independent third party laboratory tests at the Battelle Memorial Institute (BMI, Columbus, OH) and indoor and outdoor field tests at the Nevada National Security Site (NNSS). LACIS prototypes will be entering operational assessment by key government emergency response groups to determine its capabilities versus requirements.

  8. Interaction of a mixed yeast culture in an ``autotroph-heterotroph'' system with a closed atmosphere cycle and spatially separated components

    NASA Astrophysics Data System (ADS)

    Pisman, T. I.; Somova, L. A.

    The study considers an experimental model of the "autotroph-heterotroph" system with a closed atmosphere cycle, in which the heterotrophic link is a mixed yeast population. The autotrophic link is represented by the algae Chlorella vulgaris and the heterotrophic link by the yeasts Candida utilis and Candida guilliermondii. The controls are populations of Chlorella and the same yeasts isolated from the atmosphere. It has been shown that the outcome of competition in the heterotrophic link depends on the strategy of the yeast population towards the substrate and oxygen. The C. utilis population quickly utilizes the substrate as it is an r-strategist and is less sensitive to oxygen deficiency. The C. guilliermondii population consumes low concentrations of the substrate because it is a K-strategist, but it is more sensitive to oxygen deficiency. That is why, in the "autotroph-heterotroph" system with a closed gas cycle, after a considerable amount of the substrate has been consumed, the C. guilliermondii population becomes more competitive that the C. utilis population. In the culture of yeasts, isolated from the atmosphere, the C. utilis population finds itself in more favorable conditions due to oxygen deficiency. The system with a complex heterotrophic component survive longer than a system whose heterotrophic component is represented by only one yeast species. This is explained for by the positive metabolite interaction of yeasts and a more complete utilization of the substrate by a mixed culture of yeasts featuring different strategies towards the substrate.

  9. Interaction of the mixed yeast culture in the autotroph-heterotroph system with a closed gas cycle and spatially separated components

    NASA Astrophysics Data System (ADS)

    Pisman, T.; Somova, L.

    The study considers the experimental model of the "autotroph-heterotroph" system with a closed gas cycle, in which the heterotrophic link is a mixed yeast population. The autotrophic link is represented by the algae Chlorella vulgaris and the heterotrophic link by the yeasts Candida utilis and Candida guilliermondii. The controls are separate links of Chlorella and yeasts isolated from the atmosphere. It has been shown that the outcome of the competition in the heterotrophic link depends on the strategy of the yeast population towards the substrate and oxygen. The C. utilis population quickly utilizes the substrate as it is an R-strategist and is less sensitive to oxygen deficiency. The C. guilliermondii population consumes low concentrations of the substrate because it is a K-strategist, but it is more sensitive to oxygen deficiency. That is why, in the "autotroph-heterotroph" system with a closed gas cycle, after a considerable amount of the substrate has been consumed, the C. guilliermondii population becomes more competitive that the C. utilis population. In the culture of a separate yeast link, isolated from the atmosphere, the C. utilis population finds itself in more favorable conditions due to oxygen deficiency. The system with a complex heterotrophic component exists longer than the system whose heterotrophic component is represented by one yeast species. This is accounted for by the positive metabolite interaction of yeasts and a more complete utilization of the substrate by a mixed culture of yeasts featuring different strategies towards the substrate.

  10. Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System.

    PubMed

    Hudson, Lauren E; McDermott, Courtney D; Stewart, Taryn P; Hudson, William H; Rios, Daniel; Fasken, Milo B; Corbett, Anita H; Lamb, Tracey J

    2016-01-01

    The probiotic yeast Saccharomyces boulardii has been shown to ameliorate disease severity in the context of many infectious and inflammatory conditions. However, use of S. boulardii as a prophylactic agent or therapeutic delivery vector would require delivery of S. boulardii to a healthy, uninflamed intestine. In contrast to inflamed mucosal tissue, the diverse microbiota, intact epithelial barrier, and fewer inflammatory immune cells within the healthy intestine may all limit the degree to which S. boulardii contacts and influences the host mucosal immune system. Understanding the nature of these interactions is crucial for application of S. boulardii as a prophylactic agent or therapeutic delivery vehicle. In this study, we explore both intrinsic and immunomodulatory properties of S. boulardii in the healthy mucosal immune system. Genomic sequencing and morphological analysis of S. boulardii reveals changes in cell wall components compared to non-probiotic S. cerevisiae that may partially account for probiotic functions of S. boulardii. Flow cytometry and immunohistochemistry demonstrate limited S. boulardii association with murine Peyer's patches. We also show that although S. boulardii induces a systemic humoral immune response, this response is small in magnitude and not directed against S. boulardii itself. RNA-seq of the draining mesenteric lymph nodes indicates that even repeated administration of S. boulardii induces few transcriptional changes in the healthy intestine. Together these data strongly suggest that interaction between S. boulardii and the mucosal immune system in the healthy intestine is limited, with important implications for future work examining S. boulardii as a prophylactic agent and therapeutic delivery vehicle.

  11. Yeast mitochondrial glutathione is an essential antioxidant with mitochondrial thioredoxin providing a back-up system

    PubMed Central

    Gostimskaya, Irina; Grant, Chris M.

    2016-01-01

    Glutathione is an abundant, low-molecular-weight tripeptide whose biological importance is dependent upon its redox-active free sulphydryl moiety. Its role as the main determinant of thiol-redox control has been challenged such that it has been proposed to play a crucial role in iron–sulphur clusters maturation, and only a minor role in thiol redox regulation, predominantly as a back-up system for the cytoplasmic thioredoxin system. Here, we have tested the importance of mitochondrial glutathione in thiol-redox regulation. Glutathione reductase (Glr1) is an oxidoreductase which converts oxidized glutathione to its reduced form. Yeast Glr1 localizes to both the cytosol and mitochondria and we have used a Glr1M1L mutant that is constitutively localized to the cytosol to test the requirement for mitochondrial Glr1. We show that the loss of mitochondrial Glr1 specifically accounts for oxidant sensitivity of a glr1 mutant. Loss of mitochondrial Glr1 does not influence iron–sulphur cluster maturation and we have used targeted roGFP2 fluorescent probes to show that oxidant sensitivity is linked to an altered redox environment. Our data indicate mitochondrial glutathione is crucial for mitochondrial thiol-redox regulation, and the mitochondrial thioredoxin system provides a back-up system, but cannot bear the redox load of the mitochondria on its own. PMID:26898146

  12. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

  13. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  14. Identification of a putative alpha-glucan synthase essential for cell wall construction and morphogenesis in fission yeast

    PubMed Central

    Hochstenbach, Frans; Klis, Frans M.; van den Ende, Herman; van Donselaar, Elly; Peters, Peter J.; Klausner, Richard D.

    1998-01-01

    The cell wall protects fungi against lysis and determines their cell shape. Alpha-glucan is a major carbohydrate component of the fungal cell wall, but its function is unknown and its synthase has remained elusive. Here, we describe a fission yeast gene, ags1+, which encodes a putative alpha-glucan synthase. In contrast to the structure of other carbohydrate polymer synthases, the predicted Ags1 protein consists of two probable catalytic domains for alpha-glucan assembly, namely an intracellular domain for alpha-glucan synthesis and an extracellular domain speculated to cross-link or remodel alpha-glucan. In addition, the predicted Ags1 protein contains a multipass transmembrane domain that might contribute to transport of alpha-glucan across the membrane. Loss of Ags1p function in a temperature-sensitive mutant results in cell lysis, whereas mutant cells grown at the semipermissive temperature contain decreased levels of cell wall alpha-glucan and fail to maintain rod shapes, causing rounding of the cells. These findings demonstrate that alpha-glucan is essential for fission yeast morphogenesis. PMID:9689051

  15. Identification of yeast and acetic acid bacteria isolated from the fermentation and acetification of persimmon (Diospyros kaki).

    PubMed

    Hidalgo, C; Mateo, E; Mas, A; Torija, M J

    2012-05-01

    Persimmon (Diospyros kaki) is a seasonal fruit with important health benefits. In this study, persimmon use in wine and condiment production was investigated using molecular methods to identify the yeast and acetic acid bacteria (AAB) isolated from the alcoholic fermentation and acetification of the fruit. Alcoholic fermentation was allowed to occur either spontaneously, or by inoculation with a commercial Saccharomyces cerevisiae wine strain, while acetification was always spontaneous; all these processes were performed in triplicates. Non-Saccharomyces yeast species were particularly abundant during the initial and mid-alcoholic fermentation stages, but S. cerevisiae became dominant toward the end of these processes. During spontaneous fermentation, S. cerevisiae Sc1 was the predominant strain isolated throughout, while the commercial strain of S. cerevisiae was the most common strain isolated from the inoculated fermentations. The main non-Saccharomyces strains isolated included Pichia guilliermondii, Hanseniaspora uvarum, Zygosaccharomyces florentinus and Cryptococcus sp. A distinct succession of AAB was observed during the acetification process. Acetobacter malorun was abundant during the initial and mid-stages, while Gluconacetobacter saccharivorans was the main species during the final stages of these acetifications. Four additional AAB species, Acetobacter pasteurianus, Acetobacter syzygii, Gluconacetobacter intermedius and Gluconacetobacter europaeus, were also detected. We observed 28 different AAB genotypes, though only 6 of these were present in high numbers (between 25%-60%), resulting in a high biodiversity index.

  16. Emergent system identification using particle swarm optimization

    NASA Astrophysics Data System (ADS)

    Voss, Mark S.; Feng, Xin

    2001-10-01

    Complex Adaptive Structures can be viewed as a combination of Complex Adaptive Systems and fully integrated autonomous Smart Structures. Traditionally when designing a structure, one combines rules of thumb with theoretical results to develop an acceptable solution. This methodology will have to be extended for Complex Adaptive Structures, since they, by definition, will participate in their own design. In this paper we introduce a new methodology for Emergent System Identification that is concerned with combining the methodologies of self-organizing functional networks (GMDH - Alexy G. Ivakhnenko), Particle Swarm Optimization (PSO - James Kennedy and Russell C. Eberhart) and Genetic Programming (GP - John Koza). This paper will concentrate on the utilization of Particle Swarm Optimization in this effort and discuss how Particle Swarm Optimization relates to our ultimate goal of emergent self-organizing functional networks that can be used to identify overlapping internal structural models. The ability for Complex Adaptive Structures to identify emerging internal models will be a key component for their success.

  17. 49 CFR 1542.211 - Identification systems.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... secured area or SIDA continuously displays the identification medium issued to that individual on the... individual who has authorized unescorted access to secured areas and SIDA's to ascertain the authority of any... approved identification media. The procedure must— (1) Apply uniformly in secured areas, SIDAs,...

  18. 49 CFR 1542.211 - Identification systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... secured area or SIDA continuously displays the identification medium issued to that individual on the... individual who has authorized unescorted access to secured areas and SIDA's to ascertain the authority of any... approved identification media. The procedure must— (1) Apply uniformly in secured areas, SIDAs,...

  19. Molecular structure of yeast RNA polymerase III: demonstration of the tripartite transcriptive system in lower eukaryotes.

    PubMed Central

    Valenzuela, P; Hager, G L; Weinberg, F; Rutter, W J

    1976-01-01

    Homogeneous RNA polymerase III (RNA nucleotidyltransferase III) has been obtained from yeast. The subunit composition of the enzyme was examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme is composed of 12 putative subunits with molecular weights 160,000, 128,000, 82,000, 41,000, 40,500, 37,000, 34,000, 28,000, 24,000, 20,000, 14,500, and 11,000. The high-molecular-weight subunits and several of the smaller subunits of yeast RNA polymerase III are clearly different from those of enzymes I and II, indicating a distinct molecular structure. However, the molecular weights of some of the small subunits (41,000, 28,000, 24,000, and 14,500) appear to be identical to those of polymerases I and II. Thus, it is possible that the three classes of enzymes in yeast have some common subunits. As in other eukaryotes, yeast polymerase II is inhibited by relatively low concentrations of alpha-amanitin; however, contrary to what has been found in higher eukaryotes, yeast polymerase III is resistant (up to 2 mg/ml) to alpha-amanitin, while yeast polymerase I is sensitive to high concentrations of the drug (50% inhibition at 0.3 mg/ml). These results establish the existence of RNA polymerase III in yeast and provide a structural basis for the discrimination of the three functional polymerases in eukaryotes. Images PMID:772675

  20. Resolving the network of cell signaling pathways using the evolving yeast two-hybrid system

    PubMed Central

    Ratushny, Vladimir; Golemis, Erica A.

    2008-01-01

    In 1983, while investigators had identified a few human proteins as important regulators of specific biological outcomes, how these proteins acted in the cell was essentially unknown in almost all cases. 25 years on, our knowledge of the mechanistic basis of protein action has been transformed by our increasingly detailed understanding of protein-protein interactions, which have allowed us to define cellular machines. The advent of the yeast two-hybrid (Y2H) system in 1989 marked a milestone in the field of proteomics. Exploiting the modular nature of transcription factors, the Y2H system allows facile measurement of the activation of reporter genes based on interactions between two chimeric or “hybrid” proteins of interest. After a decade of service as a leading platforms for individual investigators to use in exploring the interaction properties of interesting target proteins, the Y2H system has increasing been applied in high throughput applications intended to map genome-scale protein-protein interactions for model organisms and humans. Although some significant technical limitations apply, the Y2H has made a great contribution to our general understanding of the topology of cellular signaling networks. PMID:18474041

  1. Entropy-based separation of yeast cells using a microfluidic system of conjoined spheres

    NASA Astrophysics Data System (ADS)

    Huang, Kai-Jian; Qin, S.-J.; Bai, Zhong-Chen; Zhang, Xin; Mai, John D.

    2013-11-01

    A physical model is derived to create a biological cell separator that is based on controlling the entropy in a microfluidic system having conjoined spherical structures. A one-dimensional simplified model of this three-dimensional problem in terms of the corresponding effects of entropy on the Brownian motion of particles is presented. This dynamic mechanism is based on the Langevin equation from statistical thermodynamics and takes advantage of the characteristics of the Fokker-Planck equation. This mechanism can be applied to manipulate biological particles inside a microfluidic system with identical, conjoined, spherical compartments. This theoretical analysis is verified by performing a rapid and a simple technique for separating yeast cells in these conjoined, spherical microfluidic structures. The experimental results basically match with our theoretical model and we further analyze the parameters which can be used to control this separation mechanism. Both numerical simulations and experimental results show that the motion of the particles depends on the geometrical boundary conditions of the microfluidic system and the initial concentration of the diffusing material. This theoretical model can be implemented in future biophysics devices for the optimized design of passive cell sorters.

  2. Entropy-based separation of yeast cells using a microfluidic system of conjoined spheres

    SciTech Connect

    Huang, Kai-Jian; Qin, S.-J. Bai, Zhong-Chen; Zhang, Xin; Mai, John D.

    2013-11-21

    A physical model is derived to create a biological cell separator that is based on controlling the entropy in a microfluidic system having conjoined spherical structures. A one-dimensional simplified model of this three-dimensional problem in terms of the corresponding effects of entropy on the Brownian motion of particles is presented. This dynamic mechanism is based on the Langevin equation from statistical thermodynamics and takes advantage of the characteristics of the Fokker-Planck equation. This mechanism can be applied to manipulate biological particles inside a microfluidic system with identical, conjoined, spherical compartments. This theoretical analysis is verified by performing a rapid and a simple technique for separating yeast cells in these conjoined, spherical microfluidic structures. The experimental results basically match with our theoretical model and we further analyze the parameters which can be used to control this separation mechanism. Both numerical simulations and experimental results show that the motion of the particles depends on the geometrical boundary conditions of the microfluidic system and the initial concentration of the diffusing material. This theoretical model can be implemented in future biophysics devices for the optimized design of passive cell sorters.

  3. Linear system identification - The application of Lion's identification scheme to a third order system with noisy input-output measurements

    NASA Technical Reports Server (NTRS)

    Brown, C. M., Jr.; Monopoli, R. V.

    1974-01-01

    A linear system identification technique developed by Lion is adapted for use on a third-order system with six unknown parameters and noisy input-output measurements. A digital computer is employed so that rapid identification takes place with only two state variable filters. Bias in the parameter estimates is partially eliminated by a signal-to-noise ratio testing procedure.

  4. A yeast transcription system for the 5S rRNA gene.

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

    A cell-free extract of yeast nuclei that can specifically transcribe cloned yeast 5S rRNA genes has been developed. Optima for transcription of 5S rDNA were determined and conditions of extract preparation leading to reproducible activities and specificities established. The major in vitro product has the same size and oligonucleotide composition as in vivo 5S rRNA. The in vitro transcription extract does not transcribe yeast tRNA genes. The extract does increase the transcription of tRNA genes packaged in chromatin. Images PMID:7145700

  5. Identification and antimicrobial susceptibility of bacteria and yeasts isolated from healthy dogs and dogs with otitis externa.

    PubMed

    Lyskova, P; Vydrzalova, M; Mazurova, J

    2007-12-01

    The bacterial and fungal flora of the external ear canal of dogs with otitis externa and of healthy dogs were studied. The most frequently isolated microorganism from otitic ears was Staphylococcus intermedius (58.8%), followed by Malassezia pachydermatis (30.9%), Streptococcus canis (29.9%), Proteus spp. (14.4%) and Escherichia coli (10.3%). A statistical analysis of our results showed that the prevalence of these microorganisms is significant in dogs with otitis externa. Furthermore, the antimicrobial susceptibility patterns of isolated strains were determined. Majority of all bacterial isolates were most susceptible to gentamicin. Malassezia pachydermatis, the most prevalent yeast in this study, showed an excellent level of susceptibility to all antifungal agents tested.

  6. Purification and Properties of an Esterase from the Yeast Saccharomyces cerevisiae and Identification of the Encoding Gene

    PubMed Central

    Degrassi, Giuliano; Uotila, Lasse; Klima, Raffaella; Venturi, Vittorio

    1999-01-01

    We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae. PMID:10427036

  7. Identification of Yeast Mutants Exhibiting Altered Sensitivity to Valinomycin and Nigericin Demonstrate Pleiotropic Effects of Ionophores on Cellular Processes

    PubMed Central

    Bhatia-Kissova, Ingrid; Valachovic, Martin; Klobucnikova, Vlasta; Zeiselova, Lucia; Griac, Peter; Nosek, Jozef

    2016-01-01

    Ionophores such as valinomycin and nigericin are potent tools for studying the impact of ion perturbance on cellular functions. To obtain a broader picture about molecular components involved in mediating the effects of these drugs on yeast cells under respiratory growth conditions, we performed a screening of the haploid deletion mutant library covering the Saccharomyces cerevisiae nonessential genes. We identified nearly 130 genes whose absence leads either to resistance or to hypersensitivity to valinomycin and/or nigericin. The processes affected by their protein products range from mitochondrial functions through ribosome biogenesis and telomere maintenance to vacuolar biogenesis and stress response. Comparison of the results with independent screenings performed by our and other laboratories demonstrates that although mitochondria might represent the main target for both ionophores, cellular response to the drugs is very complex and involves an intricate network of proteins connecting mitochondria, vacuoles, and other membrane compartments. PMID:27711131

  8. A simple and effective chromosome modification method for large-scale deletion of genome sequences and identification of essential genes in fission yeast

    PubMed Central

    Hirashima, Kyotaro; Iwaki, Tomoko; Takegawa, Kaoru; Giga-Hama, Yuko; Tohda, Hideki

    2006-01-01

    The technologies for chromosome modification developed to date are not satisfactorily universal, owing to the typical requirements for special enzymes and sequences. In the present report, we propose a new approach for chromosome modification in Schizosaccharomyces pombe that does not involve any special enzymes or sequences. This method, designated the ‘Latour system’, has wide applicability with extremely high efficiency, although both the basic principle and the operation are very simple. We demonstrate the ability of the Latour system to discriminate essential genes, with a long chromosomal area of 100 kb containing 33 genes deleted simultaneously and efficiently. Since no foreign sequences are retained after deletion using the Latour system, this system can be repeatedly applied at other sites. Provided that a negative selectable marker is available, the Latour system relies solely upon homologous recombination, which is highly conserved in living organisms. For this reason, it is expected that the system will be applicable to various yeasts. PMID:16434698

  9. Identification of methylated proteins in the yeast small ribosomal subunit: a role for SPOUT methyltransferases in protein arginine methylation.

    PubMed

    Young, Brian D; Weiss, David I; Zurita-Lopez, Cecilia I; Webb, Kristofor J; Clarke, Steven G; McBride, Anne E

    2012-06-26

    We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω-monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation of Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes.

  10. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis.

    PubMed

    Boretsky, Yuriy R; Pynyaha, Yuriy V; Boretsky, Volodymyr Y; Fedorovych, Dariya V; Fayura, Lyubov R; Protchenko, Olha; Philpott, Caroline C; Sibirny, Andriy A

    2011-05-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  11. Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

    PubMed Central

    Boretsky, Yuriy R.; Pynyaha, Yuriy V.; Boretsky, Volodymyr Y.; Fedorovych, Dariya V.; Fayura, Lyubov R.; Protchenko, Olha; Philpott, Caroline C.; Sibirny, Andriy A.

    2012-01-01

    Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondii Δvma1–17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondii Δfra1–45 mutant accumulated 1.8–2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1–17 and Δfes1–77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1–45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1–77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80–22. Complementation analysis revealed that Δvma1–17 and Δfra1–45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast. PMID:21261808

  12. Autonomous system for pathogen detection and identification

    SciTech Connect

    Belgrader, P.; Benett, W.; Bergman, W.; Langlois, R.; Mariella, R.; Milanovich, F.; Miles, R.; Venkateswaran, K.; Long, G.; Nelson, W.

    1998-09-24

    This purpose of this project is to build a prototype instrument that will, running unattended, detect, identify, and quantify BW agents. In order to accomplish this, we have chosen to start with the world' s leading, proven, assays for pathogens: surface-molecular recognition assays, such as antibody-based assays, implemented on a high-performance, identification (ID)-capable flow cytometer, and the polymerase chain reaction (PCR) for nucleic-acid based assays. With these assays, we must integrate the capability to: l collect samples from aerosols, water, or surfaces; l perform sample preparation prior to the assays; l incubate the prepared samples, if necessary, for a period of time; l transport the prepared, incubated samples to the assays; l perform the assays; l interpret and report the results of the assays. Issues such as reliability, sensitivity and accuracy, quantity of consumables, maintenance schedule, etc. must be addressed satisfactorily to the end user. The highest possible sensitivity and specificity of the assay must be combined with no false alarms. Today, we have assays that can, in under 30 minutes, detect and identify simulants for BW agents at concentrations of a few hundred colony-forming units per ml of solution. If the bio-aerosol sampler of this system collects 1000 Ymin and concentrates the respirable particles into 1 ml of solution with 70% processing efficiency over a period of 5 minutes, then this translates to a detection/ID capability of under 0.1 agent-containing particle/liter of air.

  13. Neural system prediction and identification challenge

    PubMed Central

    Vlachos, Ioannis; Zaytsev, Yury V.; Spreizer, Sebastian; Aertsen, Ad; Kumar, Arvind

    2013-01-01

    Can we infer the function of a biological neural network (BNN) if we know the connectivity and activity of all its constituent neurons?This question is at the core of neuroscience and, accordingly, various methods have been developed to record the activity and connectivity of as many neurons as possible. Surprisingly, there is no theoretical or computational demonstration that neuronal activity and connectivity are indeed sufficient to infer the function of a BNN. Therefore, we pose the Neural Systems Identification and Prediction Challenge (nuSPIC). We provide the connectivity and activity of all neurons and invite participants (1) to infer the functions implemented (hard-wired) in spiking neural networks (SNNs) by stimulating and recording the activity of neurons and, (2) to implement predefined mathematical/biological functions using SNNs. The nuSPICs can be accessed via a web-interface to the NEST simulator and the user is not required to know any specific programming language. Furthermore, the nuSPICs can be used as a teaching tool. Finally, nuSPICs use the crowd-sourcing model to address scientific issues. With this computational approach we aim to identify which functions can be inferred by systematic recordings of neuronal activity and connectivity. In addition, nuSPICs will help the design and application of new experimental paradigms based on the structure of the SNN and the presumed function which is to be discovered. PMID:24399966

  14. Decentralized system identification using stochastic subspace identification for wireless sensor networks.

    PubMed

    Cho, Soojin; Park, Jong-Woong; Sim, Sung-Han

    2015-04-08

    Wireless sensor networks (WSNs) facilitate a new paradigm to structural identification and monitoring for civil infrastructure. Conventional structural monitoring systems based on wired sensors and centralized data acquisition systems are costly for installation as well as maintenance. WSNs have emerged as a technology that can overcome such difficulties, making deployment of a dense array of sensors on large civil structures both feasible and economical. However, as opposed to wired sensor networks in which centralized data acquisition and processing is common practice, WSNs require decentralized computing algorithms to reduce data transmission due to the limitation associated with wireless communication. In this paper, the stochastic subspace identification (SSI) technique is selected for system identification, and SSI-based decentralized system identification (SDSI) is proposed to be implemented in a WSN composed of Imote2 wireless sensors that measure acceleration. The SDSI is tightly scheduled in the hierarchical WSN, and its performance is experimentally verified in a laboratory test using a 5-story shear building model.

  15. Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae.

    PubMed

    Eid, Rawan; Boucher, Eric; Gharib, Nada; Khoury, Chamel; Arab, Nagla T T; Murray, Alistair; Young, Paul G; Mandato, Craig A; Greenwood, Michael T

    2016-03-01

    Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress. PMID:26886577

  16. Identification of human ferritin, heavy polypeptide 1 (FTH1) and yeast RGI1 (YER067W) as pro-survival sequences that counteract the effects of Bax and copper in Saccharomyces cerevisiae.

    PubMed

    Eid, Rawan; Boucher, Eric; Gharib, Nada; Khoury, Chamel; Arab, Nagla T T; Murray, Alistair; Young, Paul G; Mandato, Craig A; Greenwood, Michael T

    2016-03-01

    Ferritin is a sub-family of iron binding proteins that form multi-subunit nanotype iron storage structures and prevent oxidative stress induced apoptosis. Here we describe the identification and characterization of human ferritin, heavy polypeptide 1 (FTH1) as a suppressor of the pro-apoptotic murine Bax sequence in yeast. In addition we demonstrate that FTH1 is a general pro-survival sequence since it also prevents the cell death inducing effects of copper when heterologously expressed in yeast. Although ferritins are phylogenetically widely distributed and are present in most species of Bacteria, Archaea and Eukarya, ferritin is conspicuously absent in most fungal species including Saccharomyces cerevisiae. An in silico analysis of the yeast proteome lead to the identification of the 161 residue RGI1 (YER067W) encoded protein as a candidate for being a yeast ferritin. In addition to sharing 20% sequence identity with the 183 residue FTH1, RGI1 also has similar pro-survival properties as ferritin when overexpressed in yeast. Analysis of recombinant protein by SDS-PAGE and by electron microscopy revealed the expected formation of higher-order structures for FTH1 that was not observed with Rgi1p. Further analysis revealed that cells overexpressing RGI1 do not show increased resistance to iron toxicity and do not have enhanced capacity to store iron. In contrast, cells lacking RGI1 were found to be hypersensitive to the toxic effects of iron. Overall, our results suggest that Rgi1p is a novel pro-survival protein whose function is not related to ferritin but nevertheless it may have a role in regulating yeast sensitivity to iron stress.

  17. [Identification system for Sildenafil in health foods].

    PubMed

    Moriyasu, T; Shigeoka, S; Kishimoto, K; Ishikawa, F; Nakajima, J; Kamimura, H; Yasuda, I

    2001-10-01

    A substantially available identification system for Sildenafil in health foods was established using 3 different analytical methods; i.e. TLC, preparative TLC/MS and HPLC/photo-diode array. Sildenafil in health foods was extracted with ethyl acetate under alkaline conditions as sample solutions for TLC and preparative TLC, and also extracted with 50% methanol and then diluted with solution of HPLC mobile phase for HPLC. The sample solution for TLC was applied to Silica gel 60 F254 plates with chloroform/methanol/28% ammonia (90:1:5, under layer) as mobile phase. Spots were located under UV radiation at 254 nm and 366 nm, and spraying dragendorff reagent. The conditions for preparative TLC were the same as these of TLC method, and samples abtained from preparative TLC were determined by MS with APCI interface, under both positive and negative modes. The HPLC analysis was carried out on a column of Cosmosil 5C18-AR (4.6 mm x 150 mm, 5 microns) with 0.05 mol/l phosphate buffer pH 3.0/acetonitrile(73:27) as mobile phase and the eluate was monitored by a photo-diode array detector. The quantitative analysis was available, when the peak of this sample on HPLC was detected at 290 nm. When this system was applied to commercial health foods, Sildenafil was identified and their contents were 25 mg-45 mg/tablet or bottle. These contents nearly correspond to that in Viagra, 25 mg, 50 mg/tablet. Therefore, there is a fear of side effects for Sildenafil, when it is taken as health foods.

  18. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System.

    PubMed

    Hoffman, Charles S; Wood, Valerie; Fantes, Peter A

    2015-10-01

    The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe.

  19. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System

    PubMed Central

    Hoffman, Charles S.; Wood, Valerie; Fantes, Peter A.

    2015-01-01

    The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe. PMID:26447128

  20. Non-linear system identification in flow-induced vibration

    SciTech Connect

    Spanos, P.D.; Zeldin, B.A.; Lu, R.

    1996-12-31

    The paper introduces a method of identification of non-linear systems encountered in marine engineering applications. The non-linearity is accounted for by a combination of linear subsystems and known zero-memory non-linear transformations; an equivalent linear multi-input-single-output (MISO) system is developed for the identification problem. The unknown transfer functions of the MISO system are identified by assembling a system of linear equations in the frequency domain. This system is solved by performing the Cholesky decomposition of a related matrix. It is shown that the proposed identification method can be interpreted as a {open_quotes}Gram-Schmidt{close_quotes} type of orthogonal decomposition of the input-output quantities of the equivalent MISO system. A numerical example involving the identification of unknown parameters of flow (ocean wave) induced forces on offshore structures elucidates the applicability of the proposed method.

  1. Offline synchronization of data acquisition systems using system identification

    NASA Astrophysics Data System (ADS)

    Maes, K.; Reynders, E.; Rezayat, A.; Roeck, G. De; Lombaert, G.

    2016-10-01

    This paper presents a technique for offline time synchronization of data acquisition systems. The technique can be applied when real-time synchronization of data acquisition systems is impossible or not sufficiently accurate. It allows for accurate synchronization based on the acquired dynamic response of the structure only, without requiring a common response or the use of a trigger signal. The synchronization is performed using the results obtained from system identification, and assumes linear dynamic behavior of the structure and proportional damping of the structural modes. A demonstration for a laboratory experiment on a cantilever steel beam shows that the proposed methodology can be used for accurate time synchronization, resulting in a significant improvement of the accuracy of the identified mode shapes.

  2. Substructure System Identification for Finite Element Model Updating

    NASA Technical Reports Server (NTRS)

    Craig, Roy R., Jr.; Blades, Eric L.

    1997-01-01

    This report summarizes research conducted under a NASA grant on the topic 'Substructure System Identification for Finite Element Model Updating.' The research concerns ongoing development of the Substructure System Identification Algorithm (SSID Algorithm), a system identification algorithm that can be used to obtain mathematical models of substructures, like Space Shuttle payloads. In the present study, particular attention was given to the following topics: making the algorithm robust to noisy test data, extending the algorithm to accept experimental FRF data that covers a broad frequency bandwidth, and developing a test analytical model (TAM) for use in relating test data to reduced-order finite element models.

  3. Automated frequency domain system identification of a large space structure

    NASA Technical Reports Server (NTRS)

    Yam, Y.; Bayard, D. S.; Hadaegh, F. Y.; Mettler, E.; Milman, M. H.

    1989-01-01

    This paper presents the development and experimental results of an automated on-orbit system identification method for large flexible spacecraft that yields estimated quantities to support on-line design and tuning of robust high performance control systems. The procedure consists of applying an input to the plant, obtaining an output, and then conducting nonparametric identification to yield the spectral estimate of the system transfer function. A parametric model is determined by curve fitting the spectral estimate to a rational transfer function. The identification method has been demonstrated experimentally on the Large Spacecraft Control Laboratory in JPL.

  4. Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System.

    PubMed

    Hudson, Lauren E; McDermott, Courtney D; Stewart, Taryn P; Hudson, William H; Rios, Daniel; Fasken, Milo B; Corbett, Anita H; Lamb, Tracey J

    2016-01-01

    The probiotic yeast Saccharomyces boulardii has been shown to ameliorate disease severity in the context of many infectious and inflammatory conditions. However, use of S. boulardii as a prophylactic agent or therapeutic delivery vector would require delivery of S. boulardii to a healthy, uninflamed intestine. In contrast to inflamed mucosal tissue, the diverse microbiota, intact epithelial barrier, and fewer inflammatory immune cells within the healthy intestine may all limit the degree to which S. boulardii contacts and influences the host mucosal immune system. Understanding the nature of these interactions is crucial for application of S. boulardii as a prophylactic agent or therapeutic delivery vehicle. In this study, we explore both intrinsic and immunomodulatory properties of S. boulardii in the healthy mucosal immune system. Genomic sequencing and morphological analysis of S. boulardii reveals changes in cell wall components compared to non-probiotic S. cerevisiae that may partially account for probiotic functions of S. boulardii. Flow cytometry and immunohistochemistry demonstrate limited S. boulardii association with murine Peyer's patches. We also show that although S. boulardii induces a systemic humoral immune response, this response is small in magnitude and not directed against S. boulardii itself. RNA-seq of the draining mesenteric lymph nodes indicates that even repeated administration of S. boulardii induces few transcriptional changes in the healthy intestine. Together these data strongly suggest that interaction between S. boulardii and the mucosal immune system in the healthy intestine is limited, with important implications for future work examining S. boulardii as a prophylactic agent and therapeutic delivery vehicle. PMID:27064405

  5. Characterization of the Probiotic Yeast Saccharomyces boulardii in the Healthy Mucosal Immune System

    PubMed Central

    Hudson, Lauren E.; McDermott, Courtney D.; Stewart, Taryn P.; Hudson, William H.; Rios, Daniel; Fasken, Milo B.; Corbett, Anita H.; Lamb, Tracey J.

    2016-01-01

    The probiotic yeast Saccharomyces boulardii has been shown to ameliorate disease severity in the context of many infectious and inflammatory conditions. However, use of S. boulardii as a prophylactic agent or therapeutic delivery vector would require delivery of S. boulardii to a healthy, uninflamed intestine. In contrast to inflamed mucosal tissue, the diverse microbiota, intact epithelial barrier, and fewer inflammatory immune cells within the healthy intestine may all limit the degree to which S. boulardii contacts and influences the host mucosal immune system. Understanding the nature of these interactions is crucial for application of S. boulardii as a prophylactic agent or therapeutic delivery vehicle. In this study, we explore both intrinsic and immunomodulatory properties of S. boulardii in the healthy mucosal immune system. Genomic sequencing and morphological analysis of S. boulardii reveals changes in cell wall components compared to non-probiotic S. cerevisiae that may partially account for probiotic functions of S. boulardii. Flow cytometry and immunohistochemistry demonstrate limited S. boulardii association with murine Peyer’s patches. We also show that although S. boulardii induces a systemic humoral immune response, this response is small in magnitude and not directed against S. boulardii itself. RNA-seq of the draining mesenteric lymph nodes indicates that even repeated administration of S. boulardii induces few transcriptional changes in the healthy intestine. Together these data strongly suggest that interaction between S. boulardii and the mucosal immune system in the healthy intestine is limited, with important implications for future work examining S. boulardii as a prophylactic agent and therapeutic delivery vehicle. PMID:27064405

  6. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

  7. Identification of regulatory elements in the AGT1 promoter of ale and lager strains of brewer's yeast.

    PubMed

    Vidgren, Virve; Kankainen, Matti; Londesborough, John; Ruohonen, Laura

    2011-08-01

    Agt1 is an interesting α-glucoside transporter for the brewing industry, as it efficiently transports maltotriose, a sugar often remaining partly unused during beer fermentation. It has been shown that on maltose the expression level of AGT1 is much higher in ale strains than in lager strains, and that glucose represses the expression, particularly in the ale strains. In the present study the regulatory elements of the AGT1 promoter of one ale and two lager strains were identified by computational methods. Promoter regions up to 1.9 kbp upstream of the AGT1 gene were sequenced from the three brewer's yeast strains and the laboratory yeast strain CEN.PK-1D. The promoter sequence of the laboratory strain was identical to the AGT1 promoter of strain S288c of the Saccharomyces Genome Database, whereas the promoter sequences of the industrial strains diverged markedly from the S288c strain. The AGT1 promoter regions of the ale and lager strains were for the most part identical to each other, except for one 22 bp deletion and two 94 and 95 bp insertions in the ale strain. Computational analyses of promoter elements revealed that the promoter sequences contained several Mig1- and MAL-activator binding sites, as was expected. However, some of the Mig1 and MAL-activator binding sites were located on the two insertions of the ale strain, and thus offered a plausible explanation for the different expression pattern of the AGT1 gene in the ale strains. Accordingly, functional analysis of A60 ale and A15 lager strain AGT1 promoters fused to GFP (encoding the green fluorescent protein) showed a significant difference in the ability of these two promoters to drive GFP expression. Under the control of the AGT1 promoter of the ale strain the emergence of GFP was strongly induced by maltose, whereas only a low level of GFP was detected with the construct carrying the AGT1 promoter of the lager strain. Thus, the extra MAL-activator binding element, present in the AGT1 promoter of

  8. Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory.

    PubMed

    Deak, Eszter; Charlton, Carmen L; Bobenchik, April M; Miller, Shelley A; Pollett, Simon; McHardy, Ian H; Wu, Max T; Garner, Omai B

    2015-01-01

    This study compared the diagnostic performance of Bruker's Microflex LT and bioMérieux's Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems. A total of 477 isolates were tested on both instruments. Discrepant results were resolved by sequencing. Overall, there was no statistically significant difference between the proportion of isolates correctly identified, miscalled or not called by each instrument. Although both systems were good at identifying yeast (66/69 to species level), the confidence level was high only to genus level for 30% of the isolates on the Bruker. Both systems performed with high accuracy when evaluated solely on Food and Drug Administration-approved organisms for each database. A user-based assessment of the 2 instruments revealed an overall preference for the Vitek MS instrument.

  9. A Method of Visualizing Three-Dimensional Distribution of Yeast in Bread Dough

    NASA Astrophysics Data System (ADS)

    Maeda, Tatsurou; Do, Gab-Soo; Sugiyama, Junichi; Oguchi, Kosei; Shiraga, Seizaburou; Ueda, Mitsuyoshi; Takeya, Koji; Endo, Shigeru

    A novel technique was developed to monitor the change in three-dimensional (3D) distribution of yeast in frozen bread dough samples in accordance with the progress of mixing process. Application of a surface engineering technology allowed the identification of yeast in bread dough by bonding EGFP (Enhanced Green Fluorescent Protein) to the surface of yeast cells. The fluorescent yeast (a biomarker) was recognized as bright spots at the wavelength of 520 nm. A Micro-Slicer Image Processing System (MSIPS) with a fluorescence microscope was utilized to acquire cross-sectional images of frozen dough samples sliced at intervals of 1 μm. A set of successive two-dimensional images was reconstructed to analyze 3D distribution of yeast. Samples were taken from each of four normal mixing stages (i.e., pick up, clean up, development, and final stages) and also from over mixing stage. In the pick up stage yeast distribution was uneven with local areas of dense yeast. As the mixing progressed from clean up to final stages, the yeast became more evenly distributed throughout the dough sample. However, the uniformity in yeast distribution was lost in the over mixing stage possibly due to the breakdown of gluten structure within the dough sample.

  10. Identification of a newly isolated erythritol-producing yeast and cloning of its erythrose [corrected] reductase genes.

    PubMed

    Deng, Huihui; Han, Ye; Liu, Yuanyuan; Jia, Wei; Zhou, Zhijiang

    2012-11-01

    A new erythritol-producing yeast (strain BH010) was isolated in this study. Analysis of the D1/D2 domain of the 26S rDNA sequence, the ITS/5.8S rDNA sequence [corrected] and the 18S rDNA sequence allowed the taxonomic position of strain BH010 to be discussed, [corrected] and it was identified and named Moniliella sp. BH010. Physiological characteristics were described. Scanning electron micrography clearly indicated that the cells were cylindrical to elliptical with an average size of 5 × 10 μm when growing in liquid medium [corrected] and that pseudohyphae and blastoconidia were observed when cultivated in agar plates. The erythrose [corrected] reductase genes were cloned, sequenced, and analyzed. BLAST analysis and multiple sequence alignment demonstrated that erythrose [corrected] reductase genes of Moniliella sp. BH010 shared very high homology with that of Trichosporonoides megachiliensis SNG-42 except for the presence of introns. The deduced amino acid sequences showed high homology to the aldo-keto reductase superfamily.

  11. Yeasts associated with the infrabuccal pocket and colonies of the carpenter ant Camponotus vicinus.

    PubMed

    Mankowski, M E; Morrell, J J

    2004-01-01

    After scanning electron microscopy indicated that the infrabuccal pockets of carpenter ants (Camponotus vicinus) contained numerous yeast-like cells, yeast associations were examined in six colonies of carpenter ants from two locations in Benton County in western Oregon. Samples from the infrabuccal-pocket contents and worker ant exoskeletons, interior galleries of each colony, and detritus and soil around the colonies were plated on yeast-extract/ malt-extract agar augmented with 1 M hydrochloric acid and incubated at 25 C. Yeasts were identified on the basis of morphological characteristics and physiological attributes with the BIOLOG(®) microbial identification system. Yeast populations from carpenter ant nest material and material surrounding the nest differed from those obtained from the infrabuccal pocket. Debaryomyces polymorphus was isolated more often from the infrabuccal pocket than from other material. This species has also been isolated from other ant species, but its role in colony nutrition is unknown.

  12. Yeasts associated with the infrabuccal pocket and colonies of the carpenter ant Camponotus vicinus.

    PubMed

    Mankowski, M E; Morrell, J J

    2004-01-01

    After scanning electron microscopy indicated that the infrabuccal pockets of carpenter ants (Camponotus vicinus) contained numerous yeast-like cells, yeast associations were examined in six colonies of carpenter ants from two locations in Benton County in western Oregon. Samples from the infrabuccal-pocket contents and worker ant exoskeletons, interior galleries of each colony, and detritus and soil around the colonies were plated on yeast-extract/ malt-extract agar augmented with 1 M hydrochloric acid and incubated at 25 C. Yeasts were identified on the basis of morphological characteristics and physiological attributes with the BIOLOG(®) microbial identification system. Yeast populations from carpenter ant nest material and material surrounding the nest differed from those obtained from the infrabuccal pocket. Debaryomyces polymorphus was isolated more often from the infrabuccal pocket than from other material. This species has also been isolated from other ant species, but its role in colony nutrition is unknown. PMID:21148849

  13. A linear discrete dynamic system model for temporal gene interaction and regulatory network influence in response to bioethanol conversion inhibitor HMF for ethanologenic yeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A linear discrete dynamic system model is constructed to represent the temporal interactions among significantly expressed genes in response to bioethanol conversion inhibitor 5-hydroxymethylfurfural for ethanologenic yeast Saccharomyces cerevisiae. This study identifies the most significant linear...

  14. Modeling of Biometric Identification System Using the Colored Petri Nets

    NASA Astrophysics Data System (ADS)

    Petrosyan, G. R.; Ter-Vardanyan, L. A.; Gaboutchian, A. V.

    2015-05-01

    In this paper we present a model of biometric identification system transformed into Petri Nets. Petri Nets, as a graphical and mathematical tool, provide a uniform environment for modelling, formal analysis, and design of discrete event systems. The main objective of this paper is to introduce the fundamental concepts of Petri Nets to the researchers and practitioners, both from identification systems, who are involved in the work in the areas of modelling and analysis of biometric identification types of systems, as well as those who may potentially be involved in these areas. In addition, the paper introduces high-level Petri Nets, as Colored Petri Nets (CPN). In this paper the model of Colored Petri Net describes the identification process much simpler.

  15. 47 CFR 25.281 - Automatic Transmitter Identification System (ATIS).

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... (ATIS). 25.281 Section 25.281 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Technical Operations § 25.281 Automatic Transmitter Identification System (ATIS). All satellite uplink transmissions carrying broadband video information shall...

  16. 47 CFR 25.281 - Automatic Transmitter Identification System (ATIS).

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... (ATIS). 25.281 Section 25.281 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Technical Operations § 25.281 Automatic Transmitter Identification System (ATIS). All satellite uplink transmissions carrying broadband video information shall...

  17. 47 CFR 25.281 - Automatic Transmitter Identification System (ATIS).

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... (ATIS). 25.281 Section 25.281 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Technical Operations § 25.281 Automatic Transmitter Identification System (ATIS). All satellite uplink transmissions carrying broadband video information shall...

  18. 47 CFR 25.281 - Automatic Transmitter Identification System (ATIS).

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... (ATIS). 25.281 Section 25.281 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) COMMON CARRIER SERVICES SATELLITE COMMUNICATIONS Technical Operations § 25.281 Automatic Transmitter Identification System (ATIS). All satellite uplink transmissions carrying broadband video information shall...

  19. High-throughput analysis of yeast replicative aging using a microfluidic system

    PubMed Central

    Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

    2015-01-01

    Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

  20. High-throughput analysis of yeast replicative aging using a microfluidic system.

    PubMed

    Jo, Myeong Chan; Liu, Wei; Gu, Liang; Dang, Weiwei; Qin, Lidong

    2015-07-28

    Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction. PMID:26170317

  1. System for expression of microsporidian methionine amino peptidase type 2 (MetAP2) in the yeast Saccharomyces cerevisiae.

    PubMed

    Upadhya, Rajendra; Zhang, Hong Shan; Weiss, Louis M

    2006-10-01

    Microsporidia are parasitic protists of all classes of vertebrates and most invertebrates. They recently emerged as important infections in various immunosuppressed and immunocompetent patient populations. They are also important veterinary and agricultural pathogens. Current therapies for microsporidiosis include benzimidazoles, which bind tubulin-inhibiting microtubule assembly, and fumagillin and its derivatives, which bind and inhibit methionine amino peptidase type 2 (MetAP2). Benzimidazoles are not active against Enterocytozoon bieneusi, the most common cause of human microsporidiosis. Fumagillin is active against most microsporidia, including E. bieneusi, but thrombocytopenia has been a problem in clinical trials. There is a pressing need for more-specific microsporidian MetAP2 inhibitors. To expedite and facilitate the discovery of safe and effective MetAP2 inhibitors, we have engineered Saccharomyces cerevisiae to be dependent on Encephalitozoon cuniculi MetAP2 (EcMetAP2) for its growth, where EcMetAP2 is harbored on an episomal uracil-selectable tetracycline-regulated plasmid. We have also constructed a leucine-selectable tetracycline-regulated expression plasmid into which any MetAP2 gene can be cloned. By utilizing a 5-fluoroorotic acid-mediated plasmid shuffle in the EcMetAP2 yeast strain, a yeast strain can be generated whose growth is dependent on MetAP2 from any organism. The level of heterologous MetAP2 gene expression can be controlled by the addition of tetracycline to the growth medium. These yeast strains should permit high-throughput screening for the identification of new inhibitors with high specificity and activity toward microsporidian MetAP2.

  2. Development of an Automatic Identification System Autonomous Positioning System.

    PubMed

    Hu, Qing; Jiang, Yi; Zhang, Jingbo; Sun, Xiaowen; Zhang, Shufang

    2015-11-11

    In order to overcome the vulnerability of the global navigation satellite system (GNSS) and provide robust position, navigation and time (PNT) information in marine navigation, the autonomous positioning system based on ranging-mode Automatic Identification System (AIS) is presented in the paper. The principle of the AIS autonomous positioning system (AAPS) is investigated, including the position algorithm, the signal measurement technique, the geometric dilution of precision, the time synchronization technique and the additional secondary factor correction technique. In order to validate the proposed AAPS, a verification system has been established in the Xinghai sea region of Dalian (China). Static and dynamic positioning experiments are performed. The original function of the AIS in the AAPS is not influenced. The experimental results show that the positioning precision of the AAPS is better than 10 m in the area with good geometric dilution of precision (GDOP) by the additional secondary factor correction technology. This is the most economical solution for a land-based positioning system to complement the GNSS for the navigation safety of vessels sailing along coasts.

  3. Development of an Automatic Identification System Autonomous Positioning System.

    PubMed

    Hu, Qing; Jiang, Yi; Zhang, Jingbo; Sun, Xiaowen; Zhang, Shufang

    2015-01-01

    In order to overcome the vulnerability of the global navigation satellite system (GNSS) and provide robust position, navigation and time (PNT) information in marine navigation, the autonomous positioning system based on ranging-mode Automatic Identification System (AIS) is presented in the paper. The principle of the AIS autonomous positioning system (AAPS) is investigated, including the position algorithm, the signal measurement technique, the geometric dilution of precision, the time synchronization technique and the additional secondary factor correction technique. In order to validate the proposed AAPS, a verification system has been established in the Xinghai sea region of Dalian (China). Static and dynamic positioning experiments are performed. The original function of the AIS in the AAPS is not influenced. The experimental results show that the positioning precision of the AAPS is better than 10 m in the area with good geometric dilution of precision (GDOP) by the additional secondary factor correction technology. This is the most economical solution for a land-based positioning system to complement the GNSS for the navigation safety of vessels sailing along coasts. PMID:26569258

  4. Development of an Automatic Identification System Autonomous Positioning System

    PubMed Central

    Hu, Qing; Jiang, Yi; Zhang, Jingbo; Sun, Xiaowen; Zhang, Shufang

    2015-01-01

    In order to overcome the vulnerability of the global navigation satellite system (GNSS) and provide robust position, navigation and time (PNT) information in marine navigation, the autonomous positioning system based on ranging-mode Automatic Identification System (AIS) is presented in the paper. The principle of the AIS autonomous positioning system (AAPS) is investigated, including the position algorithm, the signal measurement technique, the geometric dilution of precision, the time synchronization technique and the additional secondary factor correction technique. In order to validate the proposed AAPS, a verification system has been established in the Xinghai sea region of Dalian (China). Static and dynamic positioning experiments are performed. The original function of the AIS in the AAPS is not influenced. The experimental results show that the positioning precision of the AAPS is better than 10 m in the area with good geometric dilution of precision (GDOP) by the additional secondary factor correction technology. This is the most economical solution for a land-based positioning system to complement the GNSS for the navigation safety of vessels sailing along coasts. PMID:26569258

  5. A combined database related and de novo MS-identification of yeast mannose-1-phosphate guanyltransferase PSA1 interaction partners at different phases of batch cultivation

    NASA Astrophysics Data System (ADS)

    Parviainen, Ville; Joenväärä, Sakari; Peltoniemi, Hannu; Mattila, Pirkko; Renkonen, Risto

    2009-04-01

    Mass spectrometry-based proteomic research has become one of the main methods in protein-protein interaction research. Several high throughput studies have established an interaction landscape of exponentially growing Baker's yeast culture. However, many of the protein-protein interactions are likely to change in different environmental conditions. In order to examine the dynamic nature of the protein interactions we isolated the protein complexes of mannose-1-phosphate guanyltransferase PSA1 from Saccharomyces cerevisiae at four different time points during batch cultivation. We used the tandem affinity purification (TAP)-method to purify the complexes and subjected the tryptic peptides to LC-MS/MS. The resulting peak lists were analyzed with two different methods: the database related protein identification program X!Tandem and the de novo sequencing program Lutefisk. We observed significant changes in the interactome of PSA1 during the batch cultivation and identified altogether 74 proteins interacting with PSA1 of which only six were found to interact during all time points. All the other proteins showed a more dynamic nature of binding activity. In this study we also demonstrate the benefit of using both database related and de novo methods in the protein interaction research to enhance both the quality and the quantity of observations.

  6. Research of Uncertainty Reasoning in Pineapple Disease Identification System

    NASA Astrophysics Data System (ADS)

    Liu, Liqun; Fan, Haifeng

    In order to deal with the uncertainty of evidences mostly existing in pineapple disease identification system, a reasoning model based on evidence credibility factor was established. The uncertainty reasoning method is discussed,including: uncertain representation of knowledge, uncertain representation of rules, uncertain representation of multi-evidences and update of reasoning rules. The reasoning can fully reflect the uncertainty in disease identification and reduce the influence of subjective factors on the accuracy of the system.

  7. Analysis of Membrane Topology and Identification of Essential Residues for the Yeast Endoplasmic Reticulum Inositol Acyltransferase Gwt1p

    PubMed Central

    Sagane, Koji; Umemura, Mariko; Ogawa-Mitsuhashi, Kaoru; Tsukahara, Kappei; Yoko-o, Takehiko; Jigami, Yoshifumi

    2011-01-01

    Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops. PMID:21367863

  8. Parameter identification for nonlinear aerodynamic systems

    NASA Technical Reports Server (NTRS)

    Pearson, Allan E.

    1992-01-01

    Continuing work on frequency analysis for transfer function identification is discussed. A new study was initiated into a 'weighted' least squares algorithm within the context of the Fourier modulating function approach. The first phase of applying these techniques to the F-18 flight data is nearing completion, and these results are summarized.

  9. [Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

    PubMed

    Morozkina, E V; Vavilova, E A; Zatsepin, S S; Klyachko, E V; Yagudin, T A; Chulkin, A M; Dudich, I V; Semenkova, L N; Churilova, I V; Benevolenskii, S V

    2016-01-01

    A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

  10. Selection of intracellular single-domain antibodies targeting the HIV-1 Vpr protein by cytoplasmic yeast two-hybrid system.

    PubMed

    Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick

    2014-01-01

    The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system.

  11. Selection of Intracellular Single-Domain Antibodies Targeting the HIV-1 Vpr Protein by Cytoplasmic Yeast Two-Hybrid System

    PubMed Central

    Matz, Julie; Hérate, Cécile; Bouchet, Jérôme; Dusetti, Nelson; Gayet, Odile; Baty, Daniel; Benichou, Serge; Chames, Patrick

    2014-01-01

    The targeting of HIV-1 using antibodies is of high interest as molecular tools to better understand the biology of the virus or as a first step toward the design of new inhibitors targeting critical viral intracellular proteins. Small and highly stable llama-derived single-domain antibodies can often be functionally expressed as intracellular antibodies in the cytoplasm of eukaryotic cells. Using a selection method based on the Sos Recruitment System, a cytoplasmic yeast two-hybrid approach, we have isolated single-domain antibodies able to bind HIV-1 Vpr and Capside proteins in the yeast cytoplasm. One anti-Vpr single domain antibody was able to bind the HIV-1 regulatory Vpr protein in the cytoplasm of eukaryotic cells, leading to its delocalization from the nucleus to the cytoplasm. To our knowledge, this is the first description of a functional single-domain intrabody targeting HIV-1 Vpr, isolated using an in vivo cytoplasmic selection method that alleviates some limitations of the conventional yeast two-hybrid system. PMID:25436999

  12. Development and Validation of an In-House Database for Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry-Based Yeast Identification Using a Fast Protein Extraction Procedure

    PubMed Central

    De Carolis, Elena; Vella, Antonietta; Vaccaro, Luisa; Torelli, Riccardo; Posteraro, Patrizia; Ricciardi, Walter; Posteraro, Brunella

    2014-01-01

    In recent studies evaluating the usefulness of the matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS)-based identification of yeasts for the routine diagnosis of fungal infections, preanalytical sample processing has emerged as a critical step for reliable MALDI-TOF MS outcomes, especially when the Bruker Daltonics Biotyper software was used. In addition, inadequate results often occurred due to discrepancies between the methods used for clinical testing and database construction. Therefore, we created an in-house MALDI-TOF MS library using the spectra from 156 reference and clinical yeast isolates (48 species in 11 genera), which were generated with a fast sample preparation procedure. After a retrospective validation study, our database was evaluated on 4,232 yeasts routinely isolated during a 6-month period and fast prepared for MALDI-TOF MS analysis. Thus, 4,209 (99.5%) of the isolates were successfully identified to the species level (with scores of ≥2.0), with 1,676 (39.6%) having scores of >2.3. For the remaining 23 (0.5%) isolates, no reliable identification (with scores of <1.7) was obtained. Interestingly, these isolates were almost always from species uniquely represented or not included in the database. As the MALDI-TOF MS results were, except for 23 isolates, validated without additional phenotypic or molecular tests, our proposed strategy can enhance the rapidity and accuracy of MALDI-TOF MS in identifying medically important yeast species. However, while continuous updating of our database will be necessary to enrich it with more strains/species of new and emerging yeasts, the present in-house MALDI-TOF MS library can be made publicly available for future multicenter studies. PMID:24554755

  13. System identification of the Arabidopsis plant circadian system

    NASA Astrophysics Data System (ADS)

    Foo, Mathias; Somers, David E.; Kim, Pan-Jun

    2015-02-01

    The circadian system generates an endogenous oscillatory rhythm that governs the daily activities of organisms in nature. It offers adaptive advantages to organisms through a coordination of their biological functions with the optimal time of day. In this paper, a model of the circadian system in the plant Arabidopsis (species thaliana) is built by using system identification techniques. Prior knowledge about the physical interactions of the genes and the proteins in the plant circadian system is incorporated in the model building exercise. The model is built by using primarily experimentally-verified direct interactions between the genes and the proteins with the available data on mRNA and protein abundances from the circadian system. Our analysis reveals a great performance of the model in predicting the dynamics of the plant circadian system through the effect of diverse internal and external perturbations (gene knockouts and day-length changes). Furthermore, we found that the circadian oscillatory rhythm is robust and does not vary much with the biochemical parameters except those of a light-sensitive protein P and a transcription factor TOC1. In other words, the circadian rhythmic profile is largely a consequence of the network's architecture rather than its particular parameters. Our work suggests that the current experimental knowledge of the gene-to-protein interactions in the plant Arabidopsis, without considering any additional hypothetical interactions, seems to suffice for system-level modeling of the circadian system of this plant and to present an exemplary platform for the control of network dynamics in complex living organisms.

  14. Input-output identification of controlled discrete manufacturing systems

    NASA Astrophysics Data System (ADS)

    Estrada-Vargas, Ana Paula; López-Mellado, Ernesto; Lesage, Jean-Jacques

    2014-03-01

    The automated construction of discrete event models from observations of external system's behaviour is addressed. This problem, often referred to as system identification, allows obtaining models of ill-known (or even unknown) systems. In this article, an identification method for discrete event systems (DESs) controlled by a programmable logic controller is presented. The method allows processing a large quantity of observed long sequences of input/output signals generated by the controller and yields an interpreted Petri net model describing the closed-loop behaviour of the automated DESs. The proposed technique allows the identification of actual complex systems because it is sufficiently efficient and well adapted to cope with both the technological characteristics of industrial controllers and data collection requirements. Based on polynomial-time algorithms, the method is implemented as an efficient software tool which constructs and draws the model automatically; an overview of this tool is given through a case study dealing with an automated manufacturing system.

  15. A portable air jet actuator device for mechanical system identification.

    PubMed

    Belden, Jesse; Staats, Wayne L; Mazumdar, Anirban; Hunter, Ian W

    2011-03-01

    System identification of limb mechanics can help diagnose ailments and can aid in the optimization of robotic limb control parameters and designs. An interesting fluid phenomenon--the Coandă effect--is utilized in a portable actuator to provide a stochastic binary force disturbance to a limb system. The design of the actuator is approached with the goal of creating a portable device which could be deployed on human or robotic limbs for in situ mechanical system identification. The viability of the device is demonstrated by identifying the parameters of an underdamped elastic beam system with fixed inertia and stiffness and variable damping. The nonparametric compliance impulse response yielded from the system identification is modeled as a second-order system and the resultant parameters are found to be in excellent agreement with those found using more traditional system identification techniques. The current design could be further miniaturized and developed as a portable, wireless, unrestrained mechanical system identification instrument for less intrusive and more widespread use. PMID:21456788

  16. A portable air jet actuator device for mechanical system identification

    NASA Astrophysics Data System (ADS)

    Belden, Jesse; Staats, Wayne L.; Mazumdar, Anirban; Hunter, Ian W.

    2011-03-01

    System identification of limb mechanics can help diagnose ailments and can aid in the optimization of robotic limb control parameters and designs. An interesting fluid phenomenon—the Coandă effect—is utilized in a portable actuator to provide a stochastic binary force disturbance to a limb system. The design of the actuator is approached with the goal of creating a portable device which could be deployed on human or robotic limbs for in situ mechanical system identification. The viability of the device is demonstrated by identifying the parameters of an underdamped elastic beam system with fixed inertia and stiffness and variable damping. The nonparametric compliance impulse response yielded from the system identification is modeled as a second-order system and the resultant parameters are found to be in excellent agreement with those found using more traditional system identification techniques. The current design could be further miniaturized and developed as a portable, wireless, unrestrained mechanical system identification instrument for less intrusive and more widespread use.

  17. System identification methods for aircraft flight control development and validation

    NASA Technical Reports Server (NTRS)

    Tischler, Mark B.

    1995-01-01

    System-identification methods compose a mathematical model, or series of models, from measurements of inputs and outputs of dynamic systems. The extracted models allow the characterization of the response of the overall aircraft or component subsystem behavior, such as actuators and on-board signal processing algorithms. This paper discusses the use of frequency-domain system-identification methods for the development and integration of aircraft flight-control systems. The extraction and analysis of models of varying complexity from nonparametric frequency-responses to transfer-functions and high-order state-space representations is illustrated using the Comprehensive Identification from FrEquency Responses (CIFER) system-identification facility. Results are presented for test data of numerous flight and simulation programs at the Ames Research Center including rotorcraft, fixed-wing aircraft, advanced short takeoff and vertical landing (ASTOVL), vertical/short takeoff and landing (V/STOL), tiltrotor aircraft, and rotor experiments in the wind tunnel. Excellent system characterization and dynamic response prediction is achieved for this wide class of systems. Examples illustrate the role of system-identification technology in providing an integrated flow of dynamic response data around the entire life-cycle of aircraft development from initial specifications, through simulation and bench testing, and into flight-test optimization.

  18. Screening of hepatocyte proteins binding with the middle surface protein of the hepatitis B virus by the yeast two-hybrid system.

    PubMed

    Li, Zhiqun; Linghu, Enqiang; Cheng, Jun

    2014-06-01

    The effect of the middle hepatitis B virus surface protein (MHBs) remains to be elucidated. To investigate the biological function of the MHBs protein, the present study performed yeast two-hybrid screening to search for proteins that interact with the MHBs protein in hepatocytes. The bait plasmid expressing the MHBs protein was constructed by cloning the gene of the MHBs protein into pGBKT7, then the recombinant plasmid DNA was transformed into AH109 yeast (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing the liver cDNA library plasmid in 2X yeast peptone dextrose adenine (YPDA) medium. The mated diploid yeast was plated on quadruple dropout medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. Following extracting and sequencing of the plasmids from positive (blue) colonies, the sequence analysis was conducted and analyzed by bioinformatics methods. Two colonies were selected and sequenced. Among them, one was the human DNA sequence from the clone RP11-490D19 on chromosome 9 and the other was homo sapiens 12 BAC RP11-180M15 (Roswell Park Cancer Institute Human BAC Library). The yeast two-hybrid system is an effective method for identifying hepatocyte proteins that interact with MHBs. The MHBs protein binds with different proteins suggesting that it has multiple functions in vivo.

  19. Molecular cloning of amphioxus uncoupling protein and assessment of its uncoupling activity using a yeast heterologous expression system

    SciTech Connect

    Chen, Kun; Sun, Guoxun; Lv, Zhiyuan; Wang, Chen; Jiang, Xueyuan; Li, Donghai; Zhang, Chenyu

    2010-10-01

    Research highlights: {yields} Invertebrates, for example amphioxus, do express uncoupling proteins. {yields} Both the sequence and the uncoupling activity of amphioxus UCP resemble UCP2. {yields} UCP1 is the only UCP that can form dimer on yeast mitochondria. -- Abstract: The present study describes the molecular cloning of a novel cDNA fragment from amphioxus (Branchiostoma belcheri) encoding a 343-amino acid protein that is highly homologous to human uncoupling proteins (UCP), this protein is therefore named amphioxus UCP. This amphioxus UCP shares more homology with and is phylogenetically more related to mammalian UCP2 as compared with UCP1. To further assess the functional similarity of amphioxus UCP to mammalian UCP1 and -2, the amphioxus UCP, rat UCP1, and human UCP2 were separately expressed in Saccharomyces cerevisiae, and the recombinant yeast mitochondria were isolated and assayed for the state 4 respiration rate and proton leak, using pYES2 empty vector as the control. UCP1 increased the state 4 respiration rate by 2.8-fold, and the uncoupling activity was strongly inhibited by GDP, while UCP2 and amphioxus UCP only increased the state 4 respiration rate by 1.5-fold and 1.7-fold in a GDP-insensitive manner, moreover, the proton leak kinetics of amphioxus UCP was very similar to UCP2, but much different from UCP1. In conclusion, the amphioxus UCP has a mild, unregulated uncoupling activity in the yeast system, which resembles mammalian UCP2, but not UCP1.

  20. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  1. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

    SciTech Connect

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.

  2. A modular and hybrid connectionist system for speaker identification.

    PubMed

    Bennani, Y

    1995-07-01

    This paper presents and evaluates a modular/hybrid connectionist system for speaker identification. Modularity has emerged as a powerful technique for reducing the complexity of connectionist systems, and allowing a priori knowledge to be incorporated into their design. Text-independent speaker identification is an inherently complex task where the amount of training data is often limited. It thus provides an ideal domain to test the validity of the modular/hybrid connectionist approach. To achieve such identification, we develop, in this paper, an architecture based upon the cooperation of several connectionist modules, and a Hidden Markov Model module. When tested on a population of 102 speakers extracted from the DARPA-TIMIT database, perfect identification was obtained.

  3. Effect of conjugated linoleic acid on fungal delta6-desaturase activity in a transformed yeast system.

    PubMed

    Chuang, L T; Thurmond, J M; Liu, J W; Kirchner, S J; Mukerji, P; Bray, T M; Huang, Y S

    2001-02-01

    Conjugated linoleic acid (CLA; 18:2), a group of positional and geometric isomers of linoleic acid (LA; 18:2n-6), has been shown to modulate immune function through its effect on eicosanoid synthesis. This effect has been attributed to a reduced production of n-6 polyunsaturated fatty acid (PUFA), the precursor of eicosanoids. Since delta6-desaturase is the rate-limiting enzyme of the n-6 PUFA production, it is our hypothesis that CLA, which has similar chemical structure to LA, interacts directly with delta6-desaturase. A unique and simple model, i.e., baker's yeast (Saccharomyces cerevisiae) transformed with fungal delta6-desaturase gene, previously established, was used to investigate the direct effect of CLA on delta6-desaturase. This model allows LA to be converted to y-linolenic acid (GLA; 18:3n-6) but not GLA to its metabolite(s). No metabolites of CLA were found in the lipids of the yeast transformed with delta6-desaturase. The inability to convert CLA to conjugated GLA was not due to the failure of yeast cells to take up the CLA isomers. CLA mixture and individual isomers significantly inhibited the activity of delta6-desaturase of the transformed yeast in vivo. Even though its uptake by the yeast was low, CLA c9,t11 isomer was found to be the most potent inhibitor of the four isomers tested, owing to its high inhibitory effect on delta6-desaturase. Since CLA did not cause significant changes in the level of delta6-desaturase mRNA, the inhibition of GLA production could not be attributed to suppression of delta6-desaturase gene expression at the transcriptional level.

  4. Identification of cardiovascular dilution systems by contrast ultrasound.

    PubMed

    Mischi, Massimo; Jansen, Annemieke H M; Korsten, Hendrikus H M

    2007-03-01

    Indicator dilution techniques permit accurate measurements of important cardiovascular parameters, such as pulmonary blood volume (PBV) and ejection fraction (EF). However, their use is limited by the need for central catheterization. Contrast ultrasonography allows overcoming this problem. PBV and EF can be measured by a dilution system identification algorithm after detection of multiple dilution curves by an ultrasound scanner. In this paper, we present a system identification method that exploits the a priori knowledge on the dilution system and finds the optimum parameters for the parametric model representing the dilution system impulse response. No subsequent model interpolation is needed. Volume measurements show accurate in-vitro results and clinical feasibility, while 50 EF measurements in patients show a 0.88 correlation coefficient with echocardiographic biplane estimates. In conclusion, adding a priori knowledge to the system identification algorithm leads to increased accuracy and robustness of the method for PBV and EF measurements.

  5. Evaluation of the Biolog automated microbial identification system

    NASA Technical Reports Server (NTRS)

    Klingler, J. M.; Stowe, R. P.; Obenhuber, D. C.; Groves, T. O.; Mishra, S. K.; Pierson, D. L.

    1992-01-01

    Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.

  6. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  7. Yeast Infections

    MedlinePlus

    Candida is the scientific name for yeast. It is a fungus that lives almost everywhere, including in ... infection that causes white patches in your mouth Candida esophagitis is thrush that spreads to your esophagus, ...

  8. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  9. Model abstraction results using state-space system identifications

    NASA Astrophysics Data System (ADS)

    Popken, Douglas A.

    2000-06-01

    In this paper we report on state-space system identification approaches to dynamic behavioral abstraction of military simulation models. Two stochastic simulation models were identified under a variety of scenarios. The `Attrition Simulation' is a model of two opposing forces with multiple weapon system types. The `Mission Simulation' is a model of a squadron of aircraft performing battlefield air interdiction. Four system identification techniques: Maximum Entropy, Compartmental Models, Canonical State-Space Models, and Hidden Markov Models (HMM), were applied to these simulation models. The system identification techniques were evaluated on how well their resulting abstractions replicated the distributions of the simulation states as well as the decision outputs. Encouraging results were achieved by the HMM technique applied to the Attrition Simulation--and by the Maximum Entropy technique applied to the Mission Simulation.

  10. Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

    PubMed

    Calderaro, A; Martinelli, M; Motta, F; Larini, S; Arcangeletti, M C; Medici, M C; Chezzi, C; De Conto, F

    2014-08-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

  11. Application of unsymmetric block Lanczos vectors in system identification

    NASA Astrophysics Data System (ADS)

    Kim, H. M., Jr.; Craig, R. R.

    1992-10-01

    This paper demonstrates a new system identification approach of using Lanczos coordinates in place of modal coordinates. Identified experimental Lanczos vectors can be directly used in many structural dynamics analysis applications. A multi-input, multi-output frequency-domain technique was used to extract system matrices and an unsymmetric block Lanczos algorithm was used to reduce the order of the experimental model. A cantilever beam example showed promising results, indicating that a new system identification approach using Lanczos coordinates is worthy of further study.

  12. Associations of UBE2I with RAD52, UBL1, p53, and RAD51 proteins in a yeast two-hybrid system

    SciTech Connect

    Shen, Zhiyuan; Pardington-Purtymun, P.E.; Comeaux, J.C.

    1996-10-15

    The yeast RAD52-dependent pathway is involved in DNA recombination and double-strand break repair. Yeast ubiquitin-conjugating enzyme UBC9 participates in S- and M-phase cyclin degradation and mitotic control. Using the human RAD52 protein as the bait in a yeast two-hybrid system, we have identified a human homolog of yeast UBC9, designated UBE2I, that interacts with RAD52, RAD51, p53, and a ubiquitin-like protein UBL1. These interactions are UBE2I-specific, since another DNA repair-related ubiquitin-conjugating enzyme, RAD6 (UBC2), does not interact with these proteins. The interaction of UBE2I with RAD52 is mediated by RAD52`s self-association region. These results suggest that the RAD52-dependent processes, cell cycle control, p53-mediated pathway(s), and ubiquitination interact through human UBE2I. 22 refs., 3 figs.

  13. Isolation and Identification of Pathogenic Filamentous Fungi and Yeasts From Adult House Fly (Diptera: Muscidae) Captured From the Hospital Environments in Ahvaz City, Southwestern Iran.

    PubMed

    Kassiri, Hamid; Zarrin, Majid; Veys-Behbahani, Rahele; Faramarzi, Sama; Kasiri, Ali

    2015-11-01

    Musca domestica L., 1758 is capable of transferring a number of pathogenic viruses, bacteria, fungi, and parasites to animals and humans. The objective of this study was to isolate and identify medically important filamentous fungi and yeasts from adult M. domestica collected from two wards of three hospital environments in Ahvaz city, Khuzestan Province, southwestern Iran. The common house flies were caught by a sterile net. These insects were washed in a solution of 1% sodium hypochlorite for 3 min and twice in sterile distilled water for 1 min. The flies were individually crushed with sterile swabs in sterile test tubes. Then 2 ml of sterile normal saline (0.85%) was added to each tube, and the tube was centrifuged for 5 min. The supernatant was then discarded, and the remaining sediment was inoculated with a sterile swab in the Sabouraud's dextrose agar medium containing chloramphenicol. Isolation and identification of fungi were made by standard mycological methods. In this research, totally 190 M. domestica from hospital environments were captured. In total, 28 fungal species were isolated. The main fungi isolated were Aspergillus spp. (67.4%), Penicillium sp. (11.6%), Mucorales sp. (11%), Candida spp. (10.5%), and Rhodotorula sp. (8.4%). Among the house flies caught at the hospitals, about 80% were found to carry one or more medically important species of fungi. This study has established that common house flies carry pathogenic fungi in the hospital environments of Ahvaz. The control of M. domestica in hospitals is essential in order to control the nosocomial fungal infections in patients.

  14. Mutagenesis and biochemical analysis of recombinant yeast prenyltransferases.

    PubMed

    Caplin, B E; Marshall, M S

    1995-01-01

    The use of the S. cerevisiae protein prenyltransferases as a model system for general prenyltransferase study is justified by the similarity of mechanism, substrate specificity, and evolutionarily conserved substrates with the mammalian prenyltransferases. Genetic identification of potential structural genes involved in prenyltransferase activity can be easily confirmed with biochemical assays using recombinant enzyme reconstitution. Yeast FTase and GGTase I produced in E. coli are indistinguishable from the native proteins and can be studied without interference from contaminating cellular protein prenyltransferases. Structure-function analysis of the yeast prenyltransferase subunits is also simplified by the rapidity with which mutant enzymes can be analyzed in E. coli and their biological activity characterized in yeast defective for the particular subunit gene.

  15. Decentralized System Identification Using Stochastic Subspace Identification for Wireless Sensor Networks

    PubMed Central

    Cho, Soojin; Park, Jong-Woong; Sim, Sung-Han

    2015-01-01

    Wireless sensor networks (WSNs) facilitate a new paradigm to structural identification and monitoring for civil infrastructure. Conventional structural monitoring systems based on wired sensors and centralized data acquisition systems are costly for installation as well as maintenance. WSNs have emerged as a technology that can overcome such difficulties, making deployment of a dense array of sensors on large civil structures both feasible and economical. However, as opposed to wired sensor networks in which centralized data acquisition and processing is common practice, WSNs require decentralized computing algorithms to reduce data transmission due to the limitation associated with wireless communication. In this paper, the stochastic subspace identification (SSI) technique is selected for system identification, and SSI-based decentralized system identification (SDSI) is proposed to be implemented in a WSN composed of Imote2 wireless sensors that measure acceleration. The SDSI is tightly scheduled in the hierarchical WSN, and its performance is experimentally verified in a laboratory test using a 5-story shear building model. PMID:25856325

  16. New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast*

    PubMed Central

    Martin, D. Christian; Kim, Hyemin; Mackin, Nancy A.; Maldonado-Báez, Lymarie; Evangelista, Carlos C.; Beaudry, Veronica G.; Dudgeon, Drew D.; Naiman, Daniel Q.; Erdman, Scott E.; Cunningham, Kyle W.

    2011-01-01

    The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis. PMID:21252230

  17. Yeast Three-Hybrid System for the Detection of Protein-Protein Interactions.

    PubMed

    Maruta, Natsumi; Trusov, Yuri; Botella, Jose R

    2016-01-01

    Protein-protein interaction studies provide useful insights into biological processes taking place within the living cell. A number of techniques are available to unravel large structural protein complexes, functional protein modules, and temporary protein associations occurring during signal transduction. The choice of method depends on the nature of the proteins and the interaction being studied. Here we present an optimized and simplified yeast three-hybrid method for analysis of protein interactions involving three components.

  18. Yeast Mitochondria as a Model System to Study the Biogenesis of Bacterial β-Barrel Proteins.

    PubMed

    Ulrich, Thomas; Oberhettinger, Philipp; Autenrieth, Ingo B; Rapaport, Doron

    2015-01-01

    Beta-barrel proteins are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. The evolutionary conservation in the biogenesis of these proteins allows mitochondria to assemble bacterial β-barrel proteins in their functional form. In this chapter, we describe exemplarily how the capacity of yeast mitochondria to process the trimeric autotransporter YadA can be used to study the role of bacterial periplasmic chaperones in this process. PMID:26427673

  19. Yeast as a model system to study glucose-mediated signalling and response.

    PubMed

    Sanz, Pascual

    2007-01-01

    Glucose is the principal carbon and energy source for a wide variety of cells, ranging from unicellular microorganisms to higher eukaryotic cells. It is taken up by these cells and metabolized to obtain the energy necessary for cell viability. In addition, the presence of this sugar is able to adjust cellular metabolism, regulate gene expression and even influence cell growth. For this reason, glucose is considered as a "hormone". Specifically, it can trigger different signalling pathways that allow cells to adjust their gene expression programmes in response to glucose availability. Elucidating the molecular mechanisms of glucose response in eukaryotes has been greatly aided by studies conducted in the yeast Saccharomyces cerevisiae. This yeast shares with complex multicellular eukaryotes many of the signal transduction components that detect glucose, transmit the corresponding signals to the interior of the cell and make the needed adjustments to cellular metabolism and gene expression. In this manuscript, I will review the current knowledge of some aspects of glucose-mediated signalling in yeast and discuss how these results have contributed to the understanding of similar processes in mammalian cells.

  20. Identification of open quantum systems from observable time traces

    DOE PAGES

    Zhang, Jun; Sarovar, Mohan

    2015-05-27

    Estimating the parameters that dictate the dynamics of a quantum system is an important task for quantum information processing and quantum metrology, as well as fundamental physics. In our paper we develop a method for parameter estimation for Markovian open quantum systems using a temporal record of measurements on the system. Furthermore, the method is based on system realization theory and is a generalization of our previous work on identification of Hamiltonian parameters.

  1. Identification of dynamical biological systems based on random effects models.

    PubMed

    Batista, Levy; Bastogne, Thierry; Djermoune, El-Hadi

    2015-01-01

    System identification is a data-driven modeling approach more and more used in biology and biomedicine. In this application context, each assay is always repeated to estimate the response variability. The inference of the modeling conclusions to the whole population requires to account for the inter-individual variability within the modeling procedure. One solution consists in using random effects models but up to now no similar approach exists in the field of dynamical system identification. In this article, we propose a new solution based on an ARX (Auto Regressive model with eXternal inputs) structure using the EM (Expectation-Maximisation) algorithm for the estimation of the model parameters. Simulations show the relevance of this solution compared with a classical procedure of system identification repeated for each subject. PMID:26736981

  2. Numerical studies of identification in nonlinear distributed parameter systems

    NASA Technical Reports Server (NTRS)

    Banks, H. T.; Lo, C. K.; Reich, Simeon; Rosen, I. G.

    1989-01-01

    An abstract approximation framework and convergence theory for the identification of first and second order nonlinear distributed parameter systems developed previously by the authors and reported on in detail elsewhere are summarized and discussed. The theory is based upon results for systems whose dynamics can be described by monotone operators in Hilbert space and an abstract approximation theorem for the resulting nonlinear evolution system. The application of the theory together with numerical evidence demonstrating the feasibility of the general approach are discussed in the context of the identification of a first order quasi-linear parabolic model for one dimensional heat conduction/mass transport and the identification of a nonlinear dissipation mechanism (i.e., damping) in a second order one dimensional wave equation. Computational and implementational considerations, in particular, with regard to supercomputing, are addressed.

  3. Tris-sucrose buffer system: a new specially designed medium for extracellular invertase production by immobilized cells of isolated yeast Cryptococcus laurentii MT-61.

    PubMed

    Aydogan, Mehmet Nuri; Taskin, Mesut; Canli, Ozden; Arslan, Nazli Pinar; Ortucu, Serkan

    2014-01-01

    The aims of the present study were to isolate new yeasts with high extracellular (exo) invertase activity and to investigate the usability of buffer systems as invertase production media by immobilized yeast cells. Among 70 yeast isolates, Cryptococcus laurentii MT-61 had the highest exo-invertase activity. Immobilization of yeast cells was performed using sodium alginate. Higher exo-invertase activity for immobilized cells was achieved in tris-sucrose buffer system (TSBS) compared to sodium acetate buffer system and potassium phosphate buffer system. TSBS was prepared by dissolving 30 g of sucrose in 1 L of tris buffer solution. The optimum pH, temperature, and incubation time for invertase production with immobilized cells were determined as 8.0, 35 °C and 36 h in TSBS, respectively. Under optimized conditions, maximum exo-invertase activity was found to be 28.4 U/mL in sterile and nonsterile TSBS. Immobilized cells could be reused in 14 and 12 successive cycles in sterile and nonsterile TSBS without any loss in the maximum invertase activity, respectively. This is the first report which showed that immobilized microbial cells could be used as a biocatalyst for exo-invertase production in buffer system. As an additional contribution, a new yeast strain with high invertase activity was isolated.

  4. Modeling and Identification of a Large Gap Magnetic Suspension System

    NASA Technical Reports Server (NTRS)

    Cox, David E. (Editor); Groom, Nelson J. (Editor); Hsiao, Min-Hung; Huang, Jen-Kuang

    1996-01-01

    This paper presents the results of modeling and system identification efforts on the NASA Large-Angle Magnetic Suspension Test Fixture (LAMSTF). The LAMSTF consists of a cylindrical permanent magnet which is levitated above a planar array of five electromagnets mounted in a circular configuration. The analytical model is first developed and open-loop characteristics are described. The system is shown to be highly unstable and requires feedback control in order to apply system identification. Limitations on modeling accuracy due to the effect of eddy-currents on the system are discussed. An algorithm is derived to identify a state-space model for the system from input/output data acquired during closed-loop operation. The algorithm is tested on both the baseline system and a perturbed system which has an increased presence of eddy currents. It is found that for the baseline system the analytic model adequately captures the dynamics, although the identified model improves the simulation accuracy. For the system perturbed by additional unmodeled eddy-currents the analytic model is no longer adequate and a higher-order model, determined through system identification, is required to accurately predict the system's time response.

  5. Recent advances in yeast organelle and membrane proteomics.

    PubMed

    Premsler, Thomas; Zahedi, René Peiman; Lewandrowski, Urs; Sickmann, Albert

    2009-10-01

    Yeast proteome research comprises two different aspects: with respect to systemic fungal infections (fungemias), invasive candidiasis, for instance by Candida albicans, is among the most common causes of morbidity and mortality particularly in the expanding population of immunocompromised patients, which rises a high medical and pharmaceutical interest in this facultative pathogenic organism. Apart from its clinical relevance, yeast research moreover provides an indispensable source of knowledge regarding fundamental biochemical processes of eukaryotic cells. In this context, the budding yeast Saccharomyces cerevisiae is, in addition to its multiple industrial applications, one of the most extensively used microorganisms and serves as the best understood eukaryotic model system so far. Consequently, numerous studies have focused on gaining insight into the yeast proteome, with protein MS providing a very efficient technology to cope with this task since it enables both protein identification and differential quantification of cellular material. In this review we present an overview of recent advances in yeast organelle and membrane proteomics focusing on the cell wall, plasma membrane, mitochondria and vacuole.

  6. Immune System Toxicity and Immunotoxicity Hazard Identification

    EPA Science Inventory

    Exposure to chemicals may alter immune system health, increasing the risk of infections, allergy and autoimmune diseases. The chapter provides a concise overview of the immune system, host factors that affect immune system heal, and the effects that xenobiotic exposure may have ...

  7. Music identification system using MPEG-7 audio signature descriptors.

    PubMed

    You, Shingchern D; Chen, Wei-Hwa; Chen, Woei-Kae

    2013-01-01

    This paper describes a multiresolution system based on MPEG-7 audio signature descriptors for music identification. Such an identification system may be used to detect illegally copied music circulated over the Internet. In the proposed system, low-resolution descriptors are used to search likely candidates, and then full-resolution descriptors are used to identify the unknown (query) audio. With this arrangement, the proposed system achieves both high speed and high accuracy. To deal with the problem that a piece of query audio may not be inside the system's database, we suggest two different methods to find the decision threshold. Simulation results show that the proposed method II can achieve an accuracy of 99.4% for query inputs both inside and outside the database. Overall, it is highly possible to use the proposed system for copyright control. PMID:23533359

  8. Identification of nonlinear optical systems using adaptive kernel methods

    NASA Astrophysics Data System (ADS)

    Wang, Xiaodong; Zhang, Changjiang; Zhang, Haoran; Feng, Genliang; Xu, Xiuling

    2005-12-01

    An identification approach of nonlinear optical dynamic systems, based on adaptive kernel methods which are modified version of least squares support vector machine (LS-SVM), is presented in order to obtain the reference dynamic model for solving real time applications such as adaptive signal processing of the optical systems. The feasibility of this approach is demonstrated with the computer simulation through identifying a Bragg acoustic-optical bistable system. Unlike artificial neural networks, the adaptive kernel methods possess prominent advantages: over fitting is unlikely to occur by employing structural risk minimization criterion, the global optimal solution can be uniquely obtained owing to that its training is performed through the solution of a set of linear equations. Also, the adaptive kernel methods are still effective for the nonlinear optical systems with a variation of the system parameter. This method is robust with respect to noise, and it constitutes another powerful tool for the identification of nonlinear optical systems.

  9. Music Identification System Using MPEG-7 Audio Signature Descriptors

    PubMed Central

    You, Shingchern D.; Chen, Wei-Hwa; Chen, Woei-Kae

    2013-01-01

    This paper describes a multiresolution system based on MPEG-7 audio signature descriptors for music identification. Such an identification system may be used to detect illegally copied music circulated over the Internet. In the proposed system, low-resolution descriptors are used to search likely candidates, and then full-resolution descriptors are used to identify the unknown (query) audio. With this arrangement, the proposed system achieves both high speed and high accuracy. To deal with the problem that a piece of query audio may not be inside the system's database, we suggest two different methods to find the decision threshold. Simulation results show that the proposed method II can achieve an accuracy of 99.4% for query inputs both inside and outside the database. Overall, it is highly possible to use the proposed system for copyright control. PMID:23533359

  10. A network identity authentication system based on Fingerprint identification technology

    NASA Astrophysics Data System (ADS)

    Xia, Hong-Bin; Xu, Wen-Bo; Liu, Yuan

    2005-10-01

    Fingerprint verification is one of the most reliable personal identification methods. However, most of the automatic fingerprint identification system (AFIS) is not run via Internet/Intranet environment to meet today's increasing Electric commerce requirements. This paper describes the design and implementation of the archetype system of identity authentication based on fingerprint biometrics technology, and the system can run via Internet environment. And in our system the COM and ASP technology are used to integrate Fingerprint technology with Web database technology, The Fingerprint image preprocessing algorithms are programmed into COM, which deployed on the internet information server. The system's design and structure are proposed, and the key points are discussed. The prototype system of identity authentication based on Fingerprint have been successfully tested and evaluated on our university's distant education applications in an internet environment.

  11. Flight test planning and parameter extraction for rotorcraft system identification

    NASA Technical Reports Server (NTRS)

    Wang, J. C.; Demiroz, M. Y.; Talbot, P. D.

    1986-01-01

    The present study is concerned with the mathematical modelling of aircraft dynamics on the basis of an investigation conducted with the aid of the Rotor System Research Aircraft (RSRA). The particular characteristics of RSRA make it possible to investigate aircraft properties which cannot be readily studied elsewhere, for example in the wind tunnel. The considered experiment had mainly the objective to develop an improved understanding of the physics of rotor flapping dynamics and rotor loads in maneuvers. The employed approach is based on a utilization of parameter identification methodology (PID) with application to helicopters. A better understanding of the contribution of the main rotor to the overall aircraft forces and moments is also to be obtained. Attention is given to the mathematical model of a rotorcraft system, an integrated identification method, flight data processing, and the identification of RSRA mathematical models.

  12. Investigation of an expert systems approach to bacterial identification.

    PubMed

    Brammer, R J; Bryant, T N; May, J H

    1991-10-01

    An investigation was carried out to assess the feasibility of using an expert systems approach to assist in the identification of unknown isolates of bacteria. A system was developed using Lisp which utilized the knowledge stored in standard bacteriological texts. A comparison of the expert systems approach and the probabilistic approach based on Bayes Theorem was made together with the advantages and disadvantages of each approach. PMID:1747777

  13. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  14. Robust nonlinear system identification using neural-network models.

    PubMed

    Lu, S; Basar, T

    1998-01-01

    We study the problem of identification for nonlinear systems in the presence of unknown driving noise, using both feedforward multilayer neural network and radial basis function network models. Our objective is to resolve the difficulty associated with the persistency of excitation condition inherent to the standard schemes in the neural identification literature. This difficulty is circumvented here by a novel formulation and by using a new class of identification algorithms recently obtained by Didinsky et al. We show how these algorithms can be exploited to successfully identify the nonlinearity in the system using neural-network models. By embedding the original problem in one with noise-perturbed state measurements, we present a class of identifiers (under L1 and L2 cost criteria) which secure a good approximant for the system nonlinearity provided that some global optimization technique is used. In this respect, many available learning algorithms in the current neural-network literature, e.g., the backpropagation scheme and the genetic algorithms-based scheme, with slight modifications, can ensure the identification of the system nonlinearity. Subsequently, we address the same problem under a third, worst case L(infinity) criterion for an RBF modeling. We present a neural-network version of an H(infinity)-based identification algorithm from Didinsky et al and show how, along with an appropriate choice of control input to enhance excitation, under both full-state-derivative information (FSDI) and noise-perturbed full-state-information (NPFSI), it leads to satisfaction of a relevant persistency of excitation condition, and thereby to robust identification of the nonlinearity. Results from several simulation studies have been included to demonstrate the effectiveness of these algorithms.

  15. Temporal and Spatial Properties of a Yeast Multi-Cellular Amplification System Based on Signal Molecule Diffusion

    PubMed Central

    Jahn, Michael; Mölle, Annett; Rödel, Gerhard; Ostermann, Kai

    2013-01-01

    We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 × 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein. PMID:24233076

  16. Agroforestry Systems In Poland A Preliminary Identification

    NASA Astrophysics Data System (ADS)

    Borek, Robert

    2015-01-01

    This paper seeks to use state-of-the-art knowledge to depict the foundations and prospects for agroforestry systems in Poland to develop, in line with political, legal, historical and environmental conditions pertaining in the country. The main legal provisions concerning the presence of trees in agriculture are presented prior to a first-ever defining of key traditional agroforestry systems in Poland.

  17. System identification requirements for high-bandwidth rotorcraft flight control system design

    NASA Technical Reports Server (NTRS)

    Tischler, Mark B.

    1991-01-01

    The application of system identification methods to high-bandwidth rotorcraft flight control system design is examined. Flight test and modeling requirements are illustrated using flight test data from a BO-105 hingeless rotor helicopter. The proposed approach involves the identification of nonparametric (transfer function and state space) model identification. Results for the BO-105 show the need for including coupled body/rotor flapping and lead-lag dynamics in the identification model structure to allow the accurate prediction of control ssytem bandwidth limitations.

  18. On neural networks in identification and control of dynamic systems

    NASA Technical Reports Server (NTRS)

    Phan, Minh; Juang, Jer-Nan; Hyland, David C.

    1993-01-01

    This paper presents a discussion of the applicability of neural networks in the identification and control of dynamic systems. Emphasis is placed on the understanding of how the neural networks handle linear systems and how the new approach is related to conventional system identification and control methods. Extensions of the approach to nonlinear systems are then made. The paper explains the fundamental concepts of neural networks in their simplest terms. Among the topics discussed are feed forward and recurrent networks in relation to the standard state-space and observer models, linear and nonlinear auto-regressive models, linear, predictors, one-step ahead control, and model reference adaptive control for linear and nonlinear systems. Numerical examples are presented to illustrate the application of these important concepts.

  19. Early identification systems for emerging foodborne hazards.

    PubMed

    Marvin, H J P; Kleter, G A; Prandini, A; Dekkers, S; Bolton, D J

    2009-05-01

    This paper provides a non-exhausting overview of early warning systems for emerging foodborne hazards that are operating in the various places in the world. Special attention is given to endpoint-focussed early warning systems (i.e. ECDC, ISIS and GPHIN) and hazard-focussed early warning systems (i.e. FVO, RASFF and OIE) and their merit to successfully identify a food safety problem in an early stage is discussed. Besides these early warning systems which are based on monitoring of either disease symptoms or hazards, also early warning systems and/or activities that intend to predict the occurrence of a food safety hazard in its very beginning of development or before that are described. Examples are trend analysis, horizon scanning, early warning systems for mycotoxins in maize and/or wheat and information exchange networks (e.g. OIE and GIEWS). Furthermore, recent initiatives that aim to develop predictive early warning systems based on the holistic principle are discussed. The assumption of the researchers applying this principle is that developments outside the food production chain that are either directly or indirectly related to the development of a particular food safety hazard may also provide valuable information to predict the development of this hazard.

  20. Early identification systems for emerging foodborne hazards.

    PubMed

    Marvin, H J P; Kleter, G A; Prandini, A; Dekkers, S; Bolton, D J

    2009-05-01

    This paper provides a non-exhausting overview of early warning systems for emerging foodborne hazards that are operating in the various places in the world. Special attention is given to endpoint-focussed early warning systems (i.e. ECDC, ISIS and GPHIN) and hazard-focussed early warning systems (i.e. FVO, RASFF and OIE) and their merit to successfully identify a food safety problem in an early stage is discussed. Besides these early warning systems which are based on monitoring of either disease symptoms or hazards, also early warning systems and/or activities that intend to predict the occurrence of a food safety hazard in its very beginning of development or before that are described. Examples are trend analysis, horizon scanning, early warning systems for mycotoxins in maize and/or wheat and information exchange networks (e.g. OIE and GIEWS). Furthermore, recent initiatives that aim to develop predictive early warning systems based on the holistic principle are discussed. The assumption of the researchers applying this principle is that developments outside the food production chain that are either directly or indirectly related to the development of a particular food safety hazard may also provide valuable information to predict the development of this hazard. PMID:18272277

  1. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  2. 40 CFR 72.33 - Identification of dispatch system.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.33 Identification of dispatch system. (a... approval of the petition, all provisions of the Acid Rain Program applicable to an affected source or an... provision of the Acid Rain Program and shall not change the liability of the owners and operators of...

  3. 40 CFR 72.33 - Identification of dispatch system.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.33 Identification of dispatch system. (a... approval of the petition, all provisions of the Acid Rain Program applicable to an affected source or an... provision of the Acid Rain Program and shall not change the liability of the owners and operators of...

  4. 40 CFR 72.33 - Identification of dispatch system.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.33 Identification of dispatch system. (a... approval of the petition, all provisions of the Acid Rain Program applicable to an affected source or an... provision of the Acid Rain Program and shall not change the liability of the owners and operators of...

  5. 40 CFR 72.33 - Identification of dispatch system.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.33 Identification of dispatch system. (a... approval of the petition, all provisions of the Acid Rain Program applicable to an affected source or an... provision of the Acid Rain Program and shall not change the liability of the owners and operators of...

  6. 40 CFR 72.33 - Identification of dispatch system.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.33 Identification of dispatch system. (a... approval of the petition, all provisions of the Acid Rain Program applicable to an affected source or an... provision of the Acid Rain Program and shall not change the liability of the owners and operators of...

  7. Training Sessions Provide Working Knowledge of National Animal Identification System

    ERIC Educational Resources Information Center

    Glaze, J. Benton, Jr.; Ahola, Jason K.

    2010-01-01

    One in-service and two train-the-trainer workshops were conducted by University of Idaho Extension faculty, Idaho State Department of Agriculture personnel, and allied industry representatives to increase Extension educators' knowledge and awareness of the National Animal Identification System (NAIS) and related topics. Training sessions included…

  8. Screening of hepatocyte proteins binding with C‑terminally truncated surface antigen middle protein of hepatitis B virus (MHBst167) by a yeast two‑hybrid system.

    PubMed

    Li, Zhi Qun; Linghu, Enqiang; Jun, Wan; Cheng, Jun

    2014-09-01

    The function of middle hepatitis B surface protein C‑terminally truncated at amino acid position 167 (MHBst167) is not currently clear. This study aimed to screen and identify the proteins that interact with MHBst167 in hepatocytes using a yeast two‑hybrid system, and to explore the effects of MHBst167 in the development of hepatocellular carcinoma and precancerous diseases of the liver. The MHBst167 gene was amplified by polymerase chain reaction (PCR) and cloned into a pGEM‑T vector. The target region was sequenced and the constructed bait plasmid, pGBKT7‑MHBst167, was transformed into AH109 yeast cells. The transformed AH109 cells were then mated with Y187 yeast cells containing the fetal liver cDNA library plasmid using a yeast two‑hybrid system. The false positives were eliminated and the true positive clones were selected by PCR and sequencing analysis. The pGBKT7‑MHBst167 bait plasmid was successfully constructed and 66 clones grew in the selective synthetic defined media lacking leucine, tryptophan, histidine and adenine. Fifty‑two clones were identified following X‑α‑Gal selection and segregation analysis. Seven proteins were found to be expressed that could interact with MHBst167 in hepatocytes by the yeast two‑hybrid system. These results have provided novel insights into the biological functions of MHBst167.

  9. Yeast: A Research Organism for Teaching Genetics.

    ERIC Educational Resources Information Center

    Manney, Thomas R.; Manney, Monta L.

    1992-01-01

    Explains why laboratory strains of bakers yeast, Saccharomyces cerevisiae, are particularly suited for classroom science activities. Describes the sexual life cycle of yeast and the genetic system with visible mutations. Presents an overview of activities that can be done with yeast and gives a source for teachers to obtain more information. (PR)

  10. DNA barcode-based molecular identification system for fish species.

    PubMed

    Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

    2010-12-01

    In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp .

  11. Biometric identification systems: the science of transaction facilitation

    NASA Astrophysics Data System (ADS)

    Rogers, Robert R.

    1994-10-01

    The future ofthe "secure transaction" and the success ofall undertakings that depend on absolute certainty that the individuals involved really are who and what they represent themselves to be is dependent upon the successful development of absolutely accurate, low-cost and easy-to-operate Biometric Identification Systems. Whether these transactions are political, military, financial or administrative (e.g. health cards, drivers licenses, welfare entitlement, national identification cards, credit card transactions, etc.), the need for such secure and positive identification has never been greater -and yet we are only at the beginning ofan era in which we will see the emergence and proliferation of Biometric Identification Systems in nearly every field ofhuman endeavor. Proper application ofthese systems will change the way the world operates, and that is precisely the goal ofComparator Systems Corporation. Just as with the photo-copier 40 years ago and the personal computer 20 years ago, the potential applications for positive personal identification are going to make the Biometric Identification System a commonplace component in the standard practice ofbusiness, and in interhuman relationships ofall kinds. The development of new and specific application hardware, as well as the necessary algorithms and related software required for integration into existing operating procedures and newly developed systems alike, has been a more-than-a-decade-long process at Comparator -and we are now on the verge of delivering these systems to the world markets so urgently in need of them. An individual could feel extremely confident and satisfied ifhe could present his credit, debit, or ATM card at any point of sale and, after inserting his card, could simply place his finger on a glass panel and in less than a second be positively accepted as being the person that the card purported him to be; not to mention the security and satisfaction of the vendor involved in knowing that

  12. Development of pilot scale nanofiltration system for yeast industry wastewater treatment

    PubMed Central

    2014-01-01

    The treatment of the yeast industry wastewater was investigated by nanofiltration (NF) membrane process on a pilot scale. Two wastewaters were used as feed: (i) dilute wastewater with COD 2000 mg/L and (ii) concentrate wastewater with COD 8000 mg/L. The permeate flux, COD retention, color and electrical conductivity (EC) removal were evaluated in relation to trans-membrane pressure and long-term filtration. A linear growth in permeate flux was found with increasing in trans-membrane pressure for wastewaters. In addition, the COD retention, color and EC removal increased with trans-membrane pressure enhancement. The results obtained from the long-term nanofiltration of dilute wastewater indicated that the permeate flux decreased from 2300 L/day to 1250 L/day and COD retention increased from 86% to 92%. The quality of the permeate in term of COD is lower than the discharge standard in river (200 mg/L). Thus, this process is useful for treatment of wastewaters produced by yeast industry. PMID:24593865

  13. Genetic identification of yeast 18S rRNA residues required for efficient recruitment of initiator tRNA(Met) and AUG selection.

    PubMed

    Dong, Jinsheng; Nanda, Jagpreet S; Rahman, Hafsa; Pruitt, Margaret R; Shin, Byung-Sik; Wong, Chi-Ming; Lorsch, Jon R; Hinnebusch, Alan G

    2008-08-15

    High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNA(i)(Met) to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd(-) mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd(-) phenotypes that (like A928 substitutions) are suppressed by overexpressing tRNA(i)(Met). Hence, the nonconserved 928:1389 base pair in h28, plus conserved 18S rRNA residues corresponding to P-site contacts in bacterial ribosomes, are critical for efficient Met-tRNA(i)(Met) binding and AUG selection in eukaryotes.

  14. Performance metrics for the evaluation of hyperspectral chemical identification systems

    NASA Astrophysics Data System (ADS)

    Truslow, Eric; Golowich, Steven; Manolakis, Dimitris; Ingle, Vinay

    2016-02-01

    Remote sensing of chemical vapor plumes is a difficult but important task for many military and civilian applications. Hyperspectral sensors operating in the long-wave infrared regime have well-demonstrated detection capabilities. However, the identification of a plume's chemical constituents, based on a chemical library, is a multiple hypothesis testing problem which standard detection metrics do not fully describe. We propose using an additional performance metric for identification based on the so-called Dice index. Our approach partitions and weights a confusion matrix to develop both the standard detection metrics and identification metric. Using the proposed metrics, we demonstrate that the intuitive system design of a detector bank followed by an identifier is indeed justified when incorporating performance information beyond the standard detection metrics.

  15. Numerical Experimentation with Maximum Likelihood Identification in Static Distributed Systems

    NASA Technical Reports Server (NTRS)

    Scheid, R. E., Jr.; Rodriguez, G.

    1985-01-01

    Many important issues in the control of large space structures are intimately related to the fundamental problem of parameter identification. One might also ask how well this identification process can be carried out in the presence of noisy data since no sensor system is perfect. With these considerations in mind the algorithms herein are designed to treat both the case of uncertainties in the modeling and uncertainties in the data. The analytical aspects of maximum likelihood identification are considered in some detail in another paper. The questions relevant to the implementation of these schemes are dealt with, particularly as they apply to models of large space structures. The emphasis is on the influence of the infinite dimensional character of the problem on finite dimensional implementations of the algorithms. Those areas of current and future analysis are highlighted which indicate the interplay between error analysis and possible truncations of the state and parameter spaces.

  16. Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

    PubMed

    Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

    2016-02-29

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis. PMID:26854340

  17. Linear antigenic mapping of flagellin (FliC) from Salmonella enterica serovar Enteritidis with yeast surface expression system.

    PubMed

    Wang, Gaoling; Shi, Bingtian; Li, Tao; Zuo, Teng; Wang, Bin; Si, Wei; Xin, Jiuqing; Yang, Kongbin; Shi, Xuanlin; Liu, Siguo; Liu, Henggui

    2016-02-29

    Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne illness around the world and can have significant health implications in humans, poultry and other animals. Flagellin (FliC) is the primary component of bacterial flagella. It has been shown that the FliC of S. Enteritidis is a significant antigenic structure and can elicit strong humoral responses against S. Enteritidis infection in chickens. Here, we constructed a FliC antigen library using a yeast surface expression system. Yeast cells expressing FliC peptide antigens were labeled with chicken sera against S. Enteritidis and sorted using FACS. The analyses of FliC peptides revealed that the FliC linear antigenicity in chickens resided on three domains which were able to elicit strong humoral responses in vivo. Animal experiments further revealed that the antibodies elicited by these antigenic domains were able to significantly inhibit the invasion of S. Enteritidis into the liver and spleen of chickens. These findings will facilitate our better understanding of the humoral responses elicited by FliC in chickens upon infection by S. Enteritidis.

  18. 77 FR 40735 - Unique Device Identification System

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-10

    ... intended to be sterilized before each use; and stand-alone software. These types of devices have physical... Systems by Hospitals and Other Healthcare Facilities and on Statistical Methodologies to Interpret the... each use; and Stand-alone software. These devices involve unique risks to patients, and consequently...

  19. Effects of distillation system and yeast strain on the aroma profile of Albariño (Vitis vinifera L.) grape pomace spirits.

    PubMed

    Arrieta-Garay, Y; Blanco, P; López-Vázquez, C; Rodríguez-Bencomo, J J; Pérez-Correa, J R; López, F; Orriols, I

    2014-10-29

    Orujo is a traditional alcoholic beverage produced in Galicia (northwest Spain) from distillation of grape pomace, a byproduct of the winemaking industry. In this study, the effect of the distillation system (copper charentais alembic versus packed column) and the yeast strain (native yeast L1 versus commercial yeast L2) on the chemical and sensory characteristics of orujo obtained from Albariño (Vitis vinifera L.) grape pomace has been analyzed. Principal component analysis, with two components explaining 74% of the variance, is able to clearly differentiate the distillates according to distillation system and yeast strain. Principal component 1, mainly defined by C6-C12 esters, isoamyl octanoate, and methanol, differentiates L1 from L2 distillates. In turn, principal component 2, mainly defined by linear alcohols, linalool, and 1-hexenol, differentiates alembic from packed column distillates. In addition, an aroma descriptive test reveals that the distillate obtained with a packed column from a pomace fermented with L1 presented the highest positive general impression, which is associated with the highest fruity and smallest solvent aroma scores. Moreover, chemical analysis shows that use of a packed column increases average ethanol recovery by 12%, increases the concentration of C6-C12 esters by 25%, and reduces the concentration of higher alcohols by 21%. In turn, L2 yeast obtained lower scores in the alembic distillates aroma profile. In addition, with L1, 9% higher ethanol yields were achieved, and L2 distillates contained 34%-40% more methanol than L1 distillates.

  20. Development of an expert system for amino acid sequence identification.

    PubMed

    Hu, L; Saulinskas, E F; Johnson, P; Harrington, P B

    1996-08-01

    An expert system for amino acid sequence identification has been developed. The algorithm uses heuristic rules developed by human experts in protein sequencing. The system is applied to the chromatographic data of phenylthiohydantoin-amino acids acquired from an automated sequencer. The peak intensities in the current cycle are compared with those in the previous cycle, while the calibration and succeeding cycles are used as ancillary identification criteria when necessary. The retention time for each chromatographic peak in each cycle is corrected by the corresponding peak in the calibration cycle at the same run. The main improvement of our system compared with the onboard software used by the Applied Biosystems 477A Protein/Peptide Sequencer is that each peak in each cycle is assigned an identification name according to the corrected retention time to be used for the comparison with different cycles. The system was developed from analyses of ribonuclease A and evaluated by runs of four other protein samples that were not used in rule development. This paper demonstrates that rules developed by human experts can be automatically applied to sequence assignment. The expert system performed more accurately than the onboard software of the protein sequencer, in that the misidentification rates for the expert system were around 7%, whereas those for the onboard software were between 13 and 21%.

  1. Closed Loop System Identification with Genetic Algorithms

    NASA Technical Reports Server (NTRS)

    Whorton, Mark S.

    2004-01-01

    High performance control design for a flexible space structure is challenging since high fidelity plant models are di.cult to obtain a priori. Uncertainty in the control design models typically require a very robust, low performance control design which must be tuned on-orbit to achieve the required performance. Closed loop system identi.cation is often required to obtain a multivariable open loop plant model based on closed-loop response data. In order to provide an accurate initial plant model to guarantee convergence for standard local optimization methods, this paper presents a global parameter optimization method using genetic algorithms. A minimal representation of the state space dynamics is employed to mitigate the non-uniqueness and over-parameterization of general state space realizations. This control-relevant system identi.cation procedure stresses the joint nature of the system identi.cation and control design problem by seeking to obtain a model that minimizes the di.erence between the predicted and actual closed-loop performance.

  2. Fatty acid elongation in yeast--biochemical characteristics of the enzyme system and isolation of elongation-defective mutants.

    PubMed

    Dittrich, F; Zajonc, D; Hühne, K; Hoja, U; Ekici, A; Greiner, E; Klein, H; Hofmann, J; Bessoule, J J; Sperling, P; Schweizer, E

    1998-03-15

    Elongation of long-chain fatty acids was investigated in yeast mutants lacking endogenous de novo fatty acid synthesis. In this background, in vitro fatty acid elongation was dependent strictly on the substrates malonyl-CoA, NADPH and a medium-chain or long-chain acyl-CoA primer of 10 or more carbon atoms. Maximal activity was observed with primers containing 12-14 carbon atoms, while shorter-chain-length acyl-CoA were almost (octanoyl-CoA) or completely (hexanoyl-CoA, acetyl-CoA) inactive. In particular, acetyl-CoA was inactive as a primer and as extender unit. The Michaelis constants for octanoyl-CoA (0.33 mM), decanoyl-CoA (0.83 mM) lauroyl-CoA (0.05 mM), myristoyl-CoA (0.4 mM) and palmitoyl-CoA (0.13 mM) were determined and were comparable for fatty acid synthesis and elongation. In contrast, the affinity of malonyl-CoA was 17-fold lower for elongation (Km = 0.13 mM) than for the fatty acid synthase (FAS) system. With increasing chain length of the primer (> or = 12:0), fatty acid elongation becomes increasingly sensitive to substrate inhibition. Due to the activation of endogenous fatty acids, ATP exhibits a stimulatory effect at suboptimal but not at saturating substrate concentrations. In the yeast cell homogenate, the specific activity of fatty acid elongation is about 10-20-fold lower than that of de novo fatty acid synthesis. The same elongation activity is observed in respiratory competent and in mitochondrially defective cells. The products of in vitro fatty acid elongation are fatty acids of 15-17 or 22-26 carbon atoms, depending on whether tridecanoyl-CoA or stearoyl-CoA is used as a primer. In vitro, the elongation products are converted in part, by alpha-oxidation, to their odd-chain-length lower homologues or are hydrolyzed to fatty acids. In contrast, no odd-chain-length elongation products or very-long-chain fatty acids (VLCFA) shorter than 26:0 are observed in vivo. Hence, VLCFA synthesis exhibits a higher processivity in vivo than in the cell

  3. Linking yeast genetics to mammalian genomes: identification and mapping of the human homolog of CDC27 via the expressed sequence tag (EST) data base.

    PubMed Central

    Tugendreich, S; Boguski, M S; Seldin, M S; Hieter, P

    1993-01-01

    We describe a strategy for quickly identifying and positionally mapping human homologs of yeast genes to cross-reference the biological and genetic information known about yeast genes to mammalian chromosomal maps. Optimized computer search methods have been developed to scan the rapidly expanding expressed sequence tag (EST) data base to find human open reading frames related to yeast protein sequence queries. These methods take advantage of the newly developed BLOSUM scoring matrices and the query masking function SEG. The corresponding human cDNA is then used to obtain a high-resolution map position on human and mouse chromosomes, providing the links between yeast genetic analysis and mapped mammalian loci. By using these methods, a human homolog of Saccharomyces cerevisiae CDC27 has been identified and mapped to human chromosome 17 and mouse chromosome 11 between the Pkca and Erbb-2 genes. Human CDC27 encodes an 823-aa protein with global similarity to its fungal homologs CDC27, nuc2+, and BimA. Comprehensive cross-referencing of genes and mutant phenotypes described in humans, mice, and yeast should accelerate the study of normal eukaryotic biology and human disease states. Images Fig. 2 PMID:8234252

  4. Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii.

    PubMed

    Boretsky, Yuriy R; Pynyaha, Yuriy V; Boretsky, Volodymyr Y; Kutsyaba, Vasyl I; Protchenko, Olga V; Philpott, Caroline C; Sibirny, Andriy A

    2007-07-01

    Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene. PMID:17467833

  5. Material Outgassing, Identification and Deposition, MOLIDEP System

    NASA Technical Reports Server (NTRS)

    Scialdone, John J.; Montoya, Alex F.

    2002-01-01

    The outgassing tests are performed employing a modified vacuum operated Cahn analytical microbalance, identified as the MOLIDEP system. The test measures under high vacuum, the time varying Molecular mass loss of a material sample held at a chosen temperature; it Identifies the outgassing molecular components using an inline SRS 300 amu Residual Gas Analyzer (RGA) and employs a temperature controlled 10 MHz Quartz Crystal Microbalance (QCM) to measure the condensable DEPosits. Both the QCM and the RGA intercept within the conductive passage the outgassing products being evacuated by a turbomolecular pump. The continuous measurements of the mass loss, the rate of loss, the sample temperature, the rate of temperature change, the QCM temperature and the QCM recorded condensable deposits or rate of deposits are saved to an Excel spreadsheet. A separate computer controls the RGA.

  6. Prefire identification for pulse power systems

    DOEpatents

    Longmire, Jerry L.; Thuot, Michael E.; Warren, David S.

    1985-01-01

    Prefires in a high-power, high-frequency, multi-stage pulse generator are detected by a system having an EMI shielded pulse timing transmitter associated with and tailored to each stage of the pulse generator. Each pulse timing transmitter upon detection of a pulse triggers a laser diode to send an optical signal through a high frequency fiber optic cable to a pulse timing receiver which converts the optical signal to an electrical pulse. The electrical pulses from all pulse timing receivers are fed through an OR circuit to start a time interval measuring device and each electrical pulse is used to stop an individual channel in the measuring device thereby recording the firing sequence of the multi-stage pulse generator.

  7. Prefire identification for pulse-power systems

    DOEpatents

    Longmire, J.L.; Thuot, M.E.; Warren, D.S.

    1982-08-23

    Prefires in a high-power, high-frequency, multi-stage pulse generator are detected by a system having an EMI shielded pulse timing transmitter associated with and tailored to each stage of the pulse generator. Each pulse timing transmitter upon detection of a pulse triggers a laser diode to send an optical signal through a high frequency fiber optic cable to a pulse timing receiver which converts the optical signal to an electrical pulse. The electrical pulses from all pulse timing receivers are fed through an OR circuit to start a time interval measuring device and each electrical pulse is used to stop an individual channel in the measuring device thereby recording the firing sequence of the multi-stage pulse generator.

  8. System Identification in Presence of Outliers.

    PubMed

    Yu, Chao; Wang, Qing-Guo; Zhang, Dan; Wang, Lei; Huang, Jiangshuai

    2016-05-01

    The outlier detection problem for dynamic systems is formulated as a matrix decomposition problem with low rank and sparse matrices, and further recast as a semidefinite programming problem. A fast algorithm is presented to solve the resulting problem while keeping the solution matrix structure and it can greatly reduce the computational cost over the standard interior-point method. The computational burden is further reduced by proper construction of subsets of the raw data without violating low-rank property of the involved matrix. The proposed method can make exact detection of outliers in case of no or little noise in output observations. In case of significant noise, a novel approach based on under-sampling with averaging is developed to denoise while retaining the saliency of outliers, and so-filtered data enables successful outlier detection with the proposed method while the existing filtering methods fail. Use of recovered "clean" data from the proposed method can give much better parameter estimation compared with that based on the raw data.

  9. System Identification and POD Method Applied to Unsteady Aerodynamics

    NASA Technical Reports Server (NTRS)

    Tang, Deman; Kholodar, Denis; Juang, Jer-Nan; Dowell, Earl H.

    2001-01-01

    The representation of unsteady aerodynamic flow fields in terms of global aerodynamic modes has proven to be a useful method for reducing the size of the aerodynamic model over those representations that use local variables at discrete grid points in the flow field. Eigenmodes and Proper Orthogonal Decomposition (POD) modes have been used for this purpose with good effect. This suggests that system identification models may also be used to represent the aerodynamic flow field. Implicit in the use of a systems identification technique is the notion that a relative small state space model can be useful in describing a dynamical system. The POD model is first used to show that indeed a reduced order model can be obtained from a much larger numerical aerodynamical model (the vortex lattice method is used for illustrative purposes) and the results from the POD and the system identification methods are then compared. For the example considered, the two methods are shown to give comparable results in terms of accuracy and reduced model size. The advantages and limitations of each approach are briefly discussed. Both appear promising and complementary in their characteristics.

  10. A fuzzy logic system for Raman spectrum identification

    NASA Astrophysics Data System (ADS)

    Castanys, M.; Soneira, M. J.; Perez-Pueyo, R.; Ruiz-Moreno, S.

    2005-06-01

    Raman Spectroscopy is a fast, rugged analytical technique based on the Raman Effect. When monochromatic light encounters matter, most of the scattered light has the same wavelength as the incident light. However, a small fraction of the scattered light is shifted in a different wavelength by the molecular vibrations and rotations in the sample. The representation of this shifted light is called Raman spectrum, and contains many sharp bands characteristics of the sample, allowing its identification without ambiguity. In this communication, a fuzzy logic system to recognize Raman spectra of artistic pigments is presented. The identification is based on the comparison between an unknown spectrum, and pattern spectra. Frequently the comparison is made by the spectrospist by visual inspection, but this is slow and imprecise. In order to mitigate this problematic, a system based on the fuzzy logic technique to identify Raman spectra is presented. The methodology consists on implementing the comparison with the Correlation. However, a Raman spectrum is inevitably affected by noise which introduces ambiguity into the correlation values. Fuzzy Logic provides a simple way to draw conclusions from imprecise data. The fuzzy identification system is based on the following statement: when the correlation between the unidentified and the pattern is enough high, the analysed pigment is recognized as the pigment which corresponds to this pattern. The membership functions, which characterize the fuzzy sets at the input (Correlation) and output (Identified/ Not_Identified) of the system, and the inference mechanism suitable for the problem, are chosen.

  11. Effect of sterol metabolism in the yeast-Drosophila system on the frequency of radiation-induced aneuploidy in the Drosophila melanogaster oocytes

    SciTech Connect

    Savitskii, V.V.; Luchnikova, E.M.; Inge-Vechtomov, S.G.

    1986-01-01

    The effect of sterol metabolism on induced mutagenesis of Drosophila melanogaster was studied in the ecogenetic system of yeast-Drosophila. Sterol deficiency was created in Drosophila by using the biomass of live cells of Saccharomyces cerevisiae strain 9-2-P712 till mutation in locus nys/sup r1/ blocking the synthesis of ergosterol as the food. It was found that rearing of Drosophila females on the mutant yeast increases the frequency of loss and nondisjunction of X chromosomes induced in mature oocytes by X rays (1000 R). Addition of 0.1% of cholesterol solution in 10% ethanol to the yeast biomass restores the resistance of oocyte to X irradiation to the control level. The possible hormonal effect on membrane leading to increased radiation-induced aneuploidy in Drosophila and the role of sterol metabolism in determining the resistance to various damaging factors are discussed.

  12. Continuous-time system identification of a smoking cessation intervention

    NASA Astrophysics Data System (ADS)

    Timms, Kevin P.; Rivera, Daniel E.; Collins, Linda M.; Piper, Megan E.

    2014-07-01

    Cigarette smoking is a major global public health issue and the leading cause of preventable death in the United States. Toward a goal of designing better smoking cessation treatments, system identification techniques are applied to intervention data to describe smoking cessation as a process of behaviour change. System identification problems that draw from two modelling paradigms in quantitative psychology (statistical mediation and self-regulation) are considered, consisting of a series of continuous-time estimation problems. A continuous-time dynamic modelling approach is employed to describe the response of craving and smoking rates during a quit attempt, as captured in data from a smoking cessation clinical trial. The use of continuous-time models provide benefits of parsimony, ease of interpretation, and the opportunity to work with uneven or missing data.

  13. An experimental study of nonlinear dynamic system identification

    NASA Astrophysics Data System (ADS)

    Stry, Greselda I.; Mook, D. Joseph

    1990-12-01

    A technique for robust identification of nonlinear dynamic systems is developed and illustrated using both simulations and analog experiments. The technique is based on the Minimum Model Error optimal estimation approach. A detailed literature review is included in which fundamental differences between the current approach and previous work is described. The most significant feature of the current work is the ability to identify nonlinear dynamic systems without prior assumptions regarding the form of the nonlinearities, in constrast to existing nonlinear identification approaches which usually require detailed assumptions of the nonlinearities. The example illustrations indicate that the method is robust with respect to prior ignorance of the model, and with respect to measurement noise, measurement frequency, and measurement record length.

  14. Continuous-Time System Identification of a Smoking Cessation Intervention.

    PubMed

    Timms, Kevin P; Rivera, Daniel E; Collins, Linda M; Piper, Megan E

    2014-01-01

    Cigarette smoking is a major global public health issue and the leading cause of preventable death in the United States. Toward a goal of designing better smoking cessation treatments, system identification techniques are applied to intervention data to describe smoking cessation as a process of behavior change. System identification problems that draw from two modeling paradigms in quantitative psychology (statistical mediation and self-regulation) are considered, consisting of a series of continuous-time estimation problems. A continuous-time dynamic modeling approach is employed to describe the response of craving and smoking rates during a quit attempt, as captured in data from a smoking cessation clinical trial. The use of continuous-time models provide benefits of parsimony, ease of interpretation, and the opportunity to work with uneven or missing data. PMID:25382865

  15. Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

    PubMed Central

    Bivi, Nicoletta; Romanello, Milena; Harrison, Richard; Clarke, Ian; Hoyle, David C; Moro, Luigi; Ortolani, Fulvia; Bonetti, Antonella; Quadrifoglio, Franco; Tell, Gianluca; Delneri, Daniela

    2009-01-01

    Background Nitrogen-containing bisphosphonates are the elected drugs for the treatment of diseases in which excessive bone resorption occurs, for example, osteoporosis and cancer-induced bone diseases. The only known target of nitrogen-containing bisphosphonates is farnesyl pyrophosphate synthase, which ensures prenylation of prosurvival proteins, such as Ras. However, it is likely that the action of nitrogen-containing bisphosphonates involves additional unknown mechanisms. To identify novel targets of nitrogen-containing bisphosphonates, we used a genome-wide high-throughput screening in which 5,936 Saccharomyces cerevisiae heterozygote barcoded mutants were grown competitively in the presence of sub-lethal doses of three nitrogen-containing bisphosphonates (risedronate, alendronate and ibandronate). Strains carrying deletions in genes encoding potential drug targets show a variation of the intensity of their corresponding barcodes on the hybridization array over the time. Results With this approach, we identified novel targets of nitrogen-containing bisphosphonates, such as tubulin cofactor B and ASK/DBF4 (Activator of S-phase kinase). The up-regulation of tubulin cofactor B may explain some previously unknown effects of nitrogen-containing bisphosphonates on microtubule dynamics and organization. As nitrogen-containing bisphosphonates induce extensive DNA damage, we also document the role of DBF4 as a key player in nitrogen-containing bisphosphonate-induced cytotoxicity, thus explaining the effects on the cell-cycle. Conclusions The dataset obtained from the yeast screen was validated in a mammalian system, allowing the discovery of new biological processes involved in the cellular response to nitrogen-containing bisphosphonates and opening up opportunities for development of new anticancer drugs. PMID:19744312

  16. Heat shock partially dissociates the overlapping modules of the yeast protein-protein interaction network: a systems level model of adaptation.

    PubMed

    Mihalik, Ágoston; Csermely, Peter

    2011-10-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a 'stratus-cumulus' type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes. PMID:22022244

  17. Heat shock partially dissociates the overlapping modules of the yeast protein-protein interaction network: a systems level model of adaptation.

    PubMed

    Mihalik, Ágoston; Csermely, Peter

    2011-10-01

    Network analysis became a powerful tool giving new insights to the understanding of cellular behavior. Heat shock, the archetype of stress responses, is a well-characterized and simple model of cellular dynamics. S. cerevisiae is an appropriate model organism, since both its protein-protein interaction network (interactome) and stress response at the gene expression level have been well characterized. However, the analysis of the reorganization of the yeast interactome during stress has not been investigated yet. We calculated the changes of the interaction-weights of the yeast interactome from the changes of mRNA expression levels upon heat shock. The major finding of our study is that heat shock induced a significant decrease in both the overlaps and connections of yeast interactome modules. In agreement with this the weighted diameter of the yeast interactome had a 4.9-fold increase in heat shock. Several key proteins of the heat shock response became centers of heat shock-induced local communities, as well as bridges providing a residual connection of modules after heat shock. The observed changes resemble to a 'stratus-cumulus' type transition of the interactome structure, since the unstressed yeast interactome had a globally connected organization, similar to that of stratus clouds, whereas the heat shocked interactome had a multifocal organization, similar to that of cumulus clouds. Our results showed that heat shock induces a partial disintegration of the global organization of the yeast interactome. This change may be rather general occurring in many types of stresses. Moreover, other complex systems, such as single proteins, social networks and ecosystems may also decrease their inter-modular links, thus develop more compact modules, and display a partial disintegration of their global structure in the initial phase of crisis. Thus, our work may provide a model of a general, system-level adaptation mechanism to environmental changes.

  18. The Yeast Three-Hybrid System as an Experimental Platform to Identify Proteins Interacting with Small Signaling Molecules in Plant Cells: Potential and Limitations

    PubMed Central

    Cottier, Stéphanie; Mönig, Timon; Wang, Zheming; Svoboda, Jiří; Boland, Wilhelm; Kaiser, Markus; Kombrink, Erich

    2011-01-01

    Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules. PMID:22639623

  19. Rapid identification of Listeria spp.: an AOAC performance test of the MIT 1000 rapid microbial identification system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods that rapidly confirm the identification of foodborne pathogens are highly desired. The Micro Imaging Technology (MIT) 1000 Rapid Microbial Identification (RMID) System is a benchtop instrument that detects laser light scattered from individual bacterial cells in solution with an array of 35 ...

  20. Applicability of an in-House Saponin-Based Extraction Method in Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Bacterial and Fungal Species in Positively Flagged Blood Cultures

    PubMed Central

    Chien, Jung-Yien; Lee, Tai-Fen; Du, Shin-Hei; Teng, Shih-Hua; Liao, Chun-Hsing; Sheng, Wang-Hui; Teng, Lee-Jene; Hsueh, Po-Ren

    2016-01-01

    We used an in-house saponin-based extraction method to evaluate the performance of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) system for the identification of bacteria and fungi in 405 positively flagged blood culture bottles. Results obtained from MALDI-TOF/MS were compared with those obtained using conventional phenotypic identification methods. Of the 405 positively flagged blood culture bottles, 365 showed monomicrobal growth and were correctly identified to the species (72.1%) or genus (89.6%) level using the Bruker Biotyper system. The remaining 40 positively flagged blood culture bottles showed polymicrobial growth. Of them, 82.5% (n = 33) of the isolates were correctly identified to the species level and 92.5% (n = 37) to the genus level using the Bruker Biotyper system. The overall accuracy of identification to the genus level in flagged blood cultures was 89.5% for Gram-positive organisms, 93.5% for Gram-negative pathogens and 71.9% for fungi. Confidence scores were ≥1.500 for 307 (75.8%) bottles, ≥1.700 for 249 (61.5%) bottles and ≥2.000 for 142 (35.1%) bottles. None of the yeast cultures yielded scores ≥1.700. Using an identification-score cutoff of ≥1.500, the MALDI Biotyper correctly identified 99.2% of Gram-positive bacteria, 97.6% of Gram-negative bacteria and 100% of yeast isolates to the genus level and 77.6% of Gram-positive bacteria, 87.1% of Gram-negative bacteria and 100.0% of yeast isolates to the species level. The overall rate of identification using our protocol was 89.9% (364/405) for genus level identification and 73.1% (296/405) for species level identification. Yeast isolates yielded the lowest confidence scores, which compromised the accuracy of identification. Further optimization of the protein extraction procedure in positive blood cultures is needed to improve the rate of identification.

  1. Applicability of an in-House Saponin-Based Extraction Method in Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry System for Identification of Bacterial and Fungal Species in Positively Flagged Blood Cultures

    PubMed Central

    Chien, Jung-Yien; Lee, Tai-Fen; Du, Shin-Hei; Teng, Shih-Hua; Liao, Chun-Hsing; Sheng, Wang-Hui; Teng, Lee-Jene; Hsueh, Po-Ren

    2016-01-01

    We used an in-house saponin-based extraction method to evaluate the performance of the Bruker Biotyper matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) system for the identification of bacteria and fungi in 405 positively flagged blood culture bottles. Results obtained from MALDI-TOF/MS were compared with those obtained using conventional phenotypic identification methods. Of the 405 positively flagged blood culture bottles, 365 showed monomicrobal growth and were correctly identified to the species (72.1%) or genus (89.6%) level using the Bruker Biotyper system. The remaining 40 positively flagged blood culture bottles showed polymicrobial growth. Of them, 82.5% (n = 33) of the isolates were correctly identified to the species level and 92.5% (n = 37) to the genus level using the Bruker Biotyper system. The overall accuracy of identification to the genus level in flagged blood cultures was 89.5% for Gram-positive organisms, 93.5% for Gram-negative pathogens and 71.9% for fungi. Confidence scores were ≥1.500 for 307 (75.8%) bottles, ≥1.700 for 249 (61.5%) bottles and ≥2.000 for 142 (35.1%) bottles. None of the yeast cultures yielded scores ≥1.700. Using an identification-score cutoff of ≥1.500, the MALDI Biotyper correctly identified 99.2% of Gram-positive bacteria, 97.6% of Gram-negative bacteria and 100% of yeast isolates to the genus level and 77.6% of Gram-positive bacteria, 87.1% of Gram-negative bacteria and 100.0% of yeast isolates to the species level. The overall rate of identification using our protocol was 89.9% (364/405) for genus level identification and 73.1% (296/405) for species level identification. Yeast isolates yielded the lowest confidence scores, which compromised the accuracy of identification. Further optimization of the protein extraction procedure in positive blood cultures is needed to improve the rate of identification. PMID:27695442

  2. System IDentification Programs for AirCraft (SIDPAC)

    NASA Technical Reports Server (NTRS)

    Morelli, Eugene A.

    2002-01-01

    A collection of computer programs for aircraft system identification is described and demonstrated. The programs, collectively called System IDentification Programs for AirCraft, or SIDPAC, were developed in MATLAB as m-file functions. SIDPAC has been used successfully at NASA Langley Research Center with data from many different flight test programs and wind tunnel experiments. SIDPAC includes routines for experiment design, data conditioning, data compatibility analysis, model structure determination, equation-error and output-error parameter estimation in both the time and frequency domains, real-time and recursive parameter estimation, low order equivalent system identification, estimated parameter error calculation, linear and nonlinear simulation, plotting, and 3-D visualization. An overview of SIDPAC capabilities is provided, along with a demonstration of the use of SIDPAC with real flight test data from the NASA Glenn Twin Otter aircraft. The SIDPAC software is available without charge to U.S. citizens by request to the author, contingent on the requestor completing a NASA software usage agreement.

  3. Computational requirements for on-orbit identification of space systems

    NASA Technical Reports Server (NTRS)

    Hadaegh, Fred Y.

    1988-01-01

    For the future space systems, on-orbit identification (ID) capability will be required to complement on-orbit control, due to the fact that the dynamics of large space structures, spacecrafts, and antennas will not be known sufficiently from ground modeling and testing. The computational requirements for ID of flexible structures such as the space station (SS) or the large deployable reflectors (LDR) are however, extensive due to the large number of modes, sensors, and actuators. For these systems the ID algorithm operations need not be computed in real-time, only in near real-time, or an appropriate mission time. Consequently the space systems will need advanced processors and efficient parallel processing algorithm design and architectures to implement the identification algorithms in near real-time. The MAX computer currently being developed may handle such computational requirements. The purpose is to specify the on-board computational requirements for dynamic and static identification for large space structures. The computational requirements for six ID algorithms are presented in the context of three examples: the JPL/AFAL ground antenna facility, the space station (SS), and the large deployable reflector (LDR).

  4. Robust uncertainty evaluation for system identification on distributed wireless platforms

    NASA Astrophysics Data System (ADS)

    Crinière, Antoine; Döhler, Michael; Le Cam, Vincent; Mevel, Laurent

    2016-04-01

    Health monitoring of civil structures by system identification procedures from automatic control is now accepted as a valid approach. These methods provide frequencies and modeshapes from the structure over time. For a continuous monitoring the excitation of a structure is usually ambient, thus unknown and assumed to be noise. Hence, all estimates from the vibration measurements are realizations of random variables with inherent uncertainty due to (unknown) process and measurement noise and finite data length. The underlying algorithms are usually running under Matlab under the assumption of large memory pool and considerable computational power. Even under these premises, computational and memory usage are heavy and not realistic for being embedded in on-site sensor platforms such as the PEGASE platform. Moreover, the current push for distributed wireless systems calls for algorithmic adaptation for lowering data exchanges and maximizing local processing. Finally, the recent breakthrough in system identification allows us to process both frequency information and its related uncertainty together from one and only one data sequence, at the expense of computational and memory explosion that require even more careful attention than before. The current approach will focus on presenting a system identification procedure called multi-setup subspace identification that allows to process both frequencies and their related variances from a set of interconnected wireless systems with all computation running locally within the limited memory pool of each system before being merged on a host supervisor. Careful attention will be given to data exchanges and I/O satisfying OGC standards, as well as minimizing memory footprints and maximizing computational efficiency. Those systems are built in a way of autonomous operations on field and could be later included in a wide distributed architecture such as the Cloud2SM project. The usefulness of these strategies is illustrated on

  5. Effect of yeast-derived products on systemic innate immune response of broiler chickens following a lipopolysaccharide challenge.

    PubMed

    Alizadeh, M; Rodriguez-Lecompte, J C; Yitbarek, A; Sharif, S; Crow, G; Slominski, B A

    2016-10-01

    This study evaluated the effect of yeast-derived products on growth performance, serum antibody levels, and mRNA gene expression of pattern-recognition receptors, and cytokines in broiler chickens. Two hundred and sixteen one-day-old male broiler chickens (Ross-308) were randomly assigned to six dietary treatments with six replicates (cage) of 6 birds per cage. Dietary treatments consisted of a Control diet without antibiotics (C), and diets containing 11 mg/kg of "virginiamycin", 0.25% of yeast cell wall (YCW), 0.2% of a commercial product "Maxi-Gen Plus" containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 8% of distiller's dried grains with solubles (DDGS). On d 21 post-hatch blood samples were collected from 6 birds per treatment and serum sample were analyzed for antibody levels. After blood sampling, birds were injected intraperitoneally with 3 mg/kg of BW of lipopolysaccharide (LPS). The unchallenged group was fed the Control diet and injected with saline solution. Spleen samples were collected to measure the gene expression of toll-like receptors (TLR)2b, TLR4, and TLR21, macrophage mannose receptor (MMR), and cytokines including interleukin (IL)-12, IL-10, IL-4, IL-6, IL-18, and interferon (IFN)-γ. No significant difference in body weight gain, feed intake, and FCR were observed among treatments. Regarding humoral immunity, the diet supplemented with YCW increased serum immunoglobulin (Ig)A level compared with the antibiotic group; however, serum concentrations of IgG and IgM were not affected by dietary treatments. Relative gene expression of TLR2 and TLR4 was not affected by dietary treatments, whereas the expression of TLR21 and MRR was upregulated in diets containing YCW and DDGS. The diet supplemented with YCW increased the expression of all cytokines, and expression of IFN-γ was upregulated in the DDGS group. However, no significant difference was observed for cytokine gene expression in the antibiotic and

  6. Identification of novel host factors via conserved domain search: Cns1 cochaperone is a novel restriction factor of tombusvirus replication in yeast.

    PubMed

    Lin, Jing-Yi; Nagy, Peter D

    2013-12-01

    A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors. PMID:24027337

  7. Identification of Novel Protein-Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs.

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a small number of clones will dominate the selection output, making it difficult to comprehensively identify potentially important interactions due to low representation in the selection output. We have developed a novel method to address this problem. By analyzing selection outputs using high-density human exon microarrays, the full potential of selection output diversity can be revealed in one experiment. FACS-based selection using yeast surface displayed human cDNA libraries combined with exon microarray analysis of the selection outputs is a powerful way of rapidly identifying protein fragments with affinity for any soluble ligand that can be fluorescently detected, including small biological molecules and drugs. In this report we present protocols for exon microarray-based analysis of yeast surface display human cDNA library selection outputs. PMID:26060075

  8. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    EPA Science Inventory

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  9. Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

    PubMed

    Visintin, Simonetta; Alessandria, Valentina; Valente, Antonio; Dolci, Paola; Cocolin, Luca

    2016-01-01

    Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product.

  10. Impairment of the glycolytic system and actin in baker's yeast during frozen storage.

    PubMed

    Hatano, S; Udou, M; Koga, N; Honjoh, K; Miyamoto, T

    1996-01-01

    After frozen storage for 7 d, the viability and CO2 productivity of a conventional baker's yeast strain D greatly decreased. The viability of a freeze-tolerant strain, DFT, used for the frozen dough method slightly decreased after the same storage period, while the CO2 productivity greatly decreased. The CO2 productivity and DNase I inhibitory activity of actin of the cell-free extracts prepared immediately after thawing from 7-d frozen-stored cells markedly decreased in both strains. In DFT, however, the productivity and the inhibitory activity of the cell-free extract increased when the extract was prepared after incubation of the frozen-thawed cells at 30 degrees C. The increase in the inhibitory activity first occurred and then the increase in the CO2 productivity. Gel filtration patterns of actin and glycolytic enzymes were compared between cell-free extracts of both strains. Peaks of actin and activity peaks of hexokinase and pyruvate kinase decreased in the strain D after frozen storage, but only slightly in the strain DFT. After frozen storage, phosphofructokinase activity peak shifted to a lower molecular weight in strain D. PMID:8824826

  11. Protein-protein interactions in two potyviruses using the yeast two-hybrid system.

    PubMed

    Lin, Lin; Shi, Yuhong; Luo, Zhaopeng; Lu, Yuwen; Zheng, Hongying; Yan, Fei; Chen, Jiong; Chen, Jianping; Adams, M J; Wu, Yunfeng

    2009-06-01

    Interactions between all ten mature proteins of the potyviruses Soybean mosaic virus (Pinellia ternata isolate) and Shallot yellow stripe virus were investigated using yeast two-hybrid (Y2H) assays. Consistently strong self-interactions were found between the pairs of HC-Pro, VPg, NIa-Pro, NIb and CP in both viruses. Apart from the NIb, such interactions have been previously reported for some other potyviruses. The 6K1/NIa-Pro combination gave a consistently moderate to strong interaction in both directions for both viruses. This interaction occurred even when the 6K1 of SMV-P was truncated to eliminate the C-terminal motif that acts as a recognition site for cleavage by the NIa-Pro. Many other interactions occurred only in one direction or only for one of the two viruses. When taken together with other published reports, the data suggest that interactions detected by Y2H should be regarded as only preliminary indications. PMID:19189854

  12. A Systems Approach to Elucidate Heterosis of Protein Abundances in Yeast*

    PubMed Central

    Blein-Nicolas, Mélisande; Albertin, Warren; da Silva, Telma; Valot, Benoît; Balliau, Thierry; Masneuf-Pomarède, Isabelle; Bely, Marina; Marullo, Philippe; Sicard, Delphine; Dillmann, Christine; de Vienne, Dominique; Zivy, Michel

    2015-01-01

    Heterosis is a universal phenomenon that has major implications in evolution and is of tremendous agro-economic value. To study the molecular manifestations of heterosis and to find factors that maximize its strength, we implemented a large-scale proteomic experiment in yeast. We analyzed the inheritance of 1,396 proteins in 55 inter- and intraspecific hybrids obtained from Saccharomyces cerevisiae and S. uvarum that were grown in grape juice at two temperatures. We showed that the proportion of heterotic proteins was highly variable depending on the parental strain and on the temperature considered. For intraspecific hybrids, this proportion was higher at nonoptimal temperature. Unexpectedly, heterosis for protein abundance was strongly biased toward positive values in interspecific hybrids but not in intraspecific hybrids. Computer modeling showed that this observation could be accounted for by assuming concave relationships between protein abundances and their controlling factors, in line with the metabolic model of heterosis. These results point to nonlinear processes that could play a central role in heterosis. PMID:25971257

  13. Communications device identification methods, communications methods, wireless communications readers, wireless communications systems, and articles of manufacture

    DOEpatents

    Steele, Kerry D [Kennewick, WA; Anderson, Gordon A [Benton City, WA; Gilbert, Ronald W [Morgan Hill, CA

    2011-02-01

    Communications device identification methods, communications methods, wireless communications readers, wireless communications systems, and articles of manufacture are described. In one aspect, a communications device identification method includes providing identification information regarding a group of wireless identification devices within a wireless communications range of a reader, using the provided identification information, selecting one of a plurality of different search procedures for identifying unidentified ones of the wireless identification devices within the wireless communications range, and identifying at least some of the unidentified ones of the wireless identification devices using the selected one of the search procedures.

  14. Identification and Analysis of National Airspace System Resource Constraints

    NASA Technical Reports Server (NTRS)

    Smith, Jeremy C.; Marien, Ty V.; Viken, Jeffery K.; Neitzke, Kurt W.; Kwa, Tech-Seng; Dollyhigh, Samuel M.; Fenbert, James W.; Hinze, Nicolas K.

    2015-01-01

    This analysis is the deliverable for the Airspace Systems Program, Systems Analysis Integration and Evaluation Project Milestone for the Systems and Portfolio Analysis (SPA) focus area SPA.4.06 Identification and Analysis of National Airspace System (NAS) Resource Constraints and Mitigation Strategies. "Identify choke points in the current and future NAS. Choke points refer to any areas in the en route, terminal, oceanic, airport, and surface operations that constrain actual demand in current and projected future operations. Use the Common Scenarios based on Transportation Systems Analysis Model (TSAM) projections of future demand developed under SPA.4.04 Tools, Methods and Scenarios Development. Analyze causes, including operational and physical constraints." The NASA analysis is complementary to a NASA Research Announcement (NRA) "Development of Tools and Analysis to Evaluate Choke Points in the National Airspace System" Contract # NNA3AB95C awarded to Logistics Management Institute, Sept 2013.

  15. Computer-assisted skull identification system using video superimposition.

    PubMed

    Yoshino, M; Matsuda, H; Kubota, S; Imaizumi, K; Miyasaka, S; Seta, S

    1997-12-01

    This system consists of two main units, namely a video superimposition system and a computer-assisted skull identification system. The video superimposition system is comprised of the following five parts: a skull-positioning box having a monochrome CCD camera, a photo-stand having a color CCD camera, a video image mixing device, a TV monitor and a videotape recorder. The computer-assisted skull identification system is composed of a host computer including our original application software, a film recorder and a color printer. After the determination of the orientation and size of the skull to those of the facial photograph using the video superimposition system, the skull and facial photograph images are digitized and stored within the computer, and then both digitized images are superimposed on the monitor. For the assessment of anatomical consistency between the digitized skull and face, the distance between the landmarks and the thickness of soft tissue of the anthropometrical points are semi-automatically measured on the monitor. The wipe images facilitates the comparison of positional relationships between the digitized skull and face. The software includes the polynomial functions and Fourier harmonic analysis for evaluating the match of the outline such as the forehead and mandibular line in both the digitized images.

  16. Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification

    PubMed Central

    Duyvejonck, Hans; Cools, Piet; Decruyenaere, Johan; Roelens, Kristien; Noens, Lucien; Vermeulen, Stefan; Claeys, Geert; Decat, Ellen; Van Mechelen, Els; Vaneechoutte, Mario

    2015-01-01

    Aim Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. Materials and Methods A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. Results For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. Discussion Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method

  17. [Groundwater organic pollution source identification technology system research and application].

    PubMed

    Wang, Xiao-Hong; Wei, Jia-Hua; Cheng, Zhi-Neng; Liu, Pei-Bin; Ji, Yi-Qun; Zhang, Gan

    2013-02-01

    Groundwater organic pollutions are found in large amount of locations, and the pollutions are widely spread once onset; which is hard to identify and control. The key process to control and govern groundwater pollution is how to control the sources of pollution and reduce the danger to groundwater. This paper introduced typical contaminated sites as an example; then carried out the source identification studies and established groundwater organic pollution source identification system, finally applied the system to the identification of typical contaminated sites. First, grasp the basis of the contaminated sites of geological and hydrogeological conditions; determine the contaminated sites characteristics of pollutants as carbon tetrachloride, from the large numbers of groundwater analysis and test data; then find the solute transport model of contaminated sites and compound-specific isotope techniques. At last, through groundwater solute transport model and compound-specific isotope technology, determine the distribution of the typical site of organic sources of pollution and pollution status; invest identified potential sources of pollution and sample the soil to analysis. It turns out that the results of two identified historical pollution sources and pollutant concentration distribution are reliable. The results provided the basis for treatment of groundwater pollution.

  18. A numericlature of the yeasts.

    PubMed

    Griffiths, A J

    1981-01-01

    A numericlature, based on a descriptive numerical code has been compiled for the yeasts. A total of 429 yeast species are represented by 389 unique four-, six- or seven-digit numbers and of these 364 correspond to single species. It is suggested that the coding method is a valid alternative to binomial nomenclature based on a conventional hierarchical classification. It can serve as a simple reference system and can be used practically as a means of differentiating between large numbers of new isolates of yeasts. PMID:7337435

  19. Inverse source localization for EEG using system identification approach

    NASA Astrophysics Data System (ADS)

    Xanthopoulos, Petros; Yatsenko, Vitaliy; Kammerdiner, Alla; Pardalos, Panos M.

    2007-11-01

    The reconstruction of the brain current sources from scalp electric recordings (Electroen-cephalogram) also known as the inverse source localization problem is a highly underdetermined problem in the field of computational neuroscience, and this problem still remains open . In this chapter we propose an alternative formulation for the inverse electroencephalography (EEG) problem based on optimization theory. For simulation purposes, a three shell realistic head model based on an averaged magnetic resonance imaging (MRI) segmentation and Boundary Element method (BEM) is constructed. System identification methodology is employed in order to determine the parameters of the system. In the last stage the inverse problem is solved using the computed forward model.

  20. An approximation theory for the identification of linear thermoelastic systems

    NASA Technical Reports Server (NTRS)

    Rosen, I. G.; Su, Chien-Hua Frank

    1990-01-01

    An abstract approximation framework and convergence theory for the identification of thermoelastic systems is developed. Starting from an abstract operator formulation consisting of a coupled second order hyperbolic equation of elasticity and first order parabolic equation for heat conduction, well-posedness is established using linear semigroup theory in Hilbert space, and a class of parameter estimation problems is then defined involving mild solutions. The approximation framework is based upon generic Galerkin approximation of the mild solutions, and convergence of solutions of the resulting sequence of approximating finite dimensional parameter identification problems to a solution of the original infinite dimensional inverse problem is established using approximation results for operator semigroups. An example involving the basic equations of one dimensional linear thermoelasticity and a linear spline based scheme are discussed. Numerical results indicate how the approach might be used in a study of damping mechanisms in flexible structures.

  1. Biological tissue identification using a multispectral imaging system

    NASA Astrophysics Data System (ADS)

    Delporte, Céline; Sautrot, Sylvie; Ben Chouikha, Mohamed; Viénot, Françoise; Alquié, Georges

    2013-02-01

    A multispectral imaging system enabling biological tissue identifying and differentiation is presented. The measurement of β(λ) spectral radiance factor cube for four tissue types (beef muscle, pork muscle, turkey muscle and beef liver) present in the same scene was carried out. Three methods for tissue identification are proposed and their relevance evaluated. The first method correlates the scene spectral radiance factor with tissue database characteristics. This method gives detection rates ranging from 63.5 % to 85 %. The second method correlates the scene spectral radiance factor derivatives with a database of tissue β(λ) derivatives. This method is more efficient than the first one because it gives detection rates ranging from 79 % to 89 % with over-detection rates smaller than 0.2 %. The third method uses the biological tissue spectral signature. It enhances contrast in order to afford tissue differentiation and identification.

  2. Modal identification of a rotating-blade system

    SciTech Connect

    Carne, T.G.; Martinez, D.R.; Ibrahim, S.R.

    1983-01-01

    A new testing technique and the Ibrahim time domain (ITD) modal identification algorithm have been combined, resulting in a capability to estimate modal parameters for rotating blade systems. This capability has been evaluated on the Sandia two-meter, vertical-axis wind turbine. Variation in modal frequencies as a function of rotation speed has been experimentally determined from 0 rpm (parked) to 800 rpm. Excitation of the rotating turbine was provided by a scheme which suddenly released a pretensioned cable, thus plucking the turbine as it rotated. The structural response was obtained by passing the signals through slip rings. Using the measured free-decay responses as input for the ITD algorithm, the modes of the rotating turbine were determined at seven rotation speeds. The measured modal parameters were compared with analytical results obtained from a finite element analysis and with experimental results obtained from a complex exponential identification algorithm.

  3. Modal identification of a rotating-blade system

    SciTech Connect

    Carne, T.G.; Martinez, D.R.; Ibrahim, S.R.

    1983-04-01

    A new testing technique and the Ibrahim time-domain (ITD) modal identification algorithm have been combined, resulting in a capability to estimate modal parameters for rotating-blade systems. This capability has been evaluated on the Sandia two-meter, vertical-axis wind turbine. Variation in modal frequencies as a function of rotation speed has been experimentally determined from 0 rpm (parked) to 800 rpm. Excitation of the rotating turbine was provided by a scheme which suddenly released a pretensioned cable, thus plucking the turbine as it rotated. The structural response was obtained by passing the signals through slip rings. Using the measured free-decay responses as input data for the ITD algorithm, the modes of the rotating turbine were determined at seven rotation speeds. The measured modal parameters were compared with analytical results obtained from a finite element analysis and with experimental results obtained from a complex exponential identification algorithm.

  4. An in vivo detection system for transient and low-abundant protein interactions and their kinetics in budding yeast.

    PubMed

    Brezovich, Andrea; Schuschnig, Martina; Ammerer, Gustav; Kraft, Claudine

    2015-03-01

    Methylation tracking (M-Track) is a protein-proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein-protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation-specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low-abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage-enrichment system for the analysis of very low-abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning-free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids.

  5. Sensor network based vehicle classification and license plate identification system

    SciTech Connect

    Frigo, Janette Rose; Brennan, Sean M; Rosten, Edward J; Raby, Eric Y; Kulathumani, Vinod K

    2009-01-01

    Typically, for energy efficiency and scalability purposes, sensor networks have been used in the context of environmental and traffic monitoring applications in which operations at the sensor level are not computationally intensive. But increasingly, sensor network applications require data and compute intensive sensors such video cameras and microphones. In this paper, we describe the design and implementation of two such systems: a vehicle classifier based on acoustic signals and a license plate identification system using a camera. The systems are implemented in an energy-efficient manner to the extent possible using commercially available hardware, the Mica motes and the Stargate platform. Our experience in designing these systems leads us to consider an alternate more flexible, modular, low-power mote architecture that uses a combination of FPGAs, specialized embedded processing units and sensor data acquisition systems.

  6. Identification of power system load dynamics using artificial neural networks

    SciTech Connect

    Bostanci, M.; Koplowitz, J.; Taylor, C.W. |

    1997-11-01

    Power system loads are important for planning and operation of an electric power system. Load characteristics can significantly influence the results of synchronous stability and voltage stability studies. This paper presents a methodology for identification of power system load dynamics using neural networks. Input-output data of a power system dynamic load is used to design a neural network model which comprises delayed inputs and feedback connections. The developed neural network model can predict the future power system dynamic load behavior for arbitrary inputs. In particular, a third-order induction motor load neural network model is developed to verify the methodology. Neural network simulation results are illustrated and compared with the induction motor load response.

  7. Linear system identification via backward-time observer models

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Phan, Minh Q.

    1992-01-01

    Presented here is an algorithm to compute the Markov parameters of a backward-time observer for a backward-time model from experimental input and output data. The backward-time observer Markov parameters are decomposed to obtain the backward-time system Markov parameters (backward-time pulse response samples) for the backward-time system identification. The identified backward-time system Markov parameters are used in the Eigensystem Realization Algorithm to identify a backward-time state-space model, which can be easily converted to the usual forward-time representation. If one reverses time in the model to be identified, what were damped true system modes become modes with negative damping, growing as the reversed time increases. On the other hand, the noise modes in the identification still maintain the property that they are stable. The shift from positive damping to negative damping of the true system modes allows one to distinguish these modes from noise modes. Experimental results are given to illustrate when and to what extent this concept works.

  8. A hybrid system identification methodology for wireless structural health monitoring systems based on dynamic substructuring

    NASA Astrophysics Data System (ADS)

    Dragos, Kosmas; Smarsly, Kay

    2016-04-01

    System identification has been employed in numerous structural health monitoring (SHM) applications. Traditional system identification methods usually rely on centralized processing of structural response data to extract information on structural parameters. However, in wireless SHM systems the centralized processing of structural response data introduces a significant communication bottleneck. Exploiting the merits of decentralization and on-board processing power of wireless SHM systems, many system identification methods have been successfully implemented in wireless sensor networks. While several system identification approaches for wireless SHM systems have been proposed, little attention has been paid to obtaining information on the physical parameters (e.g. stiffness, damping) of the monitored structure. This paper presents a hybrid system identification methodology suitable for wireless sensor networks based on the principles of component mode synthesis (dynamic substructuring). A numerical model of the monitored structure is embedded into the wireless sensor nodes in a distributed manner, i.e. the entire model is segmented into sub-models, each embedded into one sensor node corresponding to the substructure the sensor node is assigned to. The parameters of each sub-model are estimated by extracting local mode shapes and by applying the equations of the Craig-Bampton method on dynamic substructuring. The proposed methodology is validated in a laboratory test conducted on a four-story frame structure to demonstrate the ability of the methodology to yield accurate estimates of stiffness parameters. Finally, the test results are discussed and an outlook on future research directions is provided.

  9. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

    Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of Saccharomyces cerevisiae, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

  10. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast.

    PubMed

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y; Strahl, Sabine; Clausen, Henrik

    2015-12-22

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  11. Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

    PubMed Central

    Halim, Adnan; Larsen, Ida Signe Bohse; Neubert, Patrick; Joshi, Hiren Jitendra; Petersen, Bent Larsen; Vakhrushev, Sergey Y.; Strahl, Sabine; Clausen, Henrik

    2015-01-01

    Dynamic cycling of N-Acetylglucosamine (GlcNAc) on serine and threonine residues (O-GlcNAcylation) is an essential process in all eukaryotic cells except yeast, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. O-GlcNAcylation modulates signaling and cellular processes in an intricate interplay with protein phosphorylation and serves as a key sensor of nutrients by linking the hexosamine biosynthetic pathway to cellular signaling. A longstanding conundrum has been how yeast survives without O-GlcNAcylation in light of its similar phosphorylation signaling system. We previously developed a sensitive lectin enrichment and mass spectrometry workflow for identification of the human O-linked mannose (O-Man) glycoproteome and used this to identify a pleothora of O-Man glycoproteins in human cell lines including the large family of cadherins and protocadherins. Here, we applied the workflow to yeast with the aim to characterize the yeast O-Man glycoproteome, and in doing so, we discovered hitherto unknown O-Man glycosites on nuclear, cytoplasmic, and mitochondrial proteins in S. cerevisiae and S. pombe. Such O-Man glycoproteins were not found in our analysis of human cell lines. However, the type of yeast O-Man nucleocytoplasmic proteins and the localization of identified O-Man residues mirror that of the O-GlcNAc glycoproteome found in other eukaryotic cells, indicating that the two different types of O-glycosylations serve the same important biological functions. The discovery opens for exploration of the enzymatic machinery that is predicted to regulate the nucleocytoplasmic O-Man glycosylations. It is likely that manipulation of this type of O-Man glycosylation will have wide applications for yeast bioprocessing. PMID:26644575

  12. Microchip implant system used for animal identification in laboratory rabbits, guineapigs, woodchucks and in amphibians.

    PubMed

    Mrozek, M; Fischer, R; Trendelenburg, M; Zillmann, U

    1995-07-01

    Traditional methods for animal identification have a number of drawbacks. We evaluated a new system for individual identification using microchip implants in rabbits, guineapigs, woodchucks (Marmota monax) and amphibians (Xenopus laevis, Pleurodeles waltlii). Implantation procedure and long-term observations are described. Microchip implants proved to be a practicable and reliable system for animal identification without obvious adverse effects. The applicability of electronic animal identification in comparison with common methods and with regard to animal welfare and legal aspects is discussed.

  13. Use of PCR coupled with electrospray ionization mass spectrometry for rapid identification of bacterial and yeast bloodstream pathogens from blood culture bottles.

    PubMed

    Kaleta, Erin J; Clark, Andrew E; Johnson, Desiree R; Gamage, Dulini C; Wysocki, Vicki H; Cherkaoui, Abdessalam; Schrenzel, Jacques; Wolk, Donna M

    2011-01-01

    Sepsis is among the top 10 causes of mortality in the United States. Rapid administration of antibiotics is one of the most important contributors to patient survival, yet only a limited number of methods exist for rapid identification of microbes cultivated from bloodstream infections, which can lead to sepsis. While traditional single-target molecular methods have been shown to greatly improve survival for septic patients by enabling rapid deescalation of broad-spectrum antibiotics, multiplex methods offer even greater possibilities. A novel multiplex method, PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS), was used to identify the genus and species of microorganisms found to cause human bloodstream infections. DNA was directly extracted from 234 BacT-Alert blood culture bottles, and results were compared to those obtained by clinical reference standard methods. The study results demonstrated 98.7% and 96.6% concordance at the genus and species levels, respectively. Mixtures of microbes were identified in 29 blood culture bottles, including mixed species of the same genus, as well as mixtures containing Gram-positive and Gram-negative organisms, exemplifying the PCR/ESI-MS capability to identify multiple organisms simultaneously without the need for cultivation. This study demonstrates high analytical accuracy in comparison to routine subculture of blood culture bottles and phenotypic identification of microbes. Without foreknowledge of the microorganisms potentially present, the PCR/ESI-MS methods can deliver accurate results in as little as 5 to 6 h after a positive alarm from the automated blood culture system; however, current batch mode testing limits the method's clinical utility at this time.

  14. Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase

    PubMed Central

    Chaudari, Amna; Huberman, Joel A

    2012-01-01

    Telomeres of the fission yeast,  Schizosaccharomyces pombe, are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome. PMID:24358832

  15. Identification of the Rps28 binding motif from yeast Edc3 involved in the autoregulatory feedback loop controlling RPS28B mRNA decay

    PubMed Central

    Kolesnikova, Olga; Back, Régis; Graille, Marc; Séraphin, Bertrand

    2013-01-01

    In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the auto-regulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3. PMID:23956223

  16. Terahertz imaging system performance model for concealed-weapon identification.

    PubMed

    Murrill, Steven R; Jacobs, Eddie L; Moyer, Steven K; Halford, Carl E; Griffin, Steven T; De Lucia, Frank C; Petkie, Douglas T; Franck, Charmaine C

    2008-03-20

    The U.S. Army Night Vision and Electronic Sensors Directorate (NVESD) and the U.S. Army Research Laboratory have developed a terahertz (THz) -band imaging system performance model for detection and identification of concealed weaponry. The MATLAB-based model accounts for the effects of all critical sensor and display components and for the effects of atmospheric attenuation, concealment material attenuation, and active illumination. The model is based on recent U.S. Army NVESD sensor performance modeling technology that couples system design parameters to observer-sensor field performance by using the acquire methodology for weapon identification performance predictions. This THz model has been developed in support of the Defense Advanced Research Project Agencies' Terahertz Imaging Focal-Plane Technology (TIFT) program and is currently being used to guide the design and development of a 0.650 THz active-passive imaging system. This paper will describe the THz model in detail, provide and discuss initial modeling results for a prototype THz imaging system, and outline plans to calibrate and validate the model through human perception testing. PMID:18709076

  17. Molecular identification and physiological characterization of yeasts, lactic acid bacteria and acetic acid bacteria isolated from heap and box cocoa bean fermentations in West Africa.

    PubMed

    Visintin, Simonetta; Alessandria, Valentina; Valente, Antonio; Dolci, Paola; Cocolin, Luca

    2016-01-01

    Yeast, lactic acid bacteria (LAB) and acetic acid bacteria (AAB) populations, isolated from cocoa bean heap and box fermentations in West Africa, have been investigated. The fermentation dynamicswere determined by viable counts, and 106 yeasts, 105 LAB and 82 AAB isolateswere identified by means of rep-PCR grouping and sequencing of the rRNA genes. During the box fermentations, the most abundant species were Saccharomyces cerevisiae, Candida ethanolica, Lactobacillus fermentum, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii, while S. cerevisiae, Schizosaccharomyces pombe, Hanseniaspora guilliermondii, Pichia manshurica, C. ethanolica, Hanseniaspora uvarum, Lb. fermentum, Lb. plantarum, A. pasteurianus and Acetobacter lovaniensis were identified in the heap fermentations. Furthermore, the most abundant species were molecularly characterized by analyzing the rep-PCR profiles. Strains grouped according to the type of fermentations and their progression during the transformation process were also highlighted. The yeast, LAB and AAB isolates were physiologically characterized to determine their ability to grow at different temperatures, as well as at different pH, and ethanol concentrations, tolerance to osmotic stress, and lactic acid and acetic acid inhibition. Temperatures of 45 °C, a pH of 2.5 to 3.5, 12% (v/v) ethanol and high concentrations of lactic and acetic acid have a significant influence on the growth of yeasts, LAB and AAB. Finally, the yeastswere screened for enzymatic activity, and the S. cerevisiae, H. guilliermondii, H. uvarumand C. ethanolica species were shown to possess several enzymes that may impact the quality of the final product. PMID:26425801

  18. Indirect Identification of Linear Stochastic Systems with Known Feedback Dynamics

    NASA Technical Reports Server (NTRS)

    Huang, Jen-Kuang; Hsiao, Min-Hung; Cox, David E.

    1996-01-01

    An algorithm is presented for identifying a state-space model of linear stochastic systems operating under known feedback controller. In this algorithm, only the reference input and output of closed-loop data are required. No feedback signal needs to be recorded. The overall closed-loop system dynamics is first identified. Then a recursive formulation is derived to compute the open-loop plant dynamics from the identified closed-loop system dynamics and known feedback controller dynamics. The controller can be a dynamic or constant-gain full-state feedback controller. Numerical simulations and test data of a highly unstable large-gap magnetic suspension system are presented to demonstrate the feasibility of this indirect identification method.

  19. Identification of linear systems by an asymptotically stable observer

    NASA Technical Reports Server (NTRS)

    Phan, Minh Q.; Horta, Lucas G.; Juang, Jer-Nan; Longman, Richard W.

    1992-01-01

    A formulation is presented for the identification of a linear multivariable system from single or multiple sets of input-output data. The system input-output relationship is expressed in terms of an observer, which is made asymptotically stable by an embedded eigenvalue assignment procedure. The prescribed eigenvalues for the observer may be real, complex, mixed real and complex, or zero. In this formulation, the Markov parameters of the observer are identified from input-output data. The Markov parameters of the actual system are then recovered from those of the observer and used to obtain a state space model of the system by standard realization techniques. The basic mathematical formulation is derived, and extensive numerical examples using simulated noise-free data are presented to illustrate the proposed method.

  20. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    PubMed Central

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  1. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    PubMed

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China.

  2. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    PubMed

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  3. Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome

    PubMed Central

    2010-01-01

    Background Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem. Results We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors. Conclusions Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a

  4. Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology - a reusable DNA archiving system.

    PubMed

    Borman, Andrew M; Linton, Christopher J; Miles, Sarah-Jane; Campbell, Colin K; Johnson, Elizabeth M

    2006-08-01

    Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system. PMID:16882605

  5. Methods, Systems and Apparatuses for Radio Frequency Identification

    NASA Technical Reports Server (NTRS)

    Fink, Patrick W. (Inventor); Chu, Andrew W. (Inventor); Lin, Gregory Y. (Inventor); Kennedy, Timothy F. (Inventor); Ngo, Phong H. (Inventor); Brown, Dewey T. (Inventor); Byerly, Diane (Inventor); Boose, Haley C. (Inventor)

    2015-01-01

    A system for radio frequency identification (RFID) includes an enclosure defining an interior region interior to the enclosure, and a feed for generating an electromagnetic field in the interior region in response to a signal received from an RFID reader via a radio frequency (RF) transmission line and, in response to the electromagnetic field, receiving a signal from an RFID sensor attached to an item in the interior region. The structure of the enclosure may be conductive and may include a metamaterial portion, an electromagnetically absorbing portion, or a wall extending in the interior region. Related apparatuses and methods for performing RFID are provided.

  6. Methods, Systems and Apparatuses for Radio Frequency Identification

    NASA Technical Reports Server (NTRS)

    Fink, Patrick W. (Inventor); Chu, Andrew W. (Inventor); Lin, Gregory Y. (Inventor); Kennedy, Timothy F. (Inventor); Ngo, Phong H. (Inventor); Brown, Dewey T. (Inventor); Byerly, Diane (Inventor)

    2016-01-01

    A system for radio frequency identification (RFID) includes an enclosure defining an interior region interior to the enclosure, and a feed for generating an electromagnetic field in the interior region in response to a signal received from an RFID reader via a radio frequency (RF) transmission line and, in response to the electromagnetic field, receiving a signal from an RFID sensor attached to an item in the interior region. The structure of the enclosure may be conductive and may include a metamaterial portion, an electromagnetically absorbing portion, or a wall extending in the interior region. Related apparatuses and methods for performing RFID are provided.

  7. Identification and robust control of linear parameter-varying systems

    NASA Astrophysics Data System (ADS)

    Lee, Lawton Hubert

    This dissertation deals with linear parameter-varying (LPV) systems: linear dynamic systems that depend on time-varying parameters. These systems appear in gain scheduling problems, and much recent research has been devoted to their prospective usefulness for systematic gain scheduling. We primarily focus on robust control of uncertain LPV systems and identification of LPV systems that are modelable as linear-fractional transformations (LFTs). Using parameter-dependent quadratic Lyapunov functions, linear matrix inequalities (LMIs), and scaled small-gain arguments, we define notions of stability and induced-{cal L}sb2 performance for uncertain LPV systems whose parameters and rates of parameter variation satisfy given bounds. The performance criterion involves integral quadratic constraints and implies naturally parameter-dependent induced-{cal L}sb2 norm bounds. We formulate and solve an {cal H}sb{infty}-like control problem for an LPV plant with measurable parameters and an "Output/State Feedback" structure: the feedback outputs include some noiselessly measured states. Necessary and sufficient solvability conditions reduce to LMIs that can be solved approximately using finite-dimensional convex programming. Reduced-order LPV controllers are constructed from the LMI solutions. A D-K iteration-like procedure provides robustness to structured, time-varying, parametric uncertainty. The design method is applied to a motivating example: flight control for the F-16 VISTA throughout its subsonic flight envelope. Parameter-dependent weights and {cal H}sb{infty} design principles describe the performance objectives. Closed-loop responses exhibited by nonlinear simulations indicate satisfactory flying qualities. Identification of linear-fractional LPV systems is treated using maximum-likelihood parameter estimation. Computing the gradient and Hessian of a maximum-likelihood cost function reduces to simulating one LPV filter per identified parameter. We use nonlinear

  8. A grass molecular identification system for forensic botany: a critical evaluation of the strengths and limitations.

    PubMed

    Ward, Jodie; Gilmore, Simon R; Robertson, James; Peakall, Rod

    2009-11-01

    Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA-based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single-nucleotide polymorphisms were utilized as molecular markers for subfamily to species-level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated "proof of concept" of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.

  9. The yeast PHO5 promoter: from single locus to systems biology of a paradigm for gene regulation through chromatin

    PubMed Central

    Korber, Philipp; Barbaric, Slobodan

    2014-01-01

    Chromatin dynamics crucially contributes to gene regulation. Studies of the yeast PHO5 promoter were key to establish this nowadays accepted view and continuously provide mechanistic insight in chromatin remodeling and promoter regulation, both on single locus as well as on systems level. The PHO5 promoter is a context independent chromatin switch module where in the repressed state positioned nucleosomes occlude transcription factor sites such that nucleosome remodeling is a prerequisite for and not consequence of induced gene transcription. This massive chromatin transition from positioned nucleosomes to an extensive hypersensitive site, together with respective transitions at the co-regulated PHO8 and PHO84 promoters, became a prime model for dissecting how remodelers, histone modifiers and chaperones co-operate in nucleosome remodeling upon gene induction. This revealed a surprisingly complex cofactor network at the PHO5 promoter, including five remodeler ATPases (SWI/SNF, RSC, INO80, Isw1, Chd1), and demonstrated for the first time histone eviction in trans as remodeling mode in vivo. Recently, the PHO5 promoter and the whole PHO regulon were harnessed for quantitative analyses and computational modeling of remodeling, transcription factor binding and promoter input-output relations such that this rewarding single-locus model becomes a paradigm also for theoretical and systems approaches to gene regulatory networks. PMID:25190457

  10. Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

    PubMed

    Papp, Gábor; Máté, Gábor; Mike, Nóra; Gazdag, Zoltán; Pesti, Miklós

    2016-03-01

    The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 μM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS. PMID:26752674

  11. A forward model-based validation of cardiovascular system identification

    NASA Technical Reports Server (NTRS)

    Mukkamala, R.; Cohen, R. J.

    2001-01-01

    We present a theoretical evaluation of a cardiovascular system identification method that we previously developed for the analysis of beat-to-beat fluctuations in noninvasively measured heart rate, arterial blood pressure, and instantaneous lung volume. The method provides a dynamical characterization of the important autonomic and mechanical mechanisms responsible for coupling the fluctuations (inverse modeling). To carry out the evaluation, we developed a computational model of the cardiovascular system capable of generating realistic beat-to-beat variability (forward modeling). We applied the method to data generated from the forward model and compared the resulting estimated dynamics with the actual dynamics of the forward model, which were either precisely known or easily determined. We found that the estimated dynamics corresponded to the actual dynamics and that this correspondence was robust to forward model uncertainty. We also demonstrated the sensitivity of the method in detecting small changes in parameters characterizing autonomic function in the forward model. These results provide confidence in the performance of the cardiovascular system identification method when applied to experimental data.

  12. Nonlinear damage identification of breathing cracks in Truss system

    NASA Astrophysics Data System (ADS)

    Zhao, Jie; DeSmidt, Hans

    2014-03-01

    The breathing cracks in truss system are detected by Frequency Response Function (FRF) based damage identification method. This method utilizes damage-induced changes of frequency response functions to estimate the severity and location of structural damage. This approach enables the possibility of arbitrary interrogation frequency and multiple inputs/outputs which greatly enrich the dataset for damage identification. The dynamical model of truss system is built using the finite element method and the crack model is based on fracture mechanics. Since the crack is driven by tensional and compressive forces of truss member, only one damage parameter is needed to represent the stiffness reduction of each truss member. Assuming that the crack constantly breathes with the exciting frequency, the linear damage detection algorithm is developed in frequency/time domain using Least Square and Newton Raphson methods. Then, the dynamic response of the truss system with breathing cracks is simulated in the time domain and meanwhile the crack breathing status for each member is determined by the feedback from real-time displacements of member's nodes. Harmonic Fourier Coefficients (HFCs) of dynamical response are computed by processing the data through convolution and moving average filters. Finally, the results show the effectiveness of linear damage detection algorithm in identifying the nonlinear breathing cracks using different combinations of HFCs and sensors.

  13. Benchmarking the operational search accuracy of a national identification system

    NASA Astrophysics Data System (ADS)

    Suman, Ambika; Whitaker, Geoff

    2005-03-01

    This paper reports on some of the challenges associated with setting up and conducting a full operational benchmark of a palm and fingerprint identification system, based on PITO's own recent experience in this field. The tests described were undertaken as part of the overall evaluation of suppliers tendering for a multi million pound contract to deliver a new national automated fingerprint service for the UK (known as IDENT1), as a successor to the existing systems, both in England and Wales, and in Scotland. The emphasis throughout was on 'operationally' representative testing and it was this that determined the design and scale of the tests, which PITO believes are the largest such tests of a national AFIS ever undertaken. The knowledge gained from performing these benchmark tests has provided PITO with extremely valuable experience in both the theoretical and practical issues surrounding the design and conduct of operational tests on large scale identification systems, and it is these issues that are discussed in this paper.

  14. Experimental evaluation of a modal parameter based system identification procedure

    NASA Astrophysics Data System (ADS)

    Yu, Minli; Feng, Ningsheng; Hahn, Eric J.

    2016-02-01

    Correct modelling of the foundation of a rotor bearing foundation system (RBFS) is an invaluable asset for the balancing and efficient running of turbomachinery. Numerical experiments have shown that a modal parameter based identification approach could be feasible for this purpose but there is a lack of experimental verification of the suitability of such a modal approach for even the simplest systems. In this paper the approach is tested on a simple experimental rig comprising a clamped horizontal bar with lumped masses. It is shown that apart from damping, the proposed approach can identify reasonably accurately the relevant modal parameters of the rig; and that the resulting equivalent system can predict reasonably well the frequency response of the rig. Hence, the proposed approach shows promise but further testing is required, since application to identifying the foundation of an RBFS involves the additional problem of accurately obtaining the force excitation from motion measurements.

  15. Improved solution for system identification equations by Epsilon-Decomposition

    NASA Technical Reports Server (NTRS)

    Ojalvo, Irving U.

    1990-01-01

    Matrix eigenvalue theory is used to examine the source of ill-conditioning in linear algebraic equations. This approach highlights the crucial role played by the zero and near-zero eigenvalues and corresponding eigenvectors of poorly conditioned systems. Insight gained from this approach is used to significantly improve a recently developed solution procedure called Epsilon-Decomposition (E-D). E-D is an efficient alternative to Singular Value Decomposition (SVD) for ill-conditioned systems arising in parameter estimation and system identification studies. The efficiency of the improved E-D over SVD resides in the need to only obtain the zero and near-zero eigenvalues of the coefficient matrix as opposed to all of its eigenvalues and vectors (as required by SVD). Thus, the efficiency of E-D is significant for large matrices with small rank deficiency.

  16. A Gamma Memory Neural Network for System Identification

    NASA Technical Reports Server (NTRS)

    Motter, Mark A.; Principe, Jose C.

    1992-01-01

    A gamma neural network topology is investigated for a system identification application. A discrete gamma memory structure is used in the input layer, providing delayed values of both the control inputs and the network output to the input layer. The discrete gamma memory structure implements a tapped dispersive delay line, with the amount of dispersion regulated by a single, adaptable parameter. The network is trained using static back propagation, but captures significant features of the system dynamics. The system dynamics identified with the network are the Mach number dynamics of the 16 Foot Transonic Tunnel at NASA Langley Research Center, Hampton, Virginia. The training data spans an operating range of Mach numbers from 0.4 to 1.3.

  17. Part identification in robotic assembly using vision system

    NASA Astrophysics Data System (ADS)

    Balabantaray, Bunil Kumar; Biswal, Bibhuti Bhusan

    2013-12-01

    Machine vision system acts an important role in making robotic assembly system autonomous. Identification of the correct part is an important task which needs to be carefully done by a vision system to feed the robot with correct information for further processing. This process consists of many sub-processes wherein, the image capturing, digitizing and enhancing, etc. do account for reconstructive the part for subsequent operations. Interest point detection of the grabbed image, therefore, plays an important role in the entire image processing activity. Thus it needs to choose the correct tool for the process with respect to the given environment. In this paper analysis of three major corner detection algorithms is performed on the basis of their accuracy, speed and robustness to noise. The work is performed on the Matlab R2012a. An attempt has been made to find the best algorithm for the problem.

  18. Yeasts associated with nectarines and their potential for biological control of brown rot.

    PubMed

    Janisiewicz, W J; Kurtzman, C P; Buyer, J S

    2010-07-01

    Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines ('Croce del Sud') from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast-like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov.

  19. The emergence of yeast lipidomics.

    PubMed

    Gaspar, Maria L; Aregullin, Manuel A; Jesch, Stephen A; Nunez, Lilia R; Villa-García, Manuel; Henry, Susan A

    2007-03-01

    The emerging field of lipidomics, driven by technological advances in lipid analysis, provides greatly enhanced opportunities to characterize, on a quantitative or semi-quantitative level, the entire spectrum of lipids, or lipidome, in specific cell types. When combined with advances in other high throughput technologies in genomics and proteomics, lipidomics offers the opportunity to analyze the unique roles of specific lipids in complex cellular processes such as signaling and membrane trafficking. The yeast system offers many advantages for such studies, including the relative simplicity of its lipidome as compared to mammalian cells, the relatively high proportion of structural and regulatory genes of lipid metabolism which have been assigned and the excellent tools for molecular genetic analysis that yeast affords. The current state of application of lipidomic approaches in yeast and the advantages and disadvantages of yeast for such studies are discussed in this report.

  20. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.