Evaluation of a noninvasive continuous cardiac output monitoring system based on thoracic bioreactance

Noninvasive cardiac output (CO) measurement can be useful in many clinical settings where invasive monitoring is not desired. Bioimpedance (intrabeat measurement of changes in transthoracic voltage amplitude in response to an injected high-frequency current) has been explored for this purpose but is limited in some clinical settings because of inherently low signal-to-noise ratio. Since changes in fluid content also induce changes in thoracic capacitive and inductive properties, we tested whether a noninvasive CO measurement could be obtained through measurement of the relative phase shift of an injected current (i.e., bioreactance). We constructed a prototype device that applies a 75-kHz current and determines the relative phase shift (dPhi/dt) of the recorded transthoracic voltage. CO was related to the product of peak dPhi/dt, heart rate, and ventricular ejection time. The preclinical study was done in nine open-chest pigs put on right heart bypass so that CO could be varied at known values. This was followed by a feasibility study in 27 postoperative patients who had a Swan-Ganz catheter (SGC). The measurements of noninvasive CO measurement and cardiopulmonary bypass pump correlated to each other (r = 0.84) despite the large variation in CO and temperatures. Similarly, in patients, mean CO values were 5.18 and 5.17 l/min as measured by SGC and the noninvasive CO measurement system, respectively, and were highly correlated over the range of values studied (r = 0.90). Preclinical and clinical data demonstrate the feasibility of using blood flow-related phase shifts of transthoracic electric signals to perform noninvasive continuous CO monitoring.     

Evidence for pyroelectric and piezoelectric sensory mechanisms in the insect integument.

Quantitative pyroelectric (PE) and piezoelectric (PZE) measurements were carried out on the insect integument of live Blaberus giganteus (cockroach) and on dry integument preparations of the same species. Voltage responses to optical pulses of 10--500 ms, absorbed in the live integument, were PE: interference filter measurements showed the responses to be proportional to the absorbed thermal radiation flux and independent of the wavelength. The voltage/time-course of the responses was in agreement with theoretically calculated PE signals. Voltage responses to mechanical pulses were PZE. The responses of the inner and outer integument surfaces always had opposite electric signs. The polar character of the integument was confirmed by means of a separate dielectric heating method. To explain these results, we hypothesize that the PE properties are for the most part localized in the two outermost layers (outer and inner epicuticle) of the integument, which consists mainly of polar lipids and proteins. Parallel alignment of these polar molecules perpendicular to the integument surface is very likely. PE and PZE responses, therefore, will not only occur in live insects but will also be measurable in dead, dry integument preparations as long as the polar tissue texture remains intact. Due to its polar texture, the insect integument will react to rapid changes in temperature, illumination, or uniaxial pressure in the same way as nonbiological PE materials, where the voltage responses depend on dX/dt (X, pressure or temperature). It seems clear, therefore, that the well-known physiological reactions of various arthropods to such physical outside influences may be related to the PE property of their integument.     

Self-Powered Wireless Carbohydrate/Oxygen Sensitive Biodevice Based on Radio Signal Transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.     

Bubble-test method for synthetic and bovine vascular material

A measurement system has been designed and constructed at NIST for the study of the mechanical properties of synthetic and bovine vascular materials. The measurement technique was validated on latex, where good agreement was found with the Neo-Hookean model. Measurements were also made on expanded polytetrafluoroethylene, which is commonly used for vascular grafts. The measurements of this material were carried out over a pressure range greater than would be seen in vivo. However, the strains were still small enough to effectively apply the Neo-Hookean model to these data.     

Quality assurance for polychromatic flow cytometry

This protocol outlines a three-part quality assurance program to optimize, calibrate and monitor flow cytometers used to measure cells labeled with five or more fluorochromes (a practice known as polychromatic flow cytometry). The initial steps of this program (system optimization) ensure that the instrument's lasers, mirrors and filters are optimally configured for the generation and transmission of multiple fluorescent signals. To determine the sensitivity and dynamic range of each fluorescence detector, the system is then calibrated by measuring fluorescence over a range of photomultiplier tube (PMT) voltages by determining the PMT voltage range and linearity (Steps 2-10) and validating the PMT voltage (Steps 11-17). Finally, to ensure consistent performance, we provide procedures to monitor the precision, accuracy and sensitivity of fluorescence measurements over time. All three aspects of this program should be performed upon installation, or whenever changes occur along the flow cytometer's optical path. However, only a few of these procedures need to be carried out on a routine basis.     

Phospholipid interactions in model membrane systems. I. Experiments on monolayers.

We study the lateral headgroup interactions among phosphatidylcholine (PC) molecules and among phosphatidylethanolamine (PE) molecules in monolayers and extend our previous models. In this paper, we present an extensive set of pressure-area isotherms and surface potential experiments on monolayers of phospholipids ranging from 14 to 22 carbons in length at the n-heptane/water interface, over a wide range of temperature, salt concentration, and pH on the acid side. The pressure data presented here are a considerable extension of previous data (1) to higher surface densities, comprehensively checked for monolayer loss, and include new data on PE molecules. We explore surface densities ranging from extremely low to intermediate, near to the main phase transition, in which range the surface pressures and potentials are found to be independent of the chain length. Thus, these data bear directly on the headgroup interactions. These interactions are observed to be independent of ionic strength. PC and PE molecules differ strongly in two respects: (a) the lateral repulsion among PC molecules is much stronger than for PE, and (b) the lateral repulsion among PC molecules increases strongly with temperature whereas PE interactions are almost independent of temperature. Similarly, the surface potential for PC is found to increase with temperature whereas for PE it does not. In this and the following paper we show that these data from dilute to semidilute monolayers are consistent with a theoretical model that predicts that, independent of coverage, for PC the P-N+ dipole is oriented slightly into the oil phase because of the hydrophobicity of the methyl groups, increasingly so with temperature, whereas for PE the P-N+ dipole is directed into the water phase.     

An apparatus for altering the mechanical load of the respiratory system

We describe an apparatus for altering the mechanical load against which the respiratory muscles operate in humans. A closed system incorporates a rolling seal spirometer. The spirometer piston shaft is coupled to a fast-responding linear actuator that develops force in proportion to desired command signals. The command signal may be flow (resistive loading or unloading), volume (elastic loading or unloading), constant voltage (continuous positive or negative pressure), or any external function. Combinations of loads can be applied. Logic circuits permit application of the load at specific times during the respiratory cycle, and the magnitude of the loads is continuously adjustable. Maximum pressure output is +/- 20 cmH2O. The apparatus permits loading or unloading over a range of ventilation extending from resting levels to those observed during high levels of exercise (over 100 l/min). In response to a square-wave input, pressure rises exponentially with a time constant of 20 ms.     

Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets

We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90-95% can be obtained for proteins in the molecular weight range of 10-90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem. 261, 4229-4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with QAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 40-80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85-95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem. 262, 10035-10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163, 517-525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrene-coated PVDF membranes, and sequenced ranged from 50-60% for beta-lactoglobin (10-50 pmol loaded onto SDS gels) to 20-30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.     

Effects of environmental parameters and irrigation on the turgor pressure of banana plants measured using the non‐invasive,online monitoring leaf patch clamp pressure probe

Turgor pressure provides a sensitive indicator for irrigation scheduling. Leaf turgor pressure of Musa acuminate was measured by using the so‐called leaf patch clamp pressure probe, i.e. by application of an external, magnetically generated and constantly retained clamp pressure to a leaf patch and determination of the attenuated output pressure P_p that is highly correlated with the turgor pressure. Real‐time recording of P_p values was made using wireless telemetric transmitters, which send the data to a receiver base station where data are logged and transferred to a GPRS modem linked to an Internet server. Probes functioned over several months under field and laboratory conditions without damage to the leaf patch. Measurements showed that the magnetic‐based probe could monitor very sensitively changes in turgor pressure induced by changes in microclimate (temperature, relative humidity, irradiation and wind) and irrigation. Irrigation effects could clearly be distinguished from environmental effects. Interestingly, oscillations in stomatal aperture, which occurred frequently below turgor pressures of 100 kPa towards noon at high transpiration or at high wind speed, were reflected in the P_p values. The period of pressure oscillations was comparable with the period of oscillations in transpiration and photosynthesis. Multiple probe readings on individual leaves and/or on several leaves over the entire height of the plants further emphasised the great impact of this non‐invasive turgor pressure sensor system for elucidating the dynamics of short‐ and long‐distance water transport in higher plants.     

Investigating the potential of Bacillus subtilis α‐amylase as a pressure‐temperature‐time indicator for high hydrostatic pressure pasteurization processes

The potential of Bacillus subtilis α‐amylase (BSA) as a pressure‐temperature‐time indicator (pTTI) for high pressure pasteurization processing (400–600 MPa; T_i 10–40°C; 1–15 min) was investigated. A stepwise approach was followed for the development of an enzyme‐based, extrinsic, isolated pTTI. First, based on literature data on the pressure stability, BSA was selected as a candidate indicator. Next to the accuracy and ease of the measurement of the indicator's response (residual activity) to the pressure treatment, the storage and handling stability of BSA at atmospheric pressure was verified. Second, the stability of BSA at a constant temperature (T) and time in function of pressure (p) was investigated. Solvent engineering was used to shift the inactivation window of BSA in the processing range of interest. Third, the enzyme (1 g/L BSA—MES 0.05 M pH 5.0) was kinetically calibrated under isobaric‐isothermal conditions. Time dependent changes in activity could be modeled best by a first‐order model. Except for low pressures and high temperatures, a synergistic effect between pressure and temperature could be observed. Based on the model selected to describe the combined p,T‐dependency of the inactivation rate constant, an elliptically shaped isorate contour plot could be constructed, illustrating the processing range where BSA can be used to demonstrate temperature gradients. Fourth, the validity of the kinetic model was tested successfully under dynamic conditions similar to those used in food industry. Finally, the indicator was found suitable to demonstrate nonuniformity in two‐sectional planes of a vertical, single vessel system. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Assessment of a non-invasive high-throughput classifier for behaviours associated with sleep and wake in mice

This work presents a non-invasive high-throughput system for automatically detecting characteristic behaviours in mice over extended periods of time, useful for phenotyping experiments. The system classifies time intervals on the order of 2 to 4 seconds as corresponding to motions consistent with either active wake or inactivity associated with sleep. A single Polyvinylidine Difluoride (PVDF) sensor on the cage floor generates signals from motion resulting in pressure. This paper develops a linear classifier based on robust features extracted from normalized power spectra and autocorrelation functions, as well as novel features from the collapsed average (autocorrelation of complex spectrum), which characterize transient and periodic properties of the signal envelope. Performance is analyzed through an experiment comparing results from direct human observation and classification of the different behaviours with an automatic classifier used in conjunction with this system. Experimental results from over 28.5 hours of data from 4 mice indicate a 94% classification rate relative to the human observations. Examples of sequential classifications (2 second increments) over transition regions between sleep and wake behaviour are also presented to demonstrate robust performance to signal variation and explain performance limitations.     

Modeling the thermal responses of the skin surface during hand-object interactions

The objective of this research is to analyze and model the decreases in skin temperature when the hand makes contact with an object at room temperature so that thermal feedback can be incorporated into haptic displays. A thermal model is proposed that predicts the thermal responses of the skin and object surface as well as the heat flux exchanged during hand-object interactions. The model was evaluated by comparing the theoretical predictions of temperature changes to those experimentally measured using an infrared thermal measurement system. The thermal measurement system was designed to overcome the limitations imposed by contact thermal sensors, and was able to measure skin temperature during contact, together with the contact area and contact force. The experimental results indicated that over the pressure range of 0.73-10.98 kPa, changes in skin temperature were well localized to the contact area and were affected by contact pressure. The pressure in turn influenced both thermal contact resistance and blood flow. Over the range of contact forces typically used in manual exploration, blood perfusion and metabolic heat generation do not appear to have a significant effect on the skin's thermal responses. The theoretical predictions and the measured data were consistent in characterizing the time course and amplitude of the skin temperature change during contact with differences typically being less than 1 degrees C between the two for pressures greater than 4 kPa. These findings indicate that the proposed thermal model is able to characterize and predict the skin temperature responses during hand-object interactions and could be used in a thermal display that simulates the properties of different materials.     

Oleic- and docosahexaenoic acid-containing phosphatidylethanolamines differentially phase separate from sphingomyelin

A central tenet of the lipid raft model is the existence of non-raft domains. In support of this view, we have established in model membranes that a phosphatidylethanolamine (PE)-containing docosahexaenoic acid (DHA) forms organizationally distinct non-raft domains in the presence of sphingomyelin (SM) and cholesterol (Chol). We have shown that formation of DHA-rich domains is driven by unfavorable molecular interactions between the rigid Chol molecule and the highly flexible DHA acyl chain. However, the molecular interactions between SM and the DHA-containing PE, which could also contribute to the formation of DHA-rich non-raft domains, have not been sufficiently investigated. To address this issue, we use differential scanning calorimetry (DSC) to study the phase behavior of mixtures of SM with either 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (16:0-22:6PE) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1PE), an oleic acid (OA)-containing control, over a wide range of concentrations. Deconvolution of binary DSC scans shows that both 16:0-22:6PE and 16:0-18:1PE phase separate from SM. Analysis of transition temperatures and partial phase diagrams, constructed from the DSC scans for the first time, shows that 16:0-22:6PE displays greater non-ideal mixing with SM compared to 16:0-18:1PE. Our findings support a model in which DHA- and OA-containing PEs differentially phase separate from SM over a wide range of molar ratios to initiate the formation of non-raft domains, which is greatly enhanced by DHA, but not OA, in the presence of cholesterol.     

Functional dissection of the PE domain responsible for translocation of PE_PGRS33 across the mycobacterial cell wall

PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE(Rv1818c)), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE(Rv1818c) domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation.     

Optimized conditions for pulsed field gel electrophoretic separations of DNA.

Quantitative measurement of DNA migration in gel electrophoresis requires precisely controlled homogeneous electric fields. A new electrophoresis system has allowed us to explore several parameters governing DNA migration during homogeneous field pulsed field gel (PFG) electrophoresis. Migration was measured at different switch times, temperatures, agarose concentrations, and voltage gradients. Conditions which increase DNA velocities permit separation over a wider size range, but reduce resolution. We have also varied the angle between the alternating electric fields. Reorientation angles between 105 degrees and 165 degrees give equivalent resolution, despite significant differences in DNA velocity. Separation of DNA fragments from 50 to greater than 7000 kilobases (Kb) can easily be optimized for speed and resolution based on conditions we describe.     

Improved antifouling properties of PVDF membranes modified with oppositely charged copolymer

Biofouling resulting from the attachment of microorganisms communities to the membrane surface is the major obstacle for the widespread application of membrane technology. This work develops a feasible approach to prepare an anti-biofouling poly(vinylidene fluoride) (PVDF) membrane. A copolymer that possessed oppositely charged groups was first synthesized via radical copolymerization with methyl methacrylate, 2-methacryloxy ethyltrimethyl ammonium chloride and 2-acrylamide-2-methyl propane sulphonic acid as monomers. The copolymer was blended with the PVDF powder to prepare the antifouling membrane via the immersed phase inversion method. The antifouling properties of the modified PVDF membrane were studied by X-ray photoelectron spectroscopy, field emission scanning electron microscopy, water contact angle measurement, zeta-potential measurement, protein adsorption, microbial adhesion and filtration experiments. The modified PVDF membrane showed limited adsorption and adhesion of protein bovine serum albumin and microbes (Escherichia coli and Saccharomyces cerevisiae) with increasing copolymer concentration in the casting solution. The modified PVDF membrane exhibited excellent antibiofouling properties.     

A mass spectrometry membrane probe and practical problems associated with its application in fermentation processes

A sensor-type, multi-compound monitoring system was constructed to measure several substances dissolved in a culture medium by use of a membrane inlet system coupled with a low-cost quadrupole mass spectrometer. The membrane inlet system was shown to have higher sensitivity with an even faster response than a capillary inlet system. The system was able to monitor on-line gaseous (H₂, CO₂ and O₂) and volatile (ethanol, butanol, acetone, acetic acid and ethyl acetate) compounds at concentrations in a range suitable for fermentation process monitoring applications. The effects of temperature, agitation speed and pressure on measurements using the membrane inlet system were investigated. For volatile compound measurements, temperature and pressure had an effect on the response but the agitation speed did not. A high concentration of compounds caused measurement saturation due to the space charge effect, but this could be overcome by reducing the surface area of the membrane. The system was applied to the monitoring of ethanol in a yeast cultivation. Measurements were affected by CO₂ produced during the fermentation, but this could be compensated for by means of a linear empirical equation. The system demonstrated high potentiality for application to the on-line monitoring of multiple compounds in fermentation processes.     

The technique of detecting systolic and diastolic pressure from the transducer output of a PC-based blood pressure monitoring system

A pressure to voltage transducer is used along with a cuff, in a PC-based blood pressure and pulse rate monitoring system for human body. During the blood pressure measurement cycle, the output voltage of the pressure to voltage transducer is recorded digitally using a data acquisition system. The recorded data are then analyzed using software routines to determine the blood pressure and pulse rate of the person under test. However, it is difficult to identify the points of systole and diastole correctly from the recorded data. This paper presents the technique that may be used to determine the systolic and diastolic pressure from the collected data.     

Modelling mechanosensitivity in membranes: effects of lateral tension on ionic pores in a microcystin toxin-containing membrane

Lipid bilayers of diphytanoyl lecithin (DPhL) in which a cyanobacterial toxin, microcystin-LR (MC-LR) was incorporated, were found to be a convenient model of natural mechanosensitive membranes. The effects of pressure difference, leading to lateral membrane tension, on artificial membranes formed on the tips of glass micropipettes were investigated using patch-clamp methodology. Emplacement of MC-LR from the bathing solution was enhanced by transmembrane voltage and/or pressure difference. MC-LR pores could be recorded over a wide voltage range, their opening probability being first increased and then reduced at high membrane potential. The pores exhibited several open pore conductance levels, the higher conductance states being more probable at greater lateral tensions. Ion gradient experiments established that the MC-LR pores are cation selective, but discriminate only weakly between K and Na. These results suggest that a lipid liquid crystal matrix containing monomers of multimeric pore-forming molecules could be used as a mechanical sensor and molecular switch. Offprint requests to: P. N. R. Usherwood