Toward Engineering Synthetic Microbial Metabolism

The generation of well-characterized parts and the formulation of biological design principles in synthetic biology are laying the foundation for more complex and advanced microbial metabolic engineering. Improvements in de novo DNA synthesis and codon-optimization alone are already contributing to the manufacturing of pathway enzymes with improved or novel function. Further development of analytical and computer-aided design tools should accelerate the forward engineering of precisely regulated synthetic pathways by providing a standard framework for the predictable design of biological systems from well-characterized parts. In this review we discuss the current state of synthetic biology within a four-stage framework (design, modeling, synthesis, analysis) and highlight areas requiring further advancement to facilitate true engineering of synthetic microbial metabolism

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules

BACKGROUND: In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. RESULTS: In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. CONCLUSION: The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA

Lactate: brain fuel in human traumatic brain injury: a comparison with normal healthy control subjects.

We evaluated the hypothesis that lactate shuttling helps support the nutritive needs of injured brains. To that end, we utilized dual isotope tracer [6,6-(2)H2]glucose, that is, D2-glucose, and [3-(13)C]lactate techniques involving arm vein tracer infusion along with simultaneous cerebral (arterial [art] and jugular bulb [JB]) blood sampling. Traumatic brain injury (TBI) patients with nonpenetrating brain injuries (n=12) were entered into the study following consent of patients' legal representatives. Written and informed consent was obtained from control volunteers (n=6). Patients were studied 5.7±2.2 (mean±SD) days post-injury; during periods when arterial glucose concentration tended to be higher in TBI patients. As in previous investigations, the cerebral metabolic rate for glucose (CMRgluc, i.e., net glucose uptake) was significantly suppressed following TBI (p<0.001). However, lactate fractional extraction, an index of cerebral lactate uptake related to systemic lactate supply, approximated 11% in both healthy control subjects and TBI patients. Further, neither the CMR for lactate (CMRlac, i.e., net lactate release), nor the tracer-measured cerebral lactate uptake differed between healthy controls and TBI patients. The percentages of lactate tracer taken up and released as (13)CO2 into the JB accounted for 92% and 91% for control and TBI conditions, respectively, suggesting that most cerebral lactate uptake was oxidized following TBI. Comparisons of isotopic enrichments of lactate oxidation from infused [3-(13)C]lactate tracer and (13)C-glucose produced during hepatic and renal gluconeogenesis (GNG) showed that 75-80% of (13)CO2 released into the JB was from lactate and that the remainder was from the oxidation of glucose secondarily labeled from lactate. Hence, either directly as lactate uptake, or indirectly via GNG, peripheral lactate production accounted for ∼70% of carbohydrate (direct lactate uptake+uptake of glucose from lactate) consumed by the injured brain. Undiminished cerebral lactate fractional extraction and uptake suggest that arterial lactate supplementation may be used to compensate for decreased CMRgluc following TBI

Orthogonal Expression of Metabolic Pathways

Microbial metabolism can be tailored to meet human specifications, but the degree to which these living systems can be repurposed is still unknown. Artificial biological control strategies are being developed with the goal of enabling the predictable implementation of novel biological functions (e.g., engineered metabolism). This dissertation project contributes genetic tools useful for modulating gene expression levels (extending promoters with UP elements) and isolating transcription and translation of engineered DNA from the endogenous cellular network (expression by orthogonal cellular machinery), which have been demonstrated in Escherichia coli for the production of lycopene, a 40-carbon tetraterpene carotenoid with antioxidant activity and a number of other desirable properties

Endogenous Nutritive Support after Traumatic Brain Injury: Peripheral Lactate Production for Glucose Supply via Gluconeogenesis.

We evaluated the hypothesis that nutritive needs of injured brains are supported by large and coordinated increases in lactate shuttling throughout the body. To that end, we used dual isotope tracer ([6,6-(2)H2]glucose, i.e., D2-glucose, and [3-(13)C]lactate) techniques involving central venous tracer infusion along with cerebral (arterial [art] and jugular bulb [JB]) blood sampling. Patients with traumatic brain injury (TBI) who had nonpenetrating head injuries (n=12, all male) were entered into the study after consent of patients' legal representatives. Written and informed consent was obtained from healthy controls (n=6, including one female). As in previous investigations, the cerebral metabolic rate (CMR) for glucose was suppressed after TBI. Near normal arterial glucose and lactate levels in patients studied 5.7±2.2 days (range of days 2-10) post-injury, however, belied a 71% increase in systemic lactate production, compared with control, that was largely cleared by greater (hepatic+renal) glucose production. After TBI, gluconeogenesis from lactate clearance accounted for 67.1% of glucose rate of appearance (Ra), which was compared with 15.2% in healthy controls. We conclude that elevations in blood glucose concentration after TBI result from a massive mobilization of lactate from corporeal glycogen reserves. This previously unrecognized mobilization of lactate subserves hepatic and renal gluconeogenesis. As such, a lactate shuttle mechanism indirectly makes substrate available for the body and its essential organs, including the brain, after trauma. In addition, when elevations in arterial lactate concentration occur after TBI, lactate shuttling may provide substrate directly to vital organs of the body, including the injured brain

A validation of Emotiv EPOC Flex saline for EEG and ERP research

Background Previous work has validated consumer-grade electroencephalography (EEG) systems for use in research. Systems in this class are cost-effective and easy to set up and can facilitate neuroscience outside of the laboratory. The aim of the current study was to determine if a new consumer-grade system, the Emotiv EPOC Saline Flex, was capable of capturing research-quality data. Method The Emotiv system was used simultaneously with a research-grade EEG system, Neuroscan Synamps2, to collect EEG data across 16 channels during five well-established paradigms: (1) a mismatch negativity (MMN) paradigm that involved a passive listening task in which rare deviant (1,500 Hz) tones were interspersed amongst frequent standard tones (1,000 Hz), with instructions to ignore the tones while watching a silent movie; (2) a P300 paradigm that involved an active listening task in which participants were asked to count rare deviant tones presented amongst frequent standard tones; (3) an N170 paradigm in which participants were shown images of faces and watches and asked to indicate whether the images were upright or inverted; (4) a steady-state visual evoked potential (SSVEP) paradigm in which participants passively viewed a flickering screen (15 Hz) for 2 min; and (5) a resting state paradigm in which participants sat quietly with their eyes open and then closed for 3 min each. Results The MMN components and P300 peaks were equivalent between the two systems (BF10 = 0.25 and BF10 = 0.26, respectively), with high intraclass correlations (ICCs) between the ERP waveforms (>0.81). Although the N170 peak values recorded by the two systems were different (BF10 = 35.88), ICCs demonstrated that the N170 ERP waveforms were strongly correlated over the right hemisphere (P8; 0.87–0.97), and moderately-to-strongly correlated over the left hemisphere (P7; 0.52–0.84). For the SSVEP, the signal-to-noise ratio (SNR) was larger for Neuroscan than Emotiv EPOC Flex (19.94 vs. 8.98, BF10 = 51,764), but SNR z-scores indicated a significant brain response at the stimulus frequency for both Neuroscan (z = 12.47) and Flex (z = 11.22). In the resting state task, both systems measured similar alpha power (BF10 = 0.28) and higher alpha power when the eyes were closed than open (BF10 = 32.27). Conclusions The saline version of the Emotiv EPOC Flex captures data similar to that of a research-grade EEG system. It can be used to measure reliable auditory and visual research-quality ERPs. In addition, it can index SSVEP signatures and is sensitive to changes in alpha oscillations

Incorporating forecasting and peer-to-peer negotiation frameworks into a distributed model predictive control approach for meshed electric networks

The continuous integration of renewable energy sources into a power network has caused a paradigm shift in energy generation and distribution. The intermittent nature of renewable sources affects the prices at which energy can be sold or purchased. In addition, the network is subject to operational constraints, voltage limits at each node, rated capacities for the power electronic devices, current bounds for distribution lines; these constraints coupled with intermittent renewable injections may pose a threat to system stability and performance. We propose a distributed predictive controller to handle operational constraints while minimising generation costs, and an agent based market negotiation framework to obtain suitable pricing policies, agreed among participating agents, that explicitly considers availability of energy storage in its formulation. The controller handles the problem of coupled constraints using information exchanges with its neighbours to guarantee their satisfaction. We study the effect of different forecast accuracy have on the overall performance and market behaviours. We provide a convergence analysis for both the negotiation iterations, and its interaction with the predictive controller. Lastly, We assess the impact of the information availability with the aid of testing scenarios

Incorporating forecasting and peer-to-peer negotiation frameworks into a distributed model-predictive control approach for meshed electric networks

The continuous integration of renewable energy sources into power networks is causing a paradigm shift in energy generation and distribution with regard to trading and control. The intermittent nature of renewable sources affects the pricing of energy sold or purchased. The networks are subject to operational constraints, voltage limits at each node, rated capacities for the power electronic devices, and current bounds for distribution lines. These economic and technical constraints, coupled with intermittent renewable injection, may pose a threat to system stability and performance. In this article, we propose a novel holistic approach to energy trading composed of a distributed predictive control framework to handle physical interactions, i.e., voltage constraints and power dispatch, together with a negotiation framework to determine pricing policies for energy transactions. We study the effect of forecasting generation and consumption on the overall network's performance and market behaviors. We provide a rigorous convergence analysis for both the negotiation framework and the distributed control. Finally, we assess the impact of forecasting in the proposed system with the aid of testing scenarios

The Extrasolar Planet epsilon Eridani b - Orbit and Mass

Hubble Space Telescope observations of the nearby (3.22 pc), K2 V star epsilon Eridani have been combined with ground-based astrometric and radial velocity data to determine the mass of its known companion. We model the astrometric and radial velocity measurements simultaneously to obtain the parallax, proper motion, perturbation period, perturbation inclination, and perturbation size. Because of the long period of the companion, \eps b, we extend our astrometric coverage to a total of 14.94 years (including the three year span of the \HST data) by including lower-precision ground-based astrometry from the Allegheny Multichannel Astrometric Photometer. Radial velocities now span 1980.8 -- 2006.3. We obtain a perturbation period, P = 6.85 +/- 0.03 yr, semi-major axis, alpha =1.88 +/- 0.20 mas, and inclination i = 30.1 +/- 3.8 degrees. This inclination is consistent with a previously measured dust disk inclination, suggesting coplanarity. Assuming a primary mass M_* = 0.83 M_{\sun}, we obtain a companion mass M = 1.55 +/- 0.24 M_{Jup}. Given the relatively young age of epsilon Eri (~800 Myr), this accurate exoplanet mass and orbit can usefully inform future direct imaging attempts. We predict the next periastron at 2007.3 with a total separation, rho = 0.3 arcsec at position angle, p.a. = -27 degrees. Orbit orientation and geometry dictate that epsilon Eri b will appear brightest in reflected light very nearly at periastron. Radial velocities spanning over 25 years indicate an acceleration consistent with a Jupiter-mass object with a period in excess of 50 years, possibly responsible for one feature of the dust morphology, the inner cavity

Hair coat properties of donkeys, mules and horses in a temperate climate

Background There are clear differences between donkeys and horses in their evolutionary history, physiology, behaviour and husbandry needs. Donkeys are often kept in climates they are not adapted to and as such may suffer impaired welfare unless protection from the elements is provided. Objectives We provide the first direct comparison of the hair coat properties of donkeys, mules and horses living outside, throughout the year, in the temperate climate of the UK. Study Design The weight, length and width of hair were measured, across the four seasons, as indicators of the hair coat insulation properties. Methods Hair samples were taken from 42 animals: 18 donkeys (4 females, 14 males), 16 horses (6 females, 10 males), and eight mules (5 females, 3 males), in March, June, September and December. Results Donkeys’ hair coats do not significantly differ across the seasons. All three measurements of the insulation properties of the hair samples indicate that donkeys do not grow a winter coat and that their hair coat was significantly lighter, shorter and thinner than that of horses and mules in winter. In contrast the hair coats of horses changed significantly between seasons, growing thicker in winter. Main Limitations The measurements cover only a limited range of features that contribute to the thermo-regulation of an animal. Further research is needed to assess shelter preferences by behavioural measures, and absolute heat loss via thermoimaging. Conclusions Donkeys, and to a lesser extent mules, appear not to be as adapted to colder, wet climates as horses, and may therefore require additional protection from the elements, such as access to a wind and waterproof shelter, in order for their welfare needs to be met