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PubMed2003

Miniaturization of fluorescence polarization receptor-binding assays using CyDye-labeled ligands.

Fluorescence polarization (FP) is an established technique for the study of biological interactions and is frequently used in the high-throughput screening (HTS) of potential new drug targets. This work describes the miniaturization of FP receptor assays to 1536-well formats for use in HTS. The FP assays were initially developed in 384-well microplates using CyDye-labeled nonpeptide and peptide ligands. Receptor expression levels varied from approximately 1 to 10 pmols receptor per mg protein, and ligand concentrations were in the 0.5- to 1.0-nM range. The FP assays were successfully miniaturized to 1536-well formats using Cy3B-labeled ligands, significantly reducing reagent consumption, particularly the receptor source, without compromising assay reliability. Z' factor values determined for the FP receptor assays in both 384- and 1536-well formats were found to be > 0.5, indicating the assays to be robust, reliable, and suitable for HTS purposes.

Animals↗

PubMed2004

A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation.

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.

Animals↗

PubMed1990

Oral insulin increases small intestinal mass and disaccharidase activity in the newborn miniature pig.

Factors, such as insulin, found in human and pig colostrum and mature milk likely influence small intestinal growth and development. Although pharmacologic doses of insulin injected parenterally may accelerate small intestinal development in altricial animals such as the rodent, the effects of oral insulin on intestinal development have not been studied. In the first of two studies, we randomized 2-d-old miniature piglets to receive bottle-feedings of a swine weaning milk formula with (group F + I) or without (group F) the addition of insulin. Serum glucose, insulin, and cortisol were measured before and 1 h after the first feeding the piglets received at our facility. In the second study, piglets were randomized (groups F and F + I) and fed for 6 d, after which blood samples were obtained as in the first experiment. The piglets were then killed and the small intestine removed for analysis. We found no differences between groups in serum glucose, insulin, and cortisol at both 2 and 8 d of age, both before and after feeding. In the second experiment, small intestinal weight was greater in the F + I than in the F group. Although no differences were noted between groups in the jejunum, values were greater for group F + I versus group F for ileal mucosal weight, protein, RNA, lactase, and maltase activities. No differences were found between groups in ileal DNA or sucrase activity. We conclude that the administration of oral insulin stimulated an increase in ileal mass and disaccharidase activity in the newborn miniature pig without apparent concomitant changes in serum glucose, insulin, or cortisol.

Administration, Oral↗

PubMed2006

Normothermic beating heart surgery with assistance of miniaturized bypass systems: the effects on intraoperative hemodynamics and inflammatory response.

The use of miniaturized cardiopulmonary bypass (CPB) circuits and avoidance of cardioplegic arrest are attempts to reduce the inflammatory response to cardiac surgery. We studied the effects of beating heart surgery (BHS) with assistance of simplified bypass systems (SBS) on global hemodynamics, myocardial function and the inflammatory response to CPB. We hypothesized that the use of SBS was associated with less hemodynamic instability after CPB resulting from attenuation of the inflammatory response when compared with surgery performed with a conventional CPB (cCPB) circuit. Forty-five patients undergoing coronary artery bypass grafting were prospectively studied. Fifteen patients were randomized to the use of a cCPB circuit, cold crystalloid cardioplegia, and moderate hypothermia. Two groups of 15 patients underwent BHS during normothermia with assistance of two different SBS consisting of only blood pump and oxygenator. Hemodynamic variables were assessed with transpulmonary thermodilution and transesophageal echocardiography. Plasma levels of proinflammatory and antiinflammatory mediators were measured perioperatively. After CPB, variables of global hemodynamics and systolic ventricular function did not differ among groups. Left ventricular diastolic function was impaired after CPB equally in all groups (P < 0.01 versus pre-CPB). At the end of surgery, there was more need for vasopressor (norepinephrine) support in both SBS groups than in the cCPB group (P < 0.01). After CPB, the release of interleukin (IL)-6 did not differ significantly among groups, whereas plasma levels of IL-10 were higher in the cCPB group (P < 0.01 versus SBS). The extent of myocardial necrosis (Troponin T) was comparable in all groups. We conclude that in our study, miniaturizing bypass systems and avoidance of cardioplegic arrest were not effective in improving hemodynamic performance and in attenuating the proinflammatory immune response after CPB.

Anesthesia↗

PubMed2006

Bone-mounted miniature robotic guidance for pedicle screw and translaminar facet screw placement: Part I--Technical development and a test case result.

OBJECTIVE: To introduce a new miniature robot (SpineAssist; MAZOR Surgical Technologies, Caesarea, Israel) that has been developed and tested as a surgical assistant for accurate percutaneous placement of pedicle screws and translaminar facet screws. METHODS: Virtual projections in three planes-axial, lateral, and anteroposterior-are reconstructed for each vertebra from a preoperative computed tomographic (CT) scan. On a specially designed graphic user interface with proprietary software, the surgeon plans the trajectory of the screws. Intraoperative fluoroscopic x-rays with targeting devices are then matched with the CT-based virtual images, as well as the surgeon's plan. A clamp is attached to the spinous process or a minimally invasive frame (Hover-T frame; MAZOR Surgical Technologies) is mounted to the iliac crest and one spinous process. The miniature robot is then attached to the clamp and/or frame. On the basis of combined CT scan and fluoroscopic data, the robot aligns itself to the desired entry point and trajectory, as dictated by the surgeon's preoperative plan. RESULTS: A test case in a cadaver lumbar spine was performed in which four screws and two rods were inserted, using a minimally invasive technique, combining the SpineAssist system and Hover-T frame in conjunction with the PathFinder system (Spinal Concept Inc., Austin, TX). The discrepancy between the planned and actual screw trajectories was measured by means of postprocedural CT scan. Overall, the four screws were implanted with an average deviation of 1.02 +/- 0.56 mm (range, 0-1.5 mm) from the surgeon's plan. CONCLUSION: These preliminary results confirm the system's accuracy and support its use in minimally invasive spine surgery applications.

Bone Screws↗

PubMed2005

Miniaturization (0.2 g) and evaluation of attachment techniques of telemetry transmitters.

We have developed a miniaturized very high frequency (VHF) transmitter design for radio-tagging small animals. The average mass of the circuitry is 0.084 g (range 0.081-0.087), hence, with the smallest power cell, complete tags weigh 0.2 g and have a life of 18-22 days. We further demonstrate that with such small tags the technique of attaching the device to the animal's body strongly affects the effective radiated power, and thus transmission ranges. Ideally the attachment couples the electronics to the animal's body as a ground plane for improved radiation. The transmitter allows the application of radio-tracking to be expanded to new taxa whose spatial behaviour and population dynamics are largely unknown, for example arthropods, reptiles or amphibians. The new design is also suitable for miniature implants and signal modulation with sensors.

Electronics↗

PubMed1987

Prostanoids, coronary circulation and coronary artery spasm in open chest dogs and miniature pigs.

Effects of prostanoids on coronary circulation were studied in anesthetized open chest dogs with intact coronary arteries or in closed chest Göttingen miniature pigs with denuded vessels. In the latter model, coronary artery spasm was repeatedly provoked at the previously denuded area by histamine i.c. after pretreatment with cimetidine i.v., H2-blocker. In the canine model, prostacyclin (PGI2) dilated to a greater extent the diameter of the large epicardial coronary artery than did PGE2, and both equally reduced the total coronary resistance. Vasoconstrictive effects of carbocyclic thromboxane A2, a stable analogue of thromboxane A2 (TxA2) on the epicardial coronary artery was augmented from 1.1 +/- 0.3 to 6.5 +/- 1.3% (p less than 0.01) after pretreatment with cyclooxygenase inhibitors. In case of Göttingen miniature pigs 3 months after endothelial denudation and cholesterol freeding, neither PGI2 nor indomethacin prevented histamine-induced coronary artery spasm. Thiothromboxane A2, a stable analogue of TxA2, did not provoke coronary artery spasm in the animals. Therefore, prostanoids may not be crucial to the provocation or prevention of coronary artery spasm from which myocardial ischemia ensues.

Animals↗

PubMed1991

Regression of diet-induced atherosclerosis in Göttingen miniature swine.

The regression of diet-induced atherosclerosis in Göttingen Miniature Swine was investigated after a 6-month induction period. At 1 month after feeding a high-cholesterol and high-fat diet, levels of beta-lipoprotein, total cholesterol, free fatty acids and phospholipid had increased rapidly and the high levels were maintained throughout the 6 month induction period. Morphological features at 6 months showed fatty streaks in the thoracic aorta and fibrous plaques in the abdominal aorta. After return to the conventional diet at 6 months, serum lipids decreased rapidly and maintained the baseline level throughout the 9 month regression period. Histopathological findings showed the regression of fatty streaks but the fibrous plaques did not regress. The present study therefore confirms the regression of fatty streaks in the aorta of Göttingen Miniature Swine by the administration of a cholesterol lowering diet.

Animals↗

PubMed2004

Improved isolation and culture of embryonic stem cells from Chinese miniature pig.

Pigs serve as a better research model for human beings than other species. The Chinese laboratory miniature pig is a new laboratory animal and is expected to be applicable in many medical research fields. This study was to establish effective technologies to isolate and culture ES cells in Chinese miniature pigs. For isolation of the inner cell mass from blastocysts, an enzyme-digestive method was compared with the traditional immunosurgery. Isolated ICM were cultured in three feeder cell layers: mouse embryonic fibroblasts (MEF), porcine embryonic fibroblasts (PEF) and a continuous cell line of mouse embryonic fibroblasts (STO). Microtubule activity of the three feeder cells was further examined by immunofluorescence. ICM were successfully isolated from 85% of blastocysts by the enzyme-digestive method, compared to only 40% by immunosurgery. When ICM were cultured in three feeder layers for two to three days, 75%, 65% and 20% of ICMs formed primary cell colonies in MEF, PEF and STO, respectively. Colonies were also formed during subcultures after 9, 5 and 1 passage in MEF, PEF and STO, respectively. Microtubules in STO cells were significantly fewer than those in MEF and PEF. When the ES-like cells were cultured in a differentiation medium, they differentiated to neuron-like cells and other types of cells. These results indicate that healthier ICM can be obtained with the enzyme-digestive method. Successful culture of ICM to ES-like cells has been achieved not only in MEF, but also in homologous (pig) feeder layer. The ES cells obtained in the present study were pluripotent.

Alkaline Phosphatase↗

PubMed1991

Early in vitro detection of interleukin-2-like activities by pretreatment with allogeneic blood lymphocytes in miniature swine.

In order to determine the means of monitoring the immunological status of allograft recipients in miniature swine, an assay was developed to measure interleukin (IL)-2 production in vitro by pretreatment of donor peripheral blood lymphocytes (PBL). Miniature swine were given 0 to 4 weekly intravenous transfusions of 5-10 X 10(7) donor PBL incompatible at major histocompatibility complex (MHC) and assayed in vitro for donor specific immune IL-2-like activities. The results are summarized as follows: (1) IL-2-like activity in 24 hr and 48 hr supernatants from mixed lymphocyte cultures (MLC) with MHC-incompatible PBL was detected without pretreatment. The 48 hr MLC supernatant exhibited a high IL-2-like activity compared with the 24 hr; (2) IL-2-like activity after only one transfusion with MHC-incompatible PBL was higher than that without pretreatment; (3) IL-2-like activity in 4 weekly transfusions was detectable slightly earlier than that without pretreatment or three transfusions with MHC-incompatible PBL.

Animals↗

PubMed2005

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1.

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.

Biotinylation↗

PubMed2003

Miniaturized digital holography sensor for distal three-dimensional endoscopy.

A miniaturized sensor head for endoscopic measurements based on digital holography is described. The system was developed to measure the shape and the three-dimensional deformation of objects located at places to which there is no access by common measurement systems. A miniaturized optical sensor, including a complete digital holographic interferometer with a CCD camera, is placed at the end of a flexible endoscope. The diameter of the head is smaller than 10 mm. The system enables interferometric measurements to be made at speeds of as many as five reconstructions per second, and it can be used outside the laboratory under normal environmental conditions. Shape measurements are performed with two wavelengths for contouring, and the deformation is measured by digital holographic interferometry. To obtain full three-dimensional data in displacement measurements we illuminate the object sequentially from three different illumination directions. To increase the lateral resolution we use temporal phase shifting.

Endoscopes↗

PubMed2004

Design and demonstration of a miniature catheter for a confocal microendoscope.

The fluorescence confocal microendoscope provides high-resolution, in vivo imaging of cellular pathology during optical biopsy. The confocal microendoscope employs a flexible fiber-optic catheter coupled to a custom-built slit-scan confocal microscope. The catheter consists of a fiber-optic imaging bundle linked to a miniature objective and focus assembly. The 3-mm-diameter catheter may be used on its own or routed though the instrument channel of a commercial endoscope, adding microscopic imaging capability to conventional endoscopy. The design and performance of the miniature objective and focus assembly are discussed. Primary applications of the system include diagnosis of disease in the gastrointestinal tract and female reproductive system.

Catheterization↗

PubMed2004

Miniaturization of holographic Fourier-transform spectrometers.

Wave propagation equations in the stationary-phase approximation have been used to identify the theoretical bounds of a miniature holographic Fourier-transform spectrometer (HFTS). It is demonstrated that the HFTS throughput can be larger than for a scanning Fourier-transform spectrometer. Given room- or a higher-temperature constraint, a small HFTS has the potential to outperform a small multichannel dispersive spectrograph with the same resolving power because of the size dependence of the signal-to-noise ratio. These predictions are used to analyze the performance of a miniature HFTS made from simple optical components covering a broad spectral range from the UV to the near IR. The importance of specific primary aberrations in limiting the HFTS performance has been both identified and verified.

Algorithms↗

PubMed2006

Design, assembly, and optical bench testing of a high-numerical-aperture miniature injection-molded objective for fiber-optic confocal reflectance microscopy.

The design, analysis, assembly methods, and optical-bench test results for a miniature injection-molded plastic objective lens used in a fiber-optic confocal reflectance microscope are presented. The five-lens plastic objective was tested as a stand-alone optical system before its integration into a confocal microscope for in vivo imaging of cells and tissue. Changing the spacing and rotation of the individual optical elements can compensate for fabrication inaccuracies and improve performance. The system performance of the miniature objective lens is measured by use of an industry-accepted slanted-edge modulation transfer function (MTF) metric. An estimated Strehl ratio of 0.61 and a MTF value of 0.66 at the fiber-optic bundle Nyquist frequency have been obtained. The optical bench testing system is configured to permit interactive optical alignment during testing to optimize performance. These results are part of an effort to demonstrate the manufacturability of low-cost, high-performance biomedical optics for high-resolution in vivo imaging. Disposable endoscopic microscope objectives could help in vivo confocal microscopy technology mature to permit wide-scale clinical screening and detection of early cancers and precancerous lesions.

Computer-Aided Design↗

PubMed1994

High resolution miniature trans-esophageal echocardiography is useful to evaluate atherosclerosis and the effects of alacepril in hyper-cholesterol rabbits.

This study tested the efficacy of high resolution miniature transesophageal echocardiography (m-TEE; 3.5 mm-diameter probe, 7.5 MHz, axial resolution of 0.25 mm) in the evaluation of atherosclerotic lesions of the thoracic aorta rabbits exposed to a high cholesterol diet. The effects of the ACE inhibitor Alacepril (90 mg/kg) on atherosclerosis were also assessed in normotensive, high cholesterol-diet rabbits by m-TEE, both in situ and in vitro. Oral treatment with Alacepril was begun on confirmation of atherosclerosis after a three month high cholesterol diet (group AL). Ultrasound examination reliably distinguished the lesions of atherosclerosis in situ. A three month course of Alacepril treatment decreased the systolic and diastolic blood pressures by approximately 20 and 5 mmHg, produced a regression of atherosclerosis and significantly decreased the intimal plus medial thickness compared with hyperlipidemic-controls (group HC) (1.0 + 0.3 vs 1.6 + 0.4 mm, p < 0.02). The intimal surface involvement area in group AL was marginally smaller than in group HC (57 + 23 vs 76 + 23%, p = 0.06). The high dose of Alacepril in this study did not affect the serum levels of t-cholesterol, HDL-C, LDL-C, triglycerides, or apoproteins A-I and B. These results suggest that Alacepril can produce regression of atherosclerosis, without changing circulating lipoprotein or apoprotein levels. Further miniature TEE offers high repeatability and is useful both in situ and in vitro for determining changes in arterial wall thickness and the severity of atherosclerosis.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

The hemodynamic effects of acute myocardial ischemia and reperfusion in Clawn miniature pigs.

Acute myocardial ischemia was induced by occluding the LAD in Clawn miniature pigs. Eight pigs (group 1) were subjected to 6 h ischemia and nine pigs (group 2) were subjected to 20 min ischemia, followed by reperfusion for 340 min. Three animals of the group 1 died due to ventricular fibrillation after occlusion and in group 2, four animals died due to the arrhythmia after reperfusion. Though the ischemic area of group 2 (15.6% of the ventricle) was narrower than that of group 1 (21.7%), the survival rate was lower. We supposed that ischemia-reperfusion injuries were strongly connected with the hemodynamics of group 2. Clawn miniature pigs are useful experimental animals for myocardial ischemic researches.

Animals↗

PubMed1986

[Histological study on the arterial wall of Göttingen miniature swine].

In order to clarify the histological aspects of arterial walls of miniature swine, 41 arterial segments removed from each of 17 Göttingen miniature swines/csk at 6-12 months after birth were examined by light microscopy. Every segment was classified into three types--elastic, transitional, and muscular--according to its histological architecture. Each type was identified on the basis of relative volume and disposition of elastic tissues, collagenous fibers, and smooth muscles in three coats, Tunica intima, Tunica media, Tunica externa. The distribution of each type was demonstrated in a transition from the elastic to the muscular type. The transitional type was designated as a representative type of artery in transitional regions between elastic and muscular types and had some characteristic structures like a mixture of both of them. This type also had longer regions in the back district with the heart as the starting point, such as the Aorta abdominalis, than that of the front. Every artery belonging to the three types had some different structures at different levels, respectively. It was supposed that the histological arrangement of tissues in the walls of a vessel from various parts of the arterial tree would respond properly to the functional demand.

Animals↗