text:
|Publication number
|Publication type
|Application number
|Publication date
|Apr 19, 1994
|Filing date
|Nov 30, 1992
|Priority date
|Dec 7, 1990
|Fee status
|Also published as
|Publication number
|Charles M. Finley, Charles L. Kissel
|Original Assignee
|Baker Hughes Incorporated
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (8), Non-Patent Citations (12), Referenced by (61), Classifications (26), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This is a continuation of copending application Ser. No. 07/625,302 filed on Dec. 7, 1990 now abandoned.
The invention relates to chemical reactors, and more particularly, the invention relates, in one aspect, to chemical reactors which employ a catalyst and even more particularly to catalytic chemical reactors which may be portable.
Chemical reactors are well known in the art and may take a number of different forms. For example, some of the well known types of reactors include, but are not limited to batch reactors, stirred tank reactors, continuous stirred tank reactors (CSTRs), tubular reactors, multiple-pass reactors, fixed bed reactors, fluidized bed reactors and spouted bed reactors. These last three are specific types of catalytic reactors.
Most commercial reactors are relatively large, on the order of tens of feet tall, which require that they remain permanent fixtures to the operating site. Chemical reaction devices which are portable most often refer to laboratory or bench scale equipment, which are not suitable for the production of large volumes of products. Skid- or trailer mounted reactors, are known, of course, where appreciable quantities of a certain material are required and it is also desired that the overall unit be modular or portable, but these tend not to be catalytic systems.
However, there remains a need for different kinds of portable catalytic chemical reactors which produce appreciable volumes of chemicals in good yields. It would be an additional advantage if the reactor were somewhat modular so that modules could be added to the system to adjust the capacity of the reactor system. A small reactor using aluminum tubes coated with copper as a catalyst was reported by Xing and Inoue, Kagaku Kogaku Ronbunshu, Vol. 10, No. 4, 439-45 (1984).
Accordingly, it is an object of the present invention to provide a chemical reactor useful in the volume production of products, which reactor may be portable.
It is another object of the present invention to provide a chemical reactor which may be considered as a module or cell and which may be readily interconnected in parallel with other reactors to increase the volume of product.
Another object of the present invention is to provide a chemical reactor having reaction chambers with surfaces that are relatively inert to oxidation reactions and their products.
In carrying out these and other objects of the invention, there is provided, in one form, a reactor for containing a catalyst suitable to facilitate a chemical reaction, where the reactor has at least one flat plate which, in turn, has a major surface, a first minor end, and a second minor end opposite the first minor end. Present within the flat plate are a plurality of reaction chambers oriented parallel to one another and parallel to the major surface, beginning at the first minor end at an inlet and terminating at the second minor end at an outlet. The chambers are for receiving a catalyst. The reactor additionally has at least one flat heating panel adjacent and parallel to the flat plate.
This invention provides a portable, self-contained system for generating dilute solutions of various products in significant quantities on-demand and on-site. With the system of this invention, the products are not produced and shipped in a concentrated liquid form, but instead are produced and used in a relatively short time on site in a dilute solution in a liquid compatible with the liquid to be treated.
The improved reactor of this invention can give a high yield to products from a reaction zone of about equal to or less than 18 inches long. Conventional prior art reactors are 10 to 30 times longer, making them impractical for use in portable units. While the reactor of this invention may be scaled up to produce greater volumes of product, even to the point that the reactor is no longer portable, such increase is expected to be accomplished by adding modules or cells, rather than appreciably increasing the size of the reaction chambers.
A presently preferred configuration of this inventive reactor uses an air pump, if air is one of the reactants; a storage tank for another of the reactants, if appropriate; a pump for water or other liquid into which the product is to be dissolved or absorbed, if appropriate; a high-temperature reactor thermally insulated and suitably shaped (for example, substantially in the form of a cube) for minimum consumption and loss of energy; an absorber system to collect product, if gaseous, from the reactor and place it into a liquid at high efficiency; an exhaust gas purifier; and a control system using gauges, valves, sensors, and electrical circuits for positive, safe operation. The system can be sufficiently small to be mounted on a skid or a trailer, and only the reactants, the liquid and either electricity or fuel for an electrical generator are required to permit on-site and on-demand generation of the product dissolved in a flowing liquid stream. With this invention, there is no need to produce a concentrated liquid product, thus avoiding any inherent hazards that might be present with the product in that form. The system may also be sufficiently large so that it would be part of a permanent installation.
These and other aspects of the invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 schematically shows the reactor of this invention connected to receive a mixture of two gases, and to discharge the reaction products into an absorber;
FIG. 2 is a view taken on line 2--2 of FIG. 1 showing details of the reactor in cross-section;
FIG. 3 is a perspective view of one of the parallel plates in the reactor;
FIG. 4 is an enlarged view taken in the area 4--4 of FIG. 2; and
FIG. 5 is a schematic diagram showing a circuit for controlling the temperature of the catalyst in the reactor.
It has been discovered that a relatively small, modular reactor can be devised, which may be easily moved from place to place to catalytically produce various chemical products. In one embodiment, the reactor is an apparatus for making an organic product from at least two reactants, the apparatus having a catalyst chamber with an inlet and an outlet. A permeable bed of catalyst support particles is in the chamber between the inlet and the outlet, and a catalyst which promotes the reaction of the reactants to form the product is present on at least some of the particles. A mechanism is provided for introducing the reactants through the inlet to the reaction chamber, and another mechanism is provided for removing the organic product from the reaction chamber through the outlet. Although it is conceivable that the reactor of the present invention may find utility in the production of inorganic materials, it is expected that it will find more applications in the manufacture of organic compounds.
The apparatus capable of serving as the reaction chamber can have various shapes, such as cylindrical or rectangular blocks, each containing hollow passageways capable of containing the catalyst and the reaction. These passageways may be tubular in nature, or they may be configured as to form a fixed-bed chamber, or numerous single tubes can be assembled in various arrays. Spouted beds or fluidized reactor zones could also be used. However, many of these reaction chamber types have inefficient temperature/heating characteristics, or serious channelling of unreacted starting material gases often occurs.
A preferred apparatus for making acrolein includes a reactor of this invention with at least one pair of substantially parallel plates disposed side-by-side. Each plate includes catalyst or reaction chambers which each have a respective inlet and outlet. A catalyst in each chamber promotes the reaction, for example, the oxidation of propylene to acrolein, and a mechanism is provided for introducing the reactants, such as a mixture of propylene and oxygen, into the catalyst chambers through the inlets. A heating element or panel between the plates heats the reactants and the catalyst to a temperature which causes the reactants to react with each other in the presence of the catalyst to form the reaction products. A mechanism is also provided for removing the reaction products from the outlets of the catalyst chambers.
In a preferred embodiment, each plate is in the shape of a rectangular slab having major sides many times greater than the thickness of the slab. The slab has a first minor end and a second minor end opposite the first minor end. For example, each plate may be a rectangular slab about 1' thick, with opposing major faces each being approximately square and having a dimension of about 18' on each side. A plurality of elongated and laterally spaced bores extend through each plate in a direction substantially parallel to the major faces of the plate beginning at the first minor end and terminating at the second minor end, to provide as many as 20 to 30 parallel catalyst chambers in each plate. The reaction chambers may be said to extend from one minor end to an opposing minor end on opposite sides of the plate, beginning at one end with their inlet and terminating at the other with their outlet.
A thin, panel-shaped flat heating element is sandwiched between adjacent major faces of a pair of plates to form a heated pair, or cell, the temperature of which is controlled by a thermostat mounted in a face of one of the plates adjacent the heating element. Preferably, heat insulation around the assembled plates which form the reactor limits the amount of external energy which must be applied to the apparatus. A separate respective capillary tube is connected at one end to a respective catalyst or reaction chamber inlet, and at the other end to a reactant supply pipe, which supplies a mixture of reactants to each catalyst chamber inlet through a respective capillary tube. These capillary tubes are fed by a common header containing pressurized mixed starting materials. This arrangement uses frictional drag in the tubes to control the flow rates through each of the separate reaction chambers. Although capillaries are preferred for flow control, other devices, such as orifices or adjustable valves, may also be used.
A separate elongated collection header over the outlets of the catalyst chambers in each plate collects reaction products leaving the reaction chambers. Each of the catalyst chambers is packed with a bed of catalyst which preferentially promotes the reaction, such as the oxidation of propylene to acrolein.
The presently preferred embodiment of the invention includes a plurality of pairs of parallel plates, each pair being constructed and arranged with a panel-shaped heating element as described above. A sufficient number of the pairs of plates are staked together to form an array, or a reactor, essentially in the shape of a cube to minimize heat loss. In another embodiment, the plates could be curved or flat and arranged concentrically. The space between adjacent pairs of plates is thermally insulated to provide good temperature control in each pair of plates served by a respective panel-shaped heating element.
Preferably, the plates are made of a metal selected from the group of aluminum, tantalum, titanium, tungsten, niobium or mixtures thereof, or at least the inner surfaces of the reaction chambers are made from these metals. It is additionally preferred that all internal surfaces contacted by the gases and/or liquids passing through the catalyst chambers and collection headers, absorber, if present, and conduits connecting the headers and absorbers or other pieces of equipment are made of metals selected from that group. It is possible that some tubing and conduits not subject to high temperatures may be made from inert polymeric plastics, elastomers and the like.
The cross-sectional area of each chamber is between 0.70 and 3.0 cm2, and preferably is in the range of about 1.2 to 1.7 cm2.
Suitable mechanisms are provided for sensing the temperature of the catalyst and controlling the heat supplied by the heaters to keep the catalyst temperature within its operating range. Of course, if the expected reaction temperature range exceeds the softening point of the metal used to line the reaction chambers, for example of aluminum, one should plan to use a different material selected from the above-enumerated list.
A preferred form of the invention also includes a pump for taking a stream of liquid from a source of liquid which is to be treated with the reaction product. Spray nozzles connected to the pump outlet, or other source of liquid under pressure, spray liquid into an absorber, through which the reaction products from the catalyst chambers pass. The reaction product is absorbed in the liquid stream from the liquid supply, and added to the system which is to be treated, either as a stream diluted by the system to be treated, or undiluted if the system to be treated is composed wholly of the product stream from the reactor. If gases are a product, those which are not absorbed in the liquid sprayed into the absorber pass through a catalytic purifier.
The reactor of this invention has been successfully used to catalytically produce (1) acrolein from air and propylene; (2) methacrolein from isobutylene and air; and (3) acrylonitrile from propylene and ammonia. The example of making acrolein from propylene and air will be used from time to time throughout this description as representative of an actual use of the reactor, but the invention should not be limited to this example. It is anticipated that the reactor of this invention will find utility in the production of organic chemicals other than those enumerated above.
Referring to FIG. 1, a reactor 10 in the general shape of a cube receives a gas mixture of air, as one reactant, and propylene, as the other reactant, from a plurality of horizontal and parallel supply lines 12 (see FIG. 2), each connected at respective inlet ends through a respective T-joint 14 to the discharge of a respective air pressure regulator 16, the inlet of which is connected by an air manifold line 18 to the discharge of air pump 20 having an inlet 22 connected to a filter 24, through which air is drawn.
A tank of one of the reactants, propylene for example, has a discharge line 28 connected through a primary pressure regulator 30 and a filter 32 to a gas supply line 34 connected through a secondary pressure regulator 36 to the T-joint 14. Obviously, if liquid reactants are used, these gaseous sub-systems would be replaced by the liquid tanks, supply lines, etc.
Reaction products from the upper side of the reactor are carried by a delivery pipe 38 (see FIG. 1) to an absorber 40, which includes a housing 42 divided into first, second, third and fourth absorption zones 44, 46, 48 and 50, respectively. A first vertical baffle 52 extends from the top of the housing 42 downwardly to terminate just above a pool 54 of liquid, which may be water, fuel, or other liquid which absorbs acrolein. A second vertical baffle 56 mounted in the housing parallel to, and spaced horizontally from, the first baffle extends from below the level of liquid pool 54 and a short distance below the top of the housing. A third vertical baffle 58 parallel to, and spaced from, the second baffle extends from the top of the housing down to terminate just above the surface of the liquid pool 54. Each of the baffles extends entirely across the housing in a direction perpendicular to the plane of FIG. 1 so that the three baffles divide the housing 42 into the four absorption zones 44, 46, 48 and 50.
A separate respective group of spray nozzles 60 mounted in the upper end of each absorption zone are supplied liquid from a liquid supply pipe 62 connected through a pressure regulator 63 to the discharge of a pump 64, which has as its inlet connected through a suction pipe 66 to a main body or stream of liquid flowing through a main pipe 68. Line 66 and pump 64 may be replaced by other suitable sources, such as pressurized water lines typical of fire hydrants and hose bib stand pipes. In any event, the pressure of the incoming fluid is determined by a pressure regulator 63, or other suitable flow-monitoring device.
A drain pipe 70 extends from the liquid pool 54 in the housing through a control valve 72 and a return pump 73 to return product-containing liquid in the absorber to the main pipe 68. Alternatively, the product-containing liquid may be sent to storage for future use.
An exhaust conduit 74 carries unabsorbed gases from the upper end of the fourth absorption zone 50 to a catalytic purifier 76, which oxidizes the remaining hydrocarbons and any carbon monoxide to water and carbon dioxide, which are discharged to the atmosphere. Obviously, if the product is a liquid or is condensed to a liquid, the equipment for handling the product and placing it into useful solutions or other forms will be different.
As shown in FIG. 2, the reactor 10 may be a sandwich assembly or array 80 of a plurality of flat, rectangular plates or slabs 82 disposed side-by-side so the reactor is substantially in the shape of a cube to minimize heat loss from a given volume for the reactor. A layer 83 of thermal insulating material surrounds the reactor to reduce heat loss and energy consumption. Each plate 82 has a first minor end 78 containing the inlets to the reactor plate, and a second minor end 79 opposite the first minor end containing the outlets. First minor end 78 and second minor end 79 are generally perpendicular to the major face of slab 82.
Although the plates may be of any suitable dimensions, a successful reactor has been designed in which the plates are about 18"�18"�1". The plates are assembled with their major surfaces vertically oriented to form the assembly 80 shown in FIG. 2. A plurality of vertical bores 84 extend through each plate. If the plate is about 1" thick, it has been found that bores about 1/2" in diameter, and located on about 5/8" centers form properly spaced and dimensioned reaction chambers 86 (FIG. 2).
The plates may be arranged in pairs 88 (FIG. 2), with a separate, respective, flat electrical heating panel 90 disposed between adjacent faces 92 of each plate in each pair. A pair of plates with a heating panel 90 between them may be referred to as a cell. A thin, separate, respective sheet 93 of ceramic insulation is between each face of each electric panel and the adjacent face of a plate. The thin sheet of ceramic insulation, which can be of any suitable thickness, say 0.03 to 0.07", provides a more uniform transfer of heat from the electric heating panels 90 to the entire adjacent surfaces of the plates. Electrical power leads 94 (FIGS. 2 and 5) supply power to the heater panels. Alternatively, the reactor could consist simply of one plate 82 and one adjacent flat electrical heating panel 90.
A thermocouple 95 on the inner end of a horizontal support 96 (FIGS. 4 and 5) in a horizontal groove 98 extending from about the center of one vertical edge of a plate in each pair to about the center of that plate, and opening toward the other plate in the pair, senses the temperature of the plates surrounding the respective reaction chambers 86, each of which is filled with a bed 100 of a catalyst which promotes the oxidation, or other reaction, of the reactant, such as propylene, to form the product, such as acrolein. A separate pair of electrical signal leads 101 (see FIGS. 4 and 5) extend from each respective thermocouple to a respective adjustable thermostat 102, which controls the amount of electrical power supplied from a generator 103 to a respective electrical heater panel 90 to keep the catalyst in the adjacent reaction chamber 86 at the required operating temperature.
A separate, respective loose plug 104 (FIG. 2) of quartz wool in the bottom of each reaction chamber 86 rests on the upper end of a separate, respective short nipple 105 threaded into the lower end of a respective reaction chamber to form a reaction chamber inlet. The upper end of a separate, respective capillary tube 108 is sealed at its lower end to a respective gas supply pipe 12. The capillary tubes act as flow control devices and may be of any suitable internal diameter for that purpose. Capillary tubes with an inside diameter of about 0.007" provides good flow control and distribution of reactant gas from the supply pipes 12.
A separate, elongated, rectangular header 110 rests on a separate, respective graphite gasket 112 on the upper edge of each plate and over the upper (outlet) ends of the reaction chamber in each plate to collect reaction products which flow up through the reaction chambers, through respective holes 114 in the gaskets 112, and to the delivery pipe 38 (see FIGS. 1 and 2).
The reactor plates and all other components with surfaces contacted by the reaction products should be made of a material which has good thermal conductivity, and which does not adversely affect the production of the desired product. For example, to produce acrolein by oxidizing propylene, aluminum is preferred because of its relatively low cost. Other metals, such as tantalum, titanium, tungsten, niobium or mixtures of these, may also be used. The term "mixtures" as used herein includes alloys of the above-listed metals with each other as well as other ways in which the various suitable metals may be mixed, such as simply being blended or used physically adjacent one another. Aluminum alloys of the 6000-type are preferred because they are inert, machinable, and can be welded. These are unusual materials for reactors, but have been found to be particularly advantageous for catalytic oxidation reactions. The use of ferrous metals within the reactor decreases the selectivity of the process to acrolein, if that is the reaction run. Brass, bronze and copper are also not used because they deteriorate chemically when contacted with such reactants as air and propylene. We are not aware of any prior art use of aluminum equipment, except for the single report of the use of aluminum tubes that were coated with copper as a catalyst described by Xing and Inoue, Kagaku Kogaku Ronbunshu, Vol. 10, No. 4, p. 439-45 (1984).
A separate respective panel 121 of thermal insulation between the adjacent outer faces 122 of each adjacent pair of plates provides good temperature control for each pair of plates served by a respective heating panel 90. The panels may be of any suitable material, such as sandstone, refractory material, spun glass, and the like. Each panel has sufficient thickness to reduce thermal flow between reactor pairs 88. Although asbestos, asbestos-filled materials such as magnesia, and transite can provide adequate protection against thermal convention, their use is discouraged due to possible environmental health hazards.
The operation of the reactor will now be described using the production of acrolein as illustrative only. With the reactor packed with beds of supported catalyst, as described above and shown in FIG. 2, a mixture of air and propylene in a ratio of 84:16, respectively, by volume, was fed into the inlets of the reaction chambers, which were heated to about 410� C. The pressure in the reaction chambers was between about 2 and about 5 lbs./in.2, and the flow rate per unit of cross-sectional area was about 100 ml/min./cm2. The effluent from the reaction chambers 86 flowed through the aluminum headers and the aluminum delivery pipe 38, which were surrounded by a layer of insulation (not shown) to keep the gas in them at a temperature above about 60� C. to prevent premature condensation of acrolein, and into the upper end of the first absorption zone of the absorber. The effluent was contacted in the first absorber zone by a spray of water supplied by pump 64 to the spray nozzles. The intake of the pump was connected to main pipe 68 carrying a main stream of water to be treated with acrolein. Water droplets with absorbed acrolein fell into the pool 54 in the bottom of the absorber. Unabsorbed gases passed under the first baffle 52 and into the bottom of the second absorption zone following the arrows shown in FIG. 1, rising to meet more water droplets sprayed into the top of that zone. Additional acrolein was absorbed and carried to the pool in the bottom of the absorber. The unabsorbed gases continued over the top of the second baffle into the top of the third absorption zone for additional absorption of acrolein, and passed under the third baffle to flow up through the spray of water in the fourth absorption zone, and out the exhaust conduit 74 through the catalytic purifier, 76, where any carbon monoxide and upreacted hydrocarbons were oxidized to carbon dioxide and water vapor, which were discharged into the atmosphere.
The water sprayed into the absorber scrubbed more than 99.9% of the acrolein from the reactor effluent, and carried it into the liquid pool in the bottom of the absorber, where the acrolein-rich water was removed through the drainpipe 70 and returned to the main stream of water downstream of the pump inlet.
In the example just described, the concentration of the acrolein in the water pool in the bottom of the absorber was about 0.2%, by weight, and it was returned to the main water stream at a rate to give a treated solution with between about 1 and about 15 ppm acrolein for weed control in ditches through which the treated water flowed. Approximately the same concentration would be used for treating injection wells for secondary recovery of oil, or maintenance of gas pressure in an underground formation. In either case, the reactive nature of the acrolein in the dilute solution causes the acrolein to substantially disappear within a few days.
The acrolein added to water for secondary recovery can scavenge hydrogen sulfide (H2 S) and destroy microbes (e.g., anaerobic bacteria which consume sulfur, say from calcium sulfate which may be available in the injection water or in the formation, and convert it to H2 S, a corrosive compound), which may be in either the treated water, or downhole, or both.
As noted, the water or other fluid supplied to the absorber may come from a pressurized source, in which case the pump is not required. The water to be treated can come from a source of irrigation water, or water to be injected into an underground formation, and either all of the water to be treated can be passed through the absorber, which would be operated to give the required final concentration of acrolein, or only a portion of the water may be passed through the absorber and then recombined with the rest of the water, as described above. It is possible to operate the absorber to produce a solution which contains up to about 25% acrolein by weight.
It has been determined that in building a compact, portable reactor of the type described above, and in which a highly-divided catalyst is employed, it is advantageous in one embodiment to limit the cross-sectional area of each reaction chamber 86 to about 2 cm2 or less, and to limit the flow rate of reactants to about 150 cc/min./cm2 of cross-sectional area or less. Accordingly, to obtain a given throughput of reactants for a desired output of product, a number of reaction chambers were grouped together, i.e. placed in parallel, as described above to permit accurate control of flow rate and temperature for each chamber. The number of parallel reaction chambers may be quite large to obtain a relatively large throughput of reactants and production of product, and the number of chambers may be sufficiently large as to make the reactor no longer portable. The same result cannot be obtained by simply using a reactor with a large, undivided cross-sectional area equal to the total of the group of parallel reaction chambers. The yield of product with the large, undivided cross sectional area is inevitably low and, therefore, uneconomic. In operating the reactor of this invention as described above at 410� C., with a feed mixture of 16% propylene and 84% air, the discharge from the reactor was 9.4% acrolein, resulting in a yield of approximately 68% of the propylene to acrolein.
Many modifications may be made in the apparatus of the present invention without departing from its spirit and scope, which are defined only in the appended claims. For example, one skilled in the art may find that certain geometric configurations of the flat plates, or certain alloys of the preferred metals give particularly advantageous results. From the foregoing description, it will be seen that this invention provides a portable, self-contained system for generating chemicals on demand and on-site to produce dilute, safe solutions thereof.
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text: This could go under a more scientific forum, but I think it's relevant to tank set-up so here it is. I was looking for something totally unrelated, and came across this article, which I will be able to access in full thru my library website next week. But I 'll copy here the abstract (and all other info for anyone wanting to get the full article) because as usual, the abstract (and the conclusion) are all you really need to read Titre du document / Document title Selectivity of color of shelter by Octopus vulgaris and O. aegina under different background colors Auteur(s) / Author(s) OKAMOTO*Masaru (1) ; ANRAKU*Kazuhiko (1) ; KAWAMURA*Gunzo (1) ; TANAKA*Yoshito (1) ; Affiliation(s) du ou des auteurs / Author(s) Affiliation(s) (1) Faculty of Fisheries, Kagoshima University, Kagoshima 890-0056, JAPON Résumé / Abstract Influence of background color in color selectivity of two octopuses Octopus vulgaris and O. aegina was studied in an experimental tank. Black, red, orange, yellow, green, blue, and white were used in a bright environment, and black, yellow and blue were used in a dark environment for the background color of the tank. Shelters of eight different colors (black, red, orange, yellow, green, blue, white, and transparent) were put in the tank and the location of the octopus relative to the shelter was observed and recorded every 30 minutes. It was found that both species chose the black, red and orange shelter more frequently regardless of background color, though there were some differences in the results of the two species. This difference was probably caused by the spectral sensitivity of the species and it was concluded that both octopuses prefer the dark color. Revue / Journal Title Nippon Suisan Gakkaishi* *ISSN*0021-5392* *CODEN*NSUGAF* Source / Source 2001,*vol.*67,*no4,*pp.*672-677*(24 ref.) Langue / Language Japonais Editeur / Publisher Nippon Suisan Gakkai, Tokyo, JAPON* (1932) (Revue) Mots-clés anglais / English Keywords Invertebrata ; Mollusca ; Cephalopoda ; Octopus vulgaris ; Stimulus preference ; Experimental study ; Behavior ; Shelter ; Color ; Selectivity ; Localisation / Location INIST-CNRS, Cote INIST : 2796,*35400009949976.0060 Nº notice refdoc (ud4) : 14151868 p.s. It makes sense to me based on a superfluous assumption- if they don't see red light very well, hence we use it to watch them at night, then that spectrum of color probably looks grey-ish to them which accommodates their desire to hide. So basically you can have a tank with reds and oranges which look appealing to us humans (compared to all black and dark) and it could still be a happy, secure feeling environment for the octo!

subset: commoncrawl


text:
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Previous studies demonstrated that mast cell– derived TNFα stimulation is critical to the upregulation of intercellular adhesion molecule (ICAM)-1 on human conjunctival epithelial cells (HCECs), which is an important feature of ocular allergic inflammation. Shedding of TNFR1 by TNFα-converting enzyme (TACE) is a primary mechanism for the regulation of TNFα-mediated events. This process has not been examined in HCECs. In this study, the authors examined the regulation of TNFR1 expression and shedding by TACE on primary HCECs and the IOBA-NHC conjunctival epithelial cell line.
Primary human conjunctival mast cells and epithelial cells were obtained from cadaveric conjunctival tissue. HCECs were incubated with and without activators (IgE-activated mast cell supernates, phorbol myristate acetate [PMA; to activate TACE], TNFα, and IFNγ [to upregulate TNFR1]) for 24 hours. Pretreatment with the TACE inhibitor TAPI-2 was used to inhibit shedding of TNFR1. Supernates collected from the incubations were analyzed with ELISA for soluble TNFR1 (sTNFR1). With the use of flow cytometry, cells were harvested from these experiments for analysis of TNFR1 and ICAM-1 receptor expression.
IgE-activated conjunctival mast cell supernates upregulated the expression of TNFR1. TAPI-2 inhibited the PMA-induced release of sTNFR1 receptor and enhanced the surface expression of TNFR1 in HCECs in a dose-dependent manner. Upregulation of TNFR1 expression by priming with TAPI-2 and IFNγ resulted in enhanced ICAM-1 expression in response to TNFα stimulation (significant change in the slope of the dose–response curve).
These results demonstrate that TACE promotes TNFR1 shedding in HCECs and that TNFR1 expression may be a more significant target than TNFα for intervention in ocular inflammation.
The proinflammatory cytokine TNFα is believed to play an important role in ocular surface inflammation such as conjunctivitis (allergic, bacterial, and viral) and dry eye disease.1-3 However, the regulation of TNFα on the human conjunctival surface has not been studied. It has been demonstrated in vitro that HCECs respond to recombinant TNFα with proinflammatory responses consistent with those observed in vivo, such as the upregulation of intercellular adhesion molecule (ICAM)-1 expression and increased amounts of the chemokine IL-8. However, regulation of the receptor for TNFα has not been studied in HCECs.
In ocular allergic inflammation, conjunctival mast cells are an important source of TNFα. We have previously demonstrated that IgE-activated human conjunctival mast cell supernates upregulate the expression of ICAM-1 on HCECs by a mechanism specifically dependent on TNFα released from the conjunctival mast cell.4 Our work has further suggested that conjunctival mast cell supernates render HCECs more sensitive to TNFα-mediated ICAM-1 upregulation. In these experiments, we demonstrated that TNFα in mast cell supernates could promote ICAM-1 upregulation at log (10−3) lower concentrations than recombinant TNFα alone.4 We hypothesized that IgE-activated conjunctival mast cell supernates upregulate the expression of TNFR1 on HCECs and that the upregulation of TNFR1 expression results in increased sensitivity to TNFα-mediated activation responses (e.g., upregulation of ICAM-1).
Our first experiments confirmed the first part of our hypothesis, demonstrating that supernates from IgE-activated conjunctival mast cells upregulate the expression of TNFR1 on primary HCECs. However, because purified conjunctival mast cell supernates require large amounts of cadaveric conjunctival tissues, we sought an alternative mechanism to upregulate TNFR1 expression and to test the biological significance of changes in expression of this receptor. Although TNFR1 expression has not been studied in HCECs, in other types of cells it has been demonstrated that TNFR1 is constitutively expressed and that it can, in some cases, be induced at the transcriptional level by IFNγ.5 TNFR1 is primarily regulated posttranscriptionally by the metalloprotease, TNFα-converting enzyme (TACE; also known as a disintegrin and a metalloprotease [ADAM]-17). TACE-mediated proteolytic cleavage of TNFR1 to its soluble form (sTNFR1) is one important mechanism for the down-regulation of TNFα-mediated inflammatory responses.6 TACE is naturally inhibited in vivo by tissue inhibitor of metalloprotease-3 (TIMP-3) and can be inhibited pharmaceutically by a variety of compounds. For the present study, we examined the mechanisms of TNFR1 expression and shedding in HCECs (primary and IOBA-NHC cell line) using the phorbol ester phorbol myristate acetate (PMA), which activates the protein kinase C pathway (a common pathway of HCEC activation) and stimulates reactive oxygen species (known to activate TACE) and the pharmaceutical TACE inhibitor TNFα protease inhibitor-2 (TAPI-2). With these tools we were able to examine the second part of our hypothesis, which demonstrates how changes in surface expression of TNFR1 affect the threshold of responsiveness to TNFα.
Collagenase (type I), hyaluronidase (type I-S), trypsin-EDTA, 2-(hydroxy ethyl) piperazine-N′-(2-ethane sulfonic acid) (HEPES), Triton X-100, density gradient (Percoll; Sigma Chemical, St. Louis, MO), toluidine blue, trypan blue, gentamicin, penicillin/streptomycin, amphotericin, PMA, dimethyl sulfoxide, and bovine serum albumin (BSA), were obtained from Sigma Chemical. Hanks basic salt solution (HBSS; without Ca2+, Mg2+, or phenol red) and newborn calf serum were obtained from Sigma Chemical or Life Technologies (Rockville, MD). TNFα protease inhibitor-2 (TAPI-2) was obtained from Peptides International (Louisville, KY).
Media (EpiLife) for primary cell culture and defined trypsin inhibitor were obtained from Cascade Biological (Portland, OR). Fibronectin/collagen (FNC Coating Mix) was obtained from AthenaES (Baltimore, MD). Wright staining was performed with a staining kit (Diff-Quik; Baxter Scientific Products, McGaw Park, IL). Recombinant human TNFα and IFNγ were obtained from Genzyme Diagnostics (Cambridge, MA).
The Tyrode physiological salt solution plus gelatin (TG) used in these studies consisted of 137 mM NaCl, 2.6 mM KCl, 0.35 mM NaH2PO4, 11.9 mM NaHCO3, 5.5 mM glucose, and 1 g/L mM gelatin adjusted to pH 7.4 with HCl. TGCM is TG with added CaCl2 (2 mM) and MgCl2 (1 mM). The density gradient (Percoll; Sigma Chemical) stock solution was prepared by mixing the commercial solution and 10× HEPES buffer plus dH2O to obtain an osmolality of 285 mOsm/kg H2O. The desired density of the gradient was prepared by mixing the stock solution with TG.
IOBA-NHC (normal human conjunctiva) cell line was derived from normal human conjunctival epithelium (Instituto de Oftalmobiología Aplicada [IOBA; University of Valladolid, Spain] Spanish Patent and Trade Mark Office register number M 2.537.742).7
Culture media for IOBA-NHC cells were prepared by supplementing Dulbecco modified Eagle medium (DMEM) nutrient mixture (F-12 Ham) with mouse EGF (2 ng/mL), bovine insulin (1 μg/mL), cholera toxin (0.1 μg/mL), hydrocortisone (5 μg/mL), and 10% heat-inactivated fetal bovine serum (all obtained from Sigma Chemical).
Modifications of previously reported methods for obtaining purified conjunctival mast cells and primary HCECs in monodispersed suspension were used in these studies.8 Briefly, human conjunctival tissue was obtained with prior consent from organ/tissue donors (8–10 sets of tissue per experiment were obtained through the Lion's Eye Bank of Wisconsin, Madison, WI, and the Kansas Eye Bank and Cornea Research Center, Wichita, KS, and were approved by the University of Wisconsin Human Subjects Committee). Upper and lower bulbar conjunctiva aseptically collected within 8 hours after death (average time, 4.5 hours) were transported in corneal preservation medium (Dexsol; Chiron Ophthalmics, Irvine, CA) and were stored at 4°C for up to 5 days. Eight to 10 sets of tissue weighing 4 to 5 g were used per experiment. Hyaluronidase and collagenase were used to digest tissue. The digestion process (30 minutes at 37°C on a rotating shaker) was first performed at a low concentration of enzymes (2 digests at 200 U/g in 10 mL final volume), followed by tissue digestion at a high concentration of enzymes (3–6 digests at 2000 U/g in 10 mL final volume). Each digest was followed by washing of the enzyme-treated tissue (with TGCM) over a 100-μm nylon mesh filter to collect freed cells. After the digestion procedure, the freed cells were pelleted, pooled, resuspended in TG, and layered over a single-density gradient (Percoll [Sigma Chemical]; 1.041 g/mL) and centrifuged (500g, 20 minutes). The resultant top cell layer (epithelial cells) was harvested, washed, and resuspended in media (EpiLife; Cascade Biological) without hydrocortisone, at a concentration of 1 × 106 cells/mL, and was transferred to fibronectin/collagen (FNC Coating Mix; AthenaES)–coated 24-well plates (0.5 mL/well) for culture at 37°C. Media were changed every 48 hours until confluence. Primary HCECs were passaged (10 passages) using trypsin-EDTA and a serum-free defined trypsin inhibitor. The mast cell– enriched pelleted cells were washed in TG, resuspended to a concentration of 1 × 106 cells/mL in mast cell culture medium, and transferred to a 24-well plate (0.5 mL/well) for an equilibration period (up to 72 hours at 37°C). After the equilibration period, the cell suspension was removed from the plate, layered over a double-density gradient (Percoll [Sigma Chemical]; 1.08 g/mL layered over 1.123 g/mL), and centrifuged (500g, 20 minutes). Purified conjunctival mast cell suspensions (>90%) harvested from the interface between the densities were washed and allowed to equilibrate in mast cell culture medium for 24 hours at 37°C before experimentation. Differentiation of other cells was obtained using Wright-stained cytospins.
IOBA-NHC cells were cultured in the media defined (see Reagents and Solutions) until almost confluent. Media were changed every 2 days. Cells were passaged using incubation with trypsin-EDTA, which was neutralized using DMEM supplemented with 20% newborn calf serum. For storage, the cells were frozen in media containing 20% heat-inactivated fetal bovine serum (FBS) and 10% dimethyl sulfoxide using a cell-freezing container (Mister Frosty; Nalgene Nunc International, Rochester, NY) and stored at −80°C. When needed, cells were quickly thawed in a 37°C water bath and transferred to culture media including antibiotics and extra FBS (20%). After 24 hours, the media were changed to regular culture media.
All mast cell challenges were conducted in TGCM at a concentration of 1 × 104 mast cells/mL (in duplicate). Purified conjunctival mast cells were challenged with anti-IgE antibody at a dose of 10 μg/mL (previously determined to induce mediator release from purified mast cells) or buffer (spontaneous control) for 90 minutes at 37°C.9,10 The resultant supernates were harvested and stored at −80°C until time of use.
HCECs were cultured until almost confluent (24–48 hours after passage) on 24-well plates (3–5 passages for primary HCECs). When mast cell supernates were used, the supernates (in TGCM buffer) were combined 1:1 with media and added to the wells (thus diluted 1:2) and incubated with HCEC monolayers (0.5 mL/well, 2–4 wells/treatment) for 24 hours. Unstimulated HCEC controls were incubated in a 1:1 mixture of TGCM/media for evaluation of constitutive ICAM-1 and TNFR1 expression.
In experiments examining the mechanisms of TNFR1 shedding, similar monolayers were prepared on 24-well plates. To investigate the role of endogenous TACE, TACE inhibitory compounds, TAPI-2 (10 and 100 mM), and EDTA (0.5 and 5 mM) were preincubated with the cells for 15 minutes at room temperature before activation with PMA (10 ng/mL) for 24 hours (0.25 mL/well, 4–6 wells/treatment). Where the effect of TAPI-2 and IFNγ priming on the dose–response curve to TNFα was being examined, the cells were preincubated with TAPI-2 (100 mM) as described, followed by 24-hour incubation with IFNγ (0.5 ng/mL). The cells were then washed and stimulated for 24 hours with TNFα (0.5, 5, and 50 ng/mL).
After 24-hour incubation with the various treatments, supernates were harvested and stored at −80°C for evaluation of sTNFR1 and sICAM-1 by ELISA. HCEC monolayers were harvested with trypsin-EDTA and resuspended in buffer. Cell counts were performed on the harvested cells using a Coulter counter (model ZM; Coulter Corp., Miami, FL) to confirm that cell counts did not vary significantly between cultures. To simultaneously measure ICAM-1 and TNFR1, each tube of 100 μL viable cells (not fixed) was stained with mouse anti– human ICAM-1(CD54)-APC–conjugated antibody and mouse anti– human TNFR1-PE–conjugated antibody or isotype-matched controls using the manufacturer's recommended amounts (10 μL/tube). Stained cells were incubated on ice for 30 minutes, washed, and resuspended in 300 μL/tube of buffer for analysis. Propidium iodide was added to each tube to determine viability. Resultant histograms were analyzed geometrically and based on gating of live cells only.
Data were analyzed using statistical software (Minitab, State College, PA). A general linear model analysis of variance with preplanned comparisons was used to generate two-tailed P values. P < 0.05 was considered statistically significant. Unless otherwise stated, all data are presented as the mean ± SEM of three to seven separate experiments.
The representative overlay histograms (n = 3) in Figure 1 demonstrate that stimulation of primary HCECs with supernates from IgE-activated conjunctival mast cells significantly upregulated the expression of TNFR1 (increase of 2 ± 0.4 mean fluorescence intensity [MFI] units; P = 0.03).
The goal of these experiments was to demonstrate that TAPI-2 inhibition of TACE resulted in the inhibition of shedding of sTNFR1. If positively demonstrated, we could then use TAPI-2 to increase the expression of TNFR1. Because our hypothesis was to demonstrate that increased expression of TNFR1 would result in increased sensitivity to TNFα-stimulated ICAM-1 upregulation, it was also important to examine whether TAPI-2 had any effect on the release of soluble ICAM-1 (sICAM-1). Stimulation of HCECs with PMA (which activates TACE) resulted in the significant release of sTNFR1 in the IOBA-NHC line (Fig. 2A; n = 4, P < 0.05). This was significantly inhibited by the TACE inhibitor TAPI-2, suggesting that shedding of sTNFR1 is TACE mediated (P < 0.05). Results were similar in primary cells, though unstimulated primary HCECs appeared to release higher constitutive amounts of sTNFR1 (n = 2; Fig. 2B). This inhibition was dose dependent, as shown in the representative curve (showing percentage inhibition) in Figure 3A (IOBA-NHC) and and3B3B (primary HCECs). Figure 3 also demonstrates inhibition with EDTA (positive control) that nonspecifically inhibited metalloprotease-mediated shedding by chelation of divalent metal ions (in this case, zinc). Given that ICAM-1 can also be shed in a soluble form, it was important to examine whether TAPI-2 has an effect on sICAM-1 shedding. Figure 4 (representative bar graphs showing percentage inhibition; n = 2) compares the effects of TAPI-2 and EDTA (5 mM [EDTA concentrations 5 mM promote the detachment of IOBA-NHC cells and primary HCECs]) on PMA-stimulated sTNFR1 concentrations to sICAM-1 concentrations in the same supernates. In IOBA-NHC (Fig. 4A), TAPI-2 inhibited sTNFR1 production but had a negligible effect on sICAM-1, whereas EDTA inhibited sTNFR1 and, to a lesser extent, sICAM-1. In primary HCECs (Fig. 4B), results were similar for the inhibitory effect of TAPI2 and EDTA on sTNFR1 production. However, primary HCECs appeared to be more sensitive to the EDTA inhibition of sICAM-1 release. These data were important in ruling out a direct effect of TAPI-2 on ICAM-1 in subsequent experiments.
To further support our data suggesting that increased expression of TNFR1 results in increased responsiveness to TNFα-mediated ICAM-1 expression, we had to find a way to increase TNFR1 expression on HCECs to an extent similar to that observed with IgE-activated mast cell supernates. In these experiments, we preincubated HCECs with TAPI-2, followed that by challenge with IFNγ, and examined TNFR1 expression using flow cytometry. The overlay histogram in Figure 5 demonstrates that this treatment significantly upregulated TNFR1 expression by a magnitude similar to that observed with IgE-activated mast cell supernates (increase in MFI of 2.7 ± 0.9; P = 0.04; n = 4). TAPI-2 alone slightly, though not significantly, enhanced TNFR1 expression, and IFNγ alone did not upregulate TNFR1 (data not shown). Responses were similar using the IOBA-NHC line (data not shown).
Upregulation of ICAM-1 expression in response to TNFα in unprimed versus TAPI-2/IFNγ-primed HCECs is shown in Figure 6. ICAM-1 expression in unstimulated cells was significantly greater in the IOBA-NHC cell line (82.4% ± 8.2% positive cells) than in primary HCECs (22% ± 3.2%); therefore, the data in Figure 6 are expressed as percentage positive cells greater than unstimulated cells for the purpose of comparison of the responses between the cell line and primary HCECs. The dose–response curves to TNFα in Figure 6A demonstrate that increasing TNFR1 expression using priming with IFNγ and TAPI-2 correlated with significantly enhanced sensitivity to TNFα-mediated upregulation of ICAM-1 expression on IOBA-NHC (left y-axis; percentage ICAM-1 expression greater than unstimulated cells; P < 0.05 for all comparisons of TNFα-challenged cells between unprimed and primed treatments, including slopes; n = 4). To reinforce our findings that TAPI-2/IFNγ priming does not affect the release of sICAM-1, we measured sICAM-1 released into supernates from the same experiments. These results, also shown in Figure 6A, demonstrated that though TNFα appeared to promote the release of sICAM-1, there was no effect of TAPI-2/IFNγ priming on this process (right y-axis; sICAM-1 concentration [minus unstimulated] in pg/mL). In primary HCECs, TAPI-2/IFNγ priming resulted in a similarly enhanced response to TNFα-mediated ICAM-1 upregulation (Fig. 6B; 5 ng/mL concentration of TNFα shown; n = 2). In IOBA-NHC cells and primary HCECs, priming with TAPI-2/IFNγ alone (no TNFα) resulted in a slight but nonsignificant upregulation of ICAM-1 expression.
Our data demonstrate that IgE-activated conjunctival mast cell supernates upregulate TNFR1 on HCECs. Manipulation of TNFR1 has led us to a better understanding of the mechanisms and consequences of TNFR1 regulation on HCECs. Using PMA to activate TACE and the TACE inhibitor TAPI-2, we confirmed that the regulation of TNFR1 on the cell surface involves TACE-mediated cleavage of the receptor to its soluble form. We were further able to demonstrate (using IFNγ and TAPI-2 priming to increase TNFR1 expression) that even small changes in TNFR1 can result in significantly enhanced responses to TNFα, such as ICAM-1 upregulation. This is important because, in ocular allergic inflammation, ICAM-1 expression is upregulated on the conjunctival epithelium and is believed to play an important role in the maintenance of inflammation on the ocular surface by participation in the migration and adhesion of proinflammatory leukocytes.11 In murine allergic conjunctivitis, antibodies targeting ICAM-1 have been successful at reducing symptoms and infiltration of inflammatory cells.12 Therefore, therapies targeted toward inhibiting TNFα-mediated upregulation of ICAM-1 expression could be beneficial. Our data suggest that TNFR1 expression is more important than TNFα levels in regulating these inflammatory processes on the ocular surface.
Members of the TNF family of receptors have been identified on the conjunctival epithelium in a rabbit model of ocular inflammation but have not been previously studied in HCECs.13 However, TNFα-mediated upregulation of ICAM-1 through TNFR1 has been demonstrated in human retinal pigment epithelial cells.14 There are two distinct membrane receptors for TNF (TNFRs), the p55-kDa TNFR1 (or CD120a) and the p75-kDa TNFR2 (or CD120b). Although most cells coexpress both receptor types, cellular responses for soluble TNFα seem to be dominated by the interaction with TNFR1, whereas cell surface TNFα is the prime physiological activator of TNFR2.15 TNFR1 activation appears to induce inflammation and defense against intracellular pathogens.16,17 It has further been demonstrated that the level of surface expression of TNFR1 can be an important factor in modulating the magnitude of TNFα-mediated signal transduction events.18
Although TNFα plays a pivotal role in the immune response to infection, left unchecked, TNFα-mediated inflammation can turn from beneficial to harmful. Regulation of the availability of TNFα receptors provides a protective mechanism against excessive TNFα activity. Proteolytic cleavage (facilitated by TACE and inhibited by tissue inhibitor of metallopreases-3 [TIMP-3]) results in receptor downregulation that may serve to decrease cellular sensitivity to TNFα (Fig. 7). Therapies targeting TNFα (e.g., antibody, soluble receptor) have been developed to manage chronic diseases, such as rheumatoid arthritis and inflammatory bowel disease, in which the dysregulation of TNFα appears to play a critical role in driving inflammation, but TNFα has not specifically been targeted in chronic allergic conjunctivitis. Recently, pharmaceutical TACE inhibitors have been developed to inhibit TNFα because TACE also cleaves TNFα from its inactive, proform on the cell surface to its active form.19 The compound we used in our studies, TAPI-2, is one such compound. A derivative of hydroxamic acid, TAPI-2 is known to inhibit TACE by binding to the active site of this metalloprotease, thus interfering with its zinc-dependent activation.20
Interestingly, however, our study suggests that TACE inhibitors may have some unintended consequences that may conflict with their ability to neutralize TNFα-mediated inflammation (e.g., inhibition shedding of TNFR1 and enhanced responsiveness to TNFα-mediated ICAM-1 upregulation). Furthermore, TACE has multiple additional targets that affect inflammation, including IL-6 and its receptor (IL-6R), transforming growth factor-α (TGFα), membrane-bound epidermal growth factor (EGF) family ligand, L-selectin, and macrophage colony-stimulating factor receptor.21-24 A recent study has also suggested that TACE promotes the shedding of soluble ICAM-1 receptor in endothelial cells.25 Therefore, TACE inhibitors might be expected to upregulate ICAM-1 expression as well as TNFR1 and IL-6R through the inhibition of shedding. Our studies, however, suggest that ICAM-1 shedding in HCECs is facilitated by a metalloprotease distinct from TACE (not inhibited by TAPI-2 but inhibited by EDTA). One explanation for this discrepancy could be that mechanisms of shedding of certain receptors are cell specific and vary depending on cell type. For example, another study has suggested that the matrix metalloprotease MMP-9 can regulate ICAM-1 shedding in osteoblasts26; we will examine these mechanisms further in HCECs.
Another important observation resulting from our studies is that there are some constitutive differences between primary HCECs (derived from cadaveric conjunctival tissues) compared with the spontaneously immortalized IOBA-NHC cell line. For example, constitutive expression of ICAM-1 is higher in unstimulated IOBA-NHC cells than in primary HCECs. Conversely, constitutive release of sTNFR1 is greater in primary HCECs than in the IOBA-NHC cell line. These baseline differences are important when interpreting the magnitude of responses to various stimuli. Another interesting difference observed in our studies was that IOBA-NHC cells appeared to be less sensitive to EDTA inhibition of the metalloprotease involved in the shedding of sICAM-1. Although further examination of this process is required, one interpretation could be that IOBA-NHC cells produce larger concentrations of certain metalloproteases (or divalent metal ions) than primary HCECs. These differences illustrate that though the IOBA-NHC cell line is useful for the execution of large experiments, it is essential to confirm any research using cell lines with parallel studies in primary cells. This is supported by gene expression profiles deposited in GenBank (accession number GSE8633), which compare conjunctival tissue with primary HCECs and the IOBA-NHC and ChWK cell lines (Gene Expression Omnibus Web site). These data suggested that, globally, primary HCECs clustered more closely to conjunctival tissue than either of the two cell lines and that, in general, the IOBA-NHC cell line was better correlated with primary HCECs than the ChWK cell line.
In conclusion, TNFα is increased on the ocular surface in various types of ocular surface inflammation such as chronic ocular allergic inflammation and dry eye disease.27 In these diseases, HCEC activation by TNFα, including the upregulation of HCEC receptors such as ICAM-1, is believed to play a significant role. Our in vitro studies have demonstrated that mediators from IgE-activated human conjunctival mast cells upregulate the expression of TNFR1 on HCECs. Although the mechanism through which mast cells upregulate TNFR1 needs further investigation, we have demonstrated that the magnitude of this upregulation correlates with an increased sensitivity of the HCECs to TNFα-mediated upregulation of ICAM-1. The potential use of therapies specifically targeting TNFα in ocular surface inflammation, such as dry eye disease and chronic allergic conjunctivitis, has been suggested. Although TACE inhibitors such as the one used in our study were initially developed for pharmaceutical inhibition of TACE-mediated TNFα activation, it is likely that we still do not have a comprehensive understanding of the targets of TACE and the consequences of TACE inhibition. Our study suggests that upsetting the balances of these processes may have conflicting consequences and that downregulation or blocking of TNFR1 may be a more beneficial avenue of inhibiting TNFα-mediated responses on the ocular surface than targeting TNFα itself.
The authors thank Kathy Schell (Flow Cytometry Facility of the University of Wisconsin-Madison Comprehensive Cancer Center) for her expert advice and support in setting up the flow cytometry analyses.
Supported by National Institutes of Health/National Eye Institute Grant R01EY012526 and by an unrestricted grant from Research to Prevent Blindness.
Disclosure: E.B. Cook, None; J.L. Stahl, None; F.M. Graziano, None; N.P. Barney, None
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text: I've seen a client tonight who's asked me to work on branding an event they're hosting and dealing with all associated web and print collateral. Besides the website and brand there will be an unknown quantity of vinyl banners, media advertising, posters, event guides, tickets, merchandise etc etc etc.. This is a long standing client who's usually happy to pay per job but I'm wondering if it'd be better (for me & my cash flow) to quote an all inclusive price? If so how the hell do I do that with so many anomalies!?!


text:
Michael Buble Takes Us Through All Stages of Life + Love in 'I Believe in You' [VIDEO]
This video takes us on a love struck couple's beautiful journey through life.
This video takes us on a love struck couple's beautiful journey through life.
'Stay positive...that outlook on life that you can get through something, it actually does get you through,' Doherty said.
Congratulations to Teresa Choppa! We're sending her to New York City to see Michael Buble's show at Madison Square Garden on July 8.
Michael Buble's To Be Loved tour is making a stop at Madison Square Garden in New York City this summer and we want you to go.
There can't be much better than a summer concert in the Bahamas. Well, I guess it could be a little better if the trip, accommodations, tickets and spending money are all part of a prize package that you won! You could win this package to see Michael Buble at the Atlantis Paradise Island Resort and go backstage and Meet Michael on us!
We at PopCrush present to you another exciting edition of Kelly Clarkson's 'Kelly Covers…' ! Girl, for real, we can barely keep up. As usual, Clarkson's covers are frickin' phenomenal. Is there anything this girl can't sing?
Michael Buble is one busy crooner! Not only is he the self-proclaimed "Christmas Prostitute," he's also a pretty funny guy, serving up a great comedy sketch this weekend on "Saturday Night Live."
Catch Buble's comedy in a hilarious spoof commercial promoting a "Duets" CD with Lady GaGa, Justin Bieber, Taylor Swift, Sting, Scotty McCreery and others. Then check out Michael's live performances of "Holly Jolly Christmas" and "Have Yourself A Merry Little Christmas" after the jump.
Former comic and current crooner Michael Buble combined the best of both his talents — and the best of both big December holidays — when he performed a 'Chrismukkah' duet with a TMZ staffer. He also had some harsh words for himself!
When it comes to popular music, Michael Buble is Mr. Christmas for 2011. The crooner claims the No. 1 spot on the Billboard album chart for the second consecutive week with 293,000 copies sold of his 'Christmas' album, which shows no signs of slowing down.
Michael Buble frolics in the snow in the music video for his big band version of 'Santa Claus Is Coming to Town,' a song from his new 'Christmas' album.
Kim Kardashian has taken a big public relations hit ever since the end of her marriage to Kris Humphries after 72 days suggested that their much ballyhooed and televised wedding was a complete sham. Case in point, even nice guy Canadian crooner Michael Buble has turned on the most famous of Karadashians. And you won't believe what he called Kim during a recent concert.
Nothing beats Christmas in New York City, and this holiday season you may find yourself meeting Michael Bublé in person right after seeing him perform at the Christmas tree lighting at Rockefeller Center. Cars 108 has your chance to win - just enter your email address below.


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We spent two nights in this casa particular in March 2015. The family is very nice and helpful. The room was nice and clean. The food was yummy! We had a problem with a flat tire and Jorge, Nilda's son, helped us to arrange the...More
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"Rooms have ensuite bathrooms, hot water, fans, 2 double beds. Cozy, clean."


text: Building Executable Biological Pathway Models Automatically from BioPAXTimo Willemsen, K. Anton Feenstra, Paul T. GrothLISC@ISWC2013The amount of biological data exposed in semantic formats is steadily increasing. In particular, pathway information (a model of how molecules interact within a cell) from databases such as KEGG and WikiPathways are available in a standard RDF-based format BioPAX. However, these models are descriptive and not executable in nature. Being able to simulate or… (More)


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San Francisco 49ers Custom Concept Rawlings Full Size Football Helmet!!
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This is a full size Authentic Rawlings Momentum Custom Concept San Francisco 49ers football helmet. This is a brand new helmet that has been fully customized. The helmet features a brand new matte black paint job, new black Schutt facemask and high quality 20 mil decals. This helmet is being sold for display purposes only . This helmet is not approved for or intended for game use. The buyer assumes all responsibility for any injury that may occur from wearing or misusing this helmet. Please check out my other items. Thanks for looking! PayPal only please. ��


text: Hi guys I am Ian, and I run Nutshell. We've been in business since 1997 - making leather belt cases for all types of mobile phones, PDAs and digital cameras. We look forward to being of service to the members of this community. If there's something you need in the way of a belt case to protect a device and you can't find it elsewhere, just ask. If we haven't got it, then we can most likely create it for you. Best regards


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- PX-4 .45 Custom Airsoft GBB Pistol replica
- Rotating Barrel Lockup System
- Large Piston design same as FN5-7 to provide higher air-flow & recoil energy
- Ambidextrous safety lever (also work as decocking lever)
- New Hop-Up Unit can be adjusted without disassembling the gun
All products item displayed are TOYS
- In the shop with Cash, Debit/Credit Card and Installment payable
No airsoftgun should be carried in a public place unless in transit when it should be unloaded and placed in a bag. Airsoftguns must NOT be concealed on one's person, or pointed or fired at another human or animal. Safety glasses and other protective clothing should always be worn when operating or viewing the firing of an Airsoft BB gun.
We are not responsible for any harm or accident caused by any misuse of these Airsoft guns
Although the information and recommendations on this website are presented in good faith and believed to be correct, we makes no representations or warranties as to the accuracy of the information. Information is supplied upon the condition that the persons will make their own determination as to its suitability for their purposes prior to use. In no event will we be responsible for damages of any nature whatsoever resulting from the use of or reliance upon information from this site or the products to which the information refers. We do not warrant the accuracy on this website and has no liability for any errors or omissions in the materials.
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- Gary R. Bauchan *,
- T. Austin Campbell and
- M. Azhar Hossain
A cytogenetic investigation was conducted on the tetraploid alfalfa [Medicago sativa subsp. sativa (L.) L. & L.] Chilean germplasm source PI 536534 using the combined techniques of C-banding and image analysis. Cluster and multiple correspondence analyses were utilized to compare the C-banding patterns of the Chilean germplasm source and the previously published African germplasm source. Cytogenetic analyses revealed polymorphisms for heterochromatic DNA in the 19 plants observed in detail. Abundant variability in the number, intensity, and location of constitutive heterochromatic DNA was noted; however, this variability was not sufficient to preclude recognition of homologous chromosomes. Five out of the 50 plants studied were aneuploids (2n = 4x + 1 = 33 or 2n = 4x − 1 = 31) because of the presence or absence of a chromosome with a satellite. The Chilean karyotype resembled the reference tetraploid African alfalfa karyotype; however, a reduction in the total amount of heterochromatic DNA was observed. Cluster analysis and multiple correspondence analysis based on all eight alfalfa genome chromosomes yielded no clear separation of Chilean and African germplasms. However, the analysis of C-banding patterns of Homolog 1 of Chromosome 8 in Chilean and African germplasms was effective in separating the two germplasm sources with the exception of two individuals from each germplasm source which clustered together.Please view the pdf by using the Full Text (PDF) link under 'View' to the left.
Copyright © 2002.


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Google Sites Check-Up
Many user created Google Sites have some common issues in the design and set-up of the Site that degrade the appearance, performance and usefulness to the visitors. A Google Sites Check-Up by Etter Ventures can greatly enhance the appearance and performance of your existing Site.
Common problems that can be addressed, resolved and eliminated include:
A Google Check-Up Can Implement
For additional information on our
Sites Check-Up services,
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Nearby points of interest
Distance: 0.50mi
Distance: 0.66mi
Distance: 0.69mi
Distance: 1.26mi
Distance: 0.50mi
Distance: 0.66mi
Distance: 0.69mi
Distance: 1.26mi
Nearby points of interest
Distance: 0.50mi
Distance: 0.66mi
Distance: 0.69mi
Distance: 1.26mi
Distance: 0.50mi
Distance: 0.66mi
Distance: 0.69mi
Distance: 1.26mi
About La Casa De Pedro
Come experience a true taste of Venezuelan hospitality and fine Spanish cuisine in Watertown, MA, just minutes from downtown Boston and Cambridge. Sip a Passion Fruit Mojito or one of Pedro"s Specialty Margaritas while dining on our beautiful outdoor patio. Savor Venezuelan specialties like Pabellón Criollo, Camarones al Ajillo, Paella and other lovingly created Latin American dishes. Pedro's restaurant offers semiprivate dining for large groups and functions such as business meetings, private parties, wedding receptions, anniversaries and other celebrations. Share delicious, traditionally prepared Latin American food and drinks and create warm memories with friends and family… Join us in celebrating life, love and friendship amid the upbeat tempo and tropical color of Pedro's "casa" at The Arsenal on the Charles in Watertown.
Watertown, MA 02472
Dining Style:
Casual Dining
South American, Mexican
Hours of Operation:
Brunch: Sunday: 11:30am - 3:00pm; Lunch: Daily: 11:30am - 3:00pm; Dinner: Monday - Thursday: 3:00pm - 9:00pn, Friday & Saturday: 3:00pm - 10:00pm, Sunday: 3:00pm - 8:00pm
Dress Code:
Casual Dress
Price Range:
$30 and under
Payment Options
AMEX, Diners Club, Discover, MasterCard, Visa
Phone Number:
(617) 923-8025
Live music on Friday and Saturday
Public Transit:
MBTA Bus 70 runs next to us
Executive Chef:
Pedro Alarcon
Private Party Facilities:
A great semi-private room available for your best convenience, weddings, birthday party or any event.
Private Party Contact:
Manager: (617) 923-8025
Banquet, Bar Dining, Bar/Lounge, Beer, Dancing, Entertainment, Full Bar, Gluten-free Menu, Happy Hour, Outdoor dining, Private Room, Weekend Brunch, Wheelchair Access, Wine
Special Events & Promotions:
Restaurant Photos
La Casa De Pedro Ratings and Reviews
Another terrific meal at Casa De Pedro. The Pork Osso bucco is so good that I am hard pressed to order something else. The garlic shrimp appetizer was great as were the nachos. The shrimp Kristofer was delicious again but they didn't include the coconut when we asked to take the leftovers home . even though we were stuffed one of the guys wanted coffee and dessert well I have to say that the CHURROS were to die for. The tabe we had for 6 was comfortable but awkward and the waiter had to circumnavigate the restaurant to go from 1 side to the other.
They also make very good ( if pricey) Margharitas
Over all just a great spot and when the weather warms up the patio is also great!
the meal appeared "mass" prepared and possibly microwaved or reheated. Used to eat at original casa Pedro.Possibly it was the intimacy of a smaller place ; but I believe the food was more authentic and certainly much tastier. The bar was very pleasant and the drinks well mixed. Unfortunately not worth repeating the culinary experience.
We keep trying this restaurant b/c we wanted it to be wonderful and delicious, but after four times, we are giving up. This most recent time we ate outside (only two other tables were occupied there) and from the moment we sat down things were not right. The waitress, although pleasant when addressing us, was usually absent from sight; I had to ask another server three times to get her and after the food was served (by another server) she disappeared. I later saw her both in the bathroom and the kitchen "fixing" her hair. We also didn't get our check so we had to go to the hostess to pay our tab.
On to the food - terrible! Partly b/c everything we were served was close to ice cold. I'm sure, since it was a long time between the time we ordered and the time we were served, it sat waiting but I would think it would have been under a warmer. In addition to being cold, there was an excess of oil on several dishes (and cold oil is even more yucky than warm oil). The plantain chips tasted like they were right out of a Frito-Lay bag. The calamari might have been good, but it was the coldest of all the dishes.
I love the idea of a Venezuelan restaurant in the suburbs, esp. b/c we frequently go to the New Rep right next door, but we are certainly finished giving it a second (and third, etc) chance.
Came as the crowds were leaving. Bar service at the table was excellent but slow. Again, probably the shift change, people, checking out etc. after the crowds left, the music kicked up a pace and the dancing began.
Menu huge and specials looked great. Only complaint: salads had too much cos and not enough of the rest of the good stuff.
Did wonder if our delicate stomachs the next day were related to the meal, or the Hendricks martini with Jalapeño.
Went for brunch. Had the huevos rancheros which was good but my friend's breakfast quesadilla looked better with more elements to it being served with fried plantains and black beans. 3 varieties of mimosas available. I had the traditional one that was very good
This was not a good experience. The food (bacalao) was overly spicy although the menu didn't warn it was spicy. The white sangria tasted like hard liquor although the menu only listed wine in the ingredients. We were charged $35 for the white sangria, although that is the price for champagne version and ours was supposed to be $30. At first the setting was quiet enough to talk but then a band started playing and it was impossible to visit with our friends who we don't see very often. The music would have been nice if that had been the purpose of our visit but it was overpowering otherwise!
I have eaten at Casa de Pedro many times prior to attending the theater. This past Saturday was very disappointing. The only thing good was eating outside. We were seated at 6:25pm. We were not served our food until 45 minutes after we ordered. My fish, although cooked, was barely lukewarm. The service was very poor, although the wait staff was pleasant.
I was very disappointed.
I do find the food choice and quality to be quite nice. The ambiance is authentic and eating outdoors is a real treat as you don't know you're in the middle of Watertown. Quite reasonable for a family dinner or just a night out. I return several times a season.
We live close by and eat here often. The food is consistently great, the portions large, and the wait staff very friendly. We usually get one of the seafood/fish dishes. The chicken vegetable soup is a favorite starter, although almost large enough to be a meal in itself. One of our guests decided to get a couple of appetizers instead of a main meal and ended up with more food than we did.
Went here for dinner spur of the moment after helping our daughter move. We had never had Venezuelan food and we were not disappointed. The food is excellent! The service was wonderful...our server was attentive but not intrusive. We had a very relaxing meal. Perfect way to end a busy day! Highly recommend!
Wonderful food, drink, ambiance, and staff! After being a faithful customer for the past five years, the food--like the drink--menu could be expanded to include more variety of appetizers and entrees. It would keep the "regulars" coming back more frequently !
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Selecting the best multifunctional dry bag backpack can be very tedious process requiring a lot of research. The Aqua-Quest 'The Stylin' is a great solution. This bag is great for a number of different outdoor hobbyists, including hunters, backpackers, boaters, hikers, fishers, commuters, cyclists and anyone else seeking a rainproof backpack to safely store gear.
I am confident anyone looking at watertight backpacks is getting a great value for their money with 'The Stylin'. This backpack has been tailor made to meet the needs of anyone in a challenging climatic condition. Designed and manufactured with a strong structural base and impressive weave design, this is one of the best durable backpacks in its class. If you're looking for a solid dry backpack, this is your answer.
There are so many different backpack models around that finding the right model for your needs can be confusing at times. Finding a product brand to trust is important and finding a great product to fit within your budget can be challenging. This backpack luckily delivers on both of those points to provide you with one of the best values on the market. 'The Stylin' is one of thebest durable backpacks and it is the perfect buy for a wide variety of different users.
Great Backpack for Daily Use
I normally ride my bike to work, and this backpack was purchased to be used as a carry bag to bring my lunch, jacket and laptop with me to the office. Living near the ocean, it's common for a storm to appear out of nowhere, and I need a bag that is going to keep all of my possessions dry if I happen to be on the bike. 'The Stylin' has been great so far.
This backpack also has a very sport and stylish appearance which is not only useful for carrying with me on the bike, but is also nice when walking into the office. This model has a vented back pocket and a zipped top pocket which provides convenient access to the vast storage areas.
For me, the small outside pocket is convenient to store my bike's lock/cable and its reflective stripping has been great when biking home in the evening time.
Aqua-Quest 'TheStylin' Product Features:
- Aqua-Quest 'The Stylin' Dry Bag is the best you can find anywhere. They are durable and 100% Waterproof.
- Durable and made of a RIPStop TPU laminated coating that is Oxford 400D Abrasion resistant, providing a tough strength and ability to repel moisture (click here to see more backpacks made of this material)
- A logo which reflects for safety on the road, as well as stress points which are double bar tapped. These straps are comfortably padded and have straps which are contoured for wide support.The straps easily adjust to fit nearly all body sizes.
- The front sleeve features and easy-access storage pocket and waterproof front flap-pocket.
- The size is 27.5×12 in when flat, has a capacity of up to 1800 cu in., weighs 1.9 pounds and when closed is its 21.5 x12in.
How Does It Rate Outside of the City?
I have taken this bag out one weekend mountain biking in the country, and this bag was great for the trip. It allowed me to carry all of the stuff I would need for the trail, including plenty of food and water. The quality of this bag is great, and I had no issues with the durability of the backpack – even in a rugged outdoor environment. It did not rain on this trip, but the bag held up with the moisture that is common in the morning.
I'm in Love with This Bag
After searching for a quality dry backpack, the hunt was over after testing out this dry backpack. I've used this bag for over a month now, and the experience has been exceptional the whole time. Aqua-Quest 'The Stylin' dry bag backpack is a great multi-purpose bag for anyone needed to protect their bag's contents in wet situations. As I am constantly biking around the city, having a bag that is truly water resistant is important for me. I've found that 'The Stylin' has passed all of the tests that it has run into so far. The 30L model watertight backpacks tested because of the additional space offered being needed to carry all of my daily items with me to the office. This bag has been awesome and was definitely a great buy.


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First Tutors helps you to find Stirling religious studies tutors in your local area. First Tutors profiles Stirling religious studies tutors offering popular subjects such as English, Maths and French, but also less common subjects such as Latin. Entrance exams, SATs, GCSEs and A-Levels private religious studies tuition are all catered for across Stirling. Please search for Stirling home religious studies tutors near you and select which most suits your needs.
This is the only place to discover the best Stirling religious studies tutor for your needs, whether you are looking for local religious studies tuition to boost your child's religious studies confidence, your own online religious studies education or you are seeking adult / casual religious studies education.
Our search will find Stirling religious studies tutors in your area by their charges and proximity to you. Our subjects include, but are not limited to, English, Maths and French from primary level right through to GCSE, A-Level and on to undergraduate level tuition.
If you are a Stirling religious studies tutor wishing to offer your local religious studies teaching services in Stirling or anywhere else across the UK, please register with First Tutors or find out more about us here.
Towns: Callander, Crianlarich, Denny, Doune, Dunblane, Falkirk, Grangemouth, Killin, Larbert, Lochearnhead, Stirling


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Food, or chemical-biological products storage (blood or plasma, medicines...) is a fundamental process in modern industry, with the primary purpose to preserve the characteristics of products, preventing accidental changes. All the hospitals, the medical or chemical laboratories, the Ho.Re.Ca industry (Hotellerie, Restaurant, Café) and the GDO (Great Organized Distribution) have an obligation to hold the specific conservation equipment that keeps the substances that guard unchanged: think of the blood conservation for transfusions, the conservation of human organs, or special medicines. There are also many applications in the food industry: from the display cases for wine or sweets to professional refrigerators and chillers, up to the cabinets for food maturing or drying.
We do care about all this, applying our experience in this field, providing the customer a wide range of programmable and universal electronic controllers which are used in all machines where it is required precise control of temperature and humidity, together with the possibility to manage, through advanced user interface, the different parameters that characterize and specialize the particular application.
Controller for retarder-prover cabinets
Controlled fermentation is used for bread, pizza and pastries dough through the proper management of temperature, humidity and time. In fact, the retarder proving blocks or slows down the fermentative activity of the yeast, maintains the dough in the required state for some hours, and finally automatically begins the phase of fermentation at the time established though the program.
Our controller for retarder-prover cabinets FE1030 is therefore essential in artisan and industrial workshops where leavened products are produced (pastry shops, bakeries, pizzerias), as it allows:
- the elimination or reduction of night work;
- the reduction of labor and general costs;
- less use of additives and yeast;
- a better quality of the product as long leavening process gives the bread more flavor and extend its life;
- a more streamlined and efficient production process;
- a minor impact of external climatic effects on the product.
In general, a better quality product and labor markets. Also manual programs for proving and cooling functions are provided.
The proposed kit for the application of retarder control consists of:
- a baseboard;
- a user interface board including an alphanumeric LCD display 2x24;
- a NTC temperature probe with cabling;
- a humidity probe with cabling;
- wiring for the connection between the main board and user interface board.
Electronic control board for drying and maturing cabinets
The RAL073_AA electronic controller handles the functions of professional cabinets used for drying and maturing meats and cheeses.
Possible applications:
- Refrigerated warehouses
- Distribution centres
- Restaurants, hotels, farmhouses
- Slaughterhouses, butcher shops, delicatessen
- Gastronomy
- Cheese factories
- Pasta factories
- Wineries and greenhouses
The RAL073_AA is compact and functional, since it integrates in a single electronic board the actuators and the user interface, characterized by simple and intuitive commands and a customer friendly display system.
The management software includes the performance of draining, drying and maturing phases: thanks to the rich menu that allows the setting of the main operating parameters, the user is able to customize each step and adapt the functioning of the controller on his specific needs.
The device also implements a self-diagnostic system that can detect and communicate to the user, through appropriate alarm signals, possible HACCP events (Hazard-Analysis and Critical Control Points), failure of a probe or the open door system.
Electronic controller for refrigerators, chillers and refrigerated display cases
We propose two models of controllers for refrigeration; the versatility of their functions makes them optimal for the control of:
- Professional blast chillers
- Professional refrigerators
- Refrigerated counters, display cases and showcases, buffet tables
- Blood banks, preservation of blood products and plasma
- Refrigerators and display cases for special medicines or heat-sensitive biological materials
- Wine cabinets and display cases
- Refrigerated cabinets
Our systems easily find use in the food industry (bakery, food and wine, supermarkets, hotels, catering and cold chain...) as well as in the medical and scientific sector (medical facilities, pharmacies, clinics, scientific or galenic laboratories).
GR_ABB Model
The GR_ABB electronic control system is made up of a power board, cabled on a plastic box, which interfaces itself with a commands board consisting of 6 capacitive backlighted keys and three seven segment display.
The setting menu, protected by password, allows the user to customize a wide range of parameters. The firmware supplied implements all the advanced functions needed in a professional blast chiller, including the control of chilling and freezing cycles (on temperature or time), to defrost cycle (on resistance, hot gas or ventilation) both in manual and automatic mode, the performing of appropriate tests to evaluate the correct insertion of the core probe into the product.
FE1028 Model
It is an extremely versatile hardware platform provided with three analog inputs for sensors handling, relay outputs to control up to 8 loads, Real Time Clock with battery backup for the management of the internal clock.
The system can be customized by the addition of optional components such as, for example, a RS-485 serial communication port or an external non-volatile memory for the saving of parameters and work programs. The FE1028 is characterized by a lower power consumption compared to the GR_ABB controller since it provides a very efficient switching power supply, from 100V to 230V without making changes.
The power board can also be interfaced with different types of keyboards, mechanical and capacitive, and with display devices and more advanced controls using alphanumeric displays, graphics or touch screen.
Remote monitoring and remote control: Internet of Things
AWe have really thought of everything... what if because of a nocturnal failure, a black out or a human error (such as a non-completely closed door) the system stops working as it should?
That's why we foresaw the possibility to connect your device to the network via the "Internet of Things", the set of technologies that equip the objects of their own intelligence, realizing communication channels with others devices using ethernet or Wifi connections. In this way you can easily set the parameters, the switching on and off, be notified about alarms, set passwords... all remotely from any PC, tablet or smartphone.
And if there's something we have not thought about... it does not mean that we can not do it!
Design and production, entirely Made in Italy and within the company, represent that something extra that comes from being able to customize the product according to specific customer needs.
We have the raw material... but your idea becomes our finished product.