ABSTRACT
Considering the low specific capacitance of structural solid supercapacitors, which is due to the low ion diffusivity in solid electrolytes and the small specific surface area of some structural electrodes such as carbon fiber fabrics, novel structural supercapacitor designs are proposed and evaluated in this study based on supercapacitor-functional sandwich composite materials. Typical electrochemical double layer capacitors (EDLCs) are proposed with liquid organic electrolyte 1 M TEABF4 in PC (propylene carbonate). In the innovative sandwich structured composites, supercapacitors are embedded in the skins and integrated in the honeycomb core where the aluminum faces of the core constitute the current collectors of the supercapacitor-functional core. The sandwich composite material exhibited a flexural modulus of 5.07 GPa and a flexural strength of 413.9 MPa. The EDLCs embedded in the skins increased the skin flexural modulus and strength by 47% and 56%, respectively, for embedded lateral EDLCs, and by 91% and 106%, respectively, for embedded lateral and longitudinal EDLCs. Compared to typical EDLCs with the same electrolyte, the structural supercapacitors in this study demonstrated superior specific electrode capacitance, Csp,el = 153 F g-1 for the honeycomb supercapacitor and Csp,el = 95.7 F g-1 for the skin supercapacitor, translating to overall structural composite material performance of 0.68 Wh/m2honeycomb and 30.5 W/m2honeycomb for the supercapacitor-functional honeycomb, and 0.02 Wh/m2skin and 5.4 W/m2skin for the supercapacitor-functional skin.
ABSTRACT
The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins--a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)--control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.
Subject(s)
Galactokinase/genetics , Galactose/metabolism , Gene Expression Regulation, Fungal , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription Factors/genetics , Bacterial Proteins , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Galactokinase/metabolism , Galactose/genetics , Genes, Reporter , Luminescent Proteins , Protein Binding , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
The control of transcription factor function plays an important role in the development of many processes in eukaryotes, such as drug resistance in fungi and human tumours undergoing chemotherapy. Detailed molecular mapping of the interactions between transcription factors and their protein partners can give important information about their mechanisms of action and reveal potential therapeutic targets. We devised a genetic screening system for mapping the interaction site between the Saccharomyces cerevisiae transcription factor-inhibitor pair Gal4p and Gal80p. A novel Gal4p activation domain mutant, L868K, was produced, which prevented it interacting with Gal80p. The split-ubiquitin system was used with a mutant GAL80 library in order to screen for compensatory mutants in Gal80p which would restore binding with L868K. Five single amino acid residue compensatory mutations in Gal80p which restored the interaction with Gal4p(L868K) were isolated. These compensatory mutations were specific to L868K as they were unable to restore the interaction with two other Gal4p mutants that were incapable of interacting with Gal80p. Mutations within Gal80p that were capable of compensating for Gal4p (L868K) clustered inside a Gal80p surface cleft, supporting the idea that this area is important for Gal4p binding. Our data suggest a way to generate information about interaction sites that should be applicable to any transcription factor.
Subject(s)
DNA-Binding Proteins/metabolism , Mutation , Protein Interaction Domains and Motifs , Protein Interaction Mapping/methods , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Ubiquitin/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Protein Binding , Protein Structure, Tertiary , Repressor Proteins/chemistry , Repressor Proteins/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Inflammation of synovium is one of the hallmarks of rheumatoid arthritis (RA). Analysis of synovial tissue has increased our understanding of RA pathogenesis, aided in identifying potential therapeutic targets and has been used in the response and mechanistic evaluation of antirheumatic treatments. In addition, studies are ongoing, aimed at the identification of diagnostic and prognostic biomarkers in the synovium. This paper outlines the currently used procedures for sampling and processing of synovial tissue, and presents a standardised recommendation to support multicentre translational research.
Subject(s)
Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/pathology , Biopsy/methods , Synovial Membrane/pathology , Synovitis/pathology , Arthritis, Rheumatoid/drug therapy , Arthroscopy/methods , Biopsy/standards , Clinical Trials as Topic/standards , Humans , Knee Joint/pathology , Multicenter Studies as Topic/standards , Treatment Outcome , Ultrasonography, Interventional/methodsABSTRACT
Saccharomyces cerevisiae can utilize high quality (e.g. glutamine and ammonia) as well as low quality (e.g. gamma-amino butyric acid and proline) nitrogen sources. The transcriptional activator Put3p allows yeast cells to utilize proline as a nitrogen source through expression of the PUT1 and PUT2 genes. Put3p activates high level transcription of these genes by binding proline directly. However, Put3p also responds to other lower quality nitrogen sources. As nitrogen quality decreases, Put3p exhibits an increase in phosphorylation concurrent with an increase in PUT gene expression. The proline-independent activation of the PUT genes requires both Put3p and the positively acting GATA factors, Gln3p and Gat1p. Conversely, the phosphorylation of Put3p is not dependent on GATA factor activity. Here, we find that the mutation of Put3p at amino acid Tyr-788 modulates the proline-independent activation of PUT1 through Gat1p. The phosphorylation of Put3p appears to influence the association of Gat1p, but not Gln3p, to the PUT1 promoter. Combined, our findings suggest that this may represent a mechanism through which yeast cells rapidly adapt to use proline as a nitrogen source under nitrogen limiting conditions.
Subject(s)
Antifungal Agents/pharmacology , GATA Transcription Factors/metabolism , Gene Expression Regulation, Fungal/drug effects , Proline Oxidase/biosynthesis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sirolimus/pharmacology , Transcription Factors/metabolism , GATA Transcription Factors/genetics , Gene Expression Regulation, Fungal/genetics , Mutation , Phosphorylation/drug effects , Phosphorylation/genetics , Proline/genetics , Proline/metabolism , Proline Oxidase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/geneticsABSTRACT
What does this mean for Connecticut physicians? It means that the government will subsidize EMRs for your offices, that within five years you will have to have an EMR to be paid by Medicare, that you will be paid at the same rate as doctors in more rural regions of the U.S, and that Medicare will pay you at reduced rates or not all for procedures it deems as of no benefit.
Subject(s)
Health Care Reform/organization & administration , Medicare/organization & administration , Efficiency, Organizational , Family Practice/economics , Family Practice/organization & administration , Humans , Practice Patterns, Physicians' , United StatesABSTRACT
The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.
Subject(s)
Galactokinase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription Factors/metabolism , Transcription, Genetic/physiology , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Binding Sites/physiology , Enzyme Stability/physiology , Galactokinase/genetics , Galactosephosphates/genetics , Galactosephosphates/metabolism , Kluyveromyces/enzymology , Kluyveromyces/genetics , Ligands , Mutation , Protein Binding/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
In Saccharomyces cerevisiae, the GAL genes encode the enzymes required for galactose metabolism. Regulation of these genes has served as the paradigm for eukaryotic transcriptional control over the last 50 years. The switch between inert and active gene expression is dependent upon three proteins--the transcriptional activator Gal4p, the inhibitor Gal80p, and the ligand sensor Gal3p. Here, we present a detailed spatial analysis of the three GAL regulatory proteins produced from their native genomic loci. Using a novel application of photobleaching, we demonstrate, for the first time, that the Gal3p ligand sensor enters the nucleus of yeast cells in the presence of galactose. Additionally, using Förster resonance energy transfer, we show that the interaction between Gal3p and Gal80p occurs throughout the yeast cell. Taken together, these data challenge existing models for the cellular localization of the regulatory proteins during the induction of GAL gene expression by galactose and suggest a mechanism for the induction of the GAL genes in which galactose-bound Gal3p moves from the cytoplasm to the nucleus to interact with the transcriptional inhibitor Gal80p.
Subject(s)
Galactose/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , DNA-Binding Proteins , Protein Binding , Protein Transport , Repressor Proteins/genetics , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription Factors/geneticsABSTRACT
OBJECTIVE: To compare work disability and job loss in early rheumatoid arthritis (RA) patients receiving adalimumab plus methotrexate (adalimumab + MTX) versus MTX alone. METHODS: In this multicenter, randomized, controlled trial, patients with RA for <2 years who had never taken MTX and who self-reported work impairment were randomized to adalimumab + MTX or placebo + MTX for 56 weeks. Primary outcome was job loss of any cause and/or imminent job loss at or after week 16. Secondary outcomes included disease activity, function (Health Assessment Questionnaire [HAQ] score), and RA quality of life (RAQoL) questionnaire score. Work was evaluated with work diaries and the RA Work Instability Scale. RESULTS: Although job loss during the 56-week study was significantly lower with adalimumab + MTX (14 of 75 patients) compared with MTX alone (29 of 73 patients; P=0.005), the primary end point was not met (12 of 75 versus 20 of 73 patients; P=0.092), likely owing to early drop out in the MTX group. There were significant improvements in American College of Rheumatology 20% response criteria, 28-joint Disease Activity Score, DeltaHAQ, DeltaRAQoL, and working time lost in the adalimumab + MTX group. Twenty-four serious adverse events were reported in 17 participants, with no differences between groups. CONCLUSION: Adalimumab + MTX reduced job loss and improved productivity in early RA when compared with MTX alone, which supports the early use of anti-tumor necrosis factor therapy and suggests its cost efficacy.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antirheumatic Agents/administration & dosage , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/physiopathology , Employment , Adalimumab , Adult , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal, Humanized , Antirheumatic Agents/adverse effects , Arthritis, Rheumatoid/diagnosis , Drug Therapy, Combination , Early Diagnosis , Female , Humans , Kaplan-Meier Estimate , Male , Methotrexate/administration & dosage , Methotrexate/adverse effects , Middle Aged , Treatment Outcome , Tumor Necrosis Factor-alpha/antagonists & inhibitorsABSTRACT
The enzymes of the Leloir pathway catalyze the conversion of galactose to a more metabolically useful version, glucose-6-phosphate. This pathway is required as galactose itself cannot be used for glycolysis directly. In most organisms, including the yeast Saccharomyces cerevisiae, five enzymes are required to catalyze this conversion: a galactose mutarotase, a galactokinase, a galactose-1-phosphate uridyltransferase, a UDP-galactose-4-epimerase, and a phosphoglucomutase. In yeast, the genes encoding these enzymes are tightly controlled at the level of transcription and are only transcribed under specific sets of conditions. In the presence of glucose, the genes encoding the Leloir pathway enzymes (often called the GAL genes) are repressed through the action of a transcriptional repressor Mig1p. In the presence of galactose, but in the absence of glucose, the concerted actions of three other proteins Gal4p, Gal80p, and Gal3p, and two small molecules (galactose and ATP) enable the rapid and high-level activation of the GAL genes. The precise molecular mechanism of the GAL genetic switch is controversial. Recent work on solving the three-dimensional structures of the various GAL enzymes proteins and the GAL transcriptional switch proteins affords a unique opportunity to delve into the precise, and potentially unambiguous, molecular mechanism of a highly exploited transcriptional circuit. Understanding the details of the transcriptional and metabolic events that occur in this pathway can be used as a paradigm for understanding the integration of metabolism and transcriptional control more generally, and will assist our understanding of fundamental biochemical processes and how these might be exploited.
Subject(s)
Fungal Proteins/biosynthesis , Galactose/metabolism , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Fungal/physiology , Saccharomycetales/physiology , Transcription, Genetic/physiologyABSTRACT
The GAL genes, which encode the enzymes required for normal galactose metabolism in yeast, are transcriptionally regulated by three proteins: Gal4p, an activator; Gal80p, an inhibitor; and Gal3p, a galactose sensor. These proteins control the switch between inert and active gene expression. The transcriptional activation function of Gal4p is rendered inactive in the presence of Gal80p. Here we present the three-dimensional structure of a complex between the acidic activation domain of Gal4p and Gal80p. The transactivation domain initiates with an extended region of polypeptide chain followed by two turns of an amphipathic alpha-helix. It fits into and across a deep cleft within the Gal80p dimer with the protein-protein interface defined primarily by hydrophobic interactions. A disordered loop in the apo-Gal80p structure (Asp-309 to Ser-316) becomes well-defined upon binding of the transactivation domain. This investigation provides a new molecular scaffold for understanding previous biochemical and genetic studies.
Subject(s)
Kluyveromyces/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Amino Acid Sequence , Binding Sites , DNA-Binding Proteins , Magnetic Resonance Spectroscopy , Models, Biological , Molecular Conformation , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Repressor Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid , Tumor Suppressor Protein p53/chemistryABSTRACT
The comparatively simple eukaryote Saccharomyces cerevisiae is composed of some 6000 individual genes. Specific sets of these genes can be transcribed co-ordinately in response to particular metabolic signals. The resultant integrated response to nutrient challenge allows the organism to survive and flourish in a variety of environmental conditions while minimal energy is expended upon the production of unnecessary proteins. The Zn(II)2Cys6 family of transcriptional regulators is composed of some 46 members in S. cerevisiae and many of these have been implicated in mediating transcriptional responses to specific nutrients. Gal4p, the archetypical member of this family, is responsible for the expression of the GAL genes when galactose is utilized as a carbon source. The regulation of Gal4p activity has been studied for many years, but we are still uncovering both nuances and fundamental control mechanisms that impinge on its function. In the present review, we describe the latest developments in the regulation of GAL gene expression and compare the mechanisms employed here with the molecular control of other Zn(II)2Cys6 transcriptional regulators. This reveals a wide array of protein-protein, protein-DNA and protein-nutrient interactions that are employed by this family of regulators.
Subject(s)
Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , Galactose/metabolism , Gene Expression Regulation, Fungal , Models, Biological , Protein Structure, Secondary , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Arthroscopy continues to experience a growth in interest from the rheumatology community reflecting a common desire to gain better understanding of the underlying processes in inflammatory and degenerative joint diseases. Arthroscopy provides the ability to assess the internal appearances of a joint in a well tolerated and repeatable manner, to obtain tissue samples from the principle site of pathology within the joint and thus confers on it the role of "gold standard" amongst currently available imaging techniques. The evolution of arthroscopy is reviewed together with an overview of the evidence obtained from its research application in the rheumatology. Methodology for the conduct of arthroscopy and synovial biopsy is described.
Subject(s)
Arthroscopy/methods , Rheumatic Diseases/diagnosis , Arthroscopes , Biopsy/methods , Humans , Knee Joint/pathology , Research Design , Rheumatic Diseases/pathology , Synovial Membrane/pathologyABSTRACT
Genome-scale metabolic models promise important insights into cell function. However, the definition of pathways and functional network modules within these models, and in the biochemical literature in general, is often based on intuitive reasoning. Although mathematical methods have been proposed to identify modules, which are defined as groups of reactions with correlated fluxes, there is a need for experimental verification. We show here that multivariate statistical analysis of the NMR-derived intra- and extracellular metabolite profiles of single-gene deletion mutants in specific metabolic pathways in the yeast Saccharomyces cerevisiae identified outliers whose profiles were markedly different from those of the other mutants in their respective pathways. Application of flux coupling analysis to a metabolic model of this yeast showed that the deleted gene in an outlying mutant encoded an enzyme that was not part of the same functional network module as the other enzymes in the pathway. We suggest that metabolomic methods such as this, which do not require any knowledge of how a gene deletion might perturb the metabolic network, provide an empirical method for validating and ultimately refining the predicted network structure.
Subject(s)
Magnetic Resonance Spectroscopy/methods , Metabolic Networks and Pathways , Models, Biological , Saccharomyces cerevisiae/metabolism , Genes, Fungal , Genome, Fungal , Glycolysis , Mutation , Proline/metabolism , Pyrimidines/biosynthesis , Pyrimidines/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Trehalose/biosynthesis , Trehalose/metabolismABSTRACT
OBJECTIVE: The role of alternative tumor necrosis factor (TNF) antagonist therapies in the context of failure of initial TNF antagonist therapy in patients with rheumatoid arthritis (RA) has yet to be clearly defined. The goal of this study was to determine the efficacy of etanercept in patients who failed to respond to infliximab. METHODS: Ninety-five patients with RA who failed to respond to infliximab and methotrexate were treated with etanercept (with continuation of concomitant methotrexate). Thirty-four patients never achieved a response to infliximab (primary nonresponse), 38 had an initial response to infliximab but relapsed (secondary nonresponse), and 23 demonstrated toxicity. Disease Activity Score in 28 joints (DAS28), European League Against Rheumatism (EULAR) response, and American College of Rheumatology (ACR) response were determined after 12 weeks of etanercept. RESULTS: After 12 weeks of etanercept, 38% of patients achieved an ACR 20% response (ACR20) on etanercept. Of these, 24% and 15% achieved ACR50 and ACR70 responses, respectively. In the primary infliximab nonresponse group, 42%, 30%, and 15% achieved ACR20, ACR50, and ACR70 responses, respectively; the percentages for the secondary nonresponse group were 34%, 21%, and 14%, respectively. Significant DAS28 reductions were observed in the entire cohort and nonresponse subtype groups. Sixty-one percent of the cohort achieved either a moderate or good EULAR score (67% of primary and 56% of secondary infliximab failures). No toxicity was observed in patients who stopped infliximab due to intolerance; 19 of 23 continued etanercept after week 12. CONCLUSION: This study confirms that etanercept is effective in patients who fail to respond to infliximab and suggests a higher response in patients who have never had a response to infliximab.