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The present invention provides systems for growing two or three dimensional mammalian cells within a culture medium facilitated by an electromagnetic field, and preferably, a time varying electromagnetic field. The cells and culture medium are contained within a fixed or rotating culture vessel, and the electromagnetic field is emitted from at least one electrode. In one embodiment, the electrode is spaced from the vessel. The invention further provides methods to promote neural tissue regeneration by means of culturing the neural cells in the claimed system. In one embodiment, neuronal cells are grown within longitudinally extending tissue strands extending axially along and within electrodes comprising electrically conductive channels or guides through which a time varying electrical current is conducted, the conductive channels being positioned within a culture medium.
1. A system for growing three-dimensional biological cells, comprising:
a rotating wall vessel containing a cell-rich medium;
cell carriers placed within said vessel; and
an electrical current passed through said cell carriers for producing an electromagnetic field within said cell-rich medium.
2. The system of
3. The system of
4. The system of
5. The system of
6. The system of
7. The system of
8. The system of
9. The system of
10. The system of
11. The system of
12. The system of
13. The system of
14. A method of culturing biological cells in the system of
Inoculating said cells into said vessel;
rotating said vessel to initiate the attachment of said cells to cell carriers;
applying an electrical potential to said cell carriers; and
measuring the growth of said cells.
15. The method of
16. The method of
17. The method of
18. The method of
The invention described herein was made by an employee of the United States Government and may be manufactured and used by or for the Government of the United States of America for governmental purposes without the payment of any royalties thereon or therefor.
1. Field of the Invention
The present invention relates generally to the fields of biophysics, tissue regeneration, tissue culture, and neurobiology. More specifically, the present invention relates to the use of an electromagnetic field, and preferably, a time varying electromagnetic field, for potentiation of or controlling the growth of biological cells and tissue, such as mammalian tissue. More specifically, the present invention relates to the use of an electromagnetic field for controlling the growth of neural cells and tissues. The preferred embodiment utilizes two-dimensional conducting plate electrodes and may be applied to conventional, two dimensional tissue cultures or to three-dimensional cultures. Three dimensional cultures may be achieved in actual microgravity or by rotating wall vessel technology which simulates the physical conditions of microgravity, and in other, conventional three-dimensional matrix based cultures. The electromagnetic field, preferably a time varying electromagnetic field, is achieved in the vicinity of the electrode by passing, through the electrode, a time varying current.
2. Description of the Related Art
Growth of a variety of both normal and neoplastic mammalian tissues in both mono-culture and co-culture has been established in both batch-fed and perfused rotating wall vessels (1-2), and in conventional plate or flask based culture systems. In some applications, growth of three-dimensional structure, e.g., tissues, in these culture systems has been facilitated by support of a solid matrix in the form of biocompatible polymers and microcarriers. In the case of spheroidal growth, three-dimensional structure has been achieved without matrix support (3-6). NASA rotating wall tissue culture technologies have extended this three dimensional capacity for a number of tissues and has allowed the tissue to express different genes and biomolecules. Neuronal tissue has been largely refractory, in terms of controlled growth induction and three dimensional organization, under conventional culture conditions. Actual microgravity, and to a lesser extent, rotationally simulated microgravity, have permitted some enhanced nerve growth (Lelkes et al). Attempts to electrically stimulate growth have utilized static electric fields, static magnetic fields, and the direct passage of current through the culture medium, though not the induction of a time varying electromagnetic field in the culture region.
Neuronal tissue comprises elongated nerve cells composed of elongated axons, dendrites, and nuclear areas. Axons and dendrites are chiefly responsible for transmission of neural signals over distance and longitudinal cell orientation is critical for proper tissue formation and function. The nucleus plays the typical role of directing nucleic acid synthesis for the control of cellular metabolic function, including growth. In vivo, the neuronal tissue is invariably spatially associated with a system of feeder, or glial, cells. This three dimensional spatial arrangement has not been reproduced by conventional in vitro culture. Investigators, Borgens RB et al, and others, have utilized static electric fields in an attempt to enhance nerve growth in culture. (Valentini et al) with some success to either alter embryonic development or achieve isolated nerve axon directional growth. However, actual potentiation of growth or genetic activity causing such, have not been achieved. Mechanical devices intended to help grow and orient three dimensional mammalian neuronal tissue are currently available. Fukuda et al. (7) used zones formed between stainless steel shaving blades to orient neuronal cells or axons. Additionally, electrodes charged with electrical potential were employed to enhance axon response. Aebischer (8) described an electrically-charged, implantable tubular membrane for use in regenerating severed nerves within the human body. However, none of these devices utilize channels of cell-attractive material, neither do they apply a time varying electromagnetic field, or a static electrical or magnetic field. Additionally, no use is made of simulated or actual microgravity techniques for pure neuronal, or mixed, neuronal and feeder cell cultures. The prior art is deficient in its lack of effective means for growing three dimensional mammalian neuronal tissue in the proximity of, or directly upon the surface, of a current carrying electrode (which may be bioattractive and directly adherent to the cells). Furthermore, the use of a time varying current to induce a corresponding time varying electromagnetic field, in the vicinity of the growing culture, to potentiate or spatially direct cell growth is not part of the prior art.
The present invention relates to a system and method for culturing biological cells, such as mammalian cells, within a culture medium. The cells are exposed to an electromagnetic field, which, in the preferred embodiment, is a time-varying electromagnetic field. In the preferred embodiment, this field is generated by a conductive electrode, adjacently spaced from the incubating cells, carrying a time varying electrical current. The electrode, in one case, is in direct galvanic contact with the culture media and cells, and in another case, it is placed external to the culture apparatus in a galvanically isolated condition. Preferably, a 10 hertz square wave of 1-6 milliamperes, and with nearly zero time average, is passed through the electrode, suitably from corner to opposite comer of a square metallic conductive plate. The cells, such as neurons in this case, were, in one embodiment, grown directly on the electrode surface, composed of a biocompatable material. In another embodiment, the cells were grown within a container under the influence of a time varying electromagnetic field from an electrode external of and adjacent to the container, galvanically isolated from the media and culture within the container.
The growing cells may actually be attracted and trophically supported by more supportive electrode material or coatings. Furthermore, channels may be incorporated in the culture vessel and lined with growth substrate which may be electrically conductive. In one embodiment, a time varying electromagnetic field is induced in the region of the channel by passing the time varying current through a conductor placed along the channel. This arrangement will further direct growth by the combined effect of the field and trophic materials.
In the preferred embodiment, the presence of the time varying electromagnetic field potentiates the growth of nerve and other tissue. The time varying field may be induced by either: 1) a time varying current within a conductor, or 2) a time varying voltage between fixed conductors. In one embodiment, for example, the culture is placed nearby a conductor through which a time varying current is passed, or between parallel plates upon which a time varying voltage is applied. In both cases, a time varying electromagnetic field results within the area of interest, i.e., in the region of the cell culture.
The system and process are utilized in combination with known tissue culture processes to produce enhanced cell growth, directed cell growth, and tissue formation and organization.
As will be understood from the description to follow, the system is operable to up or down regulate the activity of specific genes. In general, growth promoting genes are up regulated and growth inhibitory genes are down regulated. The effect is shown to persist for some period after termination of the applied time varying field. This persistent, growth promoting effect subsides after a period of some days, and the cells return to a growth state characterized by controls, having never been exposed to the fields. This is beneficial in certain applications, in that medical applications for clinical medical care, i.e. nerve regeneration, are therefore safer than if the "pseudo transformed" state persisted. The set of gene transformations, associated with the time varying electromagnetic field, also promote the ability of the growing tissue to adhere and thrive on substrates by the induction of genes leading to the secretion of extracellular materials favorable to the tissue microenvironment.
Several methods of producing the time varying electromagnetic field in the vicinity of the living tissue culture are encompassed. In one embodiment, an array of conductive current carrying elements (or voltaic electrodes) are arranged so as to intensify or focus the time varying electromagnetic (EM) field onto the culture. Each embodiment is characterized by a method for application of the time varying field to the target tissue, such as neuronal, for stimulation of growth, or repair or induction of changes in gene activity patterns. The term "field generator" is used herein to represent these various embodiments for generating the time varying electromagnetic fields. In its simplest form, it is a conductive electrode, placed near the target cells, through which current is directed from a controlled waveform current source.
As suggested above, in one embodiment, the field generator is in the form of a conductive channel mounted on or embedded in a disc of biocompatable material. (FIG. 11) One or more of these discs may be then placed within a rotational bioreactor so as to obtain the beneficial culture conditions associated therewith. The combination of the stimulatory electromagnetic field with the rotational environment, known to permit morphological expression beyond conventional culture, is particularly effective. This is because the induced pattern of growth enhancing genes is permitted to be ultimately expressed, as cell growth and tissue formation, without mechanical inhibition from the culture apparatus. Also the inherent growth advantages well known in the rotational systems is synergistic with the growth stimulation derived from the time varying electromagnetic field. The conditions may be further optimized by utilizing actual microgravity, in space. In this application, mechanical rotation of the cell culture vessel is not required but may be utilized to achieve mixing and sufficient mass transfer to sustain a healthy culture. Other forms of mixing may be introduced as necessary to achieve adequate mass transfer for each embodiment.
In one embodiment, illustrated in FIGS. 10 and 11, slip-ring contacts or their equivalents are electrically connected to the ends of the channels, and an external power source is provided for applying the time varying electrical current defining the waveform through the channels. In another embodiment, the channel consists of a pair of parallel, mutually spaced conductors across which a time varying voltage is applied. This also achieves the time varying electromagnetic field but restricts it to the region between the parallel electrodes, which is advantageous for directing localized growth according to a desired physical pattern. The present invention also relates to a system and method for culturing primarily two dimensional mammalian cells facilitated by a time varying electromagnetic field. The electrodes may either be in direct galvanic contact or galvanically isolated from the target cells. The present invention provides a strategy to re-engineer nerve tissue and myoneural junctions and can be used medically for axonal regeneration.
In one embodiment of the present invention, there is provided a system for growing three dimensional mammalian cells, comprising a rotating wall vessel containing a cell-rich medium and a formed cell growth substrate. A time varying electromagnetic field is applied to enhance tissue growth which may occur on a shaped substrate. The electromagnetic field may be generated by means such as by directing the current waveform directly through a conductive substrate (or substrate layer) or by projecting the field from an external antenna, or electrode adjacent to and spaced from the medium, the spacing being sufficiently small relative to the strength of the electromagnetic field to induce effectual levels of electromagnetic field within the medium, in accordance with the particular application. A time varying electromagnetic field may be emitted from a nearby plate or other suitable "antennae," or a time varying voltage may be applied across suitable electrodes (such as plates) to produce the time varying electromagnetic field. The field generation system may either be rotating with the vessel or fixed, and spaced from, the rotating vessel. The rotating wall vessel can be a rotating wall perfused vessel or a rotating wall batch-fed vessel.
The time varying electromagnetic field is advantageously produced by a varying electrical potential in the form of a square wave having a frequency of approximately ten cycles per second. In one embodiment, a current of about ten milliamps, conducted between opposite comers of a metallic conductor, produces a stimulatory time varying electromagnetic field extending several centimeters from the plate surface. In practice, the range of frequency and oscillating electromagnetic field strength is a parameter which may be selected to for achieving the desired stimulation of particular tissues, cells, or genes, and for providing the appropriate amount of up/down regulation of these genes.
In one embodiment of the present invention, the cell growth susbtrates or carriers are spherical disks containing multiple parallel channels (FIG. 10) which are coated with a bioattractive material. The bioattractive material has a longitudinal axis across which the time varying electrical potential is applied and through which a time varying current is conducted. The mammalian cells adhere to the bioattractive material and are free to orient, as they grow. Representative bioattractive materials include titanium, zirconium and platinum.
The class of mammalian cells preferably is selected from the group consisting of neuronal cells, normal human neuronal progenitor cells (NHNP), and a cell responding to the time varying electromagnetic field. It will be understood by those of ordinary skill in the art that the teachings of the present invention apply to other cell types.
In another embodiment of the present invention, there is provided a method of culturing mammalian cells in the claimed system, comprising the steps of inoculating the cells into the vessel containing a culture medium, rotating the vessel to enhance the proliferation of the cells and, in one embodiment, to initiate the attachment of the cells to microcarrier spheres or beads suspended within the culture medium, applying a time varying electromagnetic field to the culture medium, cells, and cell carriers, and measuring the growth of the cells. Preferably, the vessel is rotated at a speed from about 2 RPM to 30 RPM, and the time varying electromagnetic field is generated by a time varying current passed through a conductor with RMS value of about 1 to 1,000 ma. In one embodiment, a range of about 1 mA to 6 mA is used.
In still another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells, comprising a petri dish containing a cell culture medium and an electrode placed in the center of the petri dish. In this embodiment, the electrode serves as the field generator. Preferably, the neural cells are applied directly on the electrode. As a result, the neutral cells exhibit accelerated growth.
In yet another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells further comprising a slide placed on the electrode. Preferably, the neural cells are applied, e.g., bubbled, on the slide instead of directly contacting the electrode, and preferably, the current producing the waveform is applied at a strength range of from about 1 mA to about 100 mA, and, in one embodiment, suitably from about 1 mA to 6 mA.
In still another embodiment of the present invention, there is provided a method of treating an individual having diseased neuronal cells, comprising the steps of growing neuronal cells in the claimed two- or three- dimensional systems and transplanting the neuronal cells into the individual. Such diseases include Parkinson's disease, diseases of neuromuscular junction and Alzheimer's Disease. Neural trauma can also be treated in same methodology.
In yet another embodiment of the present invention, the time varying electromagnetic field (or electrical potential) induces cellular response including cellular control of growth and differentiation at gene level. Preferably, the cellular control of growth and differentiation is to suppress or enhance growth regulatory functions at gene level. Still preferably, the gene is associated with increased tissue and cell proliferation.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.
So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.
FIG. 1 shows a petri dish with cells in a concentrated bubble placed on the metal electrode in the center of the dish.
FIG. 2 shows normal human neuronal progenitor (NHNP) cells grown in conventional tissue culture procedures.
FIG. 3 shows the perimeter of non-waveform influenced normal human neuronal progenitor cells 24 hours after the experiment.
FIG. 4 shows neural tube formation within nornal human neuronal progenitor cells under the influence of waveform.
FIG. 5 shows neural tube generation within normal human neuronal progenitor cells under the influence of waveform.
FIG. 6 shows the composition of waveform-influenced neural tissue 24 hours after the exposure.
FIG. 7 shows the waveform-influenced normal human neuronal progenitor cells 24 hours after the exposure.
FIG. 8 shows a close-up of waveform-influenced normal human neuronal progenitor cells.
FIG. 9 shows waveform-influenced normal human neuronal progenitor cells 24 hours after the exposure.
FIG. 10 shows prototype of the system 1, consisting of silicon plates 2, fluid coupling 3, slip rings 4, a rotating wall pressure vessel (RWPV) 5, electrical conductor 6 (i.e., an electrical conductive bioattractive inlay strip), a perfusion inlet 7, perfusion outlet 8, a stand for the rotating wall pressure vessel 9 and a source of time varying electrical current 10.
FIG. 11A is a plan view of one of the disc-shaped silicon plates 2, showing a central opening 11 about which rotation occurs, the electrical conductors 6, and electrical contacts 12 connected to the strips at opposite ends thereof. FIG. 11B is a side view of the silicon plate 2, demonstrating the electrical contacts 12 and the electrical conductive strips, in the form of bioattractive inlays 6.
As used herein, the term "bioattractive material" shall refer to materials to which a cellular material will attach.
As used herein, the term "longitudinally orient" shall refer to orientation in an elongated cordlike fashion.
As used herein, the term "parallel channels" shall refer to electric channels which are designed to provide constant output to all the electrodes simultaneously.
As used herein, the term "cell carriers" shall refer to microcarrier beads, scaffolds and matrices which support the growth and/or attachment of cellular materials.
As used herein, the term "rotating wall batch-fed vessel" shall refer to slow turning lateral vessel (STLV) and high aspect rotating vessel (HARV).
As used herein, the term "Corona Effecft" shall refer to the accelerated growth pattern of neuronal cells electrically potentiated by waveform.
In one preferred embodiment, the present invention is directed to the growth of three dimensional mammalian neuronal tissue using an electrically conductive strip in the form of a channel or mold coated with a bioattractive or biocompatable material to which an electrical potential is applied to longitudinally orient the neural cells or axons as they adhere to the bioattractive material which is suspended in an axon rich medium.
A Specifically, in the present embodiment, the apparatus includes a bioreactor chamber vessel employing electrically insulative, biocompatable spherical disks of a material such as silicon. These disks rotate inside the pressure vessel. Each disk has multiple parallel channels cut into its surface. The channels have a semicircular cross-section and contain an electrically conductive inlay in the form of a channel-shaped conductive strip of a bioattractive material such as zirconium, titanium and platinum. Each channel strip 6 has an electrical contact on each longitudinal end that is used to create and control an electrical potential along the length of the strip. The vessel is filled with a medium and the disks are rotated within a medium containing axons. The cells adhere to the electrically conductive bioattractive inlay material. The desired longitudinal cell orientation and therefore the structure of the resulting tissue is affected and/or controlled by the electrical stimulus.
The present invention is also directed to the growth of two dimensional mammalian neuronal tissue using electrodes. The electrodes are either in direct contact or not in contact with the target cells.
In one embodiment of the present invention, there is provided a system for growing three dimensional mammalian cells, comprising a rotating wall vessel containing a cell-rich medium, cell carriers placed within the vessel and an electrical potential applied to the cell carrier. Preferably, the rotating wall vessel can be a rotating wall perfused vessel or a rotating wall batch-fed vessel.
In one embodiment of the present invention, the cell carriers are spherical disks containing multiple parallel channels, which are coated with a bioattractive material. More preferably, the bioattractive material has a longitudinal axis across which the electrical potential is applied. The mammalian cells adhere to the bioattractive material and are therefore oriented longitudinally upon the electrical stimulus. Representative bioattractive materials include titanium, zirconium and platinum.
In the methods of the present invention, the mammalian cell is selected from the group consisting of a neuronal cell, a normal human neuronal progenitor cell (NHNP) and a cell responding to waveform. A person having ordinary skill in this art will be able to apply the teachings of the present invention to other cell types.
In another embodiment of the present invention, there is provided a method of culturing mammalian cells in the claimed system, comprising the steps of inoculating the cells into the vessel, rotating the vessel to initiate the attachment of the cell to the cell carriers, applying an electrical potential to the cell carriers and measuring the growth of the cells. Preferably, the vessel is rotated at a speed from about 10 RPM to 30 RPM, and the electrical potential is applied at a strength range of from about 1 mA to about 6 mA.
In still another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells, comprising a petri dish containing a cell culture medium and an electrode placed in the center of the petri dish. The electrode is charged with a waveform. Preferably, the neural cells are bubbled directly on the electrode. As a result, the neutral cells exhibit accelerated growth.
In yet another embodiment of the present invention, there is provided a system for growing two-dimensional neural cells further comprising a slide placed on the electrode. Preferably, the neural cells are bubbled on the slide instead of directly contacting the electrode. Preferably, the waveform is applied at a strength range of from about 1 mA to about 6 mA.
In still yet another embodiment of the present invention, there is provided a method of treating an individual having diseased neuronal cells, comprising the steps of growing neuronal cells in the two or three dimensional systems disclosed herein and transplanting the grown neuronal cells into the individual. Such diseases include Parkinson's disease, diseases of neuromuscular junction and Alzheimer's Disease. Neural trauma can also be treated with this same methodology.
In yet another embodiment of the present invention, the waveform (or electrical potential) induces a cellular response including cellular control of growth and differentiation at gene level. Preferably, the cellular control of growth and differentiation is to suppress or enhance growth regulatory functions at gene level. Still preferably, the gene is associated with embryogenesis.
The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
Normal human neuronal progenitor cells (NHNP) were pooled from three donors. As controls, normal human neuronal progenitor cells were grown in conventional tissue culture following standard cell culturing procedures.
GTSF-2 medium with 10% FBS, Ciprofloxacin and Fungizone was used to culture the cells. 1�PBS, Collagenase, DNase and Trypsin were purchased from Clonetics. The cells were grown on 12-100 mm Petri dishes (tissue culture coated or not coated). Electrodes were made of platinum and stainless steel. A waveform generator was used to generate the waveform in a strength of 1-6 mA (AC) square wave, 10 Hz variable duty cycle.
Electrically Potentiating Cell Growth When Electrode is in Direct Contact with the Target Cells
Initially, a metal electrode was placed inside a petri dish and centered.
Normal human neuronal progenitor cells were seeded at 2�105 cells in 0.7 ml of media and carefully dropped on the electrode in a concentrated bubble (FIG. 1). Cells were incubated for 2 days. Second day after seeding is considered day 0 of the experiment. At day 0, each dish was given 15 ml of media and waveform was applied to seven electrodes. Cells were observed under a dissecting microscope and fed with 15 ml of media at day 3, and 13 ml every three days at day 6, 9 and 12. At day 14, the cells were fed again with 13 ml of media. At day 17, the cells were incubated for 10 minutes in a Collagenase/DNase cocktail, then trypsin was directly applied to the cocktail and the cells were further incubated for 3 more minutes. Before the media was added to deactivate trypsin, the cocktail mix was pipetted up and down several times. The cells were washed twice with 1�PBS, reapplied with the media and placed on ice. The cells were counted, assessed for viability.
To examine the accelerated growth of cells 48 and 72 hours after waveform was discontinued, cells were treated the same as above, except that after day 14 treatment, instead of harvesting, two dishes from the non-waveform group (control) and two dishes from the waveform group were randomly chosen and re-seeded at 9�105 cells in two new petri dishes each, with a total of four dishes. Cells from one set (#11 waveform and control #6) were counted and photographed 48 hours after seeding, and cells from the second set (#12 waveform and control #7) were counted and photographed 72 hours after seeding.
To examine accelerated growth pattern "Corona Effect" after the electrical potentiation, the same treatment was applied to the cells without harvesting. A dish each from waveform group and non-waveform group were chosen randomly. Cells still attached in sheet from were lifted off of the electrodes carefully and placed in new petri dishes with medium, and then photographed 24 hours later.
Electrically Potentiatiny Cell Growth When Electrode is not in Direct Contact with the Target Cells
Initially, a metal electrode was placed inside a petri dish and centered. A slide was carefully placed on the electrode under sterile conditions. Normal human neuronal progenitor cells were seeded at 2�105 cells in 0.7 ml of media and bubbled on the slide. Cells were incubated for 2 days. 25 ml of media were applied and two 1000 μl pipetman blue tips were placed in the dish to anchor the slide to bottom of the dish. The second day after seeding was considered day 0 of the experiment. At day 0, each dish was given 25 ml of media and waveform was applied to six of the twelve electrodes.
Cells were observed under a dissecting microscope and fed with 25 ml of media every three days at day 3, 6, 9 and 12. At day 14, the cells were fed again with 25 ml of media. At day 18, the cells were incubated for 10 minutes in a Collagenase/DNase cocktail, then typsin was directly applied to the cocktail and the cells were further incubated for 3 more minutes. Before the media was added to deactivate trypsin, the cocktail mix was pipetted up and down several times. The cells were washed twice with 1�PBS, reapplied with the media and placed on ice. The cells were counted, assessed for viability and then replated at 100,000 per plate. The remaining waveform and non waveform slides were fixed and refrigerated for staining at a later date.
Electrically Potentiated Growth of Cells
Normal human neuronal progenitor-pool cells exposed to a time varying electromagnetic field (waveform), either in direct contact or not in direct contact with the electrode, displayed an accelerated growth rate and different morphology as compared to non waveform cells (control), i.e., cells not subject to the time varying electromagnetic field (see Table 1 and Table 2, FIGS. 2-9). After the application of the time varying electromagnetic field or waveform, the cells preferentially aligned, while cells without waveform exposure showed random pattern. Cells in direct contact with the electrode remained stimulated up to at least 72 hours after waveforn was removed (Table 3); while those not in direct contact with the electrode once removed from waveform continued to experience accelerated and long term stimulation growth pattern even after 168 hours (Table 4). Viability was also higher in the cells exposed to the waveform (Table 4). Cells were suspended easily with the Collagenase/DNase then trypsin sequence.
Waveform Gene Array Display (GAD) Results
Normal Human Neural Progenitor cells or human adult astrocytes were exposed to waveform and non-waveform growth conditions for 17 days. Upon completion of the exposure period cells were harvested via trypsinization and poly-RNA was prepared from the respective groups of cells. RNA samples were quick frozen and shipped to Synteni Corporation for GAD analysis. Below are the results of a survey of the response of over 10,000 human genes. The results were divided into two categories (Table 5 and Table 6). Those genes down regulated or suppressed by the waveform and those up regulated or enhanced in activity by the waveform.
An analysis of the data indicates a significant down regulation of maturation and regulatory genes. These maturation and regulatory genes are normally associated with the differentiated or non-growth profile of normal cells. However, there is a significant up regulation of some 150 genes which are mainly associated with growth and cellular proliferation. Neither two nor three dimensional growth of neural cells has been achieved prior to this event with the positive outcome of enhanced growth and apparent gene regulatory control.
Down Regulated Genes in Descending Order (Highest to lowest)
1. Homo sapiens (clone Zap2) mRNA fragment {Incyte PD:1661837}
2. CDC28 protein kinase 2{Incyte PD:1384823}
3. Synteni: YCFR 22 {YC 22.2000.W}
4. ESTs, Moderately similar to cell growth regulating nucleolar protein LYAR [M.musculus] {Incyte PD:2233551}
5. KERATIN, TYPE II CYTOSKELETAL 7 {Incyte PD:1649959}
6. MITOTTC KINESIN-LIKE PROTEIN-1 {Incyte PD:2640427}
7. EST {Incyte PD:674714}
8. Synteni: YCFR 22 {YC 22.2000.X}
9. Synteni: YCFR 26 {YC 26.0062.N}
10. Synteni: YCFR 22 {YC 22.2000.Z}
11. Transcription factor 6-like 1 (mitochondrial transcription factor 1-like) {Incyte PD:3371995}
12. Interferon-inducible 56-KDa protein {Incyte PD:1215596}
13. EST {Incyte PD:1794375}
14. Homo sapiens mitotic feedback control protein Madp2 homolog mRNA, complete cds {Incyte PD:2414624}
15. EST {Incyte PD:151026}
16. Homo sapiens Pig3 (PIG3) mRNA, complete cds {Incyte PD:2395269}
17. General transcription factor IIIA {Incyte PD:1527070}
18. Cellular retinoic acid-binding protein [human, skin, mRNA, 735 nt] {Incyte PD:585432}
19. EST {Incyte PD:1755159}
20. Homo sapiens mRNA for KIAA0285 gene, complete cds {Incyte PD:1738053}
21. ESTs, Weakly similar to F25H5.h [C.elegans] {Incyte PD:1923567}
22. Homo sapiens mRNA expressed in osteoblast, complete cds {Incyte PD:2537863}
23. EST {Incyte PD:3204745}
24. Homo sapiens mRNA for serine/threonine protein kinase SAK {Incyte PD:2732630}
25. Homo sapiens serum-inducible kinase mRNA, complete cds {Incyte PD:1255087}
26. Carbonic anhydrase II {Incyte PD:2474163}
27. EST {Incyte PD:660376}
28. GRANCALCIN {Incyte PD:1671852}
29. N-CHIMAERIN {Incyte PD:1852659}
30. Homo sapiens Pig10 (PIG10) M3RNA, complete cds {Incyte PD:1731061}
31. Adenylosuccinate lyase {Incyte PD:1653326}
32. EST {Incyte PD:1798393}
33. Homo sapiens HP protein (HP) mRNA, complete cds {Incyte PD:30841223}
34. ESTs, Moderately similar to T10C6[C.elegans] {Incyte PD:1923186}
35. Chromosome condensation 1 {Incyte PD:3180854}
36. Calmodulin 1 (phosphorylase kinase, delta) {Incyte PD:2803306}
37. Centromere protein A (17kD) {Incyte PD:2444942}
38. V-jun avian sarcoma virus 17 oncogene homolog {Incyte PD:1920177}
39. Human glutathione-S-transferase homolog mRNA, complete cds {Incyte PD:1862232}
40. Homo sapiens gene for protein involved in sexual development, complete cds {Incyte PD:3033934}
41. EST {Incyte PD:2630992}
42. Human low-Mr GTP-binding protein (RAB32) mRNA, partial cds {Incyte PD:1662688}
43. Annexin III (lipocortin Ill) {Incyte PD:1920650}
44. Hydroxymethylbilane synthase {Incyte PD:1509204}
45. Synteni: HK 4 {HK 4.2000.Y}
46. Ribosomal protein L7a {Incyte PD:2579602}
47. Human mRNA for myosin regulatory light chain {Incyte PD:78783}
48. Ferredoxin reductase {Incyte PD:1819763}
49. Human copper transport protein HAH1 (HAH1) mRNA, complete cds {Incyte PD:2313349}
50. Human G protein gamma-11 subunit mRNA, complete cds {Incyte PD:1988432}
51. Synteni: HK 4 {HK 4.2000.W}
52. Human XIST, coding sequence a mRNA (locus DXS399E) {Incyte PD:1514318}
53. Ribosomal protein, large, P0 {Incyte PD:3511355}
54. Homo sapiens clone 23714 mRNA sequence {Incyte PD:1728368}
55. Human mRNA for Apo113 Human (MER5(Aop1-Mouse)like protein), complete cds {Incyte PD:2527879}
56. Synteni: HK 4 {HK 4.2000.Z}
57. Proteasome (prosome, macropain) subunit, beta type, 5 {Incyte PD:2503119}
58. Human PINCH protein mRNA, complete cds {Incyte PD:126888}
59. Homo sapiens peroxisome assembly protein PEX10 mRNA, complete cds {Incyte PD:998279}
60. Homo sapiens short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) mRNA, complete cds {Incyte PD:1638850}
61. Neuroblastoma RAS viral (v-ras) oncogene homolog {Incyte PD:2816984}
62. H.sapiens mRNA for b4 integrin interactor {Incyte PD:1932850}
63. Human forkhead protein FREAC-1 mRNA, complete cds {Incyte PD:1449920}
64. Human mRNA for protein D123, complete cds {Incyte PD:1920522}
65. H.sapiens mRNA for A-kinase anchoring protein AKAP95 {Incyte PD:1628787}
66. Carbonyl reductase {Incyte PD:1633249}
67. EST {Incyte PD:2060973}
68. ESTs, Highly similar to GUANINE NUCLEOTIDE-BINDING PROTEIN G(I)/G(S)/G(O) GAMMA-7 SUBUNIT [Rattus norvegicus] {Incyte PD:1640161}
69. Homo sapiens Na+/Ca+exchanger mRNA sequence {Incyte PD:2880435}
70. STRESS-ACTIVATED PROTEIN KINASE JNK1 {Incyte PD:3331719}
71. Homo sapiens leupaxin mRNA, complete cds {Incyte PD:1595756}
72. CLEAVAGE SIGNAL-1 PROTEIN {Incyte PD:2054053}
73. EST {Incyte PD:1798965}
74. Human DNA from overlapping chromosome 19 cosmids R31396, F2545 1, and R31076 containing COX6B and UPKA, genomic sequence {Incyte PD:1320685}
75. INTERFERON-INDUCED 17 KD PROTEIN {Incyte PD:2862971}
76. Human homolog of yeast IPP isomerase {Incyte PD:1526240}
77. Translation elongation factor 1 gamma {Incyte PD:3138196}
78. Tropomyosin alpha chain (skeletal muscle) {Incyte PD:1572555}
79. Aplysia ras-related homolog 9 {Incyte PD:2733928}
80. ATP SYNTHASE ALPHA CHAIN, MITOCHONDRIAL PRECURSOR {Incyte PD:3206210}
81. Homo sapiens androgen receptor associated protein 24 (ARA24) mRNA, complete cds {Incyte PD:552654}
82. Glucagon {Incyte PD:1333075}
83. Human enhancer of rudimentary homolog mRNA, complete cds {Incyte PD:1704472}
84. TRANSCRIPTIONAL ENHANCER FACTOR TEF-1 {Incyte PD:2957175}
85. Ubiquitin-like protein {Incyte PD:1754454}
86. Human RGP4 mRNA, complete cds {Incyte PD:617517}
87. Cellular retinol-binding protein {Incyte PD:1612969}
88. Ornithine decarboxylase 1 {Incyte PD:1930235}
89. EST {Incyte PD:3605632}
90. EST {Incyte PD:2057260}
91. ESTs, Weakly similar to CAMP-DEPENDENT PROTEIN KINASE TYPE 2 [Saccharomyces cerevisiae] {Incyte PD:2055611}
92. Human p37NB mRNA, complete cds {Incyte PD:1407110}
93. Human mRNA for suppressor for yeast mutant, complete cds {Incyte PD:2888814}
94. EST {Incyte PD:3142705}
95. ESTs, Weakly similar to K01H12.1 [C.elegans] {Incyte PD:56197}
96. Cell division cycle 2, G1 to S and G2 to M {Incyte PD:1525795}
97. EST {Incyte PD:1794175}
98. EST {Incyte PD:1489557}
99. ESTs, Weakly similar to PROTEIN PHOSPHATASE PP2A, 72 KD REGULATORY SUBUNIT [H.sapiens] {Incyte PD:2379045}
100. CAMP-DEPENDENT PROTEIN KINASE TYPE II-ALPHA REGULATORY CHAIN {Incyte PD:1649731}
101. ESTs, Weakly similar to transcription factor [H.sapiens] {Incyte PD:1637517}
102. ATP synthase, H+transporting, mitochondrial F1 complex, O subunit (oligomycin sensitivity conferring protein) {Incyte PD:2193246}
103. RAS-LIKE PROTEIN TC21{Incyte PD:2505425}
104. Small nuclear ribonucleoprotein polypeptides B and B1 {Incyte PD:2071473}
105. EST {Incyte PD:1922084}
106. Proliferating cell nuclear antigen {Incyte PD:2781405}
107. ESTs, Highly similar to HIGH MOBILITY GROUP-LIKE NUCLEAR PROTEIN 2[Saccharomyces cerevisiae] {Incyte PD:2669174}
108. EST {Incyte PD:1844150}
109. Human mRNA for proteasome subunit HsC10-II, complete cds {Incyte PD:1737833}
110. Homo sapiens mRNA for ST1 C2, complete cds {Incyte PD:3993007}
111. Human dual specificity phosphatase tyrosine/serine mRNA, complete cds {Incyte PD:1514573}
112. Human stimulator of TAR RNA binding (SRB) mRNA, complete cds {Incyte PD:2057162}
113. EST {Incyte PD:2507206}
114. H.sapiens mRNA for Ndr protein kinase {Incyte PD:3318571}
115. ESTs, Weakly similar to Grb2-related adaptor protein [H.sapiens] {Incyte PD:1857259}
116. ESTs, Highly similar to Tbc1 [M.musculus] {Incyte PD:1889147}
117. GTPase-activating protein ras p21 (RASA) {Incyte PD:147344}
118. Human mRNA for KIAA0123 gene, partial cds {Incyte PD:1752436}
119. Synteni: YCFR 22 {YC 22.2000.Y}
120. Human non-histone chromosomal protein (NHC) mRNA, complete cds {Incyte PD:1748670}
121. Thioredoxin {Incyte PD:2606240}
122. FATTY ACID-BINDING PROTEIN, EPIDERMAL {Incyte PD:2537805}
123. Proteasome component C2 {Incyte PD:2195309}
124. Homo sapiens heat shock protein hsp40 homolog mRNA, complete cds {Incyte PD:2844989}
125. Human amyloid precursor protein-binding protein 1 mRNA, complete cds {Incyte PD:1663083}
126. Homo sapiens DNA binding protein homolog (DRIL1) mRNA, complete cds {Incyte PD:2538333}
127. Human Has2 mRNA, complete cds {Incyte PD:3602403}
128. EST {Incyte PD:1749678}
129. Homo sapiens golgi SNARE (GS27) mRNA, complete cds {Incyte PD:3279439}
130. ESTs, Weakly similar to UBIQUITIN-ACTIVATING ENZYME E1 HOMOLOG [H.sapiens] {Incyte PD:1710472}
131. Synteni: YCFR 22 {YC 22.2000N}
132. Voltage-dependent anion channel 2 {Incyte PD:2189062}
133. Human rap2 mRNA for ras-related protein {Incyte PD:3334979}
134. Acid phosphatase 1, soluble {Incyte PD:620871}
135. Human clone 23840 mRNA, partial cds {Incyte PD:1830083}
136. Human mRNA for KIAA0008 gene, complete cds {Incyte PD:1970111}
137. H.sapiens mRNA for protein-tyrosine-phosphatase (tissue type: foreskin) {Incyte PD:444957}
138. Human B-cell receptor associated protein (hBAP) mRNA, partial cds {Incyte PD:2545562}
139. ESTs, Highly similar to ring finger protein [H.sapiens] {Incyte PD:2860918}
140. H.sapiens mRNA for CLPP {Incyte PD:2675481}
141. APOPTOSIS REGULATOR BCL-X {Incyte PD:1855683}
142. PROTEASOME COMPONENT C13 PRECURSOR {Incyte PD:2668334}
143. Sorting nexin 1 {Incyte PD:1508407}
144. Human voltage dependent anion channel form 3 mRNA, complete cds {Incyte PD:2051154}
145. H.sapiens mRNA for translin {Incyte PD:986855}
146. Human DEAD-box protein p72 (P72) mRNA, complete cds {Incyte PD:1750553}
147. Ras homolog gene family, member G (rho G) {Incyte PD:1342744}
148. EST {Incyte PD:1377794}
149. Human FEZ2 mRNA, partial cds {Incyte PD:2623268}
150. Human homolog of Drosophila discs large protein, isoform 2 (hdlg-2) mRNA, complete cds {Incyte PD:2203554}
151. ALCOHOL DEHYDROGENASE {Incyte PD:1634342}
152. 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase (hydroxymethylglutaricaciduria) {Incyte PD:1695917}
153. ENOYL-COA HYDRATASE, MITOCHONDRIAL PRECURSOR {Incyte PD:2235870}
154. Proteasome (prosome, macropain) subunit, beta type, 6 {Incyte PD:2989852}
155. INTERFERON GAMMA UP-REGULATED I-5111 PROTEIN PRECURSOR {Incyte PD:2211625}
156. Epimorphin {Incyte PD:3438987}
157. H.sapiens RY-1 mRNA for putative nucleic acid binding protein {Incyte PD:1805712}
158. EST {Incyte PD:1905120}
159. KD HOUSEKEEPING PROTEIN {Incyte PD:1819287}
160. Cytochrome c oxidase subunit VIIb {Incyte PD:2060789}
161. EST {Incyte PD:661516}
162. Homo sapiens nuclear VCP-like protein NVLp.2 (NVL.2) mRNA, complete cds {Incyte PD:1445507}
163. EST {Incyte PD:1251588}
164. EST {Incyte PD:1665871}
165. Homo sapiens inositol polyphosphate 4-phosphatase type 11-alpha mRNA, complete cds {Incyte PD:3032739}
166. Homo sapiens arsenite translocating ATPase (ASNA1) mRNA, complete cds {Incyte PD:1666094}
167. Human SnRNP core protein Sm D3 mRNA, complete cds {Incyte PD:1624865}
168. Homo sapiens clone 23777 putative taansmembrane GTPase mRNA, partial cds {Incyte PD:2554541}
169. Homo sapiens regulator of G protein signaling RGS12 (RGS) mRNA, complete cds {Incyte PD:3618382}
170. Human Ki nuclear autoantigen mRNA, complete cds {Incyte PD:1308112}
171. Homo sapiens peroxisomal phytanoyl-CoA alpha-hydroxylase (PAHX) mRNA, complete cds {Incyte PD:4073867}
172. PLACENTAL CALCIUM-BINDING PROTEIN {Incyte PD:1222317}
173. PRE-MRNA SPLICING FACTOR SF2, P32 SUBUNIT PRECURSOR {Incyte PD:1552335}
174. Human clone C4E 1.63 (CAC)n/(GTG)n repeat-containing mRNA {Incyte PD:1928789}
175. Human glioma pathogenesis-related protein (GliPR) mRNA, complete cds {Incyte PD:477045}
176. Homeo box A9 {Incyte PD:459651}
Waveform Up Regulated Genes in Ascending Order (Lowest to Highest)
1. NEUROMEDIN B PRECURSOR {Incyte PD:2754315}
2. Synteni: YCFR 21 {YC 21.0031.N}
3. ATRIAL NATRTC PEPTIDE CLEARANCE RECEPTOR PRECURSOR {Incyte PD:1353606}
4. Synteni: YCFR 85 {YC 85.2000.Y}
5. Homo sapiens CHD3mRNA, complete cds {Incyte PD:1965248}
6. EST {Incyte PD:565872}
7. Synteni: YCFR 46 Cy3 {YC 46.2000. Z}
8. ESTs, Weakly similar to metaxin [H.sapiens] {Incyte PD:1754461}
9. Plasminogen {Incyte PD:2515873}
10. Human mRNA for CC chemokine LARC precursor, complete cds {Incyte PD:2220923}
11. Synteni: YCFR 21 {YC 21.0062.N)
12. Homo sapiens Amplified in Breast Cancer (AIB1) mRNA, complete cds Incyte PD:2634478}
13. Homo sapiens clone 24640 mRNA sequence {Incyte PD:1560143}
14. Synteni: YCFR 21 {YC 21.2000.N}
15. EST {Incyte PD:143912}
16. Human transcription factor SIM2long form mRNA, complete cds {Incyte PD:996104}
17. EST {Incyte PD:2841478}
18. PUTATIVE DNA BINDING PROTEIN A20 {Incyte PD:1878791}
19. Protein tyrosine phosphatase, receptor type, mu polypeptide {Incyte PD:987736}
20. Human clone A9A2BRB5(CAC)n/(GTG)n repeat-containing mRNA {Incyte PD:1987975}
21. Endothelin converting enzyme 1 {Incyte PD:1963819}
22. BB1 {Incyte PD:1966148}
23. Pleiotrophin (heparin binding growth factor 8, neurite growth-promoting factor 1) {Incyte PD:2989411}
24. Argininosuccinate synthetase {Incyte PD:1981145}
25. Human breast epithelial antigen BA46mRNA, complete cds {Incyte PD:1319020}
26. Human clone 46690 brain expressed mRNA from chromosome X {Incyte PD:1669780}
27. Human plectin (PLEC1) mRNA, complete cds {Incyte PD:1907232}
28. Homo sapiens mRNA for calmegin, complete cds {Incyte PD:2498216}
29. EST {Incyte PD:769182}
30. Amyloid beta (A4) precursor-like protein 2 {Incyte PD:3876715}
31. Polymerase (RNA) II (DNA directed) polypeptide A (220 kD) {Incyte PD:1382059}
32. GLUCOSE TRANSPORTER TYPE 3, BRAIN {Incyte PD:2745082}
33. Homo sapiens sarco-/endoplasmic reticulum Ca-ATPase 3 (ATP2A3) mRNA, alternatively spliced, partial cds {Incyte PD:688411}
34. Human c-jun proto oncogene (JUN), complete cds, clone hCJ-1 {Incyte PD:1969563}
35. Microtubule-associated protein 1A {Incyte PD:702684}
36. Clusterin (complement lysis inhibitor, testosterone-repressed prostate message 2; apolipoprotein J) {Incyte PD:2966620}
37. NADH-CYTOCHROME B5 REDUCTASE {Incyte PD:1901142}
38. Protein-tyrosine kinase 7 {Incyte PD:996229}
39. Alpha-1 type XVI collagen {Incyte PD:1963529}
40. EST {Incyte PD:2839121}
41. Homo sapiens mRNA for DEC 1, complete cds {Incyte PD:1732479}
42. Human endogenous retroviral protease mRNA, complete cds {Incyte PD:1347636}
43. ATPase, Na+/K+transporting, alpha 1polypeptide {Incyte PD:1730609}
44. Laminin,alpha 4 {Incyte PD:1851696}
45. Hexabrachion (tenascin C, cytotactin) {Incyte PD:1453450}
46. Human mRNA for KIAA0325 gene, partial cds {Incyte PD:1995315}
47. Integrin beta-5 subunit {Incyte PD:418731}
48. Microfibrillar-associated protein 4 {Incyte PD:1659231}
49. Fibulin 1 {Incyte PD:1320658}
50. Protein serine/threonine kinase stk2 {Incyte PD:1518981}
51. ESTs, Weakly similar to HYPOTHETICAL 16.1 KD PROTEIN IN SEC 17-QCR1 INTERGENIC REGION [Saccharomyces cerevisiae] {Incyte PD:1923722}
52. Homo sapiens carbonic anhydrase precursor (CA 12) mRNA, complete cds {Incyte PD:3766382}
53. H.sapiens mRNA for SIX1 protein {Incyte PD:3208486}
54. Plasminogen activator inhibitor, type I {Incyte PD:1445767}
55. Human mRNA for SHPS-1, complete cds {Incyte PD:2180684}
56. Collagen, type V, alpha 1 {Incyte PD:1672442}
57. Homo sapiens monocarboxylate transporter (MCT3) mRNA, complete cds {Incyte PD:1343253}
58. Human follistatin gene {Incyte PD:1577614}
59. Human putative RNA binding protein (RBP56) mRNA, complete cds {Incyte PD:1907369}
60. Homo sapiens mRNA for PRP8 protein, complete cds {Incyte PD:3616229}
61. Homo sapiens CAGH13 mRNA, complete cds {Incyte PD:1432042}
62. EST {Incyte PD:2953888}
63. Intercellular adhesion molecule 1 (CD54), human rhinovirus receptor {Incyte PD:1556061}
64. Human p120E4F transcription factor mRNA, complete cds {Incyte PD:1940164}
65. Collagen, type VI, alpha 1 {Incyte PD:2672056}
66. Human mRNA for pM5 protein {Incyte PD:1578951}
67. ALZHEIMER'S DISEASE AMYLOID A4 PROTEIN PRECURSOR {Incyte PD:126370}
68. Human mRNA for KIAA0062 gene, partial cds {Incyte PD:3138128}
69. Human clone HSH1 HMG CoA synthase mRNA, partial cds {Incyte PD:1807407}
70. Filamin 1 (actin-binding protein-280) {Incyte PD:1708528}
71. Synteni: YCFR 85 {YC 85.2000.X}
72. Synteni: YCFR 46 Cy3 {YC 46.2000.W}
73. Homologue of mouse tumor rejection antigen gp96 {Incyte PD:2679349}
74. Tissue inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory) {Incyte PD:418041}
75. Human XMP mRNA, complete cds {Incyte PD:1887661}
76. Cytochrome P450, subfamily XIA (cholesterol side chain cleavage) {Incyte PD:2368282}
77. Granulin {Incyte PD:812141}
78. Human extracellular matrix protein 1 (ECM1) mRNA, complete cds {Incyte PD:1965806}
79. 78 KD GLUCOSE REGULATED PROTEIN PRECURSOR {Incyte PD:2884613}
80. Synteni: YCFR 21 {YC 21.2000.X}
81. Homo sapiens mRNA for serin protease with IGF-binding motif, complete cds {Incyte PD:1958902}
82. Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein {Incyte PD:1687060}
83. Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 {Incyte PD:1516886}
84. Hormone receptor (growth factor-inducible nuclear protein N10) {Incyte PD:1958560}
85. Fibulin 2 {Incyte PD:1901095}
86. Kinase insert domain receptor (a type III receptor tyrosine kinase) {Incyte PD:2220338}
87. Synteni: YCFR 45 {YC 45.2000.X}
88. Syndecan 4 (amphiglycan, ryudocan) {Incyte PD:3214670}
89. Synteni: YCFR 21 {YC 21.0500. N}
90. Human pre-B cell enhancing factor (PBEF) mRNA, complete cds {Incyte PD:1641590}
91. Cytochrome P450, subfamily IIC (mephenytoin 4-hydroxylase) {Incyte PD:168865}
92. Latent transforming growth factor beta binding protein 1{Incyte PD:1313183}
93. Lysyl hydroxylase {Incyte PD:1759127}
94. Human mRNA for KIAA0230 gene, partial cds {Incyte PD:1449824}
95. Human mRNA for dihydropyrimidinase related protein-2, complete cds {Incyte PD:2784546}
96. H.sapiens garp gene mRNA, complete CDS {Incyte PD:3572014}
97. EST {Incyte PD:724880}
98. ESTs, Weakly similar to TRANSMEMBRANE PROTEIN SEX PRECURSOR [H.sapiens] {Incyte PD:1511346}
99. Human contactin associated protein (Caspr) mRNA, complete cds {Incyte PD:2309047}
100. Human cysteine-rich fibroblast growth factor receptor (CFR-1) mRNA, complete cds {Incyte PD:2204871}
101. EST {Incyte PD:2580841}
102. Collagen, type V, alpha {Incyte PD:1887959}
103. H.sapiens RNA for type VI collagen alpha3 chain {Incyte PD:1314882}
104. Protein kinase C substrate 80K-H {Incyte PD:1723971}
105. Fibrillin 1 (Marfan syndrome) {Incyte PD:1448051}
106. Collagen, type XI, alpha 1 {Incyte PD:3598222}
107. H.sapiens mRNA for extracellular matrix protein collagen type )aV, C-terminus {Incyte PD:2208990}
108. Collagen, type II, alpha 1 (primary osteoarthritis, spondyloepiphyseal dysplasia, congenital) {Incyte PD:2518178}
109. ESTs, Weakly similar to unknown [S.cerevisiae] {Incyte PD:2171401}
110. EST {Incyte PD:1923572}
111. Human insulin-like growth factor binding protein 5 (IGFBP5) mRNA {Incyte PD:1686585}
112. Human mRNA for KIAA0242 gene, partial cds {Incyte PD:1940994}
113. Complement component 1, s subcomponent {Incyte PD:1904751}
114. Human chromosome 17 unknown product mRNA, complete cds {Incyte PD:2849603}
115. Homo sapiens lysosomal pepstatin insensitive protease (CLN2) mRNA, complete cds {Incyte PD:3500996}
116. Collagen, type IV, alpha 2 {Incyte PD:1906574}
117. ESTs, Moderately similar to ZINC FINGER PROTEIN HF.12 [Homo sapiens] {Incyte PD:3729155}
118. Homo sapiens stanniocalcin precursor (STC) mRNA, complete cds {Incyte PD:2222921}
119. P55-C-FOS PROTO-ONCOGENE PROTEIN {Incyte PD:341491}
120. EST {Incyte PD:2424631}
121. EST {Incyte PD:1940710}
122. Thrombospondin 1 {Incyte PD:2055534}
123. Complement component C 1r {Incyte PD:1664320}
124. REGULATOR OF G-PROTEIN SIGNALLING 2{Incyte PD:1218114}
125. INTEGRAL MEMBRANE PROTEIN E16 {Incyte PD:1911012}
126. Collagen, type I, alpha 1 {Incyte PD:782235}
127. H.sapiens mRNA for adipophilin {Incyte PD:1985104}
128. EST {Incyte PD:1979450}
129. EST {Incyte PD:690994}
130. Cathepsin D (lysosomal aspartyl protease) {Incyte PD:3940755}
131. Matrix metalloproteinase 2 (gelatinase A, 72 kD gelatinase, 72 kD type IV collagenase) {Incyte PD:1558081}
132. Cyclin D2 {Incyte PD:1618422}
133. EST {Incyte PD:2636514}
134. COMPLEMENT C3 PRECURSOR {Incyte PD:1513989}
135. Homo sapiens secreted frizzled related protein mRNA, complete cds {Incyte PD:428236}
136. INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN 3 PRECURSOR {Incyte PD:1447903}
137. Fibronectin 1 {Incyte PD:3553729}
138. Early growth response protein 1 {Incyte PD:1705208}
139. Human autoantigen DFS70 mRNA, partial cds {Incyte PD:42920}
140. Prostaglandin-endoperoxide synffiase 2 (prostaglandin G/H synthase and cyclooxygenase) {Incyte PD:3139163}
141. Synteni: YCFR 43 {YC 43.2000.W}
142. Synteni: YCFR 43 {YC 43.2000.Z}
143. Synteni: YCFR 23 {YC 23.0062.N}
144. Synteni: YCFR 43 {YC 43.2000.Y}
145. Homo sapiens phosphomevalonate kinase mRNA, complete cds {Incyte PD:1497123}
146. Synteni: YCFR 43 {YC 43.2000.X}
147. Synteni: YCFR 23 {YC 23.0031.N}
148. CARTILAGE GLYCOPROTEIN-39 PRECURSOR {Incyte PD:157510}
149. Synteni: YCFR 23 {YC 23.0125.N}
150. Synteni: YCFR 23 {YC 23.0250.N}
151. Synteni: YCFR 23 {YC 23.4000.N}
152. Human germline oligomeric matrix protein (COMP) mRNA, complete cds {Incyte PD:2636634}
Prototype of the System
FIGS. 10 and 11 are partially diagrammatic representations of one embodiment of the system 1. Spherical disks 2 of biocompatable material are arranged along the horizontal spin filter or horizontal oxygenator of a standard rotating wall perfused vessel or rotating wall batch-fed vessel 5.
The electrically conductive, bioattractive strips 6 are each suitably embedded in or affixed to each of the disks such that each disk has a positive and negative pole 12, associated with positive and negative terminals 12 connected to respective endportions of the strips, as shown in FIG. 11A. As will be more fully described, in one preferred embodiment in which an alternating current is applied to the strips, the polarity of the strips changes cyclically in correspondence with the change in polarity of the applied current. Each biocompatable disk is preferably two-sided, allowing growth of tissue on both the left and right portions of the spherical disk. Sterilization of the reactor core is effected by one of multiple sterilization procedure, either ethylene oxide sterilization, autoclave sterilization if the polymer perrnits, or in the case of the batch-fed vessel, sterilization with hydrogen peroxide. After sterilization and sufficient detoxification procedures, cellular material is seeded into the reactor at a level to be determined according to the cell line of interest.
A source of time varying current 10, suitably a laboratory current source with adjustable wave-forn output connected to a remote power source, not shown, is operable to provide a time varying current, suitably of a value of about 1 mA to about 1,000 mA, in the present embodiment. The time varying current is suitably an alternating current, as indicated above, although in other embodiments it is a pulsating DC current. The current is conducted from the source 10 along first and second conductors 13A and 13B to slip rings 4. The slip rings are non-rotatable relative to the vessel 5, and therefore rotate with the vessel during operation. Current received through conductors 13A and 13B is conducted through the associated slip rings to first and second sets of conductors, represented by first and second conductors 14A and 14B, which are preferably insulated with an insulative material, not shown, compatible with the fluids and products within the bioreactor chamber. Each conductor is mechanically connected to a respective peripheral portion of each of the respective discs, and electrically connected with an end portion of one of the conductive strips 6 (FIGS. 1A and 1B). As viewed in FIG. 10, conductor 14A and the associated slip ring are indicated to be of positive (+) polarity, and conductor 14 B and its associated slip ring are indicated to be of relatively negative (−) polarity. As suggested above, upon the current changing in polarity, conductor 14A will momentarily have a negative potential relative to conductor 14B, thereby permitting a time varying current, which in this embodiment is an alternating current, to flow through conductor 6 which. In other embodiments, the time-varying current may be in the form of a pulsating DC current, suitably a square wave or other waveform, rather than an alternating current, in which case the conductors 14A and 14B and their associated slip rings remain of the same polarity but of differing potentials.
After inoculation, the rotating wall vessel 5 is rotated at an appropriate speed and single cellular material begins to attach onto the surface of the biocompatable material 6. After initial growth of one 24- or 48-hour period, electrical stimulation, i.e., potentiation, begins via continuous low-level or pulsatile electrical flow through each disk in series.
Use of the methods of the present invention to control the proliferative rate of normal human adult astrocytes and normal human neural progenitor cells (NHNP) has been demonstrated. The procedure is applicable to, but not limited to, the control of normal human neural cells in both two-dimensional and threeimensional culture. As presented in the molecular genetic data shown in Table 5 and Table 6, many of the genetic responses in both up regulated and down regulated genes are maturation and growth regulatory in nature. An inspection reveals these genes are also primarily involved in the eipbryogenic process. Therefore it is reasonable to conclude that control over the embryogenic development process can be achieved via the presently demonstrated methodology.
As shown in Table 6, specific genes such as human germline oligomeric matrix protein, prostaglandin endoperoxide synthase 2, early growth response protein 1, and insulin like growth factor binding protein 3 precursor are highly up regulated, while Keratin Type II cytoskelatal 7, mytotic kinesin like protein 1, transcription factor 6 like 1, mytotic feedback control protein, and cellular retinoic acid binding protein are down regulated (Table 5). Each of these two sets or classes of genes are only examples from the sum of approximately 320 genes changes expressed as a consequence of exposure to electrical potentiation.
As is clearly demonstrated in the human body, the bioelectric, biochemical process of electrical nerve stimulation is a documented reality. The present invention demonstrates that the same phenomena can be potentiated in a synthetic atmosphere, i.e., in rotating wall cell culture vessels. As may be understood from the forgoing discussion, this electrical potentiation can be used for a number of purposes.
The following references were cited herein.
1. Schwarz et al., U.S. Pat. No. 4,988,623, (1991).
2. Schwarz et al., U.S. Pat. No. 5,026,650, (1991).
3. Goodwin, et al., In Vitro Cell Dev. Biol., 28A:47-60(1992).
4. Goodwin, et al., Proc. Soc. Exp. Biol. Med., 202:181-192 (1993).
5. Goodwin, et al., J. Cell Biochem., 51:301-311 (1993).
6. Goodwin, et al., In Vitro Cell Dev. Biol. Anim., 33:366-374 (1997).
7. Fukuda et al., U.S. Pat. No. 5,328,843
8. Aebischer, U.S. Pat. No. 5,030,225
Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.
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South Derbyshire youngsters get on their bikes as Tour de France begins
Posted: July 04, 2014|
AS the Tour de France got underway in Yorkshire yesterday, children at a school in South Derbyshire have also been getting on their bikes.
Pupils at Pennine Way Junior Academy have been taking part in the 'Bikeability' course, which is led by the district council and educates youngsters about being responsible when riding on roads and built-up areas. Children are being encouraged to cycle to school to help address parking issues in the area.
The scheme also teaches children to signal properly, conduct emergency stops and how to care and look after their bikes. Teacher Richard Snape, who is in charge of cycling at thev school, said: "Not only are we teaching children to cycle safely on the roads and cycle paths but also encouraging them to lead more healthy lifestyles.
"We also have a huge car parking problem around Pennine Way and we hope that encouraging more children to come to school on their bikes will ease the conjestion around the school, in the morning and at the end of the day."
South Derbyshire Bikeability coordiantor, Stedroy Baker, said:"The children at Pennine Way have been brilliant. Not only are they working hard and listening to instructions in order to keep them safe, but they're enjoying it immensly aswell. It been a fantastic two days cycle training."

subset: commoncrawl


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Mandalay Holiday Resort And Tourist Park
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TravelPod Member ReviewsMandalay Holiday Resort And Tourist Park Busselton
Well kept grounds, cool water slide
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
Lovely to have a park so well kept. Very pleasant and helpful staff
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
Lovely caravan park, great facilities although the pool was being refurbished while we were there. Assistance given parking at sites, lots of green grass. Only thing we didn't like was we were given 3 A4 pages of rules and regulations when we checked in - way over the top. All persons under 21 have to wear wristbands for the entire length of their stay in the park. We have come across lots of nice caravan parks in our travels but none have had the need to have so many rules. It spoiled the experience for us, and we're not rule breakers, it was just over the top
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
If you want a spotlessly clean, manicured and well situated caravan park, then this is for you.
The staff are some of the best we've come across and the facilities are second to none.
We cannot find a single fault!
This review is the subjective opinion of a TravelPod member and not of TravelPod.com.
TripAdvisor Reviews Mandalay Holiday Resort And Tourist Park Busselton
Travel Blogs from Busselton
Day 12 - York to Perth Airport & Busselton (350km)
Up at 7am, no-one to annoy as we left the almost deserted Motel, and we were on the road by 8.10am - a small miracle for the Dawsons, only 10 minutes late, today!
Everything was going nicely to plan, until we were 5 mins up the road heading for Perth airport, and then the diesel warning light came on - D'Oh !! The one thing we forgot to …
Getting our winery tour on!
Wow this part of WA is so beautiful just like everyone had said. So green and lush and the beaches are stunning with their aqua rolling waves. Around every corner is a winery or brewery or chocolate shop or ice creamery! Renee is in heaven-we all are for that matter-something for everyone! We are staying at the Mandalay Holiday Resort in Busselton …
Day 114
Still overcast & cool. Travelled through Nannup (timber town) heading to Busselton. Still driving through timber forests & farming land. Stopped at a little farm that make sheep cheese. They milk around 200 sheep. They had just finished milking when we got there. The cheese was great, they have won a number of awards for it. Set up camp in …
Back On The Road
... us their car for the trip so once we'd sobered up from our leaving party it was time to set off on the 5 hour drive.
We'd decided camping would be a good idea and so we'd invested heavily in a 4 man dome tent ($25) and an air bed ($27). The tent was put to the test on the first night as the heavens opened. It largely withstood the deluge but there was a little bit of water around the insides giving a ...


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Vitro G1022 DescriptionSKU: 120-G1022
120-G1022 G Series Cantilever, for 24' - 30' Long Table Tops
G Series Cantilever, for 24' - 30' long tabletops, 2' square construction, table mounting bracket included, painted black finish.
Wall blocking recommended at 11-3/4', 16-1/2' and 27' to secure the G1022 cantilever.
Price discount for quantities of 5 or more.
Contact customer service for pricing. You can question a customer service representative about this product by phone (1-800-541-8683), by Live Chat, or by the contact form at the bottom of this page.
Due to inconsistencies in colors displayed by computer monitors and printers, please consider colors and finishes displayed on this website to be a guideline only. If you are trying to match existing colors, or obtain an exact color please request a sample prior to ordering. Due to the thousands of options in finish and colors, restaurant furniture is custom manufactured to your specifications and there are no returns or refunds once an order has been placed with the manufacturer.
Vitro is a premier furniture manufacturer specializing in the restaurant, food service and hospitality industries. For most Vitro products there are an almost limitless number of colors and finishes. We provide the resources to you to get an idea of the finishes you want. If you are matching existing furniture or restaurant finishings we can obtain a sample for you from the manufacturer of the stain or fabric. As with all high quality furniture we would like to speak with you about your order to ensure you have selected the finish and other options that you want. We are happy to communicate with you via telephone or email. Once you have identified the options in which you are interested, we can then obtain a quote from Vitro for any additional cost of non standard finishes, an estimate of shipping and delivery date. All Vitro furniture is custom made to your specifications and will carry a lead time of at least 5-6 weeks. Some products and finishes may carry a lead time of 7-8 weeks or more. No returns on this item. Once the order has been placed with the factory, prior to manufacturing and/or shipping, the customer is responsible for any costs incurred if the order is modified or cancelled.
G Series Cantilver Style Base, 2" square heavy wall construction, includes table mounting bracket, black painted finish, for table tops 24" - 30" long. Wall blocking recommended at 11-3/4", 16-1/2" and 27" to secure cantilever for 30" table height.
Vitro G1022 SpecificationsPDF Spec Sheet


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Can Large MPAs Protect Tuna and Sharks?
Thu, 04 Jun 2015 14:04:29 +0000
a guest blog by David Curnick, written for the 2015 Write About Research Competition. With a global human population of over 7 billion it is becoming ever more important to manage our natural resources effectively. For centuries, the oceans have been seen as an endless bounty, ripe for harvesting. However, this simply isn't the case […]
The post Can Large MPAs Protect Tuna and Sharks? appeared first on GEE Research.Read more...
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The Maine Public Broadcasting Network's innovative Super Thursday One-Day Radio Pledge effort exceeded its spring membership drive goal on Thursday night, raising $374,000. During the 24-hour period, over 3,750 listeners donated by phone and online resulting in a pledge total that set a record for the organization. More than 18% of the callers were new members.
The 35th Boston/New England Emmy Award nominations have been announced, and five programs from the Maine Public Broadcasting Network garnered nominations. The Boston/New England Emmy Awards recognize the best work being done in Television throughout Maine, New Hampshire, Vermont, Upstate New York, Massachusetts, Rhode Island, and Connecticut.
The Maine Public Broadcasting Network announces the return of the Maine Capitol Connection on Wednesday, January 8th at 9:00 a.m. This is the second year of the free over-the-air television and cable channel devoted to providing public access to the political processes of the Maine State Legislature in Augusta. Journalist Mal Leary will again serve as the Managing Editor and Director of the channel.
The Maine Public Broadcasting Network announces the selection of Robin Rilette as MPBN"s new Music Director and host of MPBN"s Morning Classical Music program. The result of a comprehensive national search, Rilette was vetted by MPBN staff in Bangor and Portland, community leaders in Maine, and members of arts organizations across the state. Rilette is expected to join MPBN in early February and immediately take over the Morning Classical host position.
The Maine Public Broadcasting Network announces preliminary plans to launch a free over-the-air television channel devoted to covering the Maine State Legislature in Augusta, Maine. The service could potentially launch as early as February 2013 as part of a six month pilot program to test the concept. Coverage would include daily sessions of the House of Representatives and the Maine Senate as well as potentially select committee hearings and press conferences.
Founded by the University of Maine System and Colby, Bates and Bowdoin Colleges.


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thisisevenlessfunnow 26-30, M 7 Answers 3 Dec 3, 2012 in Parenting & Family
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No and I'll ask one.
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I have asked almost 5,000 questions on EP. Do YOU take a title for one of the most rated, most answered questions?I've had quite a few. Both entertaining and serious. I love EP, but I've been away for awhile. Been sick with back pain. Just want to say I've appreciated all of you during the...PrincessMaine 46-50, F 8 Answers 4 Apr 13, 2013 in WTK
lezstar 46-50, M 25 Answers 1 Jan 26, 2010
Daphney1001 18-21, M 5 Answers 2 Mar 19, 2012
Several people commented about my questions.Not offended I promise, but I don't understand what is wrong with them?i don't think they meant they were offensive, but just curious to the origins. I really don't care. Just being my curious selfIMAGINATIONATWORK 36-40 8 Answers 4 May 12, 2012
I wish EP had a section for "least answered questions". Do you try and answer ppls ? if they do not have answer yet?3grnowl 26-30 11 Answers 7 May 30, 2012
Have you ever gotten on ep with a buzz, answered questions, gotten best answer but the next day don't remember?jumpsforfun 31-35, F 3 Answers 2 Nov 8, 2012 in Hobbies
Have you answered questions without considering if it will offend the person asking? If so, did you apologize?


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Cast, creators of TV's 'Walking Dead' shuffling to Riverside
Three stars and an executive producer of AMC's horror hit series "The Walking Dead" will be live and in person at the Riverside Theater on Feb. 16.
Norman Reedus, who plays the crossbow-wielding outcast Daryl, and Steven Yeun and Lauren Cohan, who portray the romantically involved Glenn and Maggie on the apocalyptic zombie series, will join Greg Nicotero, a co-executive producer, special-effects makeup artist and episode director, for a live panel discussion. They also will answer fan questions, provide an inside look behind the scenes of the show and screen never-before-seen footage. »Read Full Article(3)
Darkness over the top - but in a good way
Justin Hawkins, frontman for British band The Darkness, had some choice words to say in a Journal Sentinel interview for those who think his glam rock group is a joke.
But let us examine some of the damning evidence at the band's Rave show Saturday, shall we? »Read Full Article
Fitzwater brings Piaf alive in Skylight's 'Onstage'
The music and life story of popular French singer Edith Piaf get under one's skin.
Singer-actress Leslie Fitzwater, who has brought Piaf to the stage several times over the course of her career, opened her one-woman "Edith Piaf Onstage" at the Skylight Music Theatre on Friday evening. »Read Full Article
First Stage's ambitious 'Promised Land' is a dream deferred
Ruth, an African-American girl in her early teens, is lost. Her father is long gone. Her overworked mother is rarely around. Her beloved brother is dead. It's 1969, and, while Ruth should be in school, she is watching TV instead.
In "To the Promised Land" - unveiled by First Stage Children's Theater on Friday night - playwright Jonathan Gillard Daly links Ruth's struggles with another coming-of-age story that unfolded in Milwaukee, where a young Golda Meir underwent her own journey of self-discovery. Daly places both girls in the same house on Walnut St., nearly 60 years apart. »Read Full Article


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A super cheap way to keep your beverages cold on the water.
Gather your supplies- a swim ring and a garbage bag. Inflate the ring, place into the garbage bag. Tie off the bag, and you're done. Will still functi...
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How to Keep Your Beverage Cold While Out and Aboutby thickneckarts
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The board of directors of the Humphrey Memorial Park and Museum has decided not to host the Gloaminfest in October and is closing the vintage home for the season early this year, instead of staying open for the Christmas season as it has in the past.
The Gloaminfest will be at St. Laurence Episcopal Church in Conifer instead.
The museum will close Oct. 1 and reopen April 1 to save money and perhaps find a new partner and regroup. The board has decided to sell an attached 14-acre property for an undisclosed amount, which could help replenish the coffers.
A group of homeowners on Bear Mountain in Evergreen has applied for a 2009 wildland-urban interface grant from the Colorado State Forest Service to help mitigate fire danger in their mountaintop neighborhood of 200 homes.
The idea is to join forces and launch a war against dead and diseased trees and to identify trees to be thinned. Participants use their own equipment and volunteer time and muscle power.
The proposal also asks for funds to educate people on FireWise principles of home protection.
County commissioners, county clerks and even the governor are urging people to either vote by mail or vote early this year, citing an unusually long ballot and a projected record turnout.
"We've had a lot of activity here in the last couple of weeks from both sides of the presidential campaign, with both Sen. Obama and Gov. Palin visiting Colorado," Gov. Bill Ritter said at a news conference Sept. 16. He said that Colorado is one of the few swing states in the national election. "It is important as a swing state that every vote count."
Jeffco Commissioner Kevin McCasky took most of the heat Sept. 15 in the first forum for the five county commission candidates.
Republican McCasky, the only incumbent, faces two challengers in District 2 in the Nov. 4 election: Democrat Jason Bane and Scott Zulauf of the Green Party. Jeffco Treasurer Faye Griffin, a Republican, and state Sen. Sue Windels, a Democrat, are battling for the District 1 commission seat, vying to replace Jim Congrove, who decided not to seek a second term.
Justice was swift for Ryan "Mad Dog" Lucas, director of Rocky Mountain Academy of Evergreen.
Lucas was arrested, tried and convicted last Wednesday of what some of his students thought was a rather heinous crime — not proclaiming enough snow days during the last school year.
His bail was set by Judge Tucker S. Gordon, a fifth-grader at the school, at $30,000.
Doors opened at 7 a.m. Sept. 15 for Sarah Palin's appearance at the Westernaires Equestrian Center at the Jefferson County Fairgrounds, but that wasn't early enough for some.
Thousands turned out to see the Alaska governor, who has electrified the campaign of Sen. John McCain since he named her as his running mate.
JEFFERSON COUNTY — An Idaho Springs man was thrown 50 feet Thursday afternoon after losing control of his motorcycle while allegedly racing another motorist on I-70.
Steven P. Smith, 39, was heading west on I-70 near Lookout Mountain when his red 1998 Honda motorcycle crossed two lanes and hit the guardrail. Police said witnesses reported that a black 2004 Porsche was racing with Smith's motorcycle. Smith was airlifted to St. Anthony Hospital with serious bodily injury and as of Friday afternoon remained in critical condition.
If you live in Evergreen and always wanted to be a firefighter, now is the time to apply for a spot in the volunteer fire department.
The department is looking to beef up the volunteer force with new recruits, and the application deadline is Nov. 1.
"Most people don't even know Evergreen Fire/Rescue consists of people like your next-door neighbor, an engineer, a truck driver or local small-business owner," said Mike Bleidl, training coordinator.
Jefferson County Open Space has recommended the county purchase 7 acres of residential-zoned vacant land adjacent to Elk Meadow Park for $600,000 and maintain it as a nature preserve.
The purchase has to be voted on and approved by the county commissioners.
The proposal was unveiled during a regular monthly meeting of the Open Space Advisory Committee in Golden on Sept. 4.
Most of the committee members agreed the land should be incorporated into Elk Meadow Park, although two voted against the purchase for cost reasons.
The Lord said to Jeremiah: "Buy a farm at Anathoth ee it will have houses, fields and vineyards."
— Jeremiah 32:8
When she returns to her adopted continent later this month, longtime Evergreen resident Pat Stephenson will bring her faith, her optimism, her unflagging commitment and — with a little help from her neighbors — enough wholesome, nutritious and desperately needed food to keep 1,000 African schoolchildren at their books for a solid year.
Copyright © 2015
Canyon Courier is your source for local news, sports, events, and information in Evergreen, Colo, and the surrounding area.


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Maldoror : the Last Film Ever Made
2 Aug 2014
Event times
Cost of entry
A rare chance to see the cult Super 8 feature film projected live on 16mm film.
AboutPremiered in Germany in April 2000, Maldoror has hypnotised and thrilled both audiences and press... a cult classic for the price of a second hand car. The action takes place somewhere behind an impossible mesh of sex, violence, emulsion, bacteria, oil, bleach, petals, glue, dirt, abrasion, acid, superstition, glare, fur, insomnia, amphetamines and deep blue cold water. At first this mesh appears to be on the screen but slowly you realise that it is in fact behind your own eyes ! Despite its subject matter Maldoror is perhaps the most realistic feature film ever made, for although the big budget multiplex features strain with every tense and twisted fibre to conjure a world of carefree spontaneity they cannot compete with the reality of filmmakers who really don¹t give a fuck. Whilst the multiplex constructs its reality with narrative, style, action and music, Maldoror uses all these techniques but also adds real human conflict, chance, technical distortion, film surface scrimshaw, creative democracy and diversity. Maldoror does not just conjure the illusion of reality, it is actually real at the same time. Its got something that the multiplex cannot offer...... WILDNESS. We have identified a gap in the market and we have turned that gap into a terrible breach. Make no mistake once you have seen it all other films are meaningless. After Maldoror, filmmaking is no longer possible; in fact, it no longer has any purpose.
Art form
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Headquartered in Houston, TX, we are a fast growing organization which has been recognized as one of the fastest growing technology companies in the US in the Deloitte Fast 500 list, the Inc. 5000 list and the Houston Business Journals Fast Tech 50.
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We provide Systems Management tools for Windows and Linux Servers.
Our products are designed to be installed and deployed within minutesyet have the ability to manage datacenters with hundreds or even thousands of distributed computer servers.
With over 5,000 customers worldwide ranging from companies with a single server, to some of the largest datacenters in the world, we provide solutions for:
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Link up your social media account to automatically promote the positions at Idera.
Get a job alert every time Idera posts a job.
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Many of the 11 million illegal immigrants living in the US are hoping the president's speech on immigration reform will give them a better chance of citizenship.
Obama has his own blueprint for immigration reform, but he is expected to back another slightly different one put together by a bipartisan group of senators, which is to be "largely consistent" with the president's plan.
Speaking from a Las Vegas high school on Tuesday around 11.15 local time, Obama will talk about an issue that led to one of the biggest re-election promises.
"It's an opportunity for all of us, I really hope the president keeps his promise and lays out a plan," said one man close to the San Ysidro port of entry in San Diego, California.
"It's great if we all can be here, but laws on this side of the border have to be respected," said a woman on the US side of the Mexican border bridge near San Ysidro.
Officials say Obama will not be revealing any legislation of his own, but is instead hoping to rally support for plan put together by the cross-party group of senators.
A number of efforts to overhaul immigration over the past two decades have failed – hampered by right-wing Republicans.
The new proposal also includes enhanced security to prevent illegal immigration in the future.
- 1Istanbul Gay pride quashed by riot police, rubber bullets and water cannon
- 2Greek debt: who will pay if Greece fails?
- 3Saakashvili warns of Odessa region's importance to Ukraine
- 4Tsipras says if Greeks approve the EU's aid plan, Syriza will resign
- 5Suspect in French beheading attack admits killing his boss
- 1euronews live TV - News | euronews : the latest international news as video on demand
- 2Greek debt crisis is "absolute supremacy of capital over humans"
- 3[Live updates] NGO flotilla bids to break Israeli blockade of Gaza
- 4Spain's first case of diphtheria in 30 years: parents of six-year-old 'oppose vaccines'
- 5[LIVE UPDATES] Greek debt deadline looms
- 6Large Hadron Collider ready to embark on an unprecedented voyage of discovery
- 7Istanbul Gay pride quashed by riot police, rubber bullets and water cannon
- 8[LIVE UPDATES] France: man decapitated, several wounded in chemical plant attack
- 9Israel prepares to repel boarders as 'Freedom Flotilla 3' tries to run Gaza blockade
- 10NewsWires : euronews : the latest international news as video on demand
- 11Greek debt summit – Live updates
- 12'Distractingly sexy' scientists hit back in lab chemistry polemic
- 13Summit up in Brussels as Greek proposals give food for thought and rumours fly
- 14Battle of Waterloo, live-tweeting 200 years on
- 15Greek PM faces day of crucial bailout talks in Brussels
- 16Citizens take power in Spain's largest cities as a political revolution sweeps the country
- 17Italy discovers biggest illegal waste dump in Europe
- 18Greek banks, stock exchange will not open on Monday, Tsipras announces
- 19Greek debt: who will pay if Greece fails?
- 20Hungary: billboard war sparks international concern
Wires > News
- 11:22 CET China says tourists attacked in Turkey during anti-China protests
- 11:00 CET Malaysia's opposition demands emergency debate on PM after graft…
- 08:12 CET China arrests man for suicide 'rumours' amid stock market rout -…
- 07:04 CET Colombia peace process now at lowest ebb – government
- 01:50 CET America celebrates July 4 with hot dogs, banners and barbecues
- 01:32 CET China lobbies hard ahead of Manila's South China Sea arbitration…
- 01:27 CET Small is beautiful, say South Koreans making plans to marry
- 01:06 CET Pope heads to South America to champion the poor and the planet


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January 14, 2014
Gladys C. Zolna has been elevated to partner at Chicago-based law firm Freeborn & Peters. Zolna is a member of the firm¿s corporate practice group, with experience involving qualified retirement plans, health and welfare benefits and executive compensation. She advises small and midsize businesses, as well as Fortune 500 companies. Zolna earned her law degree from the University of Chicago and also has a bachelor's degree from the University of Illinois at Urbana-Champaign.
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CTnow.com, 285 Broad St., Hartford, CT 06115
A Hartford Courant website


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I just stumbled upon this. I use a mixture from trueprotein thats is 75% Casien and 25% Whey, it doesn't mix well. So I threw a banana, cup of milk, and 2 scoops of protein in blender instead of a shaker thinking it would be like a drink for once..... well it turned into pudding, AND IT'S GOOD.
Try it out.
1 cup 2% milk
1 banana
1-2 scoops protein (pref. casien since it tends not to mix as well as whey) and I would suggest something that would go good with teh banana.
Next time I'll throw in some peanut butter.
Basically turned into 4g fat, 33g carb, and 58g protein in approx. 425 calories.


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Remember Bedroom Inspiration series ? If you read this blog for a while you might remember that I've started this series some time ago. Today I want to continue this series with 5 modern bedrooms that I'm sure you'll love. Now enjoy these 5 modern, simple and beautiful bedrooms. If you want more bedrooms you should also to check the whole bedroom design inspiration series starting with the last post Bedroom Inspiration #5. The pictures for this post are from spacify.
39 comments
so let me get this straight,
modern bedroom = square futon + (floors – carpet) – box spring mattress + plus blanket sprawled across bed
Link back – http://inspiredbye.wordpress.com/2007/06/27/bedroom-inspiration/
Hubs – Yes … it seems that this is the trend now .. simplicity.
umm modern = very ugly and angular
[…] that now is at number 7. If you would like to see more bedroom inspiration you can start from here, and keep follow the links from that […]
these bedrooms look sick. i love the modern look
I think the modern bedroom is good for the single, but for a family it's not practical. imagine a two year cruising in a room like that oopps!
that's bull, it's all about the colors and durability of the fabrics… you can have kids in any style of bedroom.
I absolutely LOVE the simplicity of a modern bedroom however, as an interior design student I can tell you that there are sooooo many more options out there and many other ways to arrive at a contemporary feeling bedroom without the common "trendy" goods seen in these pics.
I have a contemporary style home myself and love the spacious, conservative feel…It allows my mind to wander and inspires creativity without being stuffy and overwhelming…LOVE IT!
I love modern. I will one day have a modern home I kind them so amazing!
In fact, I dislik this style because of its shape very angular
^FAO: Hubs: This style of bed is not a "futon", nor is it called a Boxspring. This low pro style of bed is usually called "Platform bed". I personally LOVE modern and contemporary interiors and I think that all these bedroom designs have absolutly stunning lines and details. Absolutly BEAUTIFUL.
I was wondering if you would know the aproximate cost for the first bed on this page. Its white and has the red bed sheets and stuff. Please email me back
I love modern style.
I love how it's so vivid and approximate.
Theres a perfect place for everything..
I typically think "White"
And pretty plain, but I love the style
especially the fourth one down.
look at the 3 picture and the decorations, so perfect.
You could have these rooms messy and they'll still look perfect.
modern is sooo kewl its like the future in beds right before your eyes!!!! I really think modern is really kewl.Plus i have one of those modern beds mines a nice platform bed!!!
modern is very cool it reminds me of the future. You all might think different but thats ok.I really like the style but you know…. I have a platfom bed and its so cool. I like how its low to the ground but you really cant stuff anything under there.
i love minimalistic design especialy in big rooms i love the coldness and the neatness they have :)) perfect!
this is the best webiste in the world!
I think that the modern design is beautiful. It is simple, yet elegant. Unlike the old room designs, which was piling a bunch of furniture and trying to make as much textured designs as possible, and called it a work of art. Nowadays, we know that more doesn't mean better. The modern design is much more clean looking. It makes you feel more free, refreshing, and lets your spiritual energy flow. And the solid colors and simple shapes is complimented with a touch of decorative designs, that work together in harmony.
I like this design as well. True compared to what is in your home now, a modern edge might seem out of place in your bedroom. Do a little research, take a look at some modern dining sets
or some modern lighting and see what peice you may be able to incorporate in to your existing style to give you home a little modern facelift
I love bedroom #2,4,5. They look so lovely and stylish. The
modern design has its own style and I like the way they keep it
as simple as possible with a great color combination.
I love modern style.this site is too good thanks.
WOW!! yes i love MODERN STYLE …it is soo freakin stylish and elegant…whooohh..dunt i wish i could afford these in my home..lolz..
WOW!! yes i love MODERN STYLE
I think Modern design is very elegant in its own, not that opulance, but pieces standing alone make their own elegance, but quietly, like art.The sparcity of placement.Minimalism was not having much furniture.unable to afford more. Now its a style. I think maybe its a conciousness, doing with less, and look….wow…what a statement!!!!!
I have a project and I can't find a problem I have that I can solve in my project. Does anyone have any ideas of a problem around the house. (for example if I have too many shoes around the house I can make a shoe stand to help me have a neat and tidy room) There must be a problem and the design must help solve the problem. If anyone has any good ideas please reply.
this modern interior is really cool. i can see my future home lookin something like this. its great!
wow its awesome
its just ok, not that bad either. if you going to insult the design..
you just dont know what your saying. why dont you study the elements of design first. before you throw some fowl word.
half use mind's
Simple and minimalist but luxurious…………
Its up to the test of each one to like particular design.
lovely……………really wonderful
They are all awesome for people to have wonderful dreams always which may clear dought to future.
very nicedecent
nice designs
[…] Modern Bedroom – http://freshome.com/2007/06/26/modern-bedroom-inspiration-6/ […]
this modern interior is really cool. i can see my future home lookin something like this. its great!
The second and fourth one are fantastic, wish my bedroom is larger than it is now so I can decorate it like the fourth one.
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text:
Last night Maricopa County Sheriff Joe Arpaio made an appearance on Fox News' Hannity to discuss the ongoing influx of unaccompanied children and illegal immigrants in Texas and Arizona. Arpaio made the point that it would be cheaper to fly illegal immigrants back to their home countries than it is to fly them from Arizona to Texas as the federal government has been doing for weeks as Border Patrol facilities continue to be overwhelmed.
One key point Sean Hannity made in the interview is that the federal government is essentially acting as a human smuggler by transporting illegal immigrants from state-to-state. Last week, Congressman Paul Gosar sent a letter to Homeland Security Secretary Jeh Johnson demanding the administration terminate its alien smuggling policy.
"I demand the Administration immediately terminate this atrocious transfer policy. Furthermore, the administration should take the necessary steps to secure our border and should take bold measures to discourage this influx of illegal aliens from continuing," Gosar said. "The Administration's actions jeopardize our national security and embolden the cartels. Those individuals responsible for this violation of the Rule of Law need to be held accountable and I will continue to push for a congressional investigation, and if necessary a criminal investigation, into this matter."
According to 8 USC § 1324 (a)(1)(A), putting an illegal alien "in a car, bus or plane and transporting them to another location is deemed "alien smuggling" and is a crime."
|Katie Pavlich is the Editor at Townhall.com. Follow her on Twitter @katiepavlich. She is a New York Times Best Selling author. Her new book Assault and Flattery: The Truth About the Left and Their War on Women, will be published on July 8, 2014.
"Mamas, don't let your babies grow up to be brainwashed government dependents. Buy this book!" says Michelle Malkin.
"This is a thorough and gutsy book that should help set history straight." - Mark Levin
Buy Katie's book today and help us keep the pressure on the Left.
Author Photo credit: Jensen Sutta Photography
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