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PubMed2006

A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation.

This report describes the development, optimization, and implementation of a miniaturized cell-based assay for the identification of small-molecule insulin mimetics and potentiators. Cell-based assays are attractive formats for compound screening because they present the molecular targets in their cellular environment. A fluorescence resonance energy transfer (FRET) cell-based assay that measures the insulin-dependent colocalization of Akt2 fused with either cyan fluorescent protein or yellow fluorescent protein to the cellular membrane was developed. This ratiometric FRET assay was miniaturized into a robust, yet sensitive 3456-well nanoplate assay with Z' factors of approximately 0.6 despite a very small assay window (less than twofold full activation with insulin). The FRET assay was used for primary screening of a large compound collection for insulin-receptor agonists and potentiators. To prioritize compounds for further development, primary hits were tested in two additional assays, a biochemical time-resolved fluorescence resonance energy transfer assay to measure insulin-receptor phosphorylation and a translocation-based imaging assay. Results from the three assays were combined to yield 11 compounds as potential leads for the development of insulin mimetics or potentiators.

Active Transport, Cell Nucleus↗

PubMed2006

Amplification of fluorescence with packed beads to enhance the sensitivity of miniaturized detection in microfluidic chip.

This paper reports the pre-concentration of C-reactive protein (CRP) antigen with packed beads in a microfluidic chamber to enhance the sensitivity of the miniaturized fluorescence detection system for portable point-of-care testing devices. Although integrated optical systems in microfluidic chips have been demonstrated by many groups to replace bulky optical systems, the problem of low sensitivity is a hurdle for on-site clinical applications. Hence we integrated the pre-concentration module with miniaturized detection in microfluidic chips (MDMC) to improve analytical sensitivity. Cheap silicon-based photodiodes with optical filter were packaged in PDMS microfluidic chips and beads were packed by a frit structure for pre-concentration. The beads were coated with CRP antibodies to capture antigens and the concentrated antigens were eluted by an acid buffer. The pre-concentration amplified the fluorescence intensity by about 20-fold and the fluorescence signal was linearly proportional to the concentration of antigens. Then the CRP antigen was analyzed by competitive immunoassay with an MDMC. The experimental result demonstrated that the analytical sensitivity was enhanced up to 1.4 nM owing to the higher signal-to-noise ratio. The amplification of fluorescence by pre-concentration of bead-based immunoassay is expected to be one of the methods for portable fluorescence detection system.

Animals↗

PubMed2005

Miniaturized membrane-assisted solvent extraction combined with gas chromatography/electron-capture detection applied to the analysis of volatile organic compounds.

A new module of membrane-assisted solvent extraction (MASE) with miniaturized membrane bags was applied to the determination of seven volatile organic compounds (VOCs): chloroform, 1,1,1-trichloroethane, trichloroethylene, 1,1,2-trichloroethane, tetrachloroethene, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane with boiling points between 61 and 147 degrees C in aqueous samples. Different from the known procedure the new, shortened membrane bags were filled with 100 microl of an organic solvent. The membrane bags were placed in a 20 ml headspace vial and filled with 15 ml of the aqueous sample. The vial was transferred into an autosampler where it was stirred for a definite time at elevated temperature. After the extraction, 1 microl of the organic extract was transferred into the spilt/splitless injector of a GC system equipped with an electron-capture detector. This work included optimization of the membrane device, the determination of the optimized extraction conditions such as stirring rate, extraction time and the impact of salt addition. The validation of the method involved repeatability, recovery and detection limit studies, followed of its application towards real water samples. The repeatability, expressed as the relative standard deviation of the peak areas of six extractions was below 10%. The detection limits (LODs) were between 5 ng/l (tetrachloroethene) and 50 ng/l (chloroform). Calibration was performed in a range from 5 ng/l to 150 microg/l, since the concentration in the aqueous samples was expected quite various in this concentration range. Five river water samples of Bitterfeld, Saxony-Anhalt, Germany were analyzed with miniaturized-MASE and the results were compared with those obtained with Headspace-Analysis. The method can be fully automated and moreover, it allows the simultaneous determination of volatile and semi volatile compounds.

Chromatography, Gas↗

PubMed2004

Binge ethanol exposure delays development of GABAergic miniature postsynaptic currents in septal neurons.

Whole cell GABA(A)R currents of septal neurons isolated from rat pups increase rapidly during the first weeks of life when inhibitory synapses are forming. Early postnatal binge ethanol intubation on days 4-9 delays this maturational up-regulation in septal neurons isolated several days later suggesting inhibitory synapse formation could be disrupted [S.-H. Hsiao, J.L. Acevedo, D.W. DuBois, K.R. Smith, J.R. West, G.D. Frye, Early postnatal ethanol intubation blunts GABA(A) receptor upregulation and modifies 3alpha-hydroxy-5alpha-pregnan-20-one sensitivity in rat MS/DB neurons, Brain Res. Dev. Brain Res. 130 (2001) 25-40]. Surprisingly, whole cell GABA(A)R function does not increase rapidly when septal neurons are grown for the same period in vitro and is not blunted by comparable ethanol exposure of the cultures [S.-H. Hsiao, D.W. DuBois, R.C. Miranda, G.D. Frye, Critically timed ethanol exposure reduces GABA(A)R function on septal neurons developing in vivo but not in vitro, Brain Res Dev. Brain Res. 1008 (2004) 69-80]. Because GABAergic miniature postsynaptic currents (mPSCs) show parallel patterns of maturation whether cortical neurons are growing in vivo or in vitro [D.D. Dunning, C.L. Hoover, I. Soltesz, M.A. Smith, D.K. ODowd, GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons, J. Neurophysiol. 82 (1999) 3286-3297], we examined the impact of binge ethanol exposure on synaptic receptors activated by these currents in septal cultures. Binge ethanol treatment of embryonic septal neurons over 6-11 days in vitro (DIV) slightly reduced GABA(A)R-mediated mPSC amplitude and frequency, but also substantially slowed decay kinetics when mPSCs were recorded later on DIV 13-18. Decreased frequency and slowed mPSC decay kinetics after ethanol were consistent with parameters measured in immature neurons. Untreated septal neurons exhibited decreased mPSC amplitude and frequency with acute 30-100 mM ethanol, without changing decay kinetics suggesting a direct inhibition of postsynaptic receptors. Sustained inhibition of GABA(A)Rs with 100 microM picrotoxin on DIV 6-11 decreased mPSC amplitude and frequency and slowed decay kinetics similar to binge ethanol exposure. These results suggest that binge ethanol exposure delays mPSC maturation by interfering with trophic postsynaptic GABA(A)R signaling during the early development of septal neurons.

Alcohol-Induced Disorders, Nervous System↗

PubMed2006

Two types of GABAergic miniature inhibitory postsynaptic currents in mouse substantia gelatinosa neurons.

The physiological and pharmacological properties of gamma-aminobutyric acid (GABA)ergic miniature inhibitory postsynaptic currents (mIPSCs) were investigated in substantia gelatinosa neurons of mouse spinal cord using whole-cell patch clamp recordings. Two cell populations were pharmacologically identified based on the effect of propofol (10 muM) on the mIPSC decay kinetics: those exhibiting propofol-sensitive mIPSCs, with a slow decay kinetic (mIPSC(SLOW)), and those exhibiting propofol-resistant mIPSCs, with a fast decay kinetic (mIPSC(FAST)) (decay time constants of 14.2+/-0.7 and 7.4+/-0.8 ms, respectively). The frequency and amplitude of both types of mIPSCs were not affected by propofol. Miniature IPSC(FAST) showed midazolam insensitivity, while midazolam prolonged the decay phase of mIPSC(SLOW) without modulation of the frequency and amplitude. Exogenous GABA-evoked responses in the neurons with mIPSC(SLOW) were potentiated by propofol, while those in neurons with mIPSC(FAST) were unaffected by propofol. Furthermore, non-stationary noise analysis of the two kinetically and pharmacologically distinct mIPSCs revealed different conductance of GABA(A) receptor channels underlying the synaptic events. Pharmacological responses to propofol and midazolam suggested that mIPSC(FAST) and mIPSC(SLOW) in substantia gelatinosa neurons can be mediated by GABA(A) receptors with different subunit compositions.

Animals↗

PubMed2004

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry.

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.

Animals↗

PubMed2004

Chaotic dynamics of a behavior-based miniature mobile robot: effects of environment and control structure.

To study the regularity and complexity of autonomous behavior, the flow of sensory information obtained in autonomous mobile robots under various conditions was analyzed as a complex system. Sensory information time series Xn was collected from a miniature mobile robot during free navigation, and plotted on the return map, the graph of X(n+tau) vs. Xn. The plot exhibited a characteristic trajectory, representing the regularity of the time series. Correlation integral and Lyapunov exponent analysis also showed properties of deterministic chaos; the presence of fractal dimension and positive Lyapunov exponent. Analysis of sensory information obtained in the robot with three different neural controllers revealed that the autonomous robot behaves in such a way that the flow of sensory information is governed by a deterministic rule, and this pattern is unique to each controller. Furthermore, the analysis in various environments exhibited that transitions from one trajectory to another on the return map occur during the course of autonomous behavior. The fractal and Lyapunov dimensions calculated in various conditions indicate that these dimension could be utilized to quantify the complexity of autonomous behavior and the relative difficulty of tasks. Analyses at different evolutionary stage revealed that behavioral performance correlates with fractal dimension. These studies using a miniature mobile robot that allowed to idealize the experimental conditions demonstrated firmly that the complex analysis could be utilized in evaluation and optimization of autonomous systems and the behavior.

Behavior↗

PubMed2003

Reduction of glycine receptor-mediated miniature inhibitory postsynaptic currents in rat spinal lamina I neurons after peripheral inflammation.

Peripheral inflammation may induce long-lasting sensitization in the central nociceptive system. Neurons in lamina I of the spinal dorsal horn play a pivotal role in the integration and relay of pain-related information. In rats we studied whether changes in passive and active membrane properties and/or alteration of glycine receptor-mediated inhibitory control of spinal lamina I neurons may contribute to central sensitization in a model of peripheral long-lasting inflammation (complete Freund's adjuvant, hindpaw). Spontaneously occurring glycine receptor-mediated miniature inhibitory postsynaptic currents (GlyR-mediated mIPSCs) were recorded in lumbar spinal lamina I neurons. Miniature IPSC rise, decay kinetics and mean GlyR-mediated mIPSC amplitude were not affected by peripheral inflammation. The mean frequency of GlyR-mediated mIPSCs of lamina I neurons ipsilateral to the inflamed hindpaw was, however, significantly reduced by peripheral inflammation when compared with neurons from noninflamed animals. Principal passive and active membrane properties and firing patterns of spinal lamina I neurons were not changed by inflammation. These results indicate that long-lasting peripheral inflammation leads to a reduced glycinergic inhibitory control of spinal lamina I neurons by a presynaptic mechanism.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2006

Miniature piezoelectric conical transducer: fabrication, evaluation and application.

This paper reports a new type miniature-conical transducer for acoustic emission measurements. The transducer follows the basic idea and structure of a conventional NBS conical transducer, but is much compact in size and easier to use. The improvements are made possible by introducing an excimer laser micromachining method for fabricating smaller PZT conical elements, which play a key role in the transducer. Conical PZT elements with contact size less then 300 microm are laser-machined and the miniature-conical transducers are constructed. Standard quantitative acoustic emission testing is performed on a plate using the fabricated transducers and good results are observed. The transducers can be very useful in many applications involving quantitative measurements of transient elastic waves.

Journal Article↗

PubMed2000

Mixed hematopoietic chimerism induces long-term tolerance to cardiac allografts in miniature swine.

BACKGROUND: Tolerance to cardiac allografts has not been achieved in large animals using methods that are readily applicable to human recipients. We investigated the effects of mixed hematopoietic chimerism on cardiac allograft survival and chronic rejection in miniature swine METHODS: Recipients were T-cell depleted using a porcine CD3 immunotoxin, and each received either of two nonmyeloablative preparative regimens previously demonstrated to permit the establishment of stable mixed hematopoietic chimerism across MHC-matched, minor antigen-mismatched histocompatibility barriers. Five to 12 months after the chimerism was induced, hearts from the original cell donors were heterotopically transplanted into the stable mixed chimeras. RESULTS: Cardiac allografts transplanted into untreated recipients across similar minor antigen barriers were rejected within 44 days (within 21, 28, 35, 39, 44 days among individual study subjects). In contrast, hearts transplanted into the mixed chimeras were all accepted long term ( > 153, > 225, > 286, > 362 days) without immunosuppressive drugs and developed minimal vasculopathy. CONCLUSIONS: Mixed hematopoietic chimerism, established in miniature swine using clinically relevant, non-myeloablative conditioning regimens, permits long-term cardiac allograft survival without chronic immunosuppressive therapy, significant vasculopathy, or graft-versus-host disease.

Animals↗

PubMed1997

Vibrational spectra of individual millimeter-size membrane patches using miniature infrared waveguides.

We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells.

Animals↗

PubMed1999

Anisotropic mechanical properties of lamellar bone using miniature cantilever bending specimens.

The flexural modulus and work-to-fracture properties of circumferential lamellar bone from baboon tibia are presented, based on experiments with miniature cantilever bending specimens. Data is provided for specimens in three orthogonal directions that show marked anisotropy. The advantages of such miniature specimens (about 150 microm in diameter and 2 mm long) include the possibility of sampling very small volumes within a heterogeneous structure such as osteonal bone, or studying biological materials that are not available in large enough volumes for conventional mechanical analysis.

Animals↗

PubMed2000

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations.

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.

Chromatography, Liquid↗

PubMed2002

Sensitivity of a miniaturized touch probe sensor using PZT thin film vibrator.

In this paper, an improved touch probe sensor device for higher sensitivity and low contact force is reported. In order to improve the resolution, we have evaluated the sensitivity and fabricated a miniaturized sensor. The sensor transducer was 3 mm long and had higher resonance frequency. The resonance frequency of the vibrator was 937 kHz. Evaluated sensitivity was 1.0 x 10(-1) mV/nm. This value equals five times larger than that of a previous sensor. Miniaturization of the sensor device carried smaller vibration operation and higher sensitivity.

Journal Article↗

PubMed2000

Optimum contact conditions for miniaturized surface acoustic wave linear motor

This paper reports the successful operation of a 70 MHz driving surface acoustic wave (SAW) linear motor with a miniaturized stator transducer. This paper also deals with an investigation into an optimized slider design for the miniaturized SAW linear motor. The performance of three silicon type sliders, with different projection size, was compared. Output forces of the three sliders were measured with change of pre-load. It was found that the slider with smaller projection tended to produce greater output force.

Journal Article↗

PubMed1997

Reproductive measurements in Sinclair and NIH miniature pigs: a retrospective analysis.

The data presented here represent a retrospective analysis of information gathered while collecting data for other studies on miniature pigs. Two different breeds of miniature pigs, NIH and Sinclair, were used in this study. The NIH females were gilts, while Sinclair females included both gilts and sows. The pigs were checked twice a day for estrus and were mated at 12 and 24 h after the onset of estrus. One- and 2-cell stage embryos were collected on Day 2; while 4-cell, 8-cell, compact morula and blastocyst stage embryos were collected on Days 2.7, 3.5, 4.3 and 6.0, respectively. The percentage of recovery of these embryos was dependent upon the surgeon (P = 0.002) and the stage of development (P = 0.018). The number of ovulations was higher (P < 0.04) in the Sinclair sows (10.4 +/- 0.60) than in the Sinclair gilts (8.9 +/- 0.67) and in the NIH gilts (8.3 +/- 0.67). When the NIH gilts were divided into swine leukocyte antigen (SLA) haplotypes, it was found that SLA(dd) gilts (8.5 +/- 0.43) had more ovulations (P = 0.02) than SLA(ad) gilts (6.8 +/- 0.57). Some animals were treated with Regumate to synchronize estrus. The Sinclair gilts (7.8 +/- 0.28) and NIH gilts (7.7 +/- 0.27) took more days (P < 0.07) to show estrus than the Sinclair sows (6.3 +/- 0.58) after the removal of Regumate. Four of the animals had reproductive tract abnormalities; more specifically, a blind uterine horn or oviduct that was not patent with the other horn. All 4 were NIH gilts with the SLA(dd) haplotype.

Journal Article↗

PubMed1987

Phenotypic differentiation of members of the family Vibrionaceae using miniaturized biochemical tests.

Enzymatic hydrolysis of 27 different chromogenic substrates and the assimilation of 44 carbon sources by 144 strains of Vibrio species of clinical importance, Aeromonas hydrophila and Plesiomonas shigelloides were studied by standardized micromethods. Some classical biochemical tests were also performed using the test kit TTE-AS (Flow Laboratories GmbH, Meckenheim, FRG). Reading of results was done automatically by a photometer and test data were recorded and stored by a microcomputer. All species investigated could be differentiated using a set of 16 miniaturized biochemical tests which are: Indole production, esculin hydrolysis, lysine decarboxylase, ornithine decarboxylase, arginine dihydrolase, fermentation of sucrose, enzymatic hydrolysis of o-nitrophenyl-beta-D-galactopyranoside, gamma-L-glutamic acid-p-nitroanilide and the assimilation of L-arabinose, D-cellobiose, D-mannose, sucrose, D-mannitol, i-inositol, acetate and DL-lactate. Comparing the TTE-AS tests to conventional test results, 94.4% overall agreement was found. 87.6% of the miniaturized assimilation tests agreed to literature data. The described tests are easy to perform and seem to be suitable for routine laboratory use.

Aeromonas↗

PubMed1991

Confirmation of endotracheal tube placement: a miniaturized infrared qualitative CO2 detector.

STUDY OBJECTIVES: A miniaturized, infrared, solid-state, end-tidal CO2 detector was used to confirm emergency endotracheal tube (ETT) placement. DESIGN: This prospective, clinical study used a miniature, infrared, solid-state end-tidal CO2 detector to confirm ETT placement in an acute setting. SETTING: The ICU, emergency department, and hospital floor. TYPE OF PARTICIPANTS: There were 88 consecutive adult patients requiring 100 emergency intubations. MEASUREMENTS AND MAIN RESULTS: The indication for airway intervention was considered urgent in 79% and under arrest conditions in 21%. The mean number of intubation attempts was 1.83 (range, one to five) with difficulty of intubation of 6.48 and confirmation of 7.75, on a linear scale from 0 (lowest) to 10 (highest). Determination of ETT position revealed intratracheal intubation in 96% and esophageal intubation in 4%. Placement was confirmed by direct visualization or radiography in all cases. Sensitivity and specificity for ETT localization was 100% (P less than .0001). CONCLUSION: This hand-held infrared capnometer reliably confirms ETT placement under emergency conditions.

Carbon Dioxide↗