A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation

Photosynthesis, biological nitrogen fixation, and carbon dioxide assimilation are three fundamental biological processes catalyzed by photosynthetic bacteria. In the present study, it is shown that mutant strains of the nonsulfur purple photosynthetic bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides, containing a blockage in the primary CO2 assimilatory pathway, derepress the synthesis of components of the nitrogen fixation enzyme complex and abrogate normal control mechanisms. The absence of the Calvin–Benson–Bassham (CBB) reductive pentose phosphate CO2 fixation pathway removes an important route for the dissipation of excess reducing power. Thus, the mutant strains develop alternative means to remove these reducing equivalents, resulting in the synthesis of large amounts of nitrogenase even in the presence of ammonia. This response is under the control of a global two-component signal transduction system previously found to regulate photosystem biosynthesis and the transcription of genes required for CO2 fixation through the CBB pathway and alternative routes. In addition, this two-component system directly controls the ability of these bacteria to grow under nitrogen-fixing conditions. These results indicate that there is a molecular link between the CBB and nitrogen fixation process, allowing the cell to overcome powerful control mechanisms to remove excess reducing power generated by photosynthesis and carbon metabolism. Furthermore, these results suggest that the two-component system integrates the expression of genes required for the three processes of photosynthesis, nitrogen fixation, and carbon dioxide fixation.

Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Ht) protein. A hallmark of HD is the proteolytic production of an N-terminal fragment of Ht, containing the polyQ repeat, that forms aggregates in the nucleus and cytoplasm of affected neurons. Proteins with longer polyQ repeats aggregate more rapidly and cause disease at an earlier age, but the mechanism of aggregation and its relationship to disease remain unclear. To provide a new, genetically tractable model system for the study of Ht, we engineered yeast cells to express an N-terminal fragment of Ht with different polyQ repeat lengths of 25, 47, 72, or 103 residues, fused to green fluorescent protein. The extent of aggregation varied with the length of the polyQ repeat: at the two extremes, most HtQ103 protein coalesced into a single large cytoplasmic aggregate, whereas HtQ25 exhibited no sign of aggregation. Mutations that inhibit the ubiquitin/proteasome pathway at three different steps had no effect on the aggregation of Ht fragments in yeast, suggesting that the ubiquitination of Ht previously noted in mammalian cells may not inherently be required for polyQ length-dependent aggregation. Changing the expression levels of a wide variety of chaperone proteins in yeast neither increased nor decreased Ht aggregation. However, Sis1, Hsp70, and Hsp104 overexpression modulated aggregation of HtQ72 and HtQ103 fragments. More dramatically, the deletion of Hsp104 virtually eliminated it. These observations establish yeast as a system for studying the causes and consequences of polyQ-dependent Ht aggregation.

Fission Yeast Aip3p (spAip3p) Is Required for an Alternative Actin-directed Polarity Program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Δ strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

A Fas-associated protein factor, FAF1, potentiates Fas-mediated apoptosis.

Fas, a member of the tumor necrosis factor receptor family, can induce apoptosis when activated by Fas ligand binding or anti-Fas antibody crosslinking. Genetic studies have shown that a defect in Fas-mediated apoptosis resulted in abnormal development and function of the immune system in mice. A point mutation in the cytoplasmic domain of Fas (a single base change from T to A at base 786), replacing isoleucine with asparagine, abolishes the signal transducing property of Fas. Mice homozygous for this mutant allele (lprcg/lprcg mice) develop lymphadenopathy and a lupus-like autoimmune disease. Little is known about the mechanism of signal transduction in Fas-mediated apoptosis. In this study, we used the two-hybrid screen in yeast to isolate a Fas-associated protein factor, FAF1, which specifically interacts with the cytoplasmic domain of wild-type Fas but not the lprcg-mutated Fas protein. This interaction occurs not only in yeast but also in mammalian cells. When transiently expressed in L cells, FAF1 potentiated Fas-induced apoptosis. A search of available DNA and protein sequence data banks did not reveal significant homology between FAF1 and known proteins. Therefore, FAF1 is an unusual protein that binds to the wild type but not the inactive point mutant of Fas. FAF1 potentiates Fas-induced cell killing and is a candidate signal transducing molecule in the regulation of apoptosis.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

A Novel Mammalian Retromer Component, Vps26B

The mammalian retromer protein complex, which consists of three proteins - Vps26, Vps29, and Vps35 - in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

Combining an enterprise resource planning system and informality:a study of efficiency and flexibility complmentarity [sic]

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT One of the current research trends in Enterprise Resource Planning (ERP) involves examining the critical factors for its successful implementation. However, such research is limited to system implementation, not focusing on the flexibility of ERP to respond to changes in business. Therefore, this study explores a combination system, made up of an ERP and informality, intended to provide organisations with efficient and flexible performance simultaneously. In addition, this research analyses the benefits and challenges of using the system. The research was based on socio-technical system (STS) theory which contains two dimensions: 1) a technical dimension which evaluates the performance of the system; and 2) a social dimension which examines the impact of the system on an organisation. A mixed method approach has been followed in this research. The qualitative part aims to understand the constraints of using a single ERP system, and to define a new system corresponding to these problems. To achieve this goal, four Chinese companies operating in different industries were studied, all of which faced challenges in using an ERP system due to complexity and uncertainty in their business environments. The quantitative part contains a discrete-event simulation study that is intended to examine the impact of operational performance when a company implements the hybrid system in a real-life situation. Moreover, this research conducts a further qualitative case study, the better to understand the influence of the system in an organisation. The empirical aspect of the study reveals that an ERP with pre-determined business activities cannot react promptly to unanticipated changes in a business. Incorporating informality into an ERP can react to different situations by using different procedures that are based on the practical knowledge of frontline employees. Furthermore, the simulation study shows that the combination system can achieve a balance between efficiency and flexibility. Unlike existing research, which emphasises a continuous improvement in the IT functions of an enterprise system, this research contributes to providing a definition of a new system in theory, which has mixed performance and contains both the formal practices embedded in an ERP and informal activities based on human knowledge. It supports both cost-efficiency in executing business transactions and flexibility in coping with business uncertainty.This research also indicates risks of using the system, such as using an ERP with limited functions; a high cost for performing informally; and a low system acceptance, owing to a shift in organisational culture. With respect to practical contribution, this research suggests that companies can choose the most suitable enterprise system approach in accordance with their operational strategies. The combination system can be implemented in a company that needs to operate a medium amount of volume and variety. By contrast, the traditional ERP system is better suited in a company that operates a high-level volume market, while an informal system is more suitable for a firm with a requirement for a high level of variety.

Hybrid approaches to estimating freeway travel times using point traffic detector data

The accurate and reliable estimation of travel time based on point detector data is needed to support Intelligent Transportation System (ITS) applications. It has been found that the quality of travel time estimation is a function of the method used in the estimation and varies for different traffic conditions. In this study, two hybrid on-line travel time estimation models, and their corresponding off-line methods, were developed to achieve better estimation performance under various traffic conditions, including recurrent congestion and incidents. The first model combines the Mid-Point method, which is a speed-based method, with a traffic flow-based method. The second model integrates two speed-based methods: the Mid-Point method and the Minimum Speed method. In both models, the switch between travel time estimation methods is based on the congestion level and queue status automatically identified by clustering analysis. During incident conditions with rapidly changing queue lengths, shock wave analysis-based refinements are applied for on-line estimation to capture the fast queue propagation and recovery. Travel time estimates obtained from existing speed-based methods, traffic flow-based methods, and the models developed were tested using both simulation and real-world data. The results indicate that all tested methods performed at an acceptable level during periods of low congestion. However, their performances vary with an increase in congestion. Comparisons with other estimation methods also show that the developed hybrid models perform well in all cases. Further comparisons between the on-line and off-line travel time estimation methods reveal that off-line methods perform significantly better only during fast-changing congested conditions, such as during incidents. The impacts of major influential factors on the performance of travel time estimation, including data preprocessing procedures, detector errors, detector spacing, frequency of travel time updates to traveler information devices, travel time link length, and posted travel time range, were investigated in this study. The results show that these factors have more significant impacts on the estimation accuracy and reliability under congested conditions than during uncongested conditions. For the incident conditions, the estimation quality improves with the use of a short rolling period for data smoothing, more accurate detector data, and frequent travel time updates.

Hybrid materials for biomedical applications

The increased longevity of humans and the demand for a better quality of life have led to a continuous search for new implant materials. Scientific development coupled with a growing multidisciplinarity between materials science and life sciences has given rise to new approaches such as regenerative medicine and tissue engineering. The search for a material with mechanical properties close to those of human bone produced a new family of hybrid materials that take advantage of the synergy between inorganic silica (SiO4) domains, based on sol-gel bioactive glass compositions, and organic polydimethylsiloxane, PDMS ((CH3)2.SiO2)n, domains. Several studies have shown that hybrid materials based on the system PDMS-SiO2 constitute a promising group of biomaterials with several potential applications from bone tissue regeneration to brain tissue recovery, passing by bioactive coatings and drug delivery systems. The objective of the present work was to prepare hybrid materials for biomedical applications based on the PDMS-SiO2 system and to achieve a better understanding of the relationship among the sol-gel processing conditions, the chemical structures, the microstructure and the macroscopic properties. For that, different characterization techniques were used: Fourier transform infrared spectrometry, liquid and solid state nuclear magnetic resonance techniques, X-ray diffraction, small-angle X-ray scattering, smallangle neutron scattering, surface area analysis by Brunauer–Emmett–Teller method, scanning electron microscopy and transmission electron microscopy. Surface roughness and wettability were analyzed by 3D optical profilometry and by contact angle measurements respectively. Bioactivity was evaluated in vitro by immersion of the materials in Kokubos’s simulated body fluid and posterior surface analysis by different techniques as well as supernatant liquid analysis by inductively coupled plasma spectroscopy. Biocompatibility was assessed using MG63 osteoblastic cells. PDMS-SiO2-CaO materials were first prepared using nitrate as a calcium source. To avoid the presence of nitrate residues in the final product due to its potential toxicity, a heat-treatment step (above 400 °C) is required. In order to enhance the thermal stability of the materials subjected to high temperatures titanium was added to the hybrid system, and a material containing calcium, with no traces of nitrate and the preservation of a significant amount of methyl groups was successfully obtained. The difficulty in eliminating all nitrates from bulk PDMS-SiO2-CaO samples obtained by sol-gel synthesis and subsequent heat-treatment created a new goal which was the search for alternative sources of calcium. New calcium sources were evaluated in order to substitute the nitrate and calcium acetate was chosen due to its good solubility in water. Preparation solgel protocols were tested and homogeneous monolithic samples were obtained. Besides their ability to improve the bioactivity, titanium and zirconium influence the structural and microstructural features of the SiO2-TiO2 and SiO2-ZrO2 binary systems, and also of the PDMS-TiO2 and PDMS-ZrO2 systems. Detailed studies with different sol-gel conditions allowed the understanding of the roles of titanium and zirconium as additives in the PDMS-SiO2 system. It was concluded that titanium and zirconium influence the kinetics of the sol-gel process due to their different alkoxide reactivity leading to hybrid xerogels with dissimilar characteristics and morphologies. Titanium isopropoxide, less reactive than zirconium propoxide, was chosen as source of titanium, used as an additive to the system PDMS-SiO2-CaO. Two different sol-gel preparation routes were followed, using the same base composition and calcium acetate as calcium source. Different microstructures with high hydrophobicit were obtained and both proved to be biocompatible after tested with MG63 osteoblastic cells. Finally, the role of strontium (typically known in bioglasses to promote bone formation and reduce bone resorption) was studied in the PDMS-SiO2-CaOTiO2 hybrid system. A biocompatible material, tested with MG63 osteoblastic cells, was obtained with the ability to release strontium within the values reported as suitable for bone tissue regeneration.

Rough fuzzy control of SVC for power system stability enhancement

This paper presents a new approach to the design of a rough fuzzy controller for the control loop of the SVC (static VAR system) in a two area power system for stability enhancement with particular emphasis on providing effective damping for oscillatory instabilities. The performances of the rough fuzzy and the conventional fuzzy controller are compared with that of the conventional PI controller for a variety of transient disturbances, highlighting the effectiveness of the rough fuzzy controller in damping the inter-area oscillations. The effect of the rough fuzzy controller in improving the CCT (critical clearing time) of the two area system is elaborated in this paper as well.

Subcarrier pairing and relay assignment with improved user fairness in a multi-user cooperative OFDM system

We consider a joint relay selection and subcarrier allocation problem that minimizes the total system power for a multi-user, multi-relay and single source cooperative OFDM based two hop system. The system is constrained to all users having a specific subcarrier requirement (user fairness). However no specific fairness constraints for relays are considered. To ensure the optimum power allocation, the subcarriers in two hops are paired with each other. We obtain an optimal subcarrier allocation for the single user case using a similar method to what is described in [1] and modify the algorithm for multiuser scenario. Although the optimality is not achieved in multiuser case the probability of all users being served fairly is improved significantly with a relatively low cost trade off.

Pulse dynamics in a three-component system : existence analysis

In this article, we analyze the three-component reaction-diffusion system originally developed by Schenk et al. (PRL 78:3781–3784, 1997). The system consists of bistable activator-inhibitor equations with an additional inhibitor that diffuses more rapidly than the standard inhibitor (or recovery variable). It has been used by several authors as a prototype three-component system that generates rich pulse dynamics and interactions, and this richness is the main motivation for the analysis we present. We demonstrate the existence of stationary one-pulse and two-pulse solutions, and travelling one-pulse solutions, on the real line, and we determine the parameter regimes in which they exist. Also, for one-pulse solutions, we analyze various bifurcations, including the saddle-node bifurcation in which they are created, as well as the bifurcation from a stationary to a travelling pulse, which we show can be either subcritical or supercritical. For two-pulse solutions, we show that the third component is essential, since the reduced bistable two-component system does not support them. We also analyze the saddle-node bifurcation in which two-pulse solutions are created. The analytical method used to construct all of these pulse solutions is geometric singular perturbation theory, which allows us to show that these solutions lie in the transverse intersections of invariant manifolds in the phase space of the associated six-dimensional travelling wave system. Finally, as we illustrate with numerical simulations, these solutions form the backbone of the rich pulse dynamics this system exhibits, including pulse replication, pulse annihilation, breathing pulses, and pulse scattering, among others.

Integrating collaborative filtering and matching-based search for product recommendation

Currently, recommender systems (RS) have been widely applied in many commercial e-commerce sites to help users deal with the information overload problem. Recommender systems provide personalized recommendations to users and thus help them in making good decisions about which product to buy from the vast number of product choices available to them. Many of the current recommender systems are developed for simple and frequently purchased products like books and videos, by using collaborative-filtering and content-based recommender system approaches. These approaches are not suitable for recommending luxurious and infrequently purchased products as they rely on a large amount of ratings data that is not usually available for such products. This research aims to explore novel approaches for recommending infrequently purchased products by exploiting user generated content such as user reviews and product click streams data. From reviews on products given by the previous users, association rules between product attributes are extracted using an association rule mining technique. Furthermore, from product click streams data, user profiles are generated using the proposed user profiling approach. Two recommendation approaches are proposed based on the knowledge extracted from these resources. The first approach is developed by formulating a new query from the initial query given by the target user, by expanding the query with the suitable association rules. In the second approach, a collaborative-filtering recommender system and search-based approaches are integrated within a hybrid system. In this hybrid system, user profiles are used to find the target user’s neighbour and the subsequent products viewed by them are then used to search for other relevant products. Experiments have been conducted on a real world dataset collected from one of the online car sale companies in Australia to evaluate the effectiveness of the proposed recommendation approaches. The experiment results show that user profiles generated from user click stream data and association rules generated from user reviews can improve recommendation accuracy. In addition, the experiment results also prove that the proposed query expansion and the hybrid collaborative filtering and search-based approaches perform better than the baseline approaches. Integrating the collaborative-filtering and search-based approaches has been challenging as this strategy has not been widely explored so far especially for recommending infrequently purchased products. Therefore, this research will provide a theoretical contribution to the recommender system field as a new technique of combining collaborative-filtering and search-based approaches will be developed. This research also contributes to a development of a new query expansion technique for infrequently purchased products recommendation. This research will also provide a practical contribution to the development of a prototype system for recommending cars.

Yhh1p/Cft1p directly links poly(A) site recognition and RNA polymerase II transcription termination

RNA polymerase II (pol II) transcription termination requires co‐transcriptional recognition of a functional polyadenylation signal, but the molecular mechanisms that transduce this signal to pol II remain unclear. We show that Yhh1p/Cft1p, the yeast homologue of the mammalian AAUAAA interacting protein CPSF 160, is an RNA‐binding protein and provide evidence that it participates in poly(A) site recognition. Interestingly, RNA binding is mediated by a central domain composed of predicted β‐propeller‐forming repeats, which occurs in proteins of diverse cellular functions. We also found that Yhh1p/Cft1p bound specifically to the phosphorylated C‐terminal domain (CTD) of pol II in vitro and in a two‐hybrid test in vivo. Furthermore, transcriptional run‐on analysis demonstrated that yhh1 mutants were defective in transcription termination, suggesting that Yhh1p/Cft1p functions in the coupling of transcription and 3′‐end formation. We propose that direct interactions of Yhh1p/Cft1p with both the RNA transcript and the CTD are required to communicate poly(A) site recognition to elongating pol II to initiate transcription termination.