A three-hybrid system to detect RNA-protein interactions in vivo.

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.

Quantum dots-bienzyme hybrid system for the sensitive determination of glucose

In this paper, we attempt to develop a sensitive detection method for glucose with the combination of the unique optical property of quantum dots and the specificity of enzymatic reactions. With glucose and hydroquinone as substrates, benzoquinone that intensively quenches the photoluminescence of quantum dots can be produced via the catalysis of bienzyme (glucose oxidase and horseradish peroxidase) system. A relatively low detection limit of 1.0 x 10(-8) mol/L can be achieved. Two linear ranges from 1.0 x 10(-6) to 1.5 x 10(-4) M and from 1.5 x 10(-4) to 1.0 x 10(-3) M were obtained.

Modelling and simulation of a photovoltaic fuel cell hybrid system

A stand-alone power system is an autonomous system that supplies electricity to the user load without being connected to the electric grid. This kind of decentralized system is frequently located in remote and inaccessible areas. It is essential for about one third of the world population which are living in developed or isolated regions and have no access to an electricity utility grid. The most people live in remote and rural areas, with low population density, lacking even the basic infrastructure. The utility grid extension to these locations is not a cost effective option and sometimes technically not feasible. The purpose of this thesis is the modelling and simulation of a stand-alone hybrid power system, referred to as “hydrogen Photovoltaic-Fuel Cell (PVFC) hybrid system”. It couples a photovoltaic generator (PV), an alkaline water electrolyser, a storage gas tank, a proton exchange membrane fuel cell (PEMFC), and power conditioning units (PCU) to give different system topologies. The system is intended to be an environmentally friendly solution since it tries maximising the use of a renewable energy source. Electricity is produced by a PV generator to meet the requirements of a user load. Whenever there is enough solar radiation, the user load can be powered totally by the PV electricity. During periods of low solar radiation, auxiliary electricity is required. An alkaline high pressure water electrolyser is powered by the excess energy from the PV generator to produce hydrogen and oxygen at a pressure of maximum 30bar. Gases are stored without compression for short- (hourly or daily) and long- (seasonal) term. A proton exchange membrane (PEM) fuel cell is used to keep the system’s reliability at the same level as for the conventional system while decreasing the environmental impact of the whole system. The PEM fuel cell consumes gases which are produced by an electrolyser to meet the user load demand when the PV generator energy is deficient, so that it works as an auxiliary generator. Power conditioning units are appropriate for the conversion and dispatch the energy between the components of the system. No batteries are used in this system since they represent the weakest when used in PV systems due to their need for sophisticated control and their short lifetime. The model library, ISET Alternative Power Library (ISET-APL), is designed by the Institute of Solar Energy supply Technology (ISET) and used for the simulation of the hybrid system. The physical, analytical and/or empirical equations of each component are programmed and implemented separately in this library for the simulation software program Simplorer by C++ language. The model parameters are derived from manufacturer’s performance data sheets or measurements obtained from literature. The identification and validation of the major hydrogen PVFC hybrid system component models are evaluated according to the measured data of the components, from the manufacturer’s data sheet or from actual system operation. Then, the overall system is simulated, at intervals of one hour each, by using solar radiation as the primary energy input and hydrogen as energy storage for one year operation. A comparison between different topologies, such as DC or AC coupled systems, is carried out on the basis of energy point of view at two locations with different geographical latitudes, in Kassel/Germany (Europe) and in Cairo/Egypt (North Africa). The main conclusion in this work is that the simulation method of the system study under different conditions could successfully be used to give good visualization and comparison between those topologies for the overall performance of the system. The operational performance of the system is not only depending on component efficiency but also on system design and consumption behaviour. The worst case of this system is the low efficiency of the storage subsystem made of the electrolyser, the gas storage tank, and the fuel cell as it is around 25-34% at Cairo and 29-37% at Kassel. Therefore, the research for this system should be concentrated in the subsystem components development especially the fuel cell.

Mso1p: A yeast protein that functions in secretion and interacts physically and genetically with Sec1p

The yeast Sec1p protein functions in the docking of secretory transport vesicles to the plasma membrane. We previously have cloned two yeast genes encoding syntaxins, SSO1 and SSO2, as suppressors of the temperature-sensitive sec1–1 mutation. We now describe a third suppressor of sec1–1, which we call MSO1. Unlike SSO1 and SSO2, MSO1 is specific for sec1 and does not suppress mutations in any other SEC genes. MSO1 encodes a small hydrophilic protein that is enriched in a microsomal membrane fraction. Cells that lack MSO1 are viable, but they accumulate secretory vesicles in the bud, indicating that the terminal step in secretion is partially impaired. Moreover, loss of MSO1 shows synthetic lethality with mutations in SEC1, SEC2, and SEC4, and other synthetic phenotypes with mutations in several other late-acting SEC genes. We further found that Mso1p interacts with Sec1p both in vitro and in the two-hybrid system. These findings suggest that Mso1p is a component of the secretory vesicle docking complex whose function is closely associated with that of Sec1p.

Complex formation in yeast double-strand break repair: participation of Rad51, Rad52, Rad55, and Rad57 proteins.

The repair of DNA double-strand breaks in Saccharomyces cerevisiae requires genes of the RAD52 epistasis group, of which RAD55 and RAD57 are members. Here, we show that the x-ray sensitivity of rad55 and rad57 mutant strains is suppressible by overexpression of RAD51 or RAD52. Virtually complete suppression is provided by the simultaneous overexpression of RAD51 and RAD52. This suppression occurs at 23 degrees C, where these mutants are more sensitive to x-rays, as well as at 30 degrees C and 36 degrees C. In addition, a recombination defect of rad55 and rad57 mutants is similarly suppressed. Direct in vivo interactions between the Rad51 and Rad55 proteins, and between Rad55 and Rad57, have also been identified by using the two-hybrid system. These results indicate that these four proteins constitute part of a complex, a "recombinosome," to effect the recombinational repair of double-strand breaks.

Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter.

Hybrid system for plagiarism detection

The Internet boom in recent years has increased the interest in the field of plagiarism detection. A lot of documents are published on the Net everyday and anyone can access and plagiarize them. Of course, checking all cases of plagiarism manually is an unfeasible task. Therefore, it is necessary to create new systems that are able to automatically detect cases of plagiarism produced. In this paper, we introduce a new hybrid system for plagiarism detection which combines the advantages of the two main plagiarism detection techniques. This system consists of two analysis phases: the first phase uses an intrinsic detection technique which dismisses much of the text, and the second phase employs an external detection technique to identify the plagiarized text sections. With this combination we achieve a detection system which obtains accurate results and is also faster thanks to the prefiltering of the text.

Lesion detection performance: comparative analysis of low-dose CT data of the chest on two hybrid imaging systems

Incidental findings on low-dose CT images obtained during hybrid imaging are an increasing phenomenon as CT technology advances. Understanding the diagnostic value of incidental findings along with the technical limitations is important when reporting image results and recommending follow-up, which may result in an additional radiation dose from further diagnostic imaging and an increase in patient anxiety. This study assessed lesions incidentally detected on CT images acquired for attenuation correction on two SPECT/CT systems. Methods: An anthropomorphic chest phantom containing simulated lesions of varying size and density was imaged on an Infinia Hawkeye 4 and a Symbia T6 using the low-dose CT settings applied for attenuation correction acquisitions in myocardial perfusion imaging. Twenty-two interpreters assessed 46 images from each SPECT/CT system (15 normal images and 31 abnormal images; 41 lesions). Data were evaluated using a jackknife alternative free-response receiver-operating-characteristic analysis (JAFROC). Results: JAFROC analysis showed a significant difference (P < 0.0001) in lesion detection, with the figures of merit being 0.599 (95% confidence interval, 0.568, 0.631) and 0.810 (95% confidence interval, 0.781, 0.839) for the Infinia Hawkeye 4 and Symbia T6, respectively. Lesion detection on the Infinia Hawkeye 4 was generally limited to larger, higher-density lesions. The Symbia T6 allowed improved detection rates for midsized lesions and some lower-density lesions. However, interpreters struggled to detect small (5 mm) lesions on both image sets, irrespective of density. Conclusion: Lesion detection is more reliable on low-dose CT images from the Symbia T6 than from the Infinia Hawkeye 4. This phantom-based study gives an indication of potential lesion detection in the clinical context as shown by two commonly used SPECT/CT systems, which may assist the clinician in determining whether further diagnostic imaging is justified.

Hybrid system simulation of computer control applications over communication networks

Discrete event-driven simulations of digital communication networks have been used widely. However, it is difficult to use a network simulator to simulate a hybrid system in which some objects are not discrete event-driven but are continuous time-driven. A networked control system (NCS) is such an application, in which physical process dynamics are continuous by nature. We have designed and implemented a hybrid simulation environment which effectively integrates models of continuous-time plant processes and discrete-event communication networks by extending the open source network simulator NS-2. To do this a synchronisation mechanism was developed to connect a continuous plant simulation with a discrete network simulation. Furthermore, for evaluating co-design approaches in an NCS environment, a piggybacking method was adopted to allow the control period to be adjusted during simulations. The effectiveness of the technique is demonstrated through case studies which simulate a networked control scenario in which the communication and control system properties are defined explicitly.

A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

An alginate-based hybrid system for growth factor delivery in the functional repair of large bone defects

The treatment of challenging fractures and large osseous defects presents a formidable problem for orthopaedic surgeons. Tissue engineering/regenerative medicine approaches seek to solve this problem by delivering osteogenic signals within scaffolding biomaterials. In this study, we introduce a hybrid growth factor delivery system that consists of an electrospun nanofiber mesh tube for guiding bone regeneration combined with peptide-modified alginate hydrogel injected inside the tube for sustained growth factor release. We tested the ability of this system to deliver recombinant bone morphogenetic protein-2 (rhBMP-2) for the repair of critically-sized segmental bone defects in a rat model. Longitudinal [mu]-CT analysis and torsional testing provided quantitative assessment of bone regeneration. Our results indicate that the hybrid delivery system resulted in consistent bony bridging of the challenging bone defects. However, in the absence of rhBMP-2, the use of nanofiber mesh tube and alginate did not result in substantial bone formation. Perforations in the nanofiber mesh accelerated the rhBMP-2 mediated bone repair, and resulted in functional restoration of the regenerated bone. [mu]-CT based angiography indicated that perforations did not significantly affect the revascularization of defects, suggesting that some other interaction with the tissue surrounding the defect such as improved infiltration of osteoprogenitor cells contributed to the observed differences in repair. Overall, our results indicate that the hybrid alginate/nanofiber mesh system is a promising growth factor delivery strategy for the repair of challenging bone injuries.

Relative entropy rate based design for linear hybrid system models

Hybrid system representations have been applied to many challenging modeling situations. In these hybrid system representations, a mixture of continuous and discrete states is used to capture the dominating behavioural features of a nonlinear, possible uncertain, model under approximation. Unfortunately, the problem of how to best design a suitable hybrid system model has not yet been fully addressed. This paper proposes a new joint state measurement relative entropy rate based approach for this design purpose. Design examples and simulation studies are presented which highlight the benefits of our proposed design approaches.

Relative entropy rate based model selection for linear hybrid system filters of uncertain nonlinear systems

Hybrid system representations have been exploited in a number of challenging modelling situations, including situations where the original nonlinear dynamics are too complex (or too imprecisely known) to be directly filtered. Unfortunately, the question of how to best design suitable hybrid system models has not yet been fully addressed, particularly in the situations involving model uncertainty. This paper proposes a novel joint state-measurement relative entropy rate based approach for design of hybrid system filters in the presence of (parameterised) model uncertainty. We also present a design approach suitable for suboptimal hybrid system filters. The benefits of our proposed approaches are illustrated through design examples and simulation studies.

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.