Temporal metabolic partitioning of the yeast and protist cellular networks: the cell is a global scale-invariant (fractal or self-similar) multioscillator.

Britton Chance, electronics expert when a teenager, became an enthusiastic student of biological oscillations, passing on this enthusiasm to many students and colleagues, including one of us (DL). This historical essay traces BC's influence through the accumulated work of DL to DL's many collaborators. The overall temporal organization of mass-energy, information, and signaling networks in yeast in self-synchronized continuous cultures represents, until now, the most characterized example of in vivo elucidation of time structure. Continuous online monitoring of dissolved gases by direct measurement (membrane-inlet mass spectrometry, together with NAD(P)H and flavin fluorescence) gives strain-specific dynamic information from timescales of minutes to hours as does two-photon imaging. The predominantly oscillatory behavior of network components becomes evident, with spontaneously synchronized cellular respiration cycles between discrete periods of increased oxygen consumption (oxidative phase) and decreased oxygen consumption (reductive phase). This temperature-compensated ultradian clock provides coordination, linking temporally partitioned functions by direct feedback loops between the energetic and redox state of the cell and its growing ultrastructure. Multioscillatory outputs in dissolved gases with 13 h, 40 min, and 4 min periods gave statistical self-similarity in power spectral and relative dispersional analyses: i.e., complex nonlinear (chaotic) behavior and a functional scale-free (fractal) network operating simultaneously over several timescales.

Similarity-Based Segmentation of Multi-Dimensional Signals.

The segmentation of time series and genomic data is a common problem in computational biology. With increasingly complex measurement procedures individual data points are often not just numbers or simple vectors in which all components are of the same kind. Analysis methods that capitalize on slopes in a single real-valued data track or that make explicit use of the vectorial nature of the data are not applicable in such scenaria. We develop here a framework for segmentation in arbitrary data domains that only requires a minimal notion of similarity. Using unsupervised clustering of (a sample of) the input yields an approximate segmentation algorithm that is efficient enough for genome-wide applications. As a showcase application we segment a time-series of transcriptome sequencing data from budding yeast, in high temporal resolution over ca. 2.5 cycles of the short-period respiratory oscillation. The algorithm is used with a similarity measure focussing on periodic expression profiles across the metabolic cycle rather than coverage per time point.

Training paramedics in focussed echo in life support.

OBJECTIVES: The aim of this study was to determine whether paramedics can be trained to perform and interpret focussed Echo in Life Support (ELS) for the assessment of cardiac movement and the recognition of reversible causes of cardiac arrest. METHODS: This study is a prospective observational pilot study. Data were collected during a 1-day course training 11 paramedics to perform ELS scans on healthy volunteers. The students were assessed on image acquisition skills and theoretical knowledge (including interpretation). Level 1 ultrasound-trained emergency medicine physicians undertook the training and assessment. RESULTS: All paramedics could obtain images in the parasternal and subxiphoid views. When performing scans in the 10-s pulse check window, 88% of attempts in both views were successful (subxiphoid mean image quality 3.8 out of 5, parasternal 4.0). Theoretical knowledge improved (mean precourse score 54%, postcourse score 89%; P<0.001). There was no apparent association between theoretical and practical performances. At 10 weeks, theoretical knowledge was nonsignificantly reduced (82%; P=0.13) but less when compared with practical performance (75% subxiphoid success, mean quality 3.0; 25% parasternal success, mean quality 4.0). CONCLUSION: Paramedics can perform focused ELS, integrate attempts into simulated cardiac arrest scenarios and retain some of this knowledge. Further work is required to assess the feasibility of incorporating this into real-world cardiac arrest management.

Quantifying periodicity in omics data.

Oscillations play a significant role in biological systems, with many examples in the fast, ultradian, circadian, circalunar, and yearly time domains. However, determining periodicity in such data can be problematic. There are a number of computational methods to identify the periodic components in large datasets, such as signal-to-noise based Fourier decomposition, Fisher's g-test and autocorrelation. However, the available methods assume a sinusoidal model and do not attempt to quantify the waveform shape and the presence of multiple periodicities, which provide vital clues in determining the underlying dynamics. Here, we developed a Fourier based measure that generates a de-noised waveform from multiple significant frequencies. This waveform is then correlated with the raw data from the respiratory oscillation found in yeast, to provide oscillation statistics including waveform metrics and multi-periods. The method is compared and contrasted to commonly used statistics. Moreover, we show the utility of the program in the analysis of noisy datasets and other high-throughput analyses, such as metabolomics and flow cytometry, respectively.

Time resolved DNA occupancy dynamics during the respiratory oscillation uncover a global reset point in the yeast growth program.

The structural dynamics of chromatin have been implicated in the regulation of fundamental eukaryotic processes, such as DNA transcription, replication and repair. Although previous studies have revealed that the chromatin landscape, nucleosome remodeling and histone modification events are intimately tied into cellular energetics and redox state, few studies undertake defined time-resolved measurements of these state variables. Here, we use metabolically synchronous, continuously-grown yeast cultures to measure DNA occupancy and track global patterns with respect to the metabolic state of the culture. Combined with transcriptome analyses and ChIP-qPCR experiments, these paint an intriguing picture where genome-wide nucleosome focusing occurs during the recovery of energy charge, followed by clearance of the promoter regions and global transcriptional slow-down, thus indicating a nucleosome-mediated "reset point" for the cycle. The reset begins at the end of the catabolic and stress-response transcriptional programs and ends prior to the start of the anabolic and cell-growth transcriptional program, and the histones on genes from both the catabolic and anabolic superclusters are deacetylated.

The dynamics of cellular energetics during continuous yeast culture.

A plethora of data is accumulating from high throughput methods on metabolites, coenzymes, proteins, and nucleic acids and their interactions as well as the signalling and regulatory functions and pathways of the cellular network. The frozen moment viewed in a single discrete time sample requires frequent repetition and updating before any appreciation of the dynamics of component interaction becomes possible. Even then in a sample derived from a cell population, time-averaging of processes and events that occur in out-of-phase individuals blur the detailed complexity of single cell organization. Continuously-grown cultures of yeast can become spontaneously self-synchronized, thereby enabling resolution of far more detailed temporal structure. Continuous on-line monitoring by rapidly responding sensors (O2 electrode and membrane-inlet mass spectrometry for O2, CO2 and H2S; direct fluorimetry for NAD(P)H and flavins) gives dynamic information from time-scales of minutes to hours. Supplemented with capillary electophoresis and gas chromatography mass spectrometry and transcriptomics the predominantly oscillatory behaviour of network components becomes evident, with a 40 min cycle between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Highly pervasive, this ultradian clock provides a coordinating function that links mitochondrial energetics and redox balance to transcriptional regulation, mitochondrial structure and organelle remodelling, DNA duplication and cell division events. Ultimately, this leads to a global partitioning of anabolism and catabolism and the enzymes involved, mediated by a relatively simple ATP feedback loop on chromatin architecture.

A yeast metabolite extraction protocol optimised for time-series analyses.

There is an increasing call for the absolute quantification of time-resolved metabolite data. However, a number of technical issues exist, such as metabolites being modified/degraded either chemically or enzymatically during the extraction process. Additionally, capillary electrophoresis mass spectrometry (CE-MS) is incompatible with high salt concentrations often used in extraction protocols. In microbial systems, metabolite yield is influenced by the extraction protocol used and the cell disruption rate. Here we present a method that rapidly quenches metabolism using dry-ice ethanol bath and methanol N-ethylmaleimide solution (thus stabilising thiols), disrupts cells efficiently using bead-beating and avoids artefacts created by live-cell pelleting. Rapid sample processing minimised metabolite leaching. Cell weight, number and size distribution was used to calculate metabolites to an attomol/cell level. We apply this method to samples obtained from the respiratory oscillation that occurs when yeast are grown continuously.

Rapid DNA, RNA and protein extraction protocols optimized for slow continuously growing yeast cultures.

Conventional extraction protocols for yeast have been developed for relatively rapid-growing low cell density cultures of laboratory strains and often do not have the integrity for frequent sampling of cultures. Therefore, these protocols are usually inefficient for cultures under slow growth conditions or of non-laboratory strains. We have developed a combined mechanical and chemical disruption procedure using vigorous bead-beating that can consistently disrupt yeast cells (> 95%), irrespective of cell cycle and metabolic state. Using this disruption technique coupled with quenching, we have developed DNA, RNA and protein extraction protocols that are optimized for a large number of samples from slow-growing high-density industrial yeast cultures. Additionally, sample volume, the use of expensive reagents/enzymes, handling times and incubations were minimized. We have tested the reproducibility of our methods using triplicate/time-series extractions and compared these with commonly used protocols or commercially available kits. Moreover, we utilized a simple flow-cytometric approach to estimate the mitochondrial DNA copy number. Based on the results, our methods have shown higher reproducibility, yield and quality.

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification.

Gas-liquid mass transfer is often rate-limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k(L) a is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory-scale stirred tank reactor (STR) with a highly efficient aeration system (k(L) a ≈ 570 h(-1)). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k(L) a correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro-differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady-state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k(L) a since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory-scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k(L) a) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell-free and culture-based methods of parameter identification, potentially confounding the determination of the "biological enhancement" of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology.

Rapid and complete bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning in minipigs after intraosseous administration.

STUDY OBJECTIVE: Management of chemical weapon casualties includes the timely administration of antidotes without contamination of rescuers. Personal protective equipment makes intravenous access difficult but does not prevent intraosseous drug administration. We therefore measured the systemic bioavailability of antidotes for organophosphorus nerve agent and cyanide poisoning when administered by the intraosseous, intravenous, and intramuscular routes in a small study of Göttingen minipigs. METHODS: Animals were randomly allocated to sequentially receive atropine (0.12 mg/kg by rapid injection), pralidoxime (25 mg/kg by injection during 2 minutes), and hydroxocobalamin (75 mg/kg during 10 minutes) by the intravenous or intraosseous route, or atropine and pralidoxime by the intramuscular route. Plasma concentrations were measured for 6 hours to characterize the antidote concentration-time profiles for each route. RESULTS: Maximum plasma concentrations of atropine and pralidoxime occurred within 2 minutes when administered by the intraosseous route compared with 8 minutes by the intramuscular route. Maximum plasma hydroxocobalamin concentration occurred at the end of the infusion when administered by the intraosseous route. The mean area under the concentration-time curve by the intraosseous route was similar to the intravenous route for all 3 drugs and similar to the intramuscular route for atropine and pralidoxime. CONCLUSION: This study showed rapid and substantial antidote bioavailability after intraosseous administration that appeared similar to that of the intravenous route. The intraosseous route of antidote administration should be considered when intravenous access is difficult.

The yin and yang of yeast transcription: elements of a global feedback system between metabolism and chromatin.

When grown in continuous culture, budding yeast cells tend to synchronize their respiratory activity to form a stable oscillation that percolates throughout cellular physiology and involves the majority of the protein-coding transcriptome. Oscillations in batch culture and at single cell level support the idea that these dynamics constitute a general growth principle. The precise molecular mechanisms and biological functions of the oscillation remain elusive. Fourier analysis of transcriptome time series datasets from two different oscillation periods (0.7 h and 5 h) reveals seven distinct co-expression clusters common to both systems (34% of all yeast ORF), which consolidate into two superclusters when correlated with a compilation of 1,327 unrelated transcriptome datasets. These superclusters encode for cell growth and anabolism during the phase of high, and mitochondrial growth, catabolism and stress response during the phase of low oxygen uptake. The promoters of each cluster are characterized by different nucleotide contents, promoter nucleosome configurations, and dependence on ATP-dependent nucleosome remodeling complexes. We show that the ATP:ADP ratio oscillates, compatible with alternating metabolic activity of the two superclusters and differential feedback on their transcription via activating (RSC) and repressive (Isw2) types of promoter structure remodeling. We propose a novel feedback mechanism, where the energetic state of the cell, reflected in the ATP:ADP ratio, gates the transcription of large, but functionally coherent groups of genes via differential effects of ATP-dependent nucleosome remodeling machineries. Besides providing a mechanistic hypothesis for the delayed negative feedback that results in the oscillatory phenotype, this mechanism may underpin the continuous adaptation of growth to environmental conditions.

'Ivory wave' toxicity in recreational drug users; integration of clinical and poisons information services to manage legal high poisoning.

BACKGROUND: Novel psychoactive substances or 'legal highs' can be defined as psychoactive substances that have been developed to avoid existing drug control measures. Consistency of name, but with change in the content of the product, may cause harm. This could result in clusters of users being poisoned and developing unexpected physical and psychiatric symptoms. We describe such an event and the clinical phenotypes of a cluster of patients poisoned with a novel psychoactive substance in 'ivory wave' and analyze data from the National Poisons Information Service (NPIS) to estimate use across the United Kingdom. In addition, the likely active ingredient in this cluster of 'ivory wave' poisonings was identified. METHODS: An analysis of consecutive patients attending the Royal Infirmary of Edinburgh emergency department in July and August 2010 with self-reported 'ivory wave' use was performed. Over a similar time frame, poisons enquiries regarding 'ivory wave' to the UK NPIS, by telephone and via the internet-based TOXBASE(®) poisons database ( www.toxbase.org ), were analyzed. A sample of 'ivory wave' powder and biological fluids from poisoned patients were investigated to determine the active ingredient. RESULTS: Thirty four emergency attendances due to 'ivory wave' toxicity were identified. The mean +/- SD (range) age was 28.6 +/- 7.8 (16-44) years. Patients demonstrated a toxidrome which lasted several days, characterized by tachycardia (65%), tachypnoea (76%), dystonia (18%), rhabdomyolysis (96%), leucocytosis (57%), agitation (62%), hallucinations (50%), insomnia (32%) and paranoia (21%). Enquiries to NPIS suggest that 'ivory wave' poisoning occurred throughout the United Kingdom. A sample of 'ivory wave' powder was analyzed and found to contain desoxypipradrol, which was also identified in biological fluids from 4 out of 5 patients tested. DISCUSSION: A cluster of cases presenting after use of a novel psychoactive substance was identified in Edinburgh and desoxypipradrol was identified as the likely cause. It was associated with prolonged psychiatric symptoms as a key feature. This chemical was regulated in response to the wider UK outbreak, which NPIS data suggest was geographically widespread but probably short lived. CONCLUSION: Novel psychoactive substances can produce significant toxicity and data from poisons centres may be used to indirectly detect new 'legal highs' that are causing clinical toxicity.

Time-structure of the yeast metabolism in vivo.

All previous studies on the yeast metabolome have yielded a plethora of information on the components, function and organisation of low molecular mass and macromolecular components involved in the cellular metabolic network. Here we emphasise that an understanding of the global dynamics of the metabolome in vivo requires elucidation of the temporal dynamics of metabolic processes on many time-scales. We illustrate this using the 40 min oscillation in respiratory activity displayed in auto-synchronous continuously grown cultures of Saccharomyces cerevisiae, where respiration cycles between a phase of increased respiration (oxidative phase) and decreased respiration (reductive phase). Thereby an ultradian clock, i.e. a timekeeping device that runs through many cycles during one day, is involved in the co-ordination of the vast majority of events and processes in yeast. Through continuous online measurements, we first show that mitochondrial and redox physiology are intertwined to produce the temporal landscape on which cellular events occur. Next we look at the higher order processes of DNA duplication and mitochondrial structure to reveal that both events are choreographed during the respiratory cycles. Furthermore, spectral analysis using the discrete Fourier transformation of high-resolution (10 Hz) time-series of NAD(P)H confirms the existence of higher frequency components of biological origin and that these follow a scale-free architecture even in stable oscillating modes. A different signal-processing approach using discrete wavelet transformations (DWT) indicates that there is a significant contribution to the overall signal from ` ~5, ~ 10 and ~ 20-minutes cycles and the amplitudes of these cycles are phase-dependent. Further investigation (derivative of Gaussian continuous wavelet transformation) reveals that the observed 20-minutes cycles are actually confined to the reductive phase and consist of two ~15-minutes cycles. Moreover, the 5 and 10-minutes cycles are restricted to the oxidative phase of the cycle. The mitochondrial origin of these signals was confirmed by pulse-injection of the cytochrome c oxidase inhibitor H(2)S. We next discuss how these multi-oscillatory states can impinge on the apparently complex reactome (represented as a phase diagram of 1,650 chemical species that show oscillatory behaviour). We conclude that biological processes can be considerably more comprehensible when dynamic in vivo time-structure is taken into account.

Redox regulation in respiring Saccharomyces cerevisiae.

BACKGROUND: In biological systems, redox reactions are central to most cellular processes and the redox potential of the intracellular compartment dictates whether a particular reaction can or cannot occur. Indeed the widespread use of redox reactions in biological systems makes their detailed description outside the scope of one review. SCOPE OF THE REVIEW: Here we will focus on how system-wide redox changes can alter the reaction and transcriptional landscape of Saccharomyces cerevisiae. To understand this we explore the major determinants of cellular redox potential, how these are sensed by the cell and the dynamic responses elicited. MAJOR CONCLUSIONS: Redox regulation is a large and complex system that has the potential to rapidly and globally alter both the reaction and transcription landscapes. Although we have a basic understanding of many of the sub-systems and a partial understanding of the transcriptional control, we are far from understanding how these systems integrate to produce coherent responses. We argue that this non-linear system self-organises, and that the output in many cases is temperature-compensated oscillations that may temporally partition incompatible reactions in vivo. GENERAL SIGNIFICANCE: Redox biochemistry impinges on most of cellular processes and has been shown to underpin ageing and many human diseases. Integrating the complexity of redox signalling and regulation is perhaps one of the most challenging areas of biology. This article is part of a Special Issue entitled Systems Biology of Microorganisms.

The scale-free dynamics of eukaryotic cells.

Temporal organization of biological processes requires massively parallel processing on a synchronized time-base. We analyzed time-series data obtained from the bioenergetic oscillatory outputs of Saccharomyces cerevisiae and isolated cardiomyocytes utilizing Relative Dispersional (RDA) and Power Spectral (PSA) analyses. These analyses revealed broad frequency distributions and evidence for long-term memory in the observed dynamics. Moreover RDA and PSA showed that the bioenergetic dynamics in both systems show fractal scaling over at least 3 orders of magnitude, and that this scaling obeys an inverse power law. Therefore we conclude that in S. cerevisiae and cardiomyocytes the dynamics are scale-free in vivo. Applying RDA and PSA to data generated from an in silico model of mitochondrial function indicated that in yeast and cardiomyocytes the underlying mechanisms regulating the scale-free behavior are similar. We validated this finding in vivo using single cells, and attenuating the activity of the mitochondrial inner membrane anion channel with 4-chlorodiazepam to show that the oscillation of NAD(P)H and reactive oxygen species (ROS) can be abated in these two evolutionarily distant species. Taken together these data strongly support our hypothesis that the generation of ROS, coupled to redox cycling, driven by cytoplasmic and mitochondrial processes, are at the core of the observed rhythmicity and scale-free dynamics. We argue that the operation of scale-free bioenergetic dynamics plays a fundamental role to integrate cellular function, while providing a framework for robust, yet flexible, responses to the environment.

Redox rhythmicity: clocks at the core of temporal coherence.

Ultradian rhythms are those that cycle many times in a day and are therefore measured in hours, minutes, seconds or even fractions of a second. In yeasts and protists, a temperature-compensated clock with a period of about an hour (30-90 minutes) provides the time base upon which all central processes are synchronized. A 40-minute clock in yeast times metabolic, respiratory and transcriptional processes, and controls cell division cycle progression. This system has at its core a redox cycle involving NAD(P)H and dithiol-disulfide interconversions. It provides an archetype for biological time keeping on longer time scales (e.g. the daily cycles driven by circadian clocks) and underpins these rhythms, which cannot be understood in isolation. Ultradian rhythms are the foundation upon which the coherent functioning of the organism depends.

Regulation of yeast oscillatory dynamics.

When yeast cells are grown continuously at high cell density, a respiratory oscillation percolates throughout the population. Many essential cellular functions have been shown to be separated temporally during each cycle; however, the regulatory mechanisms involved in oscillatory dynamics remain to be elucidated. Through GC-MS analysis we found that the majority of metabolites show oscillatory dynamics, with 70% of the identified metabolite concentrations peaking in conjunction with NAD(P)H. Through statistical analyses of microarray data, we identified that biosynthetic events have a defined order, and this program is initiated when respiration rates are increasing. We then combined metabolic, transcriptional data and statistical analyses of transcription factor activity, identified the top oscillatory parameters, and filtered a large-scale yeast interaction network according to these parameters. The analyses and controlled experimental perturbation provided evidence that a transcriptional complex formed part of the timing circuit for biosynthetic, reductive, and cell cycle programs in the cell. This circuitry does not act in isolation because both have strong translational, proteomic, and metabolic regulatory mechanisms. Our data lead us to conclude that the regulation of the respiratory oscillation revolves around coupled subgraphs containing large numbers of proteins and metabolites, with a potential to oscillate, and no definable hierarchy, i.e., heterarchical control.

A tuneable attractor underlies yeast respiratory dynamics.

Our understanding of the molecular structure and function in the budding yeast, Saccharomyces cerevisiae, surpasses that of all other eukaryotic cells. However, the fundamental properties of the complex processes and their control systems have been difficult to reconstruct from detailed dissection of their molecular components. Spontaneous oscillatory dynamics observed in self-synchronized continuous cultures is pervasive, involves much of the cellular network, and provides unique insights into integrative cell physiology. Here, in non-invasive experiments in vivo, we exploit these oscillatory dynamics to analyse the global timing of the cellular network to show the presence of a low-order chaotic component. Although robust to a wide range of environmental perturbations, the system responds and reacts to the imposition of harsh environmental conditions, in this case low pH, by dynamic re-organization of respiration, and this feeds upwards to affect cell division. These complex dynamics can be represented by a tuneable attractor that orchestrates cellular complexity and coherence to the environment.