Applications and limitations of regulatory RNA elements in synthetic biology and biotechnology.

Synthetic biology requires the design and implementation of novel enzymes, genetic circuits or even entire cells, which can be controlled by the user. RNA-based regulatory elements have many important functional properties in this regard, such as their modular nature and their ability to respond to specific external stimuli. These properties have led to the widespread exploration of their use as gene regulation devices in synthetic biology. In this review, we focus on two major types of RNA elements: riboswitches and RNA thermometers (RNATs). We describe their general structure and function, before discussing their potential uses in synthetic biology (e.g. in the production of biofuels and biodegradable plastics). We also discuss their limitations, and novel strategies to implement RNA-based regulatory devices in biotechnological applications. We close with a description of some common model organisms used in synthetic biology, with a focus on the current applications and limitations of RNA-based regulation.

Increased electroencephalographic gamma activity reveals awakening from isoflurane anaesthesia in rats.

BACKGROUND: Animal studies often require reliable measures for anaesthetic drug effects. Lately, EEG-based depth of anaesthesia estimation has been widely applied to rat models. This study investigated the reliability of different EEG spectral properties in revealing awakening from isoflurane anaesthesia in rats. METHODS: Adult Wistar rats with previously implanted frontal epidural electrodes were anaesthetized using isoflurane. The anaesthesia was slowly lightened until awakening, as observed by the first spontaneous movement, after which anaesthesia was induced again by increasing the isoflurane concentration. EEG was recorded during the recovery and induction periods, and the spectrograms and 23 quantitative spectral parameters used in the depth of anaesthesia estimation were calculated from the signals. RESULTS: The awakening was accompanied by a decrease in EEG activity at frequencies below 25 Hz, while the activity at higher frequencies (25-150 Hz) was increased. Whereas the behaviour of parameters used to measure activity in the lower frequencies was subject to variability between animals, the increase in higher frequency activity was more consistent, resulting in a statistically significant change in the relative gamma power parameters at the moment of awakening. CONCLUSIONS: The increase in frontal relative gamma activity, especially in the 50-150 Hz frequency band, seems to be the most reliable EEG indicator for the awakening of a rat from isoflurane anaesthesia. A number of other spectral measures can also be used to detect this event. However, the role of gamma frequencies in the performance of these parameters is crucial.

Soft sensors for a sensing-actuation system with high bladder voiding efficiency.

Sensing-actuation systems can assist a bladder with lost sensation and weak muscle control. Here, we advance the relevant technology by integrating a soft and thin capacitive sensor with a shape memory alloy-based actuator to achieve a high-performance closed-loop configuration. In our design, sensors capable of continuous bladder volume detection and actuators with strong emptying force have been used. This integration has previously hindered performance due to large bladder volume changes. Our solution integrates sensing-actuation elements that are bladder compatible but do not interfere with one another, achieving real-time bladder management. The system attains a highly desirable voiding target of 71 to 100% of a rat's bladder with a volume sensitivity of 0.7 µF/liter. Our system represents an efficient voiding solution that avoids overfilling and represents a technological solution to bladder impairment treatment, serving as a model for similar soft sensor-actuator integration with other organs.

Monitoring of global cerebral ischemia using wavelet entropy rate of change.

In this paper, the subband wavelet entropy (SWE) and its time difference are proposed as two quantitative measures for analyzing and segmenting the electroencephalographic (EEG) signals. SWE for EEG subbands, namely Delta, Theta, Alpha, Beta, and Gamma, is calculated and segmented using wavelet analysis. In addition, a time difference entropy measure was calculated because it does not require a baseline and equals to zero in all clinical bands as the initial condition. Visual and quantitative results were obtained from 11 rodents that were subjected to 3, 5, and 7 min of global ischemic brain injury by asphyxic cardiac arrest. We found that the time difference of SWE is capable of amplifying the variations between clinical bands during the various stages of the recovery process and may serve as a novel analytical approach to grade and classify brain rhythms during global ischemic brain injury and recovery.

Investigation of the effects of ischemic preconditioning on the HRV response to transient global ischemia using linear and nonlinear methods.

Ischemic preconditioning (IP) has been used as a strategy to prevent cell death in various organs, including the brain and the heart. Investigation of the effects of ischemic preconditioning mostly employed models with reduced complexity, such as cell cultures, tissue slices or perfused organ preparations. Although such models can provide valuable insight into the protective mechanism of preconditioning, the functional (re)organization of the control mechanisms at the level of the living organism cannot be assessed. The purpose of the present animal model study was to evaluate the effect of global ischemic preconditioning on the heart rate variability (HRV) response to the asphyxia insult. The data consisted of 4 h RR interval measurements recorded in five preconditioned and five non-preconditioned Wistar rats. Using linear (time and frequency domain) and nonlinear (approximate entropy and parameters of Poincare plots) measures, we evaluated the dynamic time course of the HRV response to the asphyxia insult and the effect of preconditioning on the autonomic neurocardiac control. Both the linear and nonlinear parameters indicate a faster recovery of the baseline HRV corresponding to the preconditioned groups, though only the spectral analysis identifies a statistically significant difference between the two groups. For the preconditioned group, at about 90 min after the asphyxic insult, the autonomic neural balance (measured by LF/HF ratio) appears fully recovered. The small variation of the rest of the parameters indicates the necessity of further investigation including the design of a larger study with a higher statistical power. Our results show for the first time that global ischemic preconditioning influences the HRV response to the asphyxia injury. The neuroprotective effect of preconditioning translates into a faster recovery of the basal HRV and the autonomic modulation of the heart.

Cognitive workload modulation through degraded visual stimuli: a single-trial EEG study.

OBJECTIVE: Our experiments explored the effect of visual stimuli degradation on cognitive workload. APPROACH: We investigated the subjective assessment, event-related potentials (ERPs) as well as electroencephalogram (EEG) as measures of cognitive workload. MAIN RESULTS: These experiments confirm that degradation of visual stimuli increases cognitive workload as assessed by subjective NASA task load index and confirmed by the observed P300 amplitude attenuation. Furthermore, the single-trial multi-level classification using features extracted from ERPs and EEG is found to be promising. Specifically, the adopted single-trial oscillatory EEG/ERP detection method achieved an average accuracy of 85% for discriminating 4 workload levels. Additionally, we found from the spatial patterns obtained from EEG signals that the frontal parts carry information that can be used for differentiating workload levels. SIGNIFICANCE: Our results show that visual stimuli can modulate cognitive workload, and the modulation can be measured by the single trial EEG/ERP detection method.

Vulnerability of the thalamic somatosensory pathway after prolonged global hypoxic-ischemic injury.

The aim of this study was to test the hypothesis that under prolonged global ischemic injury, the somatosensory thalamus and the cortex would manifest differential susceptibility leading to varying degrees of thalamo-cortical dissociation. The thalamic electrical responses displayed increasing suppression with longer durations of ischemia leading to a significant thalamo-cortical electrical dissociation. The data also point to a selective vulnerability of the network oscillations involving the thalamic relay and reticular thalamic neurons. An adult rat model of asphyxial cardiac arrest involving three cohorts with 3 min (G1, n=5), 5 min (G2, n=5) and 7 min (G3, n=5) of asphyxia respectively was used. The cortical evoked response, as quantified by the peak amplitude at 20 ms in the cortical evoked potential, recovers to more than 60% of baseline in all the cases. The multi-unit responses to the somatosensory stimuli recorded from the thalamic ventral posterior lateral (VPL) nuclei consists typically of three components: (1). the ON response (<30 ms after stimulus), (2). the OFF response (period of inhibition, from 30 ms to 100 ms after stimulus) and (3). rhythmic spindles (beyond 100 ms after stimulus). Asphyxia has a significant effect on the VPL ON response at 30 min (P<0.025), 60 min (P<0.05) and 90 min (P<0.05) after asphyxia. Only animals in G3 show a significant suppression (P<0.05) of the VPL ON response when compared to the sham group at 30 min, 60 min and 90 min after asphyxia. There was no significant reduction in somatosensory cortical N20 (negative peak in the cortical response at 20 ms after stimulus) amplitude in any of the three groups with asphyxia indicating a thalamo-cortical dissociation in G3. Further, rhythmic spindle oscillations in the thalamic VPL nuclei that normally accompany the ON response recover either slowly after the recovery of ON response (in the case of G1 and G2) or do not recover at all (in the case of G3).We conclude that there is strong evidence for selective vulnerability of thalamic relay neurons and its network interactions with the inhibitory interneurons in the somatosensory pathway leading to a thalamo-cortical dissociation after prolonged durations of global ischemia.

Effect of varying pacing waveform shapes on propagation and hemodynamics in the rabbit heart.

The propagation characteristics of myocardium stimulated with anodal, cathodal, and equiphasic biphasic pacing pulses were examined in Langendorff-perfused rabbit hearts. Conduction velocity measurements were made using an array of bipolar extracellular electrodes, transmembrane potentials recorded using floating intracellular microelectrodes, and hemodynamics measured by fluid-filled catheter transducer systems. Anodal (A) stimulation pulses improved the electrical conduction at all the stimulus amplitudes tested in both longitudinal (e.g., 5 V 2-msec pulse: [A] 54.9 +/- 0.7 cm/sec; cathodal [C] 49.7 +/- 1.5 cm/sec) and transverse (e.g., 5 V 2 msec pulse: [A] 31.3 +/- 1.7 cm/sec; [C] 23.3 +/- 2.9 cm/sec) directions. Microelectrode recordings verified that increased conduction velocities of the anodal pulses were associated with faster upstrokes of the action potentials. The increased threshold associated with anodal pulses may be overcome by using a biphasic (B) waveform, in effect adding a second phase (e.g., 2-msec pulse: [A] 2.03 +/- 1.3 V; [C] 3.85 +/- 1.5 V; [B] 2.15 +/- 0.9 V). The conduction speeds achieved by the biphasic pulses were found to be comparable to the equivalent anodal pulses (e.g., 5 V 2-msec pulse: [B] 55.2 +/- 1.7 cm/sec longitudinal and 32.4 +/- 2.1 cm/sec transverse). It is postulated that the enhanced conduction by anodal and biphasic pulses may be due to preconditioning of the myocardium before stimulation, resulting in more vigorous action potential upstrokes. In preliminary experiments, it was observed that improved conduction elicited by these pulses also resulted in enhanced contractility as measured by shortened electromechanical delays and faster rate of rise of pressure development (dP/dtmax: [A] 25.4 +/- 0.4 mm Hg/sec; [C] 19.4 +/- 0.8 mm Hg/sec; [B] 25.7 +/- 1.2 mm Hg/sec, respectively). Use of novel hybrid pulses involving an anodal component may offer a way for implanted pacemakers to enhance the electro-mechanical response of the heart.

Effects of anodal vs. cathodal pacing on the mechanical performance of the isolated rabbit heart.

Previous studies have suggested that anodal pacing enhances electrical conduction in the heart near the pacing site. It was hypothesized that enhanced conduction by anodal pacing would also enhance ventricular pressure in the heart. Left ventricular pressure measurements were made in isolated, Langendorff-perfused rabbit hearts by means of a Millar pressure transducer with the use of a balloon catheter fixed in the left ventricle. The pressure wave was analyzed for maximum pressure (Pmax) generated in the left ventricle and the work done by the left ventricle (Parea). Eight hearts were paced with monophasic square-wave pulses of varying amplitudes (2, 4, 6, and 8 V) with 100 pulses of each waveform delivered to the epicardium. Anodal stimulation pulses showed statistically significant improvement in mechanical response at 2, 4, and 8 V. Relative to unipolar cathodal pacing, unipolar anodal pacing improved Pmax by 4.4 +/- 2.3 (SD), 5.3 +/- 3.1, 3.5 +/- 4.9, and 4.8 +/- 1.9% at 2, 4, 6, and 8 V, respectively. Unipolar anodal stimulation also improved Parea by 9.0 +/- 3.0, 12.0 +/- 6.0, 10.1 +/- 7.7, and 11.9 +/- 6.0% at 2, 4, 6, and 8 V, respectively. Improvements in Pmax and Parea indicate that an anodally paced heart has a stronger mechanical response than does a cathodally paced heart. Anodal pacing might be useful as a novel therapeutic technology to treat mechanically impaired or failed hearts.

Spectral analysis methods for neurological signals.

This paper reviews some novel spectral analysis techniques that are useful for neurological signals in general and EEG signals in particular. First, some drawbacks and limitations of the commonly used Fast Fourier transforms (FFTs) are presented, and then alternative algorithms are outlined. An auto-regressive (AR) modeling based spectral estimation procedure is presented to overcome the problems of lower resolution and 'leakage' effects inherent in the FFT algorithm. For signals which are transient in nature or rapidly time-varying, two alternative algorithms are presented. The first is an adaptive AR parameter estimation algorithm and the second is a wavelet based time-frequency representation algorithm. Finally, a Spectral Distance measure and the Itakura distance measure are presented to quantify the differences between the spectra of two signals in a succinct manner. The application and performance of all the algorithms is illustrated using electroencephalograms (EEGs) recorded in animals during hypoxic asphyxic injury to brain.

Adaptive Fourier modeling for quantification of tremor.

A new computational method for quantification of tremor, the weighted frequency Fourier linear combiner (WFLC), is presented. This technique rapidly determines the frequency and amplitude of tremor by adjusting its filter weights according to a gradient search method. It provides continual tracking of frequency and amplitude modulations over the course of a test. By quantifying time-varying characteristics, the WFLC assists in correctly interpreting the results of spectral analysis, particularly for recordings exhibiting multiple spectral peaks. It therefore supplements spectral analysis, providing a more accurate picture of tremor than spectral analysis alone. The method has been incorporated into a desktop tremor measurement system to provide clinically useful analysis of tremor recorded during handwriting and drawing using a digitizing tablet. Simulated data clearly demonstrate tracking of variations in frequency and amplitude. Clinical recordings then show specific examples of quantification of time-varying aspects of tremor.

Optical light scatter imaging of cellular and sub-cellular morphology changes in stressed rat hippocampal slices.

Optical imaging, such as transmission imaging, is used to study brain tissue injury. Transmission imaging detects cellular swelling via an increase in light transmitted by tissue slices due to a decrease in scattering particle concentration. Transmission imaging cannot distinguish sub-cellular particle size changes from cellular swelling or shrinkage. We present an optical imaging method, based on Mie scatter theory, to detect changes in sub-cellular particle size and concentration. The system uses a modified inverted microscope and a 16-bit cooled CCD camera to image tissue light scatter at two angles. Dual-angle scatter ratio imaging successfully discriminated latex microsphere suspensions of differing sizes (0.6, 0.8, 1 and 2 microm) and concentrations. We applied scatter imaging to hippocampal slices treated with 100 microM N-methyl-D-aspartate (NMDA) to model excitotoxic injury or -40 mOsm hypotonic perfusion solution to cause edema injury. We detected light scatter decreases similar to transmission imaging in the CA1 region of the hippocampus for both treatments. Using our system, we could distinguish between NMDA and hypotonic treatments on the basis of statistically significant (P<0.0003) differences in the scatter ratio measured in CA1. Scatter imaging should be useful in studying tissue injuries or activity resulting in brain tissue swelling as well as morphological changes in sub-cellular organelles such as mitochondrial swelling.

Removal of ECG interference from the EEG recordings in small animals using independent component analysis.

In experiments involving small animals, the electroencephalogram (EEG) recorded during severe injury and accompanying resuscitation exhibit the strong presence of electrocardiogram (ECG). For improved quantitative EEG (qEEG) analysis, it is therefore imperative to remove ECG interference from EEG. In this paper, we validate the use of independent component analysis (ICA) to effectively suppress the interference of ECG from EEG recordings during normal activity, asphyxia and recovery following asphyxia. Two channels of EEG from five rats were recorded continuously for 2 h. Simultaneous recording of one channel ECG was also made. Epochs of 4 s and 1 min were selected from baseline, asphyxia and recovery (every 10 min) and their independent components and power spectra were calculated. The improvement in normalized power spectrum of EEG obtained for all animals was 7.71+/-3.63 db at the 3rd minute of recovery and dropped to 1.15+/-0.60 db at 63rd minute. The application of ICA has been particularly useful when the power of EEG is low, such as that observed during early brain hypoxic-asphyxic injury. The method is also useful in situations where accurate indications of EEG signal power and frequency content are needed.

In vivo nitric oxide sensor using non-conducting polymer-modified carbon fiber.

Nitric oxide (NO) is emerging as a very important and ubiquitous gaseous messenger in the body. The response characteristics of NO sensors made of non-conducting polymer modified carbon fiber electrodes are investigated to determine their selectivity, sensitivity, and stability for in vivo use. A composite polymer, comprising Nafion, m-phenylenediamine, and resorcinol, showed the best selectivity and stability to amperometric NO detection. The non-conducting, self-limiting polymer film protects the electrode from interference and fouling by other biochemicals. Although the relative sensitivity to NO of the modified sensor is lower than that of the unmodified carbon fiber electrodes (less than 6%), the composite polymer electrode showed high selectivity against ascorbic acid (> 2000:1), nitrite (> 600:1), and dopamine (> 200:1). The stability of the NO sensor was maintained for at least 1 week. The NO sensitivity after in vivo experiments (n = 8) is 88.1 +/- 5.6% of initial sensitivity data obtained before in vivo experiments. Preliminary in vivo experiments done with this electrode are shown to capture elevated NO levels in brain following an ischemic injury.

A novel quantitative EEG injury measure of global cerebral ischemia.

OBJECTIVE: To develop a novel quantitative EEG (qEEG) based analysis method, cepstral distance (CD) and compare it to spectral distance (SD) in detecting EEG changes related to global ischemia in rats. METHODS: Adult Wistar rats were subjected to asphyxic-cardiac arrest for sham, 1, 3, 5 and 7 min (n=5 per group). The EEG signal was processed and fitted into an autoregressive (AR) model. A pre-injury baseline EEG was compared to selected data segments during asphyxia and recovery. The dissimilarities in the EEG segments were measured using CD and SD. A segment measured was considered abnormal when it exceeded 30% of baseline and its duration was used as the index of injury. A comprehensive Neurodeficit Score (NDS) at 24 h was used to assess outcome and was correlated with CD and SD measures. RESULTS: A higher correlation was found with CD and asphyxia time (r=0.81, P<0.001) compared to SD and asphyxia time (r=0.69, P<0.001). Correlation with cardiac arrest time (MAP<10 mmHg) showed that CD was superior (r=0.71, P<0.001) to SD (r=0.52, P=0.002). CD obtained during global ischemia and 90 min into recovery correlated significantly with NDS at 24 h after injury (Spearman coefficient=-0.83, P<0.005), and was more robust than the traditional SD (Spearman coefficient=-0.63, P<0.005). CONCLUSION: The novel qEEG-based injury index from CD was superior to SD in quantifying early cerebral dysfunction after cardiac arrest and in providing neurological prognosis at 24 h after global ischemia in adult rats. Studying early qEEG changes after asphyxic-cardiac arrest may provide new insights into the injury and recovery process, and present opportunities for therapy.

Detection of non-linearity in the EEG of schizophrenic patients.

OBJECTIVE: The aim of this study is to detect non-linearity in the EEG of schizophrenia with a modified method of surrogate data. We also want to identify if dimension complexity (correlation dimension using spatial embedding) could be used as a discriminating statistic to demonstrate non-linearity in the EEG. The difference between the attractor dimension of healthy subjects and schizophrenic subjects is expected to be interpreted as reflecting some mechanisms underlying brain wave by views of non-linear dynamics analysis may reflect mechanistic differences. METHODS: EEGs were recorded with 14 electrodes in 18 healthy male subjects (average age: 26.3; range: 20--35) and 18 male schizophrenic patients (average age: 30.6; range: 24--40) during a resting eye-closed state. Neither of two groups was taking medicines. All artificial epochs in the EEG records were rejected by an experienced doctor's visual inspection. RESULTS: Testing non-linearity with modified surrogate data, we showed that correlation dimension of EEG data of schizophrenia does refuse the null hypothesis that the data were resulted from a linear dynamic system. A decrease of dimension complexity was found in the EEG of schizophrenia compared with controls. We interpreted it as the result of the psychopath's dysfunction overall brain. The surrogating procedure results in a significant increase in D(s). CONCLUSIONS: Non-linearity of the EEG in schizophrenia was proven in our study. We think the correlation dimension with spatial embedding as a good discriminating statistic for testing such non-linearity. Moreover, schizophrenic patients' EEGs were compared with controls and a lower dimension complexity was found. The results of our study indicate the possibility of using the methods of non-linear time series analysis to identify the EEGs of schizophrenic patients.

Somatosensory stimulus entrains spindle oscillations in the thalamic VPL nucleus in barbiturate anesthetized rats.

This study reports the effect of external stimuli on spindle oscillations in the somatosensory thalamus of barbiturate anesthetized rats. Multi-unit responses to somatosensory stimuli were measured from the contralateral thalamic ventral posterior lateral (VPL) nucleus at different stimulus strengths and periods. Spindle oscillations could be entrained by the somatosensory stimuli at periods between 2 and 5 s. A resonance phenomenon described as a quiescent pre-stimulus period followed by entrained post-stimulus oscillations, was observed for somatosensory stimuli above the threshold for eliciting cortical evoked potentials and a stimulus period between 2 and 5 s. This study demonstrates an ascending pathway for localized modulation of spindle oscillations.

Adaptive estimation of latency changes in evoked potentials.

Changes in latency of evoked potentials (EP) may indicate clinically and diagnostically important changes in the status of the nervous system. A low signal-to-noise ratio of the EP signal makes it difficult to estimate small, transient, time-varying changes in latency, or delays. Here, we present an adaptive algorithm that estimates small delay (latency change) values even when EP signal amplitudes are time-varying. When the delay is time invariant, the adaptive algorithm produces an unbiased estimate with delay estimation error less than half of the sampling interval. A lower estimation error variance is obtained when, in a pair of signals, the adaptive algorithm delays the signal with the higher SNR. The adaptive algorithm delays the signal with the higher SNR. The adaptive delay estimation algorithm was tested on intra-operative recordings of somatosensory EP, and analysis of those recordings reveals that the anesthetic etomidate produces a step change in the amplitude and latency of the EP signals.

Applications of adaptive filtering to ECG analysis: noise cancellation and arrhythmia detection.

Several adaptive filter structures are proposed for noise cancellation and arrhythmia detection. The adaptive filter essentially minimizes the mean-squared error between a primary input, which is the noisy ECG, and a reference input, which is either noise that is correlated in some way with the noise in the primary input or a signal that is correlated only with ECG in the primary input. Different filter structures are presented to eliminate the diverse forms of noise: baseline wander, 60 Hz power line interference, muscle noise, and motion artifact. An adaptive recurrent filter structure is proposed for acquiring the impulse response of the normal QRS complex. The primary input of the filter is the ECG signal to be analyzed, while the reference input is an impulse train coincident with the QRS complexes. This method is applied to several arrhythmia detection problems: detection of P-waves, premature ventricular complexes, and recognition of conduction block, atrial fibrillation, and paced rhythm.

Adaptive Fourier estimation of time-varying evoked potentials.

An estimation procedure for dealing with time-varying evoked potentials is presented here. The evoked response is modeled as a dynamic Fourier series and the Fourier coefficients are estimated adaptively by the least mean square algorithm. Approximate expressions have been developed for the estimation error and time constant of adaptation. A procedure for optimizing the estimator performance is also presented. The effectiveness of the estimator in dealing with simulated as well as actual evoked responses is demonstrated.