Rapid on-site multiplex assays for total and toxigenic Microcystis using real-time PCR with microwave cell disruption.

A quantitative real-time polymerase chain reaction (qPCR) is a robust means by which to monitor toxin-producing cyanobacteria. However, qPCR usually requires DNA extraction, which is a time-consuming, labor-intensive pretreatment. To be able to quickly determine the potential of cyanotoxin contamination in the field, a rapid pretreatment method for DNA extraction and a portable qPCR device are needed. In this study, we applied a microwave-based method for the qPCR pretreatment and a multicolor portable qPCR with UPL and TaqMan probes to quantify toxigenic and total Microcystis. The method was tested using laboratory cultures of toxigenic Microcystis aeruginosa PCC7820. The qPCR results showed the cycle thresholds value (Ct value) correlated well with cell numbers, with detection limit at about 1,000 cells/ml. This scheme was applied in 22 environmental samples from six drinking water reservoirs (DWRs) in Taiwan. Although the results for qPCR were about four times higher than those of microscopic observation, good correlation between qPCR and microscope methods were obtained (r-square: 0.79, P < 0.01). The ratios of toxigenic Microcystis to total Microcystis in two reservoirs, Sin-Shan Reservoir and Shih-men Reservoir, were less than 10%. In three other reservoirs, Ren-Yi-Tan Reservoir, Nan-Hua Reservoir and Bao-Shan Reservoir, much higher (>46.1%) ratios were obtained. The scheme may assist quick assessment of the risk associated with toxic cyanobacteria in DWRs.

A modified "triangular pulse" stimulator for C-fibers stimulation.

A low-cost, battery-powered stimulator is described. This device generates asymmetric current pulse with fast rising phase and slower exponential decay. The current intensity and the time constant of the exponential decay can be independently and continuously varied. An example of using this stimulator to selectively activate C-fibers is demonstrated. In this case the total charge injected in one stimulation is only 67 nanocolumb, which is much smaller than that injected by conventional DC polarization technique. Detailed information about the circuit design is described.

Effect of conduction distance on amplitude and area of compound action potentials of A fiber and C fiber.

We used 24 sacral dorsal roots of the rat to analyze amplitude and area changes of biphasic and monophasic compound action potentials (CAPs) at 4 conduction distances. Both the CAPs of the A-fiber and C-fiber were analyzed. The changes were examined with the paired t test and linear regression. All the variables decreased linearly with increasing conduction distance except area of monophasic CAP, which remained constant throughout. CAP data were also compared between the S1 and S2 roots by using the pooled t test. Only area of monophasic CAP showed good correspondence with the anatomical data. Therefore, area of monophasic CAP is suggested as the best parameter for representing the functional state of a nerve.

A multichannel system for recording and analysis of cortical field potentials in freely moving rats.

A system has been developed to record and analyze the cortical electrical activity from 16 different sites in freely moving rats. The hardware includes a 16-channel amplifier system whose high input impedance, low noise, small size, light weight and shielded multistrand connecting cable allow high quality multichannel recording of field potentials. The software developed for this system consists of data acquisition, data analysis and topographic mapping of cortical-evoked potentials as well as electroencephalograms. Cortical field potentials evoked by CO2-laser stimulation were compared between wakeful and pentobarbital-treated conditions. To investigate the background interference produced by sleep spindle, three kinds of reference-free methods (the Wilson, local average and weighted average methods) were utilized to compare the coherence between field potentials obtained from two cerebral hemispheres using monopolar vs. reference-free recordings.

Algorithmic complexity as an index of cortical function in awake and pentobarbital-anesthetized rats.

This study introduces algorithmic complexity to measure characteristics of brain functions. The EEG of the rat was recorded with implanted electrodes. The normalized complexity value was relatively independent of data length, and it showed a simpler and easier calculation characteristic than other non-linear indexes. The complexity index revealed significant differences among awake, asleep, and anesthetized states. It may be useful in tracking short-term and long-term changes in brain functions, such as anesthetized depth, drug effects, or sleep-wakefulness.

Interactive program for spectral and area analysis of compound action potentials of A-fiber and C-fiber.

An interactive program was described to correct the baseline wandering of the compound action potentials (CAPs) of C-fiber, to calculate the area and the peak amplitude of CAPs, and to analyze their spectral distribution. Using this program, we found the optimal bandpass of the filter for recording CAPs to be from 10 Hz to 3 kHz.

Comparison of touch- and laser heat-evoked cortical field potentials in conscious rats.

Field potentials and multiunit activities from chronically implanted cortical electrodes were used to study tactile and nociceptive information processing from the tail of the rat. Fourteen stainless steel screws implanted in the skull were used as electrodes to record field potentials in different cortical areas. Electrical, mechanical, and laser pulses were applied to the tail to induce evoked cortical field potentials. Evoked responses were compared before and after sodium pentobarbital anesthesia (50 mg/kg, i.p.). In both electrical- and mechanical-evoked potential (EEP and MEP) studies, two major peaks were found in the conscious animal. The polarity of the late component was modified after pentobarbital anesthesia. In the laser-evoked potential (LEP) study, two distinct negative peaks were found. Both peaks were very sensitive to anesthesia. Following quantitative analysis, our data suggest that the first positive peak of EEP and MEP corresponded to the activation of the Abeta fiber, the second negative peak of MEP and the first peak of LEP corresponded to Adelta fiber activation, while the second peak of LEP corresponded to C fiber activation. The absolute magnitudes of all cortical components were positively related to the intensity of the stimulation. From spatial mapping analysis, a localized concentric source of field potential was observed in the primary somatosensory cortex (SI) only after activation of the Abeta fiber. Larger responsive cortical areas were found in response to Adelta and C fiber activation. In an intracortical recording experiment, both tactile and nociceptive stimulation evoked heightened unit activity changes at latencies corresponding to respective field potentials. We conclude that different cortical areas are involved in the processing of A and C fiber afferent inputs, and barbiturate anesthesia modifies their processing.

The application of cepstral coefficients and maximum likelihood method in EMG pattern recognition.

A new technique for classifying patterns of movement via electromyographic (EMG) signals is presented. Two methods (conventional autoregressive (AR) coefficients and cepstral coefficients) for extracting features from EMG signals and three classification algorithms (Euclidean Distance Measure (EDM), Weighted Distance Measure (WDM), and Maximum Likelihood Method (MLM)) for discriminating signals representative of broad classes of movements are described and compared. These three classifiers are derived from Bayes classifier with some assumptions, the relationship among them is discussed. The conventional MLM is modified to avoid heavy matrix inversion. Six able-bodied subjects with two pairs of surface electrodes located on bilateral sternocleidomastoid and upper trapezius muscles were studied in the experiment. The EMG signals of 20 repetitions of 10 motions were analyzed for each subject. Experimental results showed that mean recognition rate of the cepstral coefficients was at least 5% superior to that of the AR coefficients. The improvement achieved by the cepstral method was statistically significant for all the three classifiers. Reasons for the superiority of cepstral features were investigated from the feature space and frequency domain, respectively. The cepstral coefficients owned better cluster separability in feature space and they emphasized the more informative part in the frequency domain. The discrimination rate of the MLM was the highest among three classifiers. Incorporation of the cepstral features with the MLM could reduce the misclassification rate by 10.6% when compared with the combination of AR coefficients and EDM. Proper choice of five of ten motions could further raise the recognition rate to more than 95%.

The effect of electrode arrangement on spectral distance measures for discrimination of EMG signals.

The proper electrode placement in applying cepstral coefficients for electromyogram (EMG) signature discrimination was investigated. We measured EMG signals of different motions with two electrode arrangements simultaneously. Electrode pairs were located separately on dominant muscles (S-type arrangement) and closely in the region between muscles (C-type arrangement). The application of the cepstral method to signals derived from a C-type arrangement did not achieve the same discrimination as with a S-type arrangement. We used a simplified model to elucidate the poor performance in C-type signals. The bandwidth of signals obtained from S-type placement is wider than that from C-type. Narrower bandwidth decreases the importance of the more discriminative parts for both autoregressive (AR) and cepstral methods. The cepstral method is more sensitive to such variation, so the degradation in performance is more serious for the cepstral method. Second, the amplitude of C-type signal is lower than the S-type; therefore, the C-type signal is more sensitive to the disturbance of noise, especially in the high-frequency band. As high-frequency noise increases, the spectral difference between different EMG signals is gradually dominated by the low-frequency part, which is more informative. Thus, the performances of both methods are improved with increasing high-frequency noise. The improving rate of the AR method is faster than the cepstral method; therefore, its discriminative efficiency may exceed the cepstral method with C-type arrangement.