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1.  Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template 
Variable gene dosage is a major source of fluctuations in gene expression in both endogenous and synthetic circuits. Synthetic incoherent feedforward regulatory motifs using RNA interference are shown to robustly adapt to changes in DNA template amounts in mammalian cells.
Variable gene dosage is a major source of fluctuations in gene product levels in both endogenous and synthetic circuits.To mitigate gene expression variability, we designed, simulated, constructed, and tested regulatory circuits, implementing an incoherent feedforward motif.A number of control mechanisms including transcription and post-transcriptional regulation were tested in mammalian cells.Feedforward regulation displayed better adaptation than negative feedback, and circuits based on RNA interference were the most robust to variation in DNA template amounts.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Indeed, changes in gene dosage are clearly a major source of variation in cells, and yet those changes are very common in both normal processes (sex determination, ploidy change) and disease (gene amplification in cancer). In synthetic networks, the problem is exacerbated due to commonly used transient delivery methods that result in very large cell-to-cell variability in gene dosage. The basic question on gene dosage compensation in nature (Veitia et al, 2008; Acar et al, 2010) and a practical challenge of overcoming sensitivity to DNA copy number in synthetic circuits prompted us to investigate mechanisms to reduce this variability using sophisticated internal regulatory mechanisms. Indeed, the baseline expression unit in many synthetic circuits is an open-loop promoter-ORF combination. We hypothesized that some sort of internal regulation will result in ‘expression units' whose gene product (i.e. protein) output will depend only mildly on the intracellular concentration of its DNA template. In other words, we searched for architecture that would lead to ‘adaptation' of the gene product to the amount of DNA template.
By examining large body of published work, we found frequent reference to a so-called ‘incoherent feedforward' network motif (Mangan and Alon, 2003). The canonical three-node incoherent loop contains input, auxiliary regulator, and output nodes. The output is controlled directly by the input and the auxiliary regulator. The latter is also controlled by the input, introducing an additional indirect effect of the input on the output. In incoherent loops, the input controls the auxiliary regulator node in such a way that input's overall indirect action on the output via this node counteracts its direct effect. In a motif named ‘type I incoherent feedforward loop' (I1-FFL), the input's direct effect is activating, as is its control of the auxiliary node, while the auxiliary node controls the output through repression. One of the most studied properties of such motifs is their transient response to persistent stimulus, that is visually characterized as a ‘bump' or ‘pulse' (hence the name ‘pulse generator') that then goes back to the original steady state of the system (Basu et al, 2004). We hypothesized that changing DNA amount could serve as an input to an incoherent circuit if the auxiliary regulator and the output nodes are coexpressed from this DNA; in other words DNA can be viewed as an ‘activator' of both the regulator and the output. We conjectured that this might lead to adaptation to changes in DNA template.
We designed and simulated in silico a number of network architectures that all exhibit incoherent feedforward connectivity. We also compared them with the well-studied feedback loop circuit that in theory weakens but does not eliminate gene product dependency on the DNA template amount. The schematics of the circuits are shown in Figure 1.
Experimental measurement of input–output response of these circuits, or their transfer function, indeed uncovered adaptation of the output to DNA template abundance. Such adaptation has not been observed with feedback loop, as expected. Among various architectures, the post-transcriptional circuits showed faster adaptation, higher absolute expression levels and lower ‘noise' (Figure 4).
We also simulated and measured stochastic variability in the circuits by collecting all the cells with similar input values and statistically analyzing output values in those cells. We found that substantial noise component could not be accounted for by known noise sources, and concluded that the very step of negative regulation, both by a repressor LacI and by a microRNA, significantly increases cell-to-cell variability. This needs to be addressed in further studies. We also found that the negative feedback loop did not result in reduced noise as we expected, yet it did not result in noise increase as in the incoherent motif. This means that there may be effective noise reduction but it is not sufficient to produce narrow distributions of outputs for a given input.
We conclude that expression units that incorporate incoherent feedforward control of the gene product provide adaptation to the amount of DNA template and can be superior to simple combinations of constitutive promoters with ORFs. We also emphasize the relevance of our findings to the long-standing question of gene dosage compensation in cells, and note that similar incoherent architectures with microRNA negative regulators have been found in cells, suggesting that their physiological role is to curb variability in gene dosage and/or promoter strength.
Natural and synthetic biological networks must function reliably in the face of fluctuating stoichiometry of their molecular components. These fluctuations are caused in part by changes in relative expression efficiency and the DNA template amount of the network-coding genes. Gene product levels could potentially be decoupled from these changes via built-in adaptation mechanisms, thereby boosting network reliability. Here, we show that a mechanism based on an incoherent feedforward motif enables adaptive gene expression in mammalian cells. We modeled, synthesized, and tested transcriptional and post-transcriptional incoherent loops and found that in all cases the gene product adapts to changes in DNA template abundance. We also observed that the post-transcriptional form results in superior adaptation behavior, higher absolute expression levels, and lower intrinsic fluctuations. Our results support a previously hypothesized endogenous role in gene dosage compensation for such motifs and suggest that their incorporation in synthetic networks will improve their robustness and reliability.
PMCID: PMC3202791  PMID: 21811230
feedforward motifs; gene dosage and noise; mammalian cells; microRNAs; negative autoregulation
2.  Active regulation of receptor ratios controls integration of quorum-sensing signals in Vibrio harveyi 
Single-cell quantification of the input–output relation of the quorum-sensing circuit reveals how Vibrio harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity.
We identify the role of multiple feedback loops in the quorum-sensing circuit of the model bacterium, Vibrio harveyi. Single-cell microscopy and genetic analysis demonstrate that a novel feedback loop regulates receptor ratios to control the integration of multiple signals.Quantitative investigation of cells with all feedback loops present as well as mutants with specific feedback loops disrupted reveals that the multiple feedback loops expand the input dynamic range and compress the output dynamic range of signal transmission, and also control the noise level of the output.Our experimental observations can be interpreted in terms of a simple model of the quorum-sensing network. Plotting output after reparameterizing the input variables directly reveals how feedback controls receptors ratios.
Organisms detect multiple environmental cues simultaneously and use the information to coordinate their behaviors. Correctly integrating signals generally requires complex signal transduction pathways (Pawson and Scott, 2010). In addition to accurately integrating signals, regulatory circuits must ensure signal transmission fidelity. Information can be lost or corrupted by internal or external perturbations, so circuits must be designed to function robustly in the presence of such fluctuations. For example, the circadian clock in Neurospora (Virshup and Forger, 2009) and the chemotaxis network in Escherichia coli (Oleksiuk et al, 2011) accurately compensate for temperature variation. However, while signal integration and signal transmission have been addressed separately, little is known about mechanisms cells use to solve both tasks simultaneously. In this study, we report how the model bacterium Vibrio harveyi simultaneously integrates and faithfully transmits multiple chemical signals.
In a process called quorum sensing, bacteria communicate by synthesizing, releasing, and detecting signal molecules called autoinducers (AIs). To study the integration of such signals, we studied a strain of V. harveyi that integrates two AI signals into its quorum-sensing circuit: AI-1, an intra-species signal, and AI-2, a ‘universal' inter-species signal. Each signal is detected by a cognate receptor AI-1 by LuxN, and AI-2 by LuxPQ (Figure 4A). The information encoded in the two AIs is transduced through a shared signaling pathway into the master quorum-sensing regulator LuxR. In this study, the AIs serve as inputs and LuxR serves as the output of the quorum-sensing circuit. Interestingly, there are five distinct feedback loops in the V. harveyi quorum-sensing circuit (Figure 4A). How does the circuit use shared components to distinguish between the two AI inputs and what role does each feedback loop have in signal integration and transmission?
Using single-cell microscopy, we assayed the activity of the quorum-sensing circuit with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input–output relation both in cells with all feedback loops present (Figure 4A) as well as in mutants with specific feedback loops disrupted (Figure 4E, I, M, and Q). We compared the mean LuxR level (Figure 4B, F, J, N, and R) and noise level (Figure 4C, G, K, O, and S) for the input–output relation of five strains. We discovered that the LuxN feedback loop regulates receptor ratios (LuxN to LuxPQ) to control the integration of two signals. We also found that the multiple feedback loops expand the input dynamic range and compress the output dynamic range of signal transmission, and also control the noise in the output.
In summary, we used single-cell microscopy to quantify the integration of quorum-sensing signals in V. harveyi. Multiple feedback loops in the quorum-sensing circuit actively regulate receptor ratios to control signal integration, sculpt the input–output dynamic range, and regulate the noise level. This system presents a paradigm for how complex circuitry allows cells to appropriately detect and respond to multiple signals in a dynamically changing environment.
Quorum sensing is a chemical signaling mechanism used by bacteria to communicate and orchestrate group behaviors. Multiple feedback loops exist in the quorum-sensing circuit of the model bacterium Vibrio harveyi. Using fluorescence microscopy of individual cells, we assayed the activity of the quorum-sensing circuit, with a focus on defining the functions of the feedback loops. We quantitatively investigated the signaling input–output relation both in cells with all feedback loops present as well as in mutants with specific feedback loops disrupted. We found that one of the feedback loops regulates receptor ratios to control the integration of multiple signals. Together, the feedback loops affect the input–output dynamic range of signal transmission and the noise in the output. We conclude that V. harveyi employs multiple feedback loops to simultaneously control quorum-sensing signal integration and to ensure signal transmission fidelity.
PMCID: PMC3130561  PMID: 21613980
feedback loops; quorum sensing; signal integration; single-cell fluorescence microscopy
3.  Why measure cardiac output? 
Critical Care  2003;7(2):114-116.
Although cardiac output is a primary determinant of global O2 transport there are no absolute values that reflect circulatory adequacy, though very low values are of negative prognostic use. There is no agreement as to what constitutes a clinically relevant change in cardiac output. A recent clinical trial suggests that early goal-directed therapy aimed at increasing cardiac output improves survival. Thus, in some patients, measurement of cardiac output is indicated as an aid to prognosis, diagnosis and to monitor the adequacy of therapy. Gonzalez et al. compared PAC thermodilution cardiac output with indirect Fick measures of cardiac output. They found that at lower cardiac outputs (< 5 l/min) the agreement between the two techniques is good, whereas at higher flows increased differences exist between the two measures. As discussed in this commentary, this study did not address the three potential questions related to PAC monitoring of cardiac output. These questions are: can the PAC cardiac output data be used to monitor cardiac output? Do technical and physiological constraints limit the accuracy of PAC cardiac output? And; are PAC cardiac output measurement errors due to respiratory variation in pulmonary blood flow? Ways of answering each question are given.
PMCID: PMC270610  PMID: 12720554
clinical trial; circulatory shock; hemodynamic monitoring
4.  Impact of very long time output variation in the treatment of total marrow irradiation with helical tomotherapy 
Beam-on time in Total Marrow Irradiation (TMI) delivery with helical tomotherapy is more than 30 minutes. The purpose of this study was to investigate extended time output variation in tomotherapy machine without dose servo system and its impact on the dosimetry of TMI planning.
Materials and methods
The calibration procedures with 1800 seconds delivery were conducted. The slab and cylindrical phantoms were used for static and rotational output variation measurements, respectively. All measurements were performed in 0.1 second interval with an Exradin A1SL ionization chamber (Standard Imaging Inc., Madison, WI, USA) connected to the tomoelectrometer supplied by the manufacture. Simulated TMI treatment planning with a slab phantom was delivered and verified with ion chamber and EDR-2 films.
The static output variations during 30 min averaged −2.9% ± 0.2%, -3.4% ± 0.3%, and −3.4% ± 0.3% at 10 min, 20 min, and 30 min, respectively. The rotational output variations from start averaged −2.5% ± 0.7%, -3.1% ± 0.7%, and −3.5% ± 0.8% at 10 min, 20 min, and 30 min, respectively. The maximum output variation was up to 4.5%. In a TMI planning model, in which beam-on time was over 30 min, planned dose and dose measured with ion chambers in both cranial and caudal sides agreed within 3%. Film measurements in cranial and caudal sides also showed the pass rates of 97.7% and 92.2% with the criteria of 3 mm/3% in gamma analysis.
These results suggest that long TMI delivery by helical tomotherapy, even without dose servo system, does not pose a risk for significant deviations from the original treatment plan regardless of the output variation. However, very long time output variation should be checked before the first treatment.
PMCID: PMC3679448  PMID: 23688298
Radiation therapy; Tomotherapy; Total marrow irradiation; Total body irradiation; Very long time output variation
5.  Measurement of cardiac output in man with a nonrecirculating indicator 
Journal of Clinical Investigation  1971;50(7):1466-1472.
The present investigation was undertaken to evaluate the utility of constant-rate injection of a nonrecirculating indicator (H2) for the measurement of cardiac output in man. 42 patients were studied during cardiac catheterization and 8 during acute complications of arteriosclerotic heart disease, including acute myocardial infarction. Pulmonary (or systemic) arterial H2 concentration was measured chromatographically from 2.0 ml blood samples drawn during constant-rate injection of dissolved H2 into the systemic venous circulation (or left heart). The chromatograph was a thermal conductivity unit housed in a constant-temperature water bath to achieve an improved signal-to-noise ratio. Intrapulmonary H2 elimination from mixed venous blood was measured directly in 14 patients and averaged 98 ±1.5% (SD). Reproducibility of output measurements was evaluated using triplicate determinations obtained over 45-60 sec in 25 consecutive patients. Coefficients of variation (SD/Mean × 100) averaged 3.4 ±2.0%, making it possible to evaluate relatively small changes in measured output with conventional statistical tests. Individual measurements could be repeated at 10-15 sec intervals. Comparisons of H2 and direct Fick measurements were made in 14 patients; H2 outputs averaged 106 ±4% (SEM) of Fick outputs (P > 0.1). Comparisons of H2 and dye dilution measurements were performed in an additional 24 patients. Seven had angiographically-negligible valvular regurgitation and dye outputs averaged 106 ±3% of H2 outputs (P > 0.1). 17 had moderate-to-severe regurigation and dye outputs averaged 91 ±4% of H2 outputs (P < 0.05), suggesting a small but systematic error due to undetected recirculation of dye. The H2 technique appears advantageous for rapidly repeated determinations of output, for quantitation of small changes in output, and for situations in which recirculation of conventional indicators is a potentially significant problem.
PMCID: PMC292086  PMID: 4932984
6.  Coping with Variability in Small Neuronal Networks 
Experimental and corresponding modeling studies indicate that there is a 2- to 5-fold variation of intrinsic and synaptic parameters across animals while functional output is maintained. Here, we review experiments, using the heartbeat central pattern generator (CPG) in medicinal leeches, which explore the consequences of animal-to-animal variation in synaptic strength for coordinated motor output. We focus on a set of segmental heart motor neurons that all receive inhibitory synaptic input from the same four premotor interneurons. These four premotor inputs fire in a phase progression and the motor neurons also fire in a phase progression because of differences in synaptic strength profiles of the four inputs among segments. Our work tested the hypothesis that functional output is maintained in the face of animal-to-animal variation in the absolute strength of connections because relative strengths of the four inputs onto particular motor neurons is maintained across animals. Our experiments showed that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in strength of particular inputs do not correlate strongly with output phase. Further experiments measured the precise temporal pattern of the premotor inputs, the segmental synaptic strength profiles of their connections onto motor neurons, and the temporal pattern (phase progression) of those motor neurons all in the same animal for a series of 12 animals. The analysis of input and output in this sample of 12 individuals suggests that the number (four) of inputs to each motor neuron and the variability of the temporal pattern of input from the CPG across individuals weaken the influence of the strength of individual inputs. Moreover, the temporal pattern of the output varies as much across individuals as that of the input. Essentially, each animal arrives at a unique solution for how the network produces functional output.
PMCID: PMC3223479  PMID: 21724619
7.  Variations in arterial blood pressure are associated with parallel changes in FlowTrac/Vigileo®-derived cardiac output measurements: a prospective comparison study 
Critical Care  2009;13(6):R179.
The reliability of autocalibrated pressure waveform analysis by the FloTrac-Vigileo® (FTV) system for the determination of cardiac output in comparison with intermittent pulmonary arterial thermodilution (IPATD) is controversial. The present prospective comparison study was designed to determine the effects of variations in arterial blood pressure on the reliability of the FTV system in patients undergoing coronary artery bypass grafting (CABG).
Comparative measurements of cardiac output by FTV (derived from a femoral arterial line; software version 1.14) and IPATD were performed in 16 patients undergoing elective CABG in the period before institution of cardiopulmonary bypass. Measurements were performed after induction of anesthesia, after sternotomy, and during five time points during graft preparation. During graft preparation, arterial blood pressure was increased stepwise in intervals of 10 to 15 minutes by infusion of noradrenaline and lowered thereafter to baseline levels.
Mean arterial blood pressure was varied between 85 mmHg and 115 mmHg. IPATD cardiac output did not show significant changes during periods with increased arterial pressure either during sternotomy or after pharmacological manipulation. In contrast, FTV cardiac output paralleled changes in arterial blood pressure; i.e. increased significantly if blood pressure was raised and decreased upon return to baseline levels. Mean arterial blood pressure (MAP) and FTV cardiac output were closely correlated (r = 0.63 (95% confidence interval [CI]: 0.49 - 0.74), P < 0.0001) while no correlation between MAP and IPATD cardiac output was observed. Bland-Altman analyses for FTV versus IPATD cardiac output measurements revealed a bias of 0.4 l/min (8.5%) and limits of agreement from 2.1 to -1.3 l/min (42.2 to -25.3%).
Acute variations in arterial blood pressure alter the reliability of the FlowTrac/Vigileo® device with the second-generation software. This finding may help to explain the variable results of studies comparing the FTV system with other cardiac output monitoring techniques, questions the usefulness of this device for hemodynamic monitoring of patients undergoing rapid changes in arterial blood pressure, and should be kept in mind when using vasopressors during FTV-guided hemodynamic optimization.
PMCID: PMC2811929  PMID: 19900267
8.  The 24 h pattern of arterial pressure in mice is determined mainly by heart rate‐driven variation in cardiac output 
Physiological Reports  2014;2(11):e12223.
Few studies have systematically investigated whether daily patterns of arterial blood pressure over 24 h are mediated by changes in cardiac output, peripheral resistance, or both. Understanding the hemodynamic mechanisms that determine the 24 h patterns of blood pressure may lead to a better understanding of how such patterns become disturbed in hypertension and influence risk for cardiovascular events. In conscious, unrestrained C57BL/6J mice, we investigated whether the 24 h pattern of arterial blood pressure is determined by variation in cardiac output, systemic vascular resistance, or both and also whether variations in cardiac output are mediated by variations in heart rate and or stroke volume. As expected, arterial pressure and locomotor activity were significantly (P < 0.05) higher during the nighttime period compared with the daytime period when mice are typically sleeping (+12.5 ± 1.0 mmHg, [13%] and +7.7 ± 1.3 activity counts, [254%], respectively). The higher arterial pressure during the nighttime period was mediated by higher cardiac output (+2.6 ± 0.3 mL/min, [26%], P < 0.05) in association with lower peripheral resistance (−1.5 ± 0.3 mmHg/mL/min, [−13%] P < 0.05). The increased cardiac output during the nighttime was mainly mediated by increased heart rate (+80.0 ± 16.5 beats/min, [18%] P < 0.05), as stroke volume increased minimally at night (+1.6 ± 0.5 μL per beat, [6%] P < 0.05). These results indicate that in C57BL/6J mice, the 24 h pattern of blood pressure is hemodynamically mediated primarily by the 24 h pattern of cardiac output which is almost entirely determined by the 24 h pattern of heart rate. These findings suggest that the differences in blood pressure between nighttime and daytime are mainly driven by differences in heart rate which are strongly correlated with differences in locomotor activity.
In C57BL/6J mice, the 24 h pattern of blood pressure is hemodynamically mediated primarily by the 24 h pattern of cardiac output which is almost entirely determined by the 24 h pattern of heart rate. These findings suggest that the differences in blood pressure between nighttime and daytime are mainly driven by differences in heart rate which are strongly correlated with differences in locomotor activity.
PMCID: PMC4255824  PMID: 25428952
24‐h variability; hemodynamics
9.  Cardiovascular system in larval zebrafish responds to developmental hypoxia in a family specific manner 
Genetic and environmental variation are both known to influence development. Evolution of a developmental response that is optimized to the environment (adaptive plasticity) requires the existence of genetic variation for that developmental response. In complex traits composed of integrated sets of subsidiary traits, the adaptive process may be slowed by the existence of multiple possible integrated responses. This study tests for family (sibship) specific differences in plastic response to hypoxia in an integrated set of cardiovascular traits in zebrafish.
Cardiac output, which is the integrated product of several subsidiary traits, varied highly significantly between families, and families differed significantly in the degree and direction of response to developmental oxygen level. The cardiac output response to oxygen environment was entirely family specific with no significant overall trend due to oxygen level. Constituent physiological variables that contribute to cardiac output all showed significant family specific response to hypoxia. Traits that were not directly related to cardiac output, such as arterial and venous diameter, and red blood cell velocities did not respond to hypoxia in a family specific manner.
Zebrafish families vary in their plastic response to hypoxia. Genetic variation in plastic response to hypoxia may therefore provide the basic ingredient for adaptation to a variable environment. Considerable variation in the degree of familial response to hypoxia exists between different cardiovascular traits that may contribute to cardiac output. It is possible that the integration of several subsidiary traits into cardiac output allows the maintenance of genetic variance in cardiac response.
PMCID: PMC1479343  PMID: 16539736
10.  Effects of velocity on upper to lower extremity muscular work and power output ratios of intercollegiate athletes 
OBJECTIVES: Peak torque expresses a point output which may, but does not always, correlate well with full range output measures such as work or power, particularly in a rehabilitating muscle. This study evaluates isokinetic performance variables, particularly (a) flexor to extensor work and power output ratios of upper and lower extremities and (b) overall upper to lower extremity work and power ratios, in intercollegiate athletes. The purpose was to ascertain how speeds of 30 and 180 degrees/s influence agonist to antagonist ratios for torque, work, and power and to determine the effects of these speeds on upper to lower limb flexor (F), extensor (E), and combined (F + E) ratios, as a guide to rehabilitation protocols and outcomes after injury. METHODS: Twenty seven athletic men without upper or lower extremity clinical histories were tested isokinetically at slow and moderately fast speeds likely to be encountered in early stages of rehabilitation after injury. Seated knee extensor and flexor outputs, particularly work and power, were investigated, as were full range elbow extensor and flexor outputs. The subjects were morphologically similar in linearity and muscularity (coefficient of variation 4.17%) so that standardisation of isokinetic outputs to body mass effectively normalised for strength differences due to body size. Peak torque (N.m/kg), total work (J/kg), and average power (W/kg) for elbow and knee flexions and extensions were measured on a Cybex 6000 isokinetic dynamometer. With respect to the raw data, the four test conditions (F at 30 degrees/s; E at 30 degrees/s; F at 180 degrees/s; E at 180 degrees/s) were analysed by one way analysis of variance. Reciprocal (agonist to antagonist) F to E ratios of the upper and lower extremities were calculated, as were upper to lower extremity flexor, extensor, and combined (F + E) ratios. Speed related differences between the derived ratios were analysed by Student's t tests (related samples). RESULTS: At the speeds tested all torque responses exhibited velocity related decrements at rates that kept flexor to extensor ratios and upper to lower extremity ratios constant (p > 0.05) for work and power. All upper extremity relative torque, work, and power flexion responses were equal to extension responses (p > 0.05) regardless of speed. Conversely, all lower extremity relative measures of torque, work, and power of flexors were significantly lower than extensor responses. In the case of both upper and lower extremities, work and power F to E ratios were unaffected by speed. Moreover, increasing speed from 30 to 180 degrees/s had no effect on upper to lower extremity work and power ratios, whether for flexion, extension, or flexion and extension combined. CONCLUSIONS: Peak torque responses may not adequately reflect tension development through an extensive range of motion. Total work produced and mean power generated, on the other hand, are highly relevant measures of performance, and these, expressed as F to E ratios, are unaffected by speeds of 30 and 180 degrees/s, whether for upper or lower extremities or for upper to lower extremities. In this sample, regardless of speed, the upper extremity produced 55% of the work and 39% of the power of the lower extremity, when flexor and extensor outputs were combined. Injured athletes are, in the early stages of function restoration, often not able to exert tension at fast speeds. An understanding of upper to lower extremity muscular work and power ratios has important implications for muscle strengthening after injury. Knowledge of normal upper to lower extremity work and power output ratios at slow to moderately fast isokinetic speeds is particularly useful in cases of bilateral upper (or lower) extremity rehabilitation, when the performance of a contralateral limb cannot be used as a yardstick. 

PMCID: PMC1756189  PMID: 10450479
11.  Constancy and variability in the output of a central pattern generator 
Experimental and corresponding modeling studies have demonstrated a 2–5 fold variation of intrinsic and synaptic parameters across animals, while functional output is maintained. These studies have led to the hypothesis that correlated, compensatory changes in particular parameters can at least partially explain the biological variability in parameters. Using the leech heartbeat CPG, we selected three different segmental motor neurons that fire in a functional phase progression but receive input from the same four premotor interneurons. Previous work suggested that the phase progression arises because the pattern of relative strength of the four inputs varies systematically across the segmental motor neurons. Nevertheless, there was considerable animal-to-animal variation in the absolute strengths of these connections. We tested the hypothesis that functional output is maintained in the face of variation in the absolute strength of connections because relative strengths onto particular motor neurons are maintained. We found relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in relative strength of particular inputs do not correlate strongly with output phase. In parallel with this variation in synaptic strength, the firing phase of the premotor inputs to these motor neurons varies considerably across individuals. We conclude that the number (four) of inputs to each motor neuron, which each vary in strength, and the phase diversity of the temporal pattern of input from the CPG diminish the influence of individual inputs. We hypothesize that each animal arrives at a unique solution for how the network produces functional output.
PMCID: PMC3071692  PMID: 21430165
leech; synaptic strength; motor neurons; network model
12.  Role of Translational Coupling in Robustness of Bacterial Chemotaxis Pathway 
PLoS Biology  2009;7(8):e1000171.
Evolutionary selection for robustness of signaling output in the face of stochastic variations in protein expression may explain the organization of bacterial chemotaxis genes.
Chemotaxis allows bacteria to colonize their environment more efficiently and to find optimal growth conditions, and is consequently under strong evolutionary selection. Theoretical and experimental analyses of bacterial chemotaxis suggested that the pathway has been evolutionarily optimized to produce robust output under conditions of such physiological perturbations as stochastic intercellular variations in protein levels while at the same time minimizing complexity and cost of protein expression. Pathway topology in Escherichia coli apparently evolved to produce an invariant output under concerted variations in protein levels, consistent with experimentally observed transcriptional coupling of chemotaxis genes. Here, we show that the pathway robustness is further enhanced through the pairwise translational coupling of adjacent genes. Computer simulations predicted that the robustness of the pathway against the uncorrelated variations in protein levels can be enhanced by a selective pairwise coupling of individual chemotaxis genes on one mRNA, with the order of genes in E. coli ranking among the best in terms of noise compensation. Translational coupling between chemotaxis genes was experimentally confirmed, and coupled expression of these genes was shown to improve chemotaxis. Bioinformatics analysis further revealed that E. coli gene order corresponds to consensus in sequenced bacterial genomes, confirming evolutionary selection for noise reduction. Since polycistronic gene organization is common in bacteria, translational coupling between adjacent genes may provide a general mechanism to enhance robustness of their signaling and metabolic networks. Moreover, coupling between expression of neighboring genes is also present in eukaryotes, and similar principles of noise reduction might thus apply to all cellular networks.
Author Summary
All cellular networks are subject to fluctuations in the levels of their components. Robustness of the network output in the face of stochastic gene expression, or gene expression noise, is therefore essential to ensure proper function. Selection for robustness might thus have shaped much of the cellular evolution. We have used Escherichia coli chemotaxis, one of the most thoroughly studied model systems for signal transduction, to analyze the role of gene organization in robustness. Our mathematical modeling predicted that coupling the expression of chemotaxis proteins with opposing functions should buffer the output of the signaling pathway against stochastic variations in protein production. Consistent with this model, protein coexpression was indeed observed to improve chemotaxis and to be under selection during chemotaxis-driven spreading of a cell population. We show that tight coexpression is ensured by both transcriptional and translational gene coupling. We conclude that evolutionary selection for pathway robustness in the presence of gene expression noise can explain, not only the polycistronic organization of chemotaxis genes, but also the gene order within chemotaxis operons. Selection on the gene order was further confirmed by the observation of a strong bias towards specific pairwise occurrences of chemotaxis genes in sequenced prokaryotic genomes.
PMCID: PMC2716512  PMID: 19688030
13.  Sexual conflict and cooperation in butterfly reproduction: a comparative study of polyandry and female fitness. 
Most butterfly species can be characterized as capital breeders, meaning that reproductive output is strongly coupled to the amount of resources they have procured during the larval stage. Accordingly, female fecundity is generally correlated with female mass, both within and across species. However, the females of some species can be partly characterized as income breeders, in the sense that their reproductive output is dependent not only on larval-derived capital but also on resources acquired during the adult stage. These adult resources can be derived from female feeding or from male-transferred nuptial gifts. Recent studies on the within-species effects of multiple matings on female fitness show that females generally gain directly from multiple matings in terms of increased lifetime offspring production. Here, we test whether the positive effects of multiple mating on female fitness also hold at a comparative level, by conducting a laboratory study of female reproductive output in eight pierid species that differ in life-time female mating frequency. Female reproductive output, measured as cumulative egg mass divided by female mass, increased significantly with polyandry (r = 0.942, p < 0.001), demonstrating that the positive effect of mating rate on female reproductive fitness also holds between species. The positive effect of male nutrient contribution is substantial, and the per capita reproductive output is more than twice as high in the most polyandrous species as in the most monandrous ones. Hence, the positive net effect of the ejaculates is highly substantial, although males and females can have sexual interests that run counter to each other, setting the stage for sexually antagonistic coevolution, so that the various component parts of the male ejaculate-sperm, nutrients, anti-aphrodisiacs, and gonadotrophic hormones-may each correspond to a separate conflict-cooperation balance between the sexes. Two scenarios for the evolution of nuptial gifts in butterflies are discussed, one arguing that variation in larval food is the underlying factor and the other arguing that sexually antagonistic coevolution is the driving force. The two views are complementary rather than mutually exclusive, although the former hypothesis predicts that variation in female mating rate depends on variation in larval food availability, whereas the latter suggests that variation in female mating rate between species results from species-specific idiosyncrasies.
PMCID: PMC1088792  PMID: 11506678
14.  Impact of quality strategies on hospital outputs 
Quality & Safety in Health Care  2009;18(Suppl_1):i62-i68.
This study was part of the Methods of Assessing Response to Quality Improvement Strategies (MARQuIS) research project on patients crossing borders, a study to investigate quality improvement strategies in healthcare systems across the European Union (EU).
To explore the association between the implementation of quality improvement strategies in hospitals and hospitals’ success in meeting defined quality requirements that are considered intermediate outputs of the care process.
Data regarding the implementation of seven quality improvement strategies (accreditation, organisational quality management programmes, audit and internal assessment of clinical standards, patient safety systems, clinical practice guidelines, performance indicators and systems for obtaining patients’ views) and four dimensions of outputs (clinical, safety, patient-centredness and cross-border patient-centredness) were collected from 389 acute care hospitals in eight EU countries using a web-based questionnaire. In a second phase, 89 of these hospitals participated in an on-site audit by independent surveyors. Pearson correlation and linear regression models were used to explore associations and relations between quality improvement strategies and achievement of outputs.
Positive associations were found between six internal quality improvement strategies and hospital outputs. The quality improvement strategies could be reasonably subsumed under one latent index which explained about half of their variation. The analysis of outputs concluded that the outputs can also be considered part of a single construct. The findings indicate that the implementation of internal as well as external quality improvement strategies in hospitals has beneficial effects on the hospital outputs studied here.
The implementation of internal quality improvement strategies as well as external assessment systems should be promoted.
PMCID: PMC2629927  PMID: 19188464
15.  Effect of inhaled morphine on the development of breathlessness during exercise in patients with chronic lung disease. 
Thorax  1996;51(6):596-600.
BACKGROUND: Inhaled morphine has previously been shown to increase exercise endurance in patients with chronic lung disease. A similar study was performed to determine whether inhaled morphine reduces the sensation of breathlessness in this group of patients. METHODS: A randomised double blind study on the effect of nebulised morphine on both exercise induced breathlessness and maximum achievable power output using isotonic saline as a control was performed in 10 patients with stable chronic lung disease. Each subject performed a progressive exercise test (Jones' stage I) on an electrically braked cycle ergometer. The work load was increased by 10 watts per minute and subjects exercised to exhaustion. At the end of each minute of exercise patients were asked to rate their degree of breathlessness according to a modified Borg scale. All subjects were randomised to receive either inhaled morphine sulphate 1 mg/ml (5 ml) or isotonic saline (5 ml) by wet nebulisation. The effect of morphine and saline on the achieved exercise capacity and the development of breathlessness during exercise was tested on separate days. RESULTS: The mean dose of morphine inhaled was 1.24 mg. There was no difference in maximum power output achieved, minute ventilation at maximum power output, nor the degree of breathlessness at maximum power output between the groups treated with morphine and placebo. The degree of breathlessness was related to the power output achieved during exercise by a power function relationship (mean r: morphine = 0.86, saline = 0.87). However, there was a wide variation in the sensation for any given power output in both groups. There was no difference in the group mean slopes (morphine = 1.15, saline = 1.00) or intercepts (morphine = 0.07, saline = 0.15) in this relationship between the morphine and saline treatment groups. CONCLUSIONS: In patients with severe chronic lung disease inhaled morphine in the doses used in this study does not relieve exercise induced breathlessness nor does it increase maximum power output achieved during progressive exercise.
PMCID: PMC1090489  PMID: 8693440
By a method of permanent intubation the entire output of bile from dogs has been obtained in a sterile state over long periods of time, and studied quantitatively. The secretion of the first few days after the initial operation is scanty, contains more pigment than that secreted later, and is sometimes so viscid as to cause temporary obstruction within the collecting tube. The small amount and thick consistency of the fluid are referable to direct injury of the liver and ducts, while the abundant bilirubin is derived, in part at least, from the hemoglobin of extravasated blood. In some animals in which there was accidental mercuric chloride poisoning at the time of operation a suppression of the bile followed which, in one instance, was complete during the 48 hours before death. In a dog developing mixed infection of the biliary tract, an almost colorless fluid, glairy as white of egg, gradually took the place of the bile. Not until a week or 10 days after operation does the bile acquire the character which it maintains later. The quantity of this later bile, as measured in successive 24 hour specimens, is greatly less than that recorded by previous investigators, a difference attributable to disturbing influences inevitable to the method of collection they employed. By our method, it averaged from 3.5 to 9.5 cc. per kilo of dog in 24 hours, though transient variations from 1 cc. up to 14 cc. were encountered. Some dogs give consistently far more bile than others. The frequently recorded effects of fasting to lessen the rate of secretion, and of a meat diet to increase it more than a carbohydrate one, were noted often. Contrary to expectation, vigorous exercise does not act to increase the quantity of the bile. During hot weather this may sink greatly, although the animal remains in good condition; and during intercurrent diseases unassociated with jaundice, the flow may almost cease. One of the best of cholagogues, bile by mouth, fails of effect under such circumstances. It acts best when the animal is healthy, the weather not oppressive, and the food intake abundant. The bilirubin output, after the immediate effects of the operation have worn off, remains nearly constant from day to day, though often exhibiting slow, wave-like variations, each extending over a week or more. These slow changes are synchronous with similar alterations in the hemoglobin percentage of the circulating blood. A recognition of them is important to studies of the bilirubin yield. A mild anemia, absent in controls maintained under like conditions, regularly develops in unexercised dogs completely deprived of bile, despite their apparent health, and as it does so the bilirubin output falls off. Prior to the development of the anemia the output averages about 7.5 mg. per kilo in 24 hours, a yield which closely corresponds with that recorded by previous workers. Vigorous exercise of animals previously sedentary causes an increased pigment output presumably as result of increased blood destruction. Since the daily pigment output is approximately constant, whereas the fluid quantity undergoes frequent and great changes, it follows that the pigment concentration must vary inversely as the fluid quantity, and vary greatly. It does both. No matter how large or small the 24 hour specimen of bile may be within "normal" limits, therein will be found the customary quota of pigment. When, under pathological conditions, the bile flow almost ceases, the pigment concentration becomes extremely great. A similar reciprocal relationship between concentration and fluid quantity would seem to hold for the mucinous element of the bile. Scanty biles are ropy as a rule, and copious ones are watery. Temporary obstruction was produced by clamping the outlet tube from the common duct, and the bile yield following upon its relief was studied. Secretion was for a time far more copious than usual, with a low pigment content per cubic centimeter, and tended to remain so until the accumulated pigment had been voided. As much extra bilirubin is put forth after 24 hours of obstruction as if the liver continued to manufacture the pigment without interruption during it. Obstruction for this length of time appears to cause no subjective disturbance in most instances, though bilirubinuria develops. Even this sign of bile retention may be lacking when obstruction endures but 12 hours. The physiological and clinical significance of these facts is briefly discussed.
PMCID: PMC2128361  PMID: 19868735
17.  Age-Dependent Terminal Declines in Reproductive Output in a Wild Bird 
PLoS ONE  2012;7(7):e40413.
In many iteroparous species individual fitness components, such as reproductive output, first increase with age and then decline during late-life. However, individuals differ greatly in reproductive lifespan, but reproductive declines may only occur in the period just before their death as a result of an age-independent decline in physiological condition. To fully understand reproductive senescence it is important to investigate to what extent declines in late-life reproduction can be explained by age, time until death, or both. However, the study of late-life fitness performance in natural populations is challenging as the exact birth and death dates of individuals are often not known, and most individuals succumb to extrinsic mortality before reaching old age. Here, we used an exceptional long-term longitudinal dataset of individuals from a natural, closed, and predator-free population of the Seychelles warbler (Acrocephalus sechellensis) to investigate reproductive output, both in relation to age and to the time until the death of an individual (reverse-age approach). We observed an initial age-dependent increase in reproductive output that was followed by a decline in old age. However, we found no significant decline in reproductive output in the years directly preceding death. Although post-peak reproductive output declined with age, this pattern differed between terminal and non-terminal reproductive attempts, and the age-dependence of the terminal breeding attempt explained much of the variation in age-specific reproductive output. In fact, terminal declines in reproductive output were steeper in very old individuals. These results indicate that not only age-dependent, but also age-independent factors, such as physiological condition, need to be considered to understand reproductive senescence in wild-living animals.
PMCID: PMC3391264  PMID: 22792307
18.  Haematocrit and red blood cell transport in preterm infants: an observational study 
AIMS—To test whether cardiac output acts as a compensatory response to changes in haematocrit.
METHODS—A cohort of 38 preterm infants (27-31 weeks' gestation) was studied with repeated Doppler measurements of left ventricular output during the 1st month of life. Red blood cell transport was calculated when the duct was closed.
RESULTS—Multiple regression analysis showed that left ventricular output correlated negatively with haematocrit when the duct was closed (n = 84) and when it was open (n = 59). The influence of an increase of 10% in haematocrit absolute value on mean (SD) left ventricular output was estimated at −55 (11) ml/kg/min. Mean (SD) red blood cell transport was 132 (30) ml/kg/min with a mean (SD) intra-individual variability of 20% (8.8%). Red blood cell transport was increased more frequently by left ventricular output than by haematocrit. Haematocrit and left ventricular output but not red blood cell transport were dependent on postnatal age.
CONCLUSION—These results suggest that in preterm infants cardiac output adaptation is effective in attenuating the effects of red blood cell mass variations on systemic oxygen carrying capacity.

PMCID: PMC1721062  PMID: 10685990
19.  A Radiometric Study of Factors Affecting Drug Output of Jet Nebulizers 
Jet nebulizers show an unreasonable variation in drug output and nebulization rates that leads to clinical and regulatory problems. Current evaluation methods appear inadequate for the purpose. Our objective was to evaluate Technetium-99m radiometry to study nebulizer parameters and the factors influencing it quantitatively. Drug output, output rate and residual mass and the effect of excipient, temperature, surface tension, air-jet speed, and equipment brand and aging were studied. Though nebulization of radiolabeled drugs followed first-order kinetics, the rates were significantly different; the heaviest drug (Tc-99m colloid) and Tc-99m salbutamol had the least nebulization. Nebulization rate for the first minute was invariably higher than the mean rate signifying the concentration effect of the solute. Drug residue was 35-75%. Drug output of different nebulizer chamber and air compressor brands was different to the extent of 270% and 180% respectively. ‘Aging’ of fluid chamber, cold drug fluid and obstruction in air-jet resulted in significant reduction in output, while addition of 2% saline as excipient did not change the output rate. Addition of ethyl alcohol resulted in a maximum of 260% enhancement (with Tc-99m salbutamol), while further reduction in surface tension was counterproductive irrespective of the drug used. We conclude that radiometry can provide valuable parametric information on the performance of different jet nebulizers.
PMCID: PMC2883224  PMID: 20582187
Jet nebulizer performance; radiometry; Technetium-99m
20.  Regulation of bile acid synthesis in man. Presence of a diurnal rhythm. 
Journal of Clinical Investigation  1983;72(6):1930-1936.
Regulation of bile acid synthesis in man is incompletely understood, in part because of difficulty in making measurements over short time periods when the enterohepatic circulation is intact. We investigated the possibility of a diurnal rhythm of bile acid synthesis in three human subjects given [26-14C]cholesterol. When this isotope of cholesterol, which is randomly labeled in the 26 and 27 positions, is converted to bile acid, the 14C is released as propionic acid randomly labeled in the 1 and 3 positions. The labeled propionic acid is then oxidized to 14CO2, output of which is a function of bile acid synthesis. However, delays in transit of the 14C through propionic acid and CO2-HCO-3 pools would shift the phase and dampen the amplitude of 14CO2 output relative to an existing diurnal rhythm of bile acid synthesis. Therefore, using constant infusion methods, we determined the turnover constants for conversion to 14CO2 of [1-14C]propionic acid and [3-14C]propionic acid to be 0.36-0.59 h-1 and 0.14-0.16 h-1, respectively. Using these constants and modeling the diurnal rhythm as a cosine function, we determined that amplitude of 14CO2 output from [26-14C]cholesterol was reduced 35% and acrophase was delayed 2.4-3.0 h relative to the diurnal rhythm of bile acid synthesis. None of the diurnal rhythm in 14CO2 output from [26-14C]cholesterol resulted from diurnal variation in propionic acid or CO2-HCO-3 metabolism since constant infusion of [1-14C]propionic acid and [3-14C]propionic acid for 30 h revealed no diurnal variation in output of 14CO2. These studies demonstrate for the first time that humans with an intact enterohepatic circulation have a diurnal rhythm of bile acid synthesis with an amplitude of +/- 35-55% around mean synthesis, and an acrophase at about 9 a.m.
PMCID: PMC437033  PMID: 6417166
21.  In-vivo validation of a new non-invasive continuous ventricular stroke volume monitoring system in an animal model 
Critical Care  2011;15(4):R165.
Recently, a non-invasive, continuous ventricular stroke volume monitoring system using skin electrodes has been developed. In contrast to impedance-based methods, the new technique (ventricular field recognition) enables measurement of changes in ventricular volume. A prototype using this new method was built (the hemologic cardiac profiler, HCP) and validated against a reference method in a pig model during variations in cardiac output.
In six Dalland pigs, cardiac output was simultaneously measured with the HCP (CO-HCP), and an invasive ultrasonic flow-probe around the ascending aorta (CO-FP). Variations in CO were achieved by change in ventricular loading conditions, cardiac pacing, and dobutamine administration. Data were analysed according to Bland-Altman analysis and Pearson's correlation.
Pearson's correlation between the CO-HCP and the CO-FP was r = 0.978. Bland-Altman analysis showed a bias of - 0.114 L/minute, and a variability of the bias (2 standard deviations, 2SD) of 0.55 L/minute.
The results of the present study demonstrate that CO-HCP is comparable to CO-FP in an animal model of cardiac output measurements during a wide variation of CO. Therefore, the HCP has the potential to become a clinical applicable cardiac output monitor.
PMCID: PMC3387602  PMID: 21745380
22.  Optimisation of positive and expiratory pressure for maximal delivery of oxygen to tissues using oesophageal Doppler ultrasonography. 
BMJ : British Medical Journal  1989;298(6684):1350-1353.
OBJECTIVE--To assess oesophageal Doppler ultrasonography as a convenient means of optimising positive end expiratory pressure for maximal delivery of oxygen to tissues. DESIGN--Measurements of blood flow, arterial oxygen saturation, and cardiac output by thermodilution (when available) at baseline and at 20-30 minutes after each incremental increase (2.5-5.0 cm H2O) in positive and expiratory pressure to a maximum of 20.0 cm H2O. If the cardiac output fell by more than 15% measurements were repeated after stepwise decreases in positive end expiratory pressure. No other manoeuvre such as endotracheal suction or changing ventilator settings, drug or fluid dosage, or the patient's position was performed for at least one hour before the start of the study or during it. SETTING--Intensive care unit. PARTICIPANTS--10 Patients being mechanically ventilated for acute respiratory failure who had stable haemodynamic and blood gas values and required a fractional inspired oxygen concentration of greater than or equal to 0.45. They were assessed on a total of 11 occasions. INTERVENTIONS--Incremental increases in positive end expiratory pressure followed when indicated by stepwise decreases. END POINT--The positive end expiratory pressure providing maximal delivery of oxygen to tissues. MEASUREMENTS and MAIN RESULTS--Arterial oxygen saturation increased with positive end expiratory pressure in all patients by an average of 6.1%. In nine of the 11 studies, however, cardiac output fell by 15% to 30% after the second increment. On the two other occasions cardiac output and oxygen delivery rose by up to 54%. Positive end expiratory pressure was decreased on seven occasions; there was considerable individual variation in the time taken for cardiac output to rise and arterial oxygen saturation to fall. In six patients good agreement was seen between the results from Doppler ultrasonography and thermodilution, the mean of the differences being -0.3% with narrow limits of agreement (-14.4% to 13.9%). CONCLUSIONS--Oesophageal Doppler ultrasonography is a rapid, safe, and reliable technique for optimising positive end expiratory pressure to obtain maximal delivery of oxygen to tissues. The results show the need to consider haemodynamic consequences when altering positive end expiratory pressure.
PMCID: PMC1836635  PMID: 2502250
23.  Model Sensitivity and Use of the Comparative Finite Element Method in Mammalian Jaw Mechanics: Mandible Performance in the Gray Wolf 
PLoS ONE  2011;6(4):e19171.
Finite Element Analysis (FEA) is a powerful tool gaining use in studies of biological form and function. This method is particularly conducive to studies of extinct and fossilized organisms, as models can be assigned properties that approximate living tissues. In disciplines where model validation is difficult or impossible, the choice of model parameters and their effects on the results become increasingly important, especially in comparing outputs to infer function. To evaluate the extent to which performance measures are affected by initial model input, we tested the sensitivity of bite force, strain energy, and stress to changes in seven parameters that are required in testing craniodental function with FEA. Simulations were performed on FE models of a Gray Wolf (Canis lupus) mandible. Results showed that unilateral bite force outputs are least affected by the relative ratios of the balancing and working muscles, but only ratios above 0.5 provided balancing-working side joint reaction force relationships that are consistent with experimental data. The constraints modeled at the bite point had the greatest effect on bite force output, but the most appropriate constraint may depend on the study question. Strain energy is least affected by variation in bite point constraint, but larger variations in strain energy values are observed in models with different number of tetrahedral elements, masticatory muscle ratios and muscle subgroups present, and number of material properties. These findings indicate that performance measures are differentially affected by variation in initial model parameters. In the absence of validated input values, FE models can nevertheless provide robust comparisons if these parameters are standardized within a given study to minimize variation that arise during the model-building process. Sensitivity tests incorporated into the study design not only aid in the interpretation of simulation results, but can also provide additional insights on form and function.
PMCID: PMC3084775  PMID: 21559475
24.  The auxin signalling network translates dynamic input into robust patterning at the shoot apex 
We provide a comprehensive expression map of the different genes (TIR1/AFBs, ARFs and Aux/IAAs) involved in the signalling pathway regulating gene transcription in response to auxin in the shoot apical meristem (SAM).We demonstrate a relatively simple structure of this pathway using a high-throughput yeast two-hybrid approach to obtain the Aux/IAA-ARF full interactome.The topology of the signalling network was used to construct a model for auxin signalling and to predict a role for the spatial regulation of auxin signalling in patterning of the SAM.We used a new sensor to monitor the input in the auxin signalling pathway and to confirm the model prediction, thus demonstrating that auxin signalling is essential to create robust patterns at the SAM.
The plant hormone auxin is a key morphogenetic signal involved in the control of cell identity throughout development. A striking example of auxin action is at the shoot apical meristem (SAM), a population of stem cells generating the aerial parts of the plant. Organ positioning and patterning depends on local accumulations of auxin in the SAM, generated by polar transport of auxin (Vernoux et al, 2010). However, it is still unclear how auxin is distributed at cell resolution in tissues and how the hormone is sensed in space and time during development. A complex ensemble of 29 Aux/IAAs and 23 ARFs is central to the regulation of gene transcription in response to auxin (for review, see Leyser, 2006; Guilfoyle and Hagen, 2007; Chapman and Estelle, 2009). Protein–protein interactions govern the properties of this transduction pathway (Del Bianco and Kepinski, 2011). Limited interaction studies suggest that, in the absence of auxin, the Aux/IAA repressors form heterodimers with the ARF transcription factors, preventing them from regulating target genes. In the presence of auxin, the Aux/IAA proteins are targeted to the proteasome by an SCF E3 ubiquitin ligase complex (Chapman and Estelle, 2009; Leyser, 2006). In this process, auxin promotes the interaction between Aux/IAA proteins and the TIR1 F-box of the SCF complex (or its AFB homologues) that acts as an auxin co-receptor (Dharmasiri et al, 2005a, 2005b; Kepinski and Leyser, 2005; Tan et al, 2007). The auxin-induced degradation of Aux/IAAs would then release ARFs to regulate transcription of their target genes. This includes activation of most of the Aux/IAA genes themselves, thus establishing a negative feedback loop (Guilfoyle and Hagen, 2007). Although this general scenario provides a framework for understanding gene regulation by auxin, the underlying protein–protein network remains to be fully characterized.
In this paper, we combined experimental and theoretical analyses to understand how this pathway contributes to sensing auxin in space and time (Figure 1). We first analysed the expression patterns of the ARFs, Aux/IAAs and TIR1/AFBs genes in the SAM. Our results demonstrate a general tendency for most of the 25 ARFs and Aux/IAAs detected in the SAM: a differential expression with low levels at the centre of the meristem (where the stem cells are located) and high levels at the periphery of the meristem (where organ initiation takes place). We also observed a similar differential expression for TIR1/AFB co-receptors. To understand the functional significance of the distribution of ARFs and Aux/IAAs in the SAM, we next investigated the global structure of the Aux/IAA-ARF network using a high-throughput yeast two-hybrid approach and uncover a rather simple topology that relies on three basic generic features: (i) Aux/IAA proteins interact with themselves, (ii) Aux/IAA proteins interact with ARF activators and (iii) ARF repressors have no or very limited interactions with other proteins in the network.
The results of our interaction analysis suggest a model for the Aux/IAA-ARF signalling pathway in the SAM, where transcriptional activation by ARF activators would be negatively regulated by two independent systems, one involving the ARF repressors, the other the Aux/IAAs. The presence of auxin would remove the inhibitory action of Aux/IAAs, but leave the ARF repressors to compete with ARF activators for promoter-binding sites. To explore the regulatory properties of this signalling network, we developed a mathematical model to describe the transcriptional output as a function of the signalling input that is the combinatorial effect of auxin concentration and of its perception. We then used the model and a simplified view of the meristem (where the same population of Aux/IAAs and ARFs exhibit a low expression at the centre and a high expression in the peripheral zone) for investigating the role of auxin signalling in SAM function. We show that in the model, for a given ARF activator-to-repressor ratio, the gene induction capacity increases with the absolute levels of ARF proteins. We thus predict that the differential expression of the ARFs generates differences in auxin sensitivities between the centre (low sensitivity) and the periphery (high sensitivity), and that the expression of TIR1/AFB participates to this regulation (prediction 1). We also use the model to analyse the transcriptional response to rapidly changing auxin concentrations. By simulating situations equivalent either to the centre or the periphery of our simplified representation of the SAM, we predict that the signalling pathway buffers its response to the auxin input via the balance between ARF activators and repressors, in turn generated by their differential spatial distributions (prediction 2).
To test the predictions from the model experimentally, we needed to assess both the input (auxin level and/or perception) and the output (target gene induction) of the signalling cascade. For measuring the transcriptional output, the widely used DR5 reporter is perfectly adapted (Figure 5) (Ulmasov et al, 1997; Sabatini et al, 1999; Benkova et al, 2003; Heisler et al, 2005). For assaying pathway input, we designed DII-VENUS, a novel auxin signalling sensor that comprises a constitutively expressed fusion of the auxin-binding domain (termed domain II or DII) (Dreher et al, 2006; Tan et al, 2007) of an IAA to a fast-maturating variant of YFP, VENUS (Figure 5). The degradation patterns from DII-VENUS indicate a high auxin signalling input both in flower primordia and at the centre of the SAM. This is in contrast to the organ-specific expression pattern of DR5::VENUS (Figure 5). These results indicate that the signalling pathway limits gene activation in response to auxin at the meristem centre and confirm the differential sensitivity to auxin between the centre and the periphery (prediction 1). We further confirmed the buffering capacities of the signalling pathway (prediction 2) by carrying out live imaging experiments to monitor DII-VENUS and DR5::VENUS expression in real time (Figure 5). This analysis reveals the presence of important temporal variations of DII-VENUS fluorescence, while DR5::VENUS does not show such global variations. Our approach thus provides evidence that the Aux/IAA-ARF pathway has a key role in patterning in the SAM, alongside the auxin transport system. Our results illustrate how the tight spatio-temporal regulation of both the distribution of a morphogenetic signal and the activity of the downstream signalling pathway provides robustness to a dynamic developmental process.
A comprehensive expression and interaction map of auxin signalling factors in the Arabidopsis shoot apical meristem is constructed and used to derive a mathematical model of auxin signalling, from which key predictions are experimentally confirmed.
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
PMCID: PMC3167386  PMID: 21734647
auxin; biosensor; live imaging; ODE; signalling
25.  Prediction of fluid responsiveness using respiratory variations in left ventricular stroke area by transoesophageal echocardiographic automated border detection in mechanically ventilated patients 
Critical Care  2006;10(6):R171.
Left ventricular stroke area by transoesophageal echocardiographic automated border detection has been shown to be strongly correlated to left ventricular stroke volume. Respiratory variations in left ventricular stroke volume or its surrogates are good predictors of fluid responsiveness in mechanically ventilated patients. We hypothesised that respiratory variations in left ventricular stroke area (ΔSA) can predict fluid responsiveness.
Eighteen mechanically ventilated patients undergoing coronary artery bypass grafting were studied immediately after induction of anaesthesia. Stroke area was measured on a beat-to-beat basis using transoesophageal echocardiographic automated border detection. Haemodynamic and echocardiographic data were measured at baseline and after volume expansion induced by a passive leg raising manoeuvre. Responders to passive leg raising manoeuvre were defined as patients presenting a more than 15% increase in cardiac output.
Cardiac output increased significantly in response to volume expansion induced by passive leg raising (from 2.16 ± 0.79 litres per minute to 2.78 ± 1.08 litres per minute; p < 0.01). ΔSA decreased significantly in response to volume expansion (from 17% ± 7% to 8% ± 6%; p < 0.01). ΔSA was higher in responders than in non-responders (20% ± 5% versus 10% ± 5%; p < 0.01). A cutoff ΔSA value of 16% allowed fluid responsiveness prediction with a sensitivity of 92% and a specificity of 83%. ΔSA at baseline was related to the percentage increase in cardiac output in response to volume expansion (r = 0.53, p < 0.01).
ΔSA by transoesophageal echocardiographic automated border detection is sensitive to changes in preload, can predict fluid responsiveness, and can quantify the effects of volume expansion on cardiac output. It has potential clinical applications.
PMCID: PMC1794488  PMID: 17163985

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