237 research outputs found
Asymptotically stable phase synchronization revealed by autoregressive circle maps
A new type of nonlinear time series analysis is introduced, based on phases,
which are defined as polar angles in spaces spanned by a finite number of
delayed coordinates. A canonical choice of the polar axis and a related
implicit estimation scheme for the potentially underlying auto-regressive
circle map (next phase map) guarantee the invertibility of reconstructed phase
space trajectories to the original coordinates. The resulting Fourier
approximated, Invertibility enforcing Phase Space map (FIPS map) is well suited
to detect conditional asymptotic stability of coupled phases. This rather
general synchronization criterion unites two existing generalisations of the
old concept and can successfully be applied e.g. to phases obtained from ECG
and airflow recordings characterizing cardio-respiratory interaction.Comment: PDF file, 232 KB, 24 pages, 3 figures; cheduled for Phys. Rev. E
(Nov) 200
Integrating DGSs and GATPs in an Adaptative and Collaborative Blended-Learning Web-Environment
The area of geometry with its very strong and appealing visual contents and
its also strong and appealing connection between the visual content and its
formal specification, is an area where computational tools can enhance, in a
significant way, the learning environments.
The dynamic geometry software systems (DGSs) can be used to explore the
visual contents of geometry. This already mature tools allows an easy
construction of geometric figures build from free objects and elementary
constructions. The geometric automated theorem provers (GATPs) allows formal
deductive reasoning about geometric constructions, extending the reasoning via
concrete instances in a given model to formal deductive reasoning in a
geometric theory.
An adaptative and collaborative blended-learning environment where the DGS
and GATP features could be fully explored would be, in our opinion a very rich
and challenging learning environment for teachers and students.
In this text we will describe the Web Geometry Laboratory a Web environment
incorporating a DGS and a repository of geometric problems, that can be used in
a synchronous and asynchronous fashion and with some adaptative and
collaborative features.
As future work we want to enhance the adaptative and collaborative aspects of
the environment and also to incorporate a GATP, constructing a dynamic and
individualised learning environment for geometry.Comment: In Proceedings THedu'11, arXiv:1202.453
Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation
<p>Abstract</p> <p>Background</p> <p>The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments.</p> <p>Results</p> <p>We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system <it>E.coli </it>BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the <it>E.coli </it>cells via a phosphate limitation.</p> <p>Conclusions</p> <p>The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant <it>E.coli</it>-strains.</p
Switchable Gene Expression in Escherichia coli Using a Miniaturized Photobioreactor
We present a light-switchable gene expression system for both inducible and switchable control of gene expression at a single cell level in Escherichia coli using a previously constructed light-sensing system. The lambda cl repressor gene with an LVA degradation tag was expressed under the control of the ompC promoter on the chromosome. The green fluorescent protein (GFP) gene fused to a lambda repressor-repressible promoter was used as a reporter. This light-switchable system allows rapid and reversible induction or repression of expression of the target gene at any desired time. This system also ensures homogenous expression across the entire cell population. We also report the design of a miniaturized photobioreactor to be used in combination with the light-switchable gene expression system. The miniaturized photobioreactor helps to reduce unintended induction of the light receptor due to environmental disturbances and allows precise control over the duration of induction. This system would be a good tool for switchable, homogenous, strong, and highly regulatable expression of target genes over a wide range of induction times. Hence, it could be applied to study gene function, optimize metabolic pathways, and control biological systems both spatially and temporally.open0
Directed evolution of bright mutants of an oxygen-independent flavin-binding fluorescent protein from Pseudomonas putida
Genetic Transformation of an Obligate Anaerobe, P. gingivalis for FMN-Green Fluorescent Protein Expression in Studying Host-Microbe Interaction
The recent introduction of “oxygen-independent” flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) is of major interest to both eukaryotic and prokaryotic microbial biologists. Accordingly, we demonstrate for the first time that an obligate anaerobe, the successful opportunistic pathogen of the oral cavity, Porphyromonas gingivalis, can be genetically engineered for expression of the non-toxic green FbFP. The resulting transformants are functional for studying dynamic bacterial processes in living host cells. The visualization of the transformed P. gingivalis (PgFbFP) revealed strong fluorescence that reached a maximum emission at 495 nm as determined by fluorescence microscopy and spectrofluorometry. Human primary gingival epithelial cells (GECs) were infected with PgFbFP and the bacterial invasion of host cells was analyzed by a quantitative fluorescence microscopy and antibiotic protection assays. The results showed similar levels of intracellular bacteria for both wild type and PgFbFP strains. In conjunction with organelle specific fluorescent dyes, utilization of the transformed strain provided direct and accurate determination of the live/metabolically active P. gingivalis' trafficking in the GECs over time. Furthermore, the GECs were co-infected with PgFbFP and the ATP-dependent Clp serine protease-deficient mutant (ClpP-) to study the differential fates of the two strains within the same host cells. Quantitative co-localization analyses displayed the intracellular PgFbFP significantly associated with the endoplasmic reticulum network, whereas the majority of ClpP- organisms trafficked into the lysosomes. Hence, we have developed a novel and reliable method to characterize live host cell-microbe interactions and demonstrated the adaptability of FMN-green fluorescent protein for studying persistent host infections induced by obligate anaerobic organisms
Parallel use of shake flask and microtiter plate online measuring devices (RAMOS and BioLector) reduces the number of experiments in laboratory-scale stirred tank bioreactors
Background
Conventional experiments in small scale are often performed in a Black Box fashion, analyzing only the product concentration in the final sample. Online monitoring of relevant process characteristics and parameters such as substrate limitation, product inhibition and oxygen supply is lacking. Therefore, fully equipped laboratory-scale stirred tank bioreactors are hitherto required for detailed studies of new microbial systems. However, they are too spacious, laborious and expensive to be operated in larger number in parallel. Thus, the aim of this study is to present a new experimental approach to obtain dense quantitative process information by parallel use of two small-scale culture systems with online monitoring capabilities: Respiration Activity MOnitoring System (RAMOS) and the BioLector device.
Results
The same mastermix (medium plus microorganisms) was distributed to the different small-scale culture systems: 1) RAMOS device; 2) 48-well microtiter plate for BioLector device; and 3) separate shake flasks or microtiter plates for offline sampling. By adjusting the same maximum oxygen transfer capacity (OTRmax), the results from the RAMOS and BioLector online monitoring systems supplemented each other very well for all studied microbial systems (E. coli, G. oxydans, K. lactis) and culture conditions (oxygen limitation, diauxic growth, auto-induction, buffer effects).
Conclusions
The parallel use of RAMOS and BioLector devices is a suitable and fast approach to gain comprehensive quantitative data about growth and production behavior of the evaluated microorganisms. These acquired data largely reduce the necessary number of experiments in laboratory-scale stirred tank bioreactors for basic process development. Thus, much more quantitative information is obtained in parallel in shorter time.Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities
Expression profiling of hypothetical genes in Desulfovibrio vulgaris leads to improved functional annotation
Hypothetical (HyP) and conserved HyP genes account for >30% of sequenced bacterial genomes. For the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, 347 of the 3634 genes were annotated as conserved HyP (9.5%) along with 887 HyP genes (24.4%). Given the large fraction of the genome, it is plausible that some of these genes serve critical cellular roles. The study goals were to determine which genes were expressed and provide a more functionally based annotation. To accomplish this, expression profiles of 1234 HyP and conserved genes were used from transcriptomic datasets of 11 environmental stresses, complemented with shotgun LC–MS/MS and AMT tag proteomic data. Genes were divided into putatively polycistronic operons and those predicted to be monocistronic, then classified by basal expression levels and grouped according to changes in expression for one or multiple stresses. One thousand two hundred and twelve of these genes were transcribed with 786 producing detectable proteins. There was no evidence for expression of 17 predicted genes. Except for the latter, monocistronic gene annotation was expanded using the above criteria along with matching Clusters of Orthologous Groups. Polycistronic genes were annotated in the same manner with inferences from their proximity to more confidently annotated genes. Two targeted deletion mutants were used as test cases to determine the relevance of the inferred functional annotations
Clinical characteristics of patients with familial amyotrophic lateral sclerosis carrying the pathogenic GGGGCC hexanucleotide repeat expansion of C9ORF72
A large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on chromosome 9p21, has been recently reported to be responsible for similar to 40% of familial amyotrophic lateral sclerosis cases of European ancestry. The aim of the current article was to describe the phenotype of amyotrophic lateral sclerosis cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial amyotrophic lateral sclerosis. We collected DNA and analysed phenotype data for 141 index Italian familial amyotrophic lateral sclerosis cases (21 of Sardinian ancestry) and 41 German index familial amyotrophic lateral sclerosis cases. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy, 12 (57.1%) patients of Sardinian ancestry and nine (22.0%) of the 41 German index familial amyotrophic lateral sclerosis cases. The disease was maternally transmitted in 27 (49.1%) pedigrees and paternally transmitted in 28 (50.9%) pedigrees (P = non-significant). On average, children developed disease 7.0 years earlier than their parents [children: 55.8 years (standard deviation 7.9), parents: 62.8 (standard deviation 10.9); P = 0.003]. Parental phenotype influenced the type of clinical symptoms manifested by the child: of the 13 cases where the affected parent had an amyotrophic lateral sclerosis-frontotemporal dementia or frontotemporal dementia, the affected child also developed amyotrophic lateral sclerosis-frontotemporal dementia in nine cases. When compared with patients carrying mutations of other amyotrophic lateral sclerosis-related genes, those with C9ORF72 expansion had commonly a bulbar onset (42.2% compared with 25.0% among non-C9ORF72 expansion cases, P = 0.03) and cognitive impairment (46.7% compared with 9.1% among non-C9ORF72 expansion cases, P = 0.0001). Median survival from symptom onset among cases carrying C9ORF72 repeat expansion was 3.2 years lower than that of patients carrying TARDBP mutations (5.0 years; 95% confidence interval: 3.6-7.2) and longer than those with FUS mutations (1.9 years; 95% confidence interval: 1.7-2.1). We conclude that C9ORF72 hexanucleotide repeat expansions were the most frequent mutation in our large cohort of patients with familial amyotrophic lateral sclerosis of Italian, Sardinian and German ancestry. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for similar to 60% of familial amyotrophic lateral sclerosis in Italy. Patients with C9ORF72 hexanucleotide repeat expansions present some phenotypic differences compared with patients with mutations of other genes or with unknown mutations, namely a high incidence of bulbar-onset disease and comorbidity with frontotemporal dementia. Their pedigrees typically display a high frequency of cases with pure frontotemporal dementia, widening the concept of familial amyotrophic lateral sclerosis
FUS Transgenic Rats Develop the Phenotypes of Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration
Fused in Sarcoma (FUS) proteinopathy is a feature of frontotemporal lobar dementia (FTLD), and mutation of the fus gene segregates with FTLD and amyotrophic lateral sclerosis (ALS). To study the consequences of mutation in the fus gene, we created transgenic rats expressing the human fus gene with or without mutation. Overexpression of a mutant (R521C substitution), but not normal, human FUS induced progressive paralysis resembling ALS. Mutant FUS transgenic rats developed progressive paralysis secondary to degeneration of motor axons and displayed a substantial loss of neurons in the cortex and hippocampus. This neuronal loss was accompanied by ubiquitin aggregation and glial reaction. While transgenic rats that overexpressed the wild-type human FUS were asymptomatic at young ages, they showed a deficit in spatial learning and memory and a significant loss of cortical and hippocampal neurons at advanced ages. These results suggest that mutant FUS is more toxic to neurons than normal FUS and that increased expression of normal FUS is sufficient to induce neuron death. Our FUS transgenic rats reproduced some phenotypes of ALS and FTLD and will provide a useful model for mechanistic studies of FUS–related diseases
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