An automated rotisserie system for processing Western blots.

An apparatus to automate completely the processing of Western blots is described. The prototype is based on a popular rotisserie system design. The incubation chamber consists of an inner cylinder that rotates inside an outer cylinder (incubation chamber). The blot is contained in the inner cylinder. Two magnets are mounted at one end of the inner cylinder, and rotation of the inner cylinder is effected by two magnets mounted on a motor drive outside the incubation chamber. Movement of chemicals into and out of the incubation chamber is driven pneumatically, and the entire process is controlled by a computer. Processing a blot for chemiluminescent detection takes 7 h to complete without human intervention. The quality of the resulting image is comparable to or better than a blot using manual processing. In addition, the prototype is capable of re-collecting all three antisera for future use.     

An automated rotisserie system for processing Western blots

An apparatus to automate completely the processing of Western blots is described. The prototype is based on a popular rotisserie system design. The incubation chamber consists of an inner cylinder that rotates inside an outer cylinder (incubation chamber). The blot is contained in the inner cylinder. Two magnets are mounted at one end of the inner cylinder, and rotation of the inner cylinder is effected by two magnets mounted on a motor drive outside the incubation chamber. Movement of chemicals into and out of the incubation chamber is driven pneumatically, and the entire process is controlled by a computer. Processing a blot for chemiluminescent detection takes 7 h to complete without human intervention. The quality of the resulting image is comparable to or better than a blot using manual processing. In addition, the prototype is capable of re-collecting all three antisera for future use.     

A Portable System for Measuring the Photosynthesis and Transpiration of Graminaceous Leaves

A portable system has been developed for measuring rates ofphotosynthesis and transpiration of Graminaceous leaves; itcomprises a transparent chamber, into which the leaves are sealed,and a gas supply. The chamber is made from poly 4-methylpent-1-ene(PMP), chosen for its transparency and small water absorption.The leaf is sealed into the chamber by an inflatable rubberseal. The chamber contains a humidity sensor, photocell, thermistor,and a fan which is positioned to give efficient gas-mixing andrapid thermal equilibration. The chamber is surrounded by aPerspex tube to absorb part of the incident infrared radiation.Another fan blows ambient air between the chamber and the Perspextube to counteract solar heating of the chamber wall. The gas supply to the chamber comes from a gas cylinder viaa dilutor. The dilutor contains four small orifices arrangedin parallel. Each orifice is mounted in the inlet of a two-waygas valve: one way goes to the leaf chamber directly, the othervia a CO₂ absorber. The total gas flow-rate is independent ofvalve position, and the CO₂ concentration entering the chambercan be varied from zero to the cylinder concentration by switchingthe valves. The system has been tested both in the laboratory and in thefield, with satisfactory results.     

An autoclavable electronic interface for germfree isolators

An electronic interface was designed and fabricated for use with germfree isolators. It allows a variety of sensors or electrical instruments inside an isolator to be readily connected to monitoring or activating devices on the outside of an isolator. It was built into a 25 cm diameter cylinder whose open ends were loosely covered by an inner and outer connector panel. These two panels were wired together through an airtight dividing wall placed transversely across the middle of the cylinder. The interface was mounted in an entry port of a stainless steel isolator and steam sterilized with the isolator. It has proven to be a useful, contamination-free, durable device.     

Laboratory scale photobioreactor

Summary A photobioreactor using three concentric glass cylinders, with a light source mounted on the axis within them is described. The space between two innermost cylinders is used as a waterjacket, while the culture of phototrophic microorganisms is in the chamber between the outer cylinder and the middle one. This chamber is also equipped with a stirrer. Rhodobacter capsulatus has been grown in the device at biomass concentration up to 550 mg/l without light limitation.     

A repeated sampling bone chamber methodology for the evaluation of tissue differentiation and bone adaptation around titanium implants under controlled mechanical conditions

A repeated sampling bone chamber methodology was developed for the study of the influence of the mechanical environment on skeletal tissue differentiation and bone adaptation around titanium implants. Via perforations, bone grows into the implanted outer bone chamber, containing an inner bone chamber with a central test implant. An actuator—easily mounted on the outer bone chamber—allows a controlled mechanical stimulation of the test implant. After each experiment, the inner bone chamber—with its content—can be harvested and analysed. A new inner bone chamber with a central implant can be inserted consecutively in the outer bone chamber and a new experiment can start. Pilot studies led to a reliable surgical protocol and showed the applicability of the methodology, offering the possibility to study skeletal tissue differentiation and adaptation around implants under well-controlled mechanical conditions, and this protected from external loading. Repeated sampling of the bone chamber allows conducting several experiments within the same animal at the same site, thereby excluding subject- and site-dependent variability and reducing the amount of experimental animals.     

Parabiotic Chamber for Organ Cultures: Improved Model

A modified parabiotic chamber for organ cultures is described. It consists of a modified organ culture dish, glass cover slip, a cylinder of semipermeable membrane, and a stainless-steel mesh filter support. Silicone and agar seals were used. Diffusion occurred rapidly and with consistent rates at 37 C when a ratio of 5:2 reservoir to inner chamber volume was used. The device is simple to construct and assemble and allows for continuous observation while maintaining a large surface area available for diffusion. The volumes of the reservoir, inner chamber, and agar base can be varied, and the chamber may be used with a wide variety of cell and organ culture systems.     

A portable chamber for measuring algal primary production in streams

This paper describes a portable chamber that measures net primary production of stream periphyton using a ¹⁴C uptake method. The unique feature is that substrates are moved through water at a velocity of 20 cm s ⁻¹ rather than moving water over substrates. The chamber consists of a plexiglass cylinder that is 9 cm in height and 15 cm in diameter. On the top of the cylinder is a DC gearmotor powered by a 12 volt, deep cycle, marine battery. The motor turns a shaft that rotates a 13.3 cm plexiglass plate at a velocity of 20 cm s ⁻¹ . Small tiles (3.2 cm × 3.2 cm × 0.5 cm) that have natural algal assemblages are mounted on the rotating plate. After adding 500 ml of filtered stream water and 185 kBq (5 μCi) NaH¹⁴CO₃ to the chamber, the chambers are placed along a stream margin for 5 h. Measurements of ¹⁴C uptake by algae on the tiles provide estimates of net primary production (NPP). To assess the sensitivity and practicality of the chamber, algal primary production was measured in open and closed canopy sections of Kingsley Creek, Randallsville, New York. In autumn, primary production was higher in the open than closed canopy section and NPP was lower in spring in both sections probably because of scouring of algae due to snowmelt.     

The Growth Physiology of Coleoptiles in Continuous-Record Vessels: Anomalous Kinetics of Cycloheximide Action on Auxin-Controlled Growth

The interacting effects of auxin and cycloheximide upon the growth of Triticum coleoptiles depends upon the incubation conditions. In the growth vessel developed by Evans and Ray (1969) the action of cycloheximide is severe and the growth kinetics show a rapid nullification of auxin action by the inhibitor; hormone action is also totally prevented by a relatively short period of pre-treatment with cycloheximide. The kinetics of response to the two substances are quite different when coleoptiles are floating on the test solutions; here, cycloheximide is much less severe in its action. These differences are due to a heightened sensitivity to cycloheximide induced by exclusion of the air bubble from the coleoptile cylinder in the Evans and Ray chamber. The outer surface of the coleoptile wall is a less efficient respiratory surface than the inner one. When coleoptiles are filled with a stagnant solution insufficient oxygen can be taken up through the inner surface and the tissue therefore suffers oxygen deprivation. This exacerbates the action of cycloheximide as a potent respiratory inhibitor. The results raise questions concerning the use of conditions in which coleoptile sections are filled and submerged, which is a feature of recently introduced continuous-record growth vessels. In particular, interpretation of the interactions between cycloheximide and auxin under such conditions must be made cautiously.     

Design of a Robotized Magnetic Platform for Targeted Drug Delivery in the Cochlea

Inner ear disorders' treatment remains challenging due to anatomical barriers. Robotic assistance seems to be a promising approach to enhance inner ear treatments and, more particularly, lead to effective targeted drug delivery into the human cochlea. In this paper we present a combination of a micro-macro system that was designed and realized in order to efficiently control the navigation of magnetic nanoparticles in an open-loop scheme throughout the cochlea, considering that the magnetic particles cannot be located in real time.In order to respect the anatomical constraints, we established the characteristics that the new platform must present then proceeded to the design of the latter. The developed system is composed of a magnetic actuator that aims to guide nanoparticles into the cochlea. Mounted on a robotic manipulator, it ensures its positioning around the patient's head. The magnetic device integrates four parallelepiped-rectangle permanent magnets. Their arrangement in space, position and orientation, allows the creation of an area of convergence of magnetic forces where nanoparticles can be pushed/pulled to. To ensure the reachability of the desired orientations and positions, a 3 DOF robot based on a Remote Centre of Motion (RCM) mechanism was developed. It features three concurrent rotational joints that generate a spherical workspace around the head. The control of the latter is based on kinematic models.A prototype of this platform was realized to validate the actuation process. Both magnetic actuator and robotic manipulator were realized using an additive manufacturing approach. We also designed a virtual human head with a life-size cochlea inside. A laser was mounted on the end effector to track the positioning of the actuator. This permitted to experimentally prove the capacity of the robotic system to reach the desired positions and orientations in accordance with the medical needs.This promising robotic approach, makes it possible to overcome anatomical barriers and steer magnetic nanoparticles to a targeted location in the inner ear and, more precisely, inside the cochlea.     

Interembryonic regions of the uterus of the viviparous lizard Mabuya brachypoda (Squamata: Scincidae)

Analysis of the structure and physiology of the uterine incubation chambers of viviparous squamates has provided insight concerning adaptations for gestation. However, the literature addressing the biology of the interembryonic regions of the uterus is very limited, presumably because it has been assumed that this area has little role in the development and support of embryos in viviparous squamates. This study was undertaken to examine the histology of the interembryonic regions of Mabuya brachypoda, a viviparous lizard with microlecithal ova and consequently substantial matrotrophic activity. The incubation chambers are oval, distended zones of the uterus, adjacent to the interembryonic regions. The wall of the interembryonic regions includes: mucosa, formed by a cuboidal or columnar epithelium with ciliated and nonciliated cells, and a lamina propria of vascularized connective tissue containing abundant acinar glands; myometrial smooth muscle consisting of inner circular and outer longitudinal layers; and serosa. The segment of the interembryonic region adjacent to the incubation chamber forms a transitional segment that displays folds of the mucosa that protrude into the uterine lumen. The limit of the incubation chamber is well defined by the long mucosal folds of the transitional segment. Long and thin extensions of extraembryonic membranes are present in the lumen of the transitional segment, outside of the incubation chamber region. The presence of abundant uterine glands and extraembryonic membranes in the interembryonic regions during gestation suggests uterine secretory activity and histotrophic transfer of nutrients to embryos in these regions.     

Quantitative examination of a perfusion microscope for the study of osmotic response of cells

The perfusion microscope was developed for the study of the osmotic response of cells. In this microscope, the cells are immobilized in a transparent chamber mounted on the stage and exposed to a variety of milieus by perfusing the chamber with solutions of different concentrations. The concentration of the supplied solution is controlled using two variable-speed syringe pumps, which supply an isotonic solution and a hypertonic solution. Before using this system to characterize the osmotic response of cells, the change in the concentration of NaCl solution flowing through the chamber is examined quantitatively using a laser interferometer and an image processing technique. The NaCl concentration is increased from an isotonic condition to a hypertonic condition abruptly or gradually at a given constant rate, and decreased from a hypertonic condition to an isotonic condition. It is confirmed that the concentration is nearly uniform in the cross direction at the middle of the chamber, and the change in the NaCl concentration is reproducible. The average rate of increase or decrease in the measured concentration agrees fairly well with the given rate when the concentration is changed gradually at a constant rate. The rate of the abrupt change is also determined to be the highest limit achieved by the present method. As the first application of using the perfusion microscope for biological studies, the volume change of cells after exposure to a hypertonic solution is measured. Then, the hydraulic conductivity of the cell membrane is determinedfrom the comparison of the volume change between the experiment and the theoretical estimation for the measured change in the NaCl concentration of the perfused solution.     

Mitochondrial membrane potential and intracellular ATP content after transient experimental ischemia in the cultured hippocampal neuron

Ischemia limits the delivery of oxygen and glucose to cells and disturbs the maintenance of mitochondrial membrane potential (MMP). MMP regulates the production of high-energy phosphate and apoptotic cascading. Thus, MMP is an important parameter determining the fate of neurons. Differences in the time course of MMP according to the grading of the ischemic impact have not been clarified. MMP and intracellular ATP contents were monitored before and after short-term oxygen-glucose deprivation. A primary hippocampal culture seeded in a 35 mm fenestrated dish for fluorescence microscopy was mounted in a sealed chamber for an anaerobic incubation. A continuous flow of 100% nitrogen into the chamber and a replacement of glucose-free medium allowed the condition of oxygen-glucose deprivation (OGD), thereby extrapolating ischemia. MMP was evaluated by the fluorescence of a voltage-dependent dye, JC-1, under fluorescence microscopy. The intracellular ATP content was evaluated in a hippocampal culture seeded in a 96-well plate by the luciferin-luciferase reaction after a designated period of OGD. During OGD, MMP decreased to 0.72+/-0.03 (normalized JC-1 fluorescence), then increased to the hyperpolarized level 1.99+/-0.12 during 60 min reoxygenation after 30 min OGD. MMP after 60 min OGD decreased and recovered occasionally during reoxygenation. After 90 min OGD and reoxygenation, MMP was reduced and never recovered. The intracellular ATP content was 8.1+/-6.6 and 3.2+/-1.9% after 30 min OGD and 30 min reoxygenation following 30 min OGD, respectively; 60 min OGD did not significantly change these levels (7.1+/-5.8, 2.6+/-0.5%). Hyperpolarization after OGD did not accompany ATP production. This observation suggests the inhibition of electron reentry into an inner membrane during reoxygenation and the disturbance of FoF1-ATP synthase. This pathological finding of an energy-producing system after OGD may provide a clue to explain post-ischemic energy failure.     

Trap for tephritid fruit fly parasites

A sticky trap for capturing the adults of tephritid fruit fly parasites was designed. This trap consisted of an outer and inner cylinder made of hardware cloth. Host fruits of fruit flies were placed in the inner cylinder and the outer one was sprayed with tanglefoot. Fruit fly adults and parasites attracted to the fruit were captured on the sticky outer cylinder.     

A newly designed tensile tester for cells and its application to fibroblasts

A tensile test system for cells has been designed and applied to fibroblasts from the rabbit patellar tendon. It consists of a thermostatic test chamber, an inverted fluorescence microscope, micromanipulators, a direct drive linear actuator, a cantilever-type load cell, and a video dimension analyzer (VDA). The test chamber and the microscope are mounted on a vibration isolator. A cell floated in Hanks' balanced salt solution of 37 degrees C is gripped with a pair of micropipettes which have very fine tips (outer diameter = 20 approximately 30 microm, inner diameter = 3 approximately 5 microm) and are coated with a cell adhesive, Cell-Tak, at their ends. One of the micropipettes is fixed to the load cell; the other one is attached to the linear actuator which is used to stretch the cell. Load applied to the cell is measured with the load cell, while elongation of the cell is determined with the VDA using the images of the ends of the micropipettes as markers. The measurement accuracy of the load cell was +/-0.05 microN. All the fibroblasts tested were firmly attached to the micropipettes during tensile testing, and showed local non-uniform deformation. The maximum load and elongation to failure of the cells were 0.9+/-0.2 microN and 86+/-24 microm, respectively.     

Spherical body formation in the spirochaete Brachyspira hyodysenteriae

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.     

Radial histophysiologic gradient culture chamber: Rationale and preparation

Summary Histophysiologic gradient culture methods reconstitute important spatial relationships that occur in nature between a parenchyma and its supporting stroma. At the epithelial-stromal interface, epithelia are firmly attached to the stromal substrate, initiation of renewal takes place, and metabolites are exchanged by a process of diffusion between epithelium and substrate. Other spatial imperatives characteristic of stratified epithelium are high density of cells, gradients of maturation, and continuity of epithelia along the entire course of the stromal-parenchymal interface. In radial gradient culture these relationships of epithelial cells, and supporting substrates are reconstituted. The culture chamber consists of a thin-walled cylinder, 2 to 3 mm in diameter and 3 cm long. The wall is a transparent collagen membrane in whose substance is embedded a reinforcing nylon mesh. To prepare a culture, one end of the cylinder is ligated, 1 or 2 particulate inocula are inserted in the open end of the cylinder, guided toward the ligature, and the open end is ligated. Subsequently, during incubation in a container with medium, the explants attach and proliferate. Proliferation and migration result in the cylinder being completely lined by a complex organoid tissue with structural characteristics of the original tissue. The tissue patterns in radial gradient culture of two human cell lines, RT-4, a bladder cancer, and 87×50, and ovarian cancer, are illustrated.     

Process development of starch hydrolysis using mixing characteristics of Taylor vortices

In food industries, enzymatic starch hydrolysis is an important process that consists of two steps: gelatinization and saccharification. One of the major difficulties in designing the starch hydrolysis process is the sharp change in its rheological properties. In this study, Taylor–Couette flow reactor was applied to continuous starch hydrolysis process. The concentration of reducing sugar produced via enzymatic hydrolysis was evaluated by varying operational variables: rotational speed of the inner cylinder, axial velocity (reaction time), amount of enzyme, and initial starch content in the slurry. When Taylor vortices were formed in the annular space, efficient hydrolysis occurred because Taylor vortices improved the mixing of gelatinized starch with enzyme. Furthermore, a modified inner cylinder was proposed, and its mixing performance was numerically investigated. The modified inner cylinder showed higher potential for enhanced mixing of gelatinized starch and the enzyme than the conventional cylinder.     

In vitro effects of Clostridium perfringens type D epsilon toxin on water and ion transport in ovine and caprine intestine

Clostridium perfringens type D produces enterotoxaemia in sheep, goats and other animals. The disease is caused by C. perfringens epsilon toxin, and while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by sheep than goat intestine. In an attempt to clarify this, we examined the in vitro effects of epsilon toxin on sheep and goat intestine. Pieces of intestinal mucosa from recently slaughtered animals were mounted in a modified Ussing-type chamber where net water flux (J(w)), short-circuit current (I(sc)) and tissue conductance (G(t)) were simultaneously recorded. After 70 min of incubation with epsilon toxin a reduction in absorptive J(w) and an increase in I(sc) and G(t) were observed in colonic tissues of both sheep and goats, but no alterations were registered in the ileum of either species. These in vitro results show that epsilon toxin affects the transport function of the colonic mucosa but it does not seem to produce any transport alteration in the ileum mucosa.