Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.Life Technologies, Inc.United States. Defense Advanced Research Projects Agency (DARPA CLIO N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210

Five-class differential diagnostics of neurodegenerative diseases using random undersampling boosting

Differentiating between different types of neurodegenerative diseases is not only crucial in clinical practice when treatment decisions have to be made, but also has a significant potential for the enrichment of clinical trials. The purpose of this study is to develop a classification framework for distinguishing the four most common neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobe degeneration, Dementia with Lewy bodies and vascular dementia, as well as patients with subjective memory complaints. Different biomarkers including features from images (volume features, region-wise grading features) and non-imaging features (CSF measures) were extracted for each subject. In clinical practice, the prevalence of different dementia types is imbalanced, posing challenges for learning an effective classification model. Therefore, we propose the use of the RUSBoost algorithm in order to train classifiers and to handle the class imbalance training problem. Furthermore, a multi-class feature selection method based on sparsity is integrated into the proposed framework to improve the classification performance. It also provides a way for investigating the importance of different features and regions. Using a dataset of 500 subjects, the proposed framework achieved a high accuracy of 75.2% with a balanced accuracy of 69.3% for the five-class classification using ten-fold cross validation, which is significantly better than the results using support vector machine or random forest, demonstrating the feasibility of the proposed framework to support clinical decision making

Monotonicity, frustration, and ordered response: an analysis of the energy landscape of perturbed large-scale biological networks

<p>Abstract</p> <p>Background</p> <p>For large-scale biological networks represented as signed graphs, the index of frustration measures how far a network is from a monotone system, i.e., how incoherently the system responds to perturbations.</p> <p>Results</p> <p>In this paper we find that the frustration is systematically lower in transcriptional networks (modeled at functional level) than in signaling and metabolic networks (modeled at stoichiometric level). A possible interpretation of this result is in terms of energetic cost of an interaction: an erroneous or contradictory transcriptional action costs much more than a signaling/metabolic error, and therefore must be avoided as much as possible. Averaging over all possible perturbations, however, we also find that unlike for transcriptional networks, in the signaling/metabolic networks the probability of finding the system in its least frustrated configuration tends to be high also in correspondence of a moderate energetic regime, meaning that, in spite of the higher frustration, these networks can achieve a globally ordered response to perturbations even for moderate values of the strength of the interactions. Furthermore, an analysis of the energy landscape shows that signaling and metabolic networks lack energetic barriers around their global optima, a property also favouring global order.</p> <p>Conclusion</p> <p>In conclusion, transcriptional and signaling/metabolic networks appear to have systematic differences in both the index of frustration and the transition to global order. These differences are interpretable in terms of the different functions of the various classes of networks.</p

Reversal of the ΔdegP Phenotypes by a Novel rpoE Allele of Escherichia coli

RseA sequesters RpoE (σE) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σE to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σE regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σE levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σE-dependent RybB::LacZ construct showed only a weak activation of the σE pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σE and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrAL222Q, it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σE-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σE-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σE levels

The use of opioids at the end of life: knowledge level of pharmacists and cooperation with physicians

Contains fulltext : 96464.pdf (publisher's version ) (Open Access)PURPOSE: What is the level of knowledge of pharmacists concerning pain management and the use of opioids at the end of life, and how do they cooperate with physicians? METHODS: A written questionnaire was sent to a sample of community and hospital pharmacists in the Netherlands. The questionnaire was completed by 182 pharmacists (response rate 45%). RESULTS: Pharmacists were aware of the most basic knowledge about opioids. Among the respondents, 29% erroneously thought that life-threatening respiratory depression was a danger with pain control, and 38% erroneously believed that opioids were the preferred drug for palliative sedation. One in three responding pharmacists did not think his/her theoretical knowledge was sufficient to provide advice on pain control. Most pharmacists had working agreements with physicians on euthanasia (81%), but fewer had working agreements on palliative sedation (46%) or opioid therapy (25%). Based on the experience of most of responding pharmacists (93%), physicians were open to unsolicited advice on opioid prescriptions. The majority of community pharmacists (94%) checked opioid prescriptions most often only after dispensing, while it was not a common practice among the majority of hospital pharmacists (68%) to check prescriptions at all. CONCLUSIONS: Although the basic knowledge of most pharmacists was adequate, based on the responses to the questionnaire, there seems to be a lack of knowledge in several areas, which may hamper pharmacists in improving the quality of care when giving advice to physicians and preventing or correcting mistakes if necessary. If education is improved, a more active role of the pharmacist may improve the quality of end-of-life pharmacotherapy

The use of opioids at the end of life: the knowledge level of Dutch physicians as a potential barrier to effective pain management

<p>Abstract</p> <p>Background</p> <p>Pain is still one of the most frequently occurring symptoms at the end of life, although it can be treated satisfactorily in most cases if the physician has adequate knowledge. In the Netherlands, almost 60% of the patients with non-acute illnesses die at home where end of life care is coordinated by the general practitioner (GP); about 30% die in hospitals (cared for by clinical specialists), and about 10% in nursing homes (cared for by elderly care physicians).</p> <p>The research question of this study is: what is the level of knowledge of Dutch physicians concerning pain management and the use of opioids at the end of life?</p> <p>Methods</p> <p>A written questionnaire was sent to a random sample of physicians of specialties most often involved in end of life care in the Netherlands. The questionnaire was completed by 406 physicians, response rate 41%.</p> <p>Results</p> <p>Almost all physicians were aware of the most basal knowledge about opioids, e.g. that it is important for treatment purposes to distinguish nociceptive from neuropathic pain (97%). Approximately half of the physicians (46%) did not know that decreased renal function raises plasma concentration of morphine(-metabolites) and 34% of the clinical specialists erroneously thought opioids are the favoured drug for palliative sedation.</p> <p>Although 91% knew that opioids titrated against pain do not shorten life, 10% sometimes or often gave higher dosages than needed with the explicit aim to hasten death. About half felt sometimes or often pressured by relatives to hasten death by increasing opioiddosage.</p> <p>The large majority (83%) of physicians was interested in additional education about subjects related to the end of life, the most popular subject was opioid rotation (46%).</p> <p>Conclusions</p> <p>Although the basic knowledge of physicians was adequate, there seemed to be a lack of knowledge in several areas, which can be a barrier for good pain management at the end of life. From this study four areas emerge, in which it seems likely that an improvement can improve the quality of pain management at the end of life for many patients in the Netherlands: 1)palliative sedation; 2)expected effect of opioids on survival; and 3) opioid rotation.</p

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552