Measuring electrical impedance of organs--instrumental equipment for research and clinical use

An apparatus for measuring the impedance of intact biological organs or parts of organs in the frequency range of 10 Hz to 10 MHz is described. In this range impedance exhibits a large dispersion, which is dependent on tissue structures. The time course of alterations of electrical impedance such as occur during ischemia can be recorded with this equipment. Five specimens in five measuring chambers can be examined simultaneously at different temperatures. In the second part of the article, a portable impedance meter for measuring the modulus of impedance near 200 Hz, the phase of impedance at 5 kHz and the local temperature at the measuring point, is described. These parameters permit an intra-operative evaluation of the changing state of ischemic organs. Sterilizable probes with four surface electrodes and an integrated temperature sensor permit atraumatic measurements at the organ surface. The measurement itself is harmless to the tissue.     

On-line evaluation of ultrasonic integrated backscatter

Although it is already known that reflected ultrasonic signals (backscatter) are changed by the structure of the tissue through which they pass, clinicians are still awaiting a practical instrument in which information from backscatter reflections will serve as a diagnostic aid additional to that provided by conventional ultrasonic scans.The equipment described here is both small and fast, and is integrated into a normal ultrasound installation. No new operating procedures have to be learned. The integrated backscatter is calculated on-line and presented on an LED as tissue characterization parameters. In order to minimize noise due to physical movement of the heart during an investigation of the myocardium, the analysis is synchronized with the ECG; and as an aid to the user, the normal system VDU displays both the ECG and the activating trigger pulse derived from the R-wave peak. An A-scan display has been used but this could readily be adapted for B-scan operation and single line analysis.Tests with backscattering models and standard instrumentation have shown no significant difference between results using time domain or frequency domain analysis.     

A convenient apparatus and tracking dye for anaerobic analytical polyacrylamide-gel electrophoresis

An anaerobic modification of conventional polyscrylamide-gel electrophoretic equipment is described. The modified apparatus has been applied to the separation of Azotobacter vinelandii nitrogenase components and should prove useful in the analysis of other O₂-sensitive proteins. Electrophoresis in reducing gels can be followed with a dithionite-resistant tracking dye, potassium gualazulene-1-sulfonate.     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA? kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

DE-3L型妇产科电脑综合治疗仪用于产后的效果观察

目的探讨DE-3L型妇产科电脑综合治疗仪应用于产后泌乳与机体康复效果。方法将我院收治的231例顺产妇依据产后康复方式差异进行分组,治疗组采用DE-3L型妇产科电脑综合治疗仪治疗,对照组产后进行母乳喂养宣教联合盆底肌训练,比较两组产后泌乳、子宫复旧与恶露量及盆底组织康复情况。结果治疗组促泌乳、子宫复旧与恶露量减少及盆底肌肉肌力恢复情况均明显优于对照组(P0.05)。结论 DE-3L型妇产科电脑综合治疗仪可有效促进产妇产后泌乳、恶露排出与盆底组织的康复,值得临床应用。     

High throughput preparation of fly genomic DNA in 96-well format using a paint-shaker

Sample homogenization is an essential step for genomic DNA extraction, with multiple downstream applications in Molecular Biology. Genotyping hundreds or thousands of samples requires an automation of this homogenization step, and high throughput homogenizer equipment currently costs 7000 euros or more. We present an apparatus for homogenization of individual Drosophila adult flies in 96-well micro-titer dishes, which was built from a small portable paint-shaker (F5 portable paint-shaker, Ushake). Single flies are disrupted in each well that contains extraction buffer and a 4-mm metal ball. Our apparatus can hold up to five 96-well micro-titer plates. Construction of the homogenizer apparatus takes about 3–4 days, and all equipment can be obtained from a home improvement store. The total material cost is approximately 700 euros including the paint-shaker. We tested the performance of our apparatus using the ZR-96 Quick-gDNA™ kit (Zymo Research) homogenization buffer and achieved nearly complete tissue homogenization after 15 minutes of shaking. PCR tests did not detect any cross contamination between samples of neighboring wells. We obtained on average 138 ng of genomic DNA per fly, and DNA quality was adequate for standard PCR applications. In principle, our tissue homogenizer can be used for isolation of DNA suitable for library production and high throughput genotyping by Multiplexed Shotgun Genotyping (MSG), as well as RNA isolation from single flies. The sample adapter can also hold and shake other items, such as centrifuge tubes (15–50 mL) or small bottles.     

A Modified Freezing-Drying Apparatus for Tissues

The preparation of tissues by the freezing-drying technic is preferred for many histochemical studies because of the rapid fixation, avoidance of deleterious action of chemical fixation and extraction by aqueous and lipid solvents in fixation, dehydration and clearing; to facilitate this procedure a freezing-drying apparatus has been constructed which permits cutting of sections within five hours after the fresh tissue is obtained. Liquid nitrogen provides a most efficient moisture trap and in conjunction with a heating element provides any desired temperature down to — 80°C. during tissue drying. Paraffin infiltration is started without disassembling the equipment and completed in a few minutes. Most stains and histochemical reactions for enzyme and other substances can be applied directly to sections of frozen-dried tissue cut from the same blocks.     

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.     

Accelerated net efflux of 3-O-[14C]methylglucose in isolated fat cells

A flow-tube apparatus suited for measurement of rapid efflux of sugars from adipocytes is described. Due to heterogeneity of fat cell populations, a conventional analysis of the time-course of net efflux of 3-O-methylglucose based on the integrated rate equation can produce gross errors in estimates of kinetic parameters. The half-saturation constant and maximum transport capacity for 3-O-methylglucose transport were found to be about 3-fold higher for net efflux than for equilibrium exchange flux, both in insulin-stimulated and non-stimulated adipocytes. This suggests asymmetric kinetic parameters for 3-O-methylglucose transport.     

A simple and effective method for isolating RNA from alfalfa pollen

Extraction of RNA from alfalfa pollen using conventional grinding devices resulted in low yields of degraded RNA; degradation appeared to be related to the length of time required to break open the pollen grains with such devices. A glass syringe was modified to provide the restricted, shearing environment necessary for rapidly rupturing this type of tissue. This apparatus would be useful for extracting a variety of molecules from pollen of any species, but is particularly valuable for species which produce low amounts of relatively small pollen grains.     

Influence of magnetic fields on calcium salts crystal formation: an explanation of the ‘pulsed electromagnetic field’ technique for bone healing

In the search for a mechanism by means of which a magnetic field deparalyses non-unions and enhances bone tissue formation, the influence of continuous magnetic fields on the formation of calcium phosphate crystal seeds has been investigated. From this perspective, an explanation is given of a working mode in conventional equipment for pulsed electromagnetic field treatment; this is compared with multifunction equipment.     

莼菜(Brasenia schreberi)表皮腺毛细胞高尔基体中一种内含物的初步研究(简报)

在不经过任何特殊处理的常规生物样品中,高尔基体扁囊(Saccules)及囊泡(Vacuoles)中的内含物在电镜下常为低电子密度,而最近我们在莼菜(Brasenia schreberi)叶柄及叶片的表皮腺毛细胞中观察到带有高电子密度的高尔基体内含物。在扁囊中,这些内含物多呈波浪形(图版Ⅰ,图1)。这种特殊形态的高尔基体内含物以及这种未经任何特殊处理而显示出高尔基体中某些物质的现象是前人没有报道过的,本文就这种内含物的结构、性质以及其染色机制进行了初步探讨。     

Swelling response of Golgi apparatus cisternae in cells treated with monensin is reduced by cell injury.

The effect of mechanical stress on Golgi apparatus was examined in thin slices of rat liver. The findings should be of relevance both to electron microscopists who routinely mince tissue, and to biochemists who homogenize tissues to isolate membranous components. The swelling response of Golgi apparatus to monensin was used as an assay because the swelling response is distinct and is thought to result from a well-characterized metabolic process, namely the acidification of vesicles. The results showed that the swelling response was compromised by monensin as far away as 6-7 cells from a cut surface even though other aspects of cell ultrastructure were not altered from normal. The monensin-induced swelling response was also evaluated in isolated Golgi apparatus and found to be similar to that with tissue. Thus, mechanical stress such as commonly used to mince tissue or isolate tissue components, appears to markedly alter Golgi apparatus function compared to the situation in vivo. In this example, the altered response of Golgi apparatus to monensin indicated that some aspects associated with the ATP-dependent proton-pumping machinery of the trans-most cisternae and trans Golgi network were compromised.     

An apparatus for anesthetizing small laboratory animals.

A simple apparatus was designed for simultaneous general anesthesia of a number of small laboratory animals with spontaneous respiration. Anesthesia is consistent over many hr. The apparatus consists of an oxygen tank and vaporizer, a glass distribution bottle, and a non-return circuit of separate inspiratory and expiratory valves for each animal. The equipment is inexpensive to construct and can be rapidly adjusted for exact control of gas flow and anesthetic depth.     

A model system for evaluating shear in the design of stirred fermentors

A model system, utilizing shear-sensitive protozoa, has been developed for characterizing the disruptive forces in agitated systems. The model system gives a measure of the maximum shear stresses in the apparatus being tested, and is particularly useful when tissue fragility is a factor in fermentor design. The time dependency of protozoan disruption is shown and discussed. Breakdown data in conventional stirred vessels and a laminar shear device are presented and discussed.     

Method and apparatus for soft tissue material parameter estimation using tissue tagged Magnetic Resonance Imaging

We describe an experimental method and apparatus for the estimation of constitutive parameters of soft tissue using Magnetic Resonance Imaging (MRI), in particular for the estimation of passive myocardial material properties. MRI tissue tagged images were acquired with simultaneous pressure recordings, while the tissue was cyclically deformed using a custom built reciprocating pump actuator A continuous three-dimensional (3D) displacement field was reconstructed from the imaged tag motion. Cavity volume changes and local tissue microstructure were determined from phase contrast velocity and diffusion tensor MR images, respectively. The Finite Element Method (FEM) was used to solve the finite elasticity problem and obtain the displacement field that satisfied the applied boundary conditions and a given set of material parameters. The material parameters which best fit the FEM predicted displacements to the displacements reconstructed from the tagged images were found by nonlinear optimization. The equipment and method were validated using inflation of a deformable silicon gel phantom in the shape of a cylindrical annulus. The silicon gel was well described by a neo-Hookian material law with a single material parameter C1=8.71+/-0.06kPa, estimated independently using a rotational shear apparatus. The MRI derived parameter was allowed to vary regionally and was estimated as C1 =8.80+/-0.86kPa across the model. Preliminary results from the passive inflation of an isolated arrested pig heart are also presented, demonstrating the feasibility of the apparatus and method for isolated heart preparations. FEM based models can therefore estimate constitutive parameters accurately and reliably from MRI tagging data.     

Simplified low-cost methodology to establish,histologically process and analyze three-dimensional cancer cell spheroid arrays

Differently of two-dimensional cell culture, three-dimensional (3D) multicellular spheroid model allows cells to establish cell-cell/cell-matrix interactions over the entire cell surface, more closely mimicking tumor microenvironments and cellular subpopulations with specific standards of morphology, differentiation and gene expression. Thenceforth several methodologies involving or the 3D cell aggregates generation or its histological processing and analysis have emerged, but in general they are laborious, expensive and complex to set up as a routine technique. Thus, we developed a complete methodology, detailing a simple, accessible and low-cost step by step, including 1) the 3D cell aggregate generation using hanging drop technique; 2) providing a simple way to assess morphological parameters of generated spheroids; followed by 3) a multiple and organized histological processing, keeping several individual spheroids inside an agarose apparatus, maintaining a known order and position of each ones, similar to tissue microarray principle; 4) until the last step, where it is allowed a simultaneous histological composition analysis of several spheroid slices, organized side by side, in a same block section, through conventional stainings or 5) immunostaining against different molecular markers. Therefore, the present methodology aims to popularize 3D cell culture, allowing to make this a regular technique in basic cell biology research, once all steps are performed without using onerous reagents, materials or equipment. In addition to bring the agarose apparatus as a simple low cost novelty, allowing high-throughput analysis of several spheroids simultaneously in an organized manner.     

Concentration of elements in mitotic chromatin as measured by x-ray microanalysis

Unfixed frozen-dried and uncoated tissue sections of the mouse duodenum were placed on carbon planchets and analyzed in a scanning electron microscope fitted with energy dispersive X-ray equipment. Computer analysis of the X-ray spectra allowed elemental microanalysis of the nucleus, cytoplasm, and mitotic chromatin regions in the cryptal and villus enterocytes. The peak to continuum ratio of S, Cl, K, and Ca were higher in mitotic chromatin than any of the other sites measured. The redistribution of Ca at mitosis is postulated to help explain both chromosome condensation and assembly of the mitotic spindle apparatus.     

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.     

To engineer is to create: the link between engineering and regeneration

Tissue engineering is a radically different approach to reconstruction of the body following degenerative diseases, trauma or chronic debilitating conditions. Although there have been some successes, tissue engineering is not yet delivering significant progress in terms of clinical outcomes and commercialization. Part of the problem is that we have failed to understand what tissue engineering really means and to appreciate that engineering is synonymous with creation. These processes involve many different phases and there has been minimal integration of these phases within tissue-engineering paradigms. The conventional concept, based upon a temporal sequence from sourcing cells through to the incorporation of generated tissue into a host, has to be transformed by a systems engineering approach in which all biological and technological phases, and the inter-relationships between them, are fully integrated. It might be that real success will not be achieved until systems biology is superimposed onto this systems engineering paradigm.