text:
Electroporation is performed in a controlled manner in individual and multiple biological cells present in biological tissue by monitoring the electrical impedance, defined herein as the ratio of current to voltage in the electroporation cell. The impedance detects the onset of electroporation in the biological cell(s), and this information is used to control the intensity and duration of the voltage to assure that electroporation has occurred without destroying the cell(s). This is applicable to electroporation in general.
1. A method for performing electroporation in a controlled manner, comprising:
(a) applying a voltage across a tissue comprising a plurality of biological cells;
(b) continuously detecting the ratio of electric current through the tissue to voltage across the tissue as an indication of degree of electroporation of the biological cells; and
(c) adjusting the applied voltage in accordance with changes in current-to-voltage ratio to achieve a controlled degree of electroporation averaged over the biological cells, thereby achieving a controlled averaged degree of electroporation of the biological cells in the tissue.
2. The method of
step (b) comprises continuously detecting the current-to-voltage ratio as an indication of an onset of electroporation of the biological cells, and
step (c) comprises adjusting duration of the applied voltage in accordance with the current-to-voltage ratio to achieve a controlled degree of electroporation.
3. The method of
voltage is applied between two electrodes in a flow-through channel, and the electrodes are positioned to apply voltage in a direction transverse to a direction of flow through the channel;
step (a) comprises continuously passing the tissue through the channel;
step (b) comprises further correlating the current-to-voltage ratio with the presence of the biological cell between the electrodes; and
step (c) comprises adjusting the magnitude of the voltage while the biological cells are between the electrodes based on an averaged degree of electroporation of the biological cells in the tissue.
4. A method for the infusion of biological cells in a tissue with a chemical substance by electroporation in a manner that permits detection of onset of and control of the electroporation, comprising:
(a) securing biological cells in the tissue in an electrical cell containing a liquid with the chemical substance dissolved therein, the electrical cell containing a barrier to electric current, the barrier arranged such that, when a voltage is imposed across the electrical cell, the barrier restricts electric current flow to a flowpath passing through the biological cells while permitting substantially no electric current to bypass the biological cells in the tissue;
(b) imposing a voltage across the electrical cell and monitoring relative values of current passing through the electrical cell and of imposed voltage as an indication of occurrence of electroporation of biological cells in the tissue.
5. The method of
6. The method of
7. The method of
8. The method of
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10. The method of
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12. The method of
13. A device for the infusion of a grouping of biological cells of a tissue with a chemical substance by electroporation, comprising:
an electric cell containing an internal support to hold a grouping of biological cells of a tissue and an internal barrier of a material substantially impermeable to electric current, the baffler positioned to restrict electric current flow in the electric cell to a flowpath crossing the internal support and through biological cells held thereby; and
means for imposing a voltage across the electric cell and for monitoring relative values of current and voltage as an indication of occurrence and degree of electroporation in biological cells held thereby.
14. The device of
15. The device of
16. A method, comprising:
(a) identifying a grouping of biological cells in a tissue of a living mammal and applying a voltage across the cells;
(b) continuously detecting a ratio of electric current through the cells to voltage across the cells as an indication of degree of electroporation of the biological cells; and
(c) adjusting a determined magnitude of the applied voltage in accordance with changes in detected magnitude of the current-to-voltage ratio to achieve a controlled degree of electroporation of the grouping of cells in the tissue.
17. The method of
18. The method of
19. The method of
20. A method, comprising:
(a) placing biological tissue in an electrically conductive medium and applying a voltage across the medium;
(b) continuously detecting the ratio of electric current through the medium to voltage across the medium as an indication of degree of electroporation of cells of the biological tissue; and
(c) adjusting a magnitude of the applied voltage in accordance with changes in magnitude of the current-to-voltage ratio to achieve a controlled degree of electroporation of cells of the biological tissue.
This application is a continuation-in-part of application Ser. No. 10/079,940, filed Feb. 19, 2002, now U.S. Pat. No. 6,927,049, which is a continuation-in-part of application Ser. No. 09/618,951, filed Jul. 19, 2000, now U.S. Pat. No. 6,482,619, which is a continuation-in-part of application Ser. No. 09/358,510, filed Jul. 21, 1999, now U.S. Pat. No. 6,300,108 issued Oct. 9, 2001, all of which are incorporated herein by reference and to which are claimed priority under 35 USC �120.
This invention resides in the fields of electroporation and mass transfer across cell membranes and more specifically to electroporation of cells in tissue.
Electroporation is a technique that is used for introducing chemical species into biological cells, and is performed by exposing the cells to an electric potential that traverses the cell membrane. While its mechanism is not fully understood, electroporation is believed to involve the breakdown of the cell membrane lipid bilayer leading to the formation of transient or permanent pores in the membrane that permit the chemical species to enter the cell by diffusion. The electric potential is typically applied in pulses, and whether the pore formation is reversible or irreversible depends on such parameters as the amplitude, length, shape and repetition rate of the pulses, in addition to the type and development stage of the cell. As a method of introducing chemical species into cells, electroporation offers numerous advantages: it is simple to use; it can be used to treat whole populations of cells simultaneously; it can be used to introduce essentially any macromolecule into a cell; it can be used with a wide variety of primary or established cell lines and is particularly effective with certain cell lines; and it can be used on both prokaryotic and eukaryotic cells without major modifications or adaptations to cell type and origin. Electroporation is currently used on cells in suspension or in culture, as well as cells in tissues and organs.
Electroporation is currently performed by placing one or more cells, in suspension or in tissue, between two electrodes connected to a generator that emits pulses of a high-voltage electric field. The pore formation, or permealization, of the membrane occurs at the cell poles, which are the sites on the cell membranes that directly face the electrodes and thus the sites at which the transmembrane potential is highest. Unfortunately, the degree of permealization occurring in electroporation varies with the cell type and also varies among cells in a given population. Furthermore, since the procedure is performed in large populations of cells whose properties vary among the individual cells in the population, the electroporation conditions can only be selected to address the average qualities of the cell population; the procedure as currently practiced cannot be adapted to the specific characteristics of individual cells. Of particular concern is that under certain conditions, electroporation can induce irreversible pore formation and cell death. A high electric field, for example, may thus produce an increase in transfection efficiency in one portion of a cell population while causing cell death in another. A further problem with known methods of electroporation is that the efficiency of transfection by electroporation can at times be low. In the case of DNA, for example, a large amount of DNA is needed in the surrounding medium to achieve effective transformation of the cell.
Many of the problems identified above are a consequence of the fact that the process of electroporation in both individual cells and tissues cannot be controlled in real time. There are no means at present to ascertain in real time when a cell enters a state of electroporation. As a result, the outcome of an electroporation protocol can only be determined through the eventual consequences of the mass transfer process and its effect on the cell. These occur long after the mass transfer under electroporation has taken place. These and other deficiencies of current methods of electroporation are addressed by the present invention.
Also relevant to the present invention are current techniques for the study and control of mass transfer across cell membranes. Knowledge of mass transfer across cell membranes in nature, both in cells that are functioning normally and in diseased cells, is valuable in the study of certain diseases. In addition, the ability to modify and control mass transfer across cell membranes is a useful tool in conducting research and therapy in modem biotechnology and medicine. The introduction or removal of chemical species such as DNA or proteins from the cell to control the function, physiology, or behavior of the cell provides valuable information regarding both normal and abnormal physiological processes of the cell.
The most common method of effecting and studying mass transfer across a cell membrane is to place the cell in contact with a solution that contains the compound that is to be transported across the membrane, either with or without electroporation. This bulk transfer method does not permit precise control or measurement of the mass transfer across the membrane. The composition of the solution at specific sites is not known and is variable. In addition, when an electric field is present, the local field intensity will vary from one point to another. Furthermore, the surface of the cell that is exposed to the solution is not well defined. Cell surface areas vary among cells in a given population, and this leads to significant differences among the cells in the amount of mass transfer. For these reasons, the amount of mass transfer achieved by bulk transfer processes is not uniform among cells, and the actual amount transferred for any particular cell cannot be determined.
Attempts made so far to overcome the limitations of bulk transfer techniques include techniques for treating individual cells that include either the mechanical injection (microinjection) of chemical compounds through the cell membrane or electroporation with microelectrodes. In injection techniques, the membrane is penetrated with a needle to deliver a chemical agent, localizing the application of the chemical agent to a small region close to the point of injection. This requires manipulation of the cell with the human hand, a technique that is difficult to perform, labor-intensive, and not readily reproducible. Electroporation with microelectrodes suffers these problems as well as the lack of any means to detect the onset of electroporation in an individual cell. These problems are likewise addressed by the present invention.
As indicted above electroporation is used in biotechnology and medicine for introducing molecules that normally do not penetrate the cell membrane into the cell. This is done by applying electrical pulses across the cell membrane. For a certain range of amplitudes and times of application the electrical pulses can reversible permeabilize the cell membrane, while pulses below that range have no effect on the membrane and above that range induce irreversible permeabilization. In general electroporation was a trial and error procedure, with the optimal electrical pulse parameters for electroporation chosen by evaluating the biological outcome of the procedure on the cell. In our earlier patents such as U.S. Pat. No. 6,482,619 we showed that cell permeabilization by electroporation can be detected by measuring electrical currents through the cells—under the assumption that the permeabilization of the cell membrane will also cause a change in the ionic flux through the cell. This in turn can be used to detect, control and optimize electroporation protocols, in real time. Using a single cell microelectromechanical chip we have provided examples of how this procedure can be used with single cells. Exactly the same concept can be also applied for controlling electroporation in a multitude of cells through experiments with cells in a confluent cell layer and with cells in tissues and such is exemplified below.
The present invention arises in part from the discovery that the onset and extent of electroporation in a biological cell and a group of cells in tissue can be correlated to changes in the electrical impedance (which term is used herein to mean the ratio of current to voltage) of a conductive medium that includes the biological cell or tissue. An increase in the current-to-voltage ratio across a group of biological cells occurs when membranes of the cells become permeable due to pore formation. Likewise, a decrease in the current-to-voltage ratio through a flowing conductive fluid occurs when the fluid draws biological cells into the region between the electrodes in a flow-through electric cell. Thus, by monitoring the impedance of the biological cells in a tissue, one can detect the point in time in which pore formation of the cells on average occurs, as well as the relative degree of cell membrane permeability due to the pore formation. This information can then be used to establish that on average the cells of the tissue have in fact undergone electroporation, or to control the electroporation process by governing the selection of the voltage magnitude. The invention provides the simultaneous electroporation of multitudes of cells, since it provides a direct indication of the actual occurrence of electroporation and an indication of the degree of electroporation averaged over the multitude. The discovery is likewise useful in the electroporation of biological tissue (masses of biological cells with contiguous membranes) for the same reasons.
The benefits of this process include a high level of control over the onset and degree of electroporation, together with a more detailed knowledge of the occurrence and degree of permeability created in particular cell masses. When applied to cells this process assures that on average, the cells of the tissue are indeed rendered permeable and are indeed transformed by the introduction of chemical species and this can be observed and measured in real time. The process also offers the ability to increase the efficiency of electroporation by avoiding variations in the electrical environment that would destroy some cells while having an insufficient effect on others.
In some of its more specific embodiments, the present invention involves the use of an electrical cell in which a an extracted tissue sample can be placed and that contains a barrier that directs the electric current flow and hence the ion flow through a flow path that passes through the tissue sample while permitting substantially no electric current to bypass the tissue sample. In some of these embodiments, the invention involves the use of an apparatus containing two liquid-retaining chambers separated by a barrier that is substantially impermeable to an electric current. The barrier contains an opening that is smaller than the tissue sample such that the tissue sample once lodged in the opening will plug or close the opening. To achieve electroporation, the tissue sample is secured over the opening by mechanical or chemical means, preferably in a reversible manner so that the tissue sample can later be removed without damage to the tissue. Once the tissue sample is secured over the opening, a voltage is imposed between the two chambers and across the tissue sample residing in the opening. The passage of current between the chambers is thus restricted to a path passing through the opening and hence through the tissue sample. By monitoring the current-voltage relation in the electric cell, the onset of electroporation is detected and the degree of pore formation is controlled, to both assure that electroporation is, on average, occurring and to prevent, on average, excessive pore formation and cell death. The user is thus afforded a highly precise knowledge and control of the condition of and the flux across an average of the cells of the tissue.
In another series of embodiments, this invention is useful in the diffusive transport of chemical species into or out of cells of a tissue. In these embodiments, the tissue sample is again divided into two chambers separated by a barrier, and the tissue sample is lodged across an opening in the barrier in such a manner that the passage of liquid around the tissue sample from one chamber to the other is substantially prevented. A liquid solution of the species to be introduced into cells of the tissue is placed in one or both of the chambers. The concentration of the species in the solution differs from that in cells of the tissue (either higher or lower, depending on whether one seeks to introduce or remove the species from the cells), or the concentration in one chamber differs from that in the other chamber.
In preferred methods of applying this invention to diffusive transport, the solutions in the two chambers differ in concentration such that the driving force for the diffusive transport is between the two chambers themselves rather than between the chambers and the interior of cells of the tissue. Knowledge and controlled monitoring of the concentrations in each of the two chambers on a periodic or continuous basis as the diffusion proceeds, together with the precise knowledge of the dimensions of the opening, enables the user to precisely observe and control, on average, the rate and amount of the species that enters an average cell of the tissue. The diffusion time can be controlled by imposing stepwise changes in the concentrations in either or both of the chambers, thereby imposing or removing the concentration differential. An application of particular interest is the combination of this type of diffusive transport of a chemical species with controlled electroporation as described in the preceding paragraph.
Each of the various embodiments of this invention may be used with different sizes of tissue samples. The apparatus described above can be adapted for use with different sizes of tissue samples by arranging the barrier such that the current or diffusive transport will be restricted to a flow path that passes through all of the cells of the sample while preventing bypass around the cells of the sample.
A further application of the concepts of this invention is the electroporation of biological cells suspended in a flowing liquid. Electrodes are placed in fixed positions in the flow channel, and a voltage is imposed between the electrodes while current passing between the electrodes is monitored. Biological cells entering the region between the electrodes will lower the current, the impedance serving as an indication of the presence of one or more cells in the region, and optionally also as a signal to initiate the application of a higher voltage sufficient to achieve electroporation.
Among the advantages that this invention offers relative to the prior art are the ability to treat tissue samples individually and to adapt the treatment conditions to the needs of individual tissue samples. In embodiments where voltage is applied, the monitoring of the impedance affords the user knowledge of the presence or absence of pores and shows the progress of the pore formation and whether irreversible pore formation that might lead to cell death has occurred. An advantage of the barrier-and-opening apparatus is its highly efficient use of electrical energy by virtue of its restriction of the current to a current flow path passing through the opening. A still further advantage is the ability of the apparatus and method to be integrated into an automated system whereby the condition of each tissue sample is monitored by instrumentation and individual tissue sample are lodged in the opening and then removed at times governed by the monitored conditions.
An aspect of the invention is a method whereby cells of tissue are electroporated while measuring and/or observing changes in electrical impedance in real time and noting increases as a beginning to electroporation and adjusting current to avoid as much as possible irreversible cellular damage.
A feature of the invention is that the magnitude of electrical current during electroporation of cells of tissue becomes dependent on the degree of electroporation of the cells so that current is adjusted within a range predetermined to obtain a desired degree of electroporation minimizing cellular damage.
Another feature of the invention is that measuring (in real time) current through a circuit gives a measurement dependent on the overall and average degree of electroporation that the cells in the tissue between the electrodes experience.
Another aspect of the invention is that precise electrical resistance of the tissue is calculated from cross-time voltage measurement with probe electrodes and cross-current measurement with the circuit attached to electroporation electrodes.
These and further features, advantages and objects of the invention will be better understood from the description that follows.
The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:
Before the present methods, treatments and devices are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pulse" includes a plurality of such pulses and reference to "the sample" includes reference to one or more samples and equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
In an electroporation protocol cells are: a) grown on a substrate, b) separated from the substrate through trypsinization, c) introduced into a cell suspension in which they float d) than the cell suspension is electroporated without real time control between electrodes, with pulses determined through a trial and error procedure, e) after electroporation the cells are seeded on a cell growth substrate and f) the cells are grown to determine through biochemical markers if the electroporation was successful. This is a six step procedure. Cell electroporation can be detected in real time for individual cells as well as a multitude of cells in a layer. This basic concept is applied here for an electroporation procedure that detects and controls electroporation in real time in a multitude of cells grown on a substrate. This facilitates electroporation with real time control of a layer of cells on the substrate directly without the need to trypsinize the cells, electroporate in a suspension and than seeding for further growth on the substrate.
The apparatus used for electroporation of a multitude of cells in a single layer is shown in
In a typical electroporation procedure for cells grown in a cell layer, the cells are grown to a desire confluence on the porous insert as described by the Millipore company instructions. During an electroporation protocol, the cell insert is introduced between the electrodes 3 and 4, as shown in
In tissue electroporation, which is done either in vitro or in vivo, the tissue which one desires to electroporate is introduced between two electrodes 3 and 4 of
In a typical electroporation procedure for tissue the tissue sample 1 is placed between the two electrodes 3 and 4 as shown in
Once the cells are electroporated, there shall be increased electrical current flow through the cells and the magnitude of the electrical current becomes dependent on the degree of electroporation of the cells in tissue. This cross-cell electrical current can be measured with via the electrodes 5–9 and can be used to monitor the process of electroporation of the cell membranes. The two probe electrodes 5–9 connected to the amplifier 19 can be used to cancel out the voltage drops at the electroporate electrodes interfaces and provide accurate readings on the voltage across the tissue.
Precise electrical resistance of the tissue is thus calculated from cross-tissue voltage measurement with the probe electrodes 5–9 and cross-current measurement. The resistance measurement reveals the degree of electroporation of the cells in tissue sample 1 since cell membrane resistance is directly dependent on the extent of membrane electroporation. In addition to monitoring the electroporation, the electrical current measurement as well as membrane resistance measurement can be used as feedback for fine-tuning of electroporation pulses to achieve highly controlled electroporation of the cells in tissue in the sample 1.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Cells—Two types of cells were examined: Mouse skin fibroblasts (NIH 3T3 cell line) and primary skeletal satellite cells. Satellite cells were isolated and expanded. The cells were plated on membrane inserts (Millicel-PCF, # PIHP03050 Millipore, USA) and incubated for 5 days in culture medium (DMEM, 10% Fetal calf serum, 1% L-glutamine, antibiotics, Biological industries, Bet Haeemek, Israel) in 6 wells plates until desired confluence was achieved.
Tissue—Liver was obtained from Spraque Dewley rats (250–350 gr), and used within 10 minutes from resection. The liver was removed from the anesthetized rat and sliced with a scalpel to a thickness ranging from 1 mm to 4 mm. Then a disk of liver was obtained by pressing a sharp circular tube onto the sample to trim the excess tissue. The resulting sample can then placed in a device as shown in
Cells—Inserts with adherent layers of cells can be placed into the configuration shown in
Tissue—The tissue layer can be placed between the measurement electrodes. Then PBS should be added to ensure good contact between the electrodes and the tissue.
To assess the efficacy of transgene delivery and expression, 2 plasmids: CMV-GFP and MSV-MyoD may be used. The GFP reporter gene may be introduced to cultured fibroblasts or to satellite cells to assess the efficiency of gene delivery. MSV-MyoD may be introduced to cultured fibroblasts to test gene-based myogenic conversion.
Inserts may be placed into the system in
Transgene Expression Gene delivery efficiency may be determined, 3 days after electroporation by the extent of the expression of GFP reporter gene, with a fluorescent microscope. Treated fibroblasts or satellite cells can be trypsinized (trypsin EDTA solution, Biological industries, Bet Haeemek, Israel) and centrifugated at 1800 rpm for 10 minutes at RT. The pellet can be suspended in cold PBS with glucose (2.5 gr/L), and cytospinned at 500 rpm for 15 minutes on glass microscope slides. GFP expression may be observed under green filter fluorescence microscope.
The MyoD-treated fibroblasts may be incubated for six hours in medium DMEM with 10% FCS, then, shifted to differentiation (low serum) medium (DMEM medium supplemented with 2% FCS) for three days.
A typical three steps electrical pulse may be used to study the process of electroporation in cell layers and tissue samples. It consists of three steps. The first pulse is a low amplitude pulse that cannot produce electroporation and is used to probe the typical electrical impedance of the cells or tissue prior to electroporation. The second pulse was varied in amplitude until a change in the electrical impedance of the cells was detected to determine the occurrence of electroporation. The occurrence of electroporation results in a decrease in the electrical impedance of the cells—while electrical pulses which do not produce electroporation will not affect the electrical impedance of the cells. It should be noted that while the polarity of the pulse was chosen to be negative, relative to the electrode in which the plasmids were introduced from electrophoretic considerations to facilitate insertion of the charged plasmids into the cells. The third electrical pulse has the same amplitude as the first. The impedance measured during the third pulse may be used to determine if the electroporation was reversible or not. The effect of several three pulse steps, separated by various intervals of time may studied.
The above Examples demonstrate the potential for electrical impedance tomography (EIT) to monitor electroporation by incorporating the conductivity change of a single cell during electroporation into an electrical model representing the whole tissue. However, cells in tissue inside the body are surrounded by extracellular conductive fluid and we now show that the bulk properties of tissue are measurably affected by electroporation. This is done here by quantifying the normalized change in conductivity of rat liver tissue ex vivo as a function of an exposed electric field. Parameters such as pulse duration, number of pulses, and time from sacrifice are investigated as a function of an applied electric field. The results show that the cells of the tissue are controllably electropermeabilized, and that the conductivity of tissue increases substantially and predictably and shows that electrical impedance tomography (BIT) can produce an image of the electroporated area.
These ex vivo experiments are designed to quantify the normalized conductivity change of tissue due to sets of electrical pulses, typical to in vivo EGT and ECT. In general, 6–12 pulses are applied at a frequency of 1 Hz, combining higher electric fields with short pulse lengths (100 μs) for ECT and lower fields with longer pulses (>1 ms) for gene therapy. The normalized conductivity change is measured as opposed to the absolute values of tissue resistivity due to the large variation in tissue properties from animal to animal.
The system used is a simple single-frequency bio-impedance measurement system for rectangular tissue slices. The experimental apparatus is shown in
The electroporation system of
To quantify the impedance change of tissue sample 1 undergoing electroporation, the system of
This three-electrode configuration enables one to verify their experimental measurements through three different electrode combinations. The first and last electrodes are not used for measurements because the contact resistance would cause uncertainty. One injecting pair and one measuring pair (at any given time) is common in bioimpedance measurement systems and has been shown to dramatically improve results over single pair systems, which inject and measure through the same electrodes.
The current through the system of
Male Wistar rats of 200 g were anesthetized and sacrificed. After ventral laparotomy, livers were excised and sliced using a scalpel, approximately 2.5 cm in length, 1–2 cm in width and 0.2–0.4 cm in thickness. Unless otherwise stated, experiments were conducted within 90 minutes from animal sacrifice with the tissue stored in 0.9%
NaCl at room temperature until use. Size of sample, lag from time of sacrifice to data acquisition, and all voltage parameters were recorded for each sample. Liver tissue was chosen because it is macroscopically isotropic and, therefore, relatively homogenous, which minimizes any issues with the orientation of the electrodes with respect to the tissue. Also, there are no contractions during in vivo reversible electroporation with this organ, and at this stage it was preferable not to deal with corrections required for these contractions. Although only liver tissue was used for the experiments, other tissue samples could easily have been used with this device.
It also should be noted that the applied electroporation voltage-to-distance ratio in any experimental setup does not necessarily correlate with the electric field exposed to the tissue because of the voltage drop associated with the contact impedance between the electrode and the tissue. The voltage was applied across the entire length of the tissue, which should account for most of the voltage drop and, when necessary, electrocardiograph paste was applied between the tissue and the electrode to ensure good electrical contact. Therefore, we assume that this discrepancy is small and will use the terms interchangeably.
Initial experiments were conducted using eight 100 us pulses at a frequency of 1 Hz with electric field varying from 0 to 500V/cm; these parameters are typical to ECT. The resistance of the tissue is calculated using Ohm's law by measuring the potential drop across the measuring electrodes and the current through the tissue (through the 2 kOhm resistor). These measurements are taken before and after delivery of the electroporation pulses. Since the electric pulse is applied across the entire tissue, the change in equivalent resistance corresponds to the change in resistivity. As shown in
An index of the results is shown below in Table 6.1.
The initial trials with 100 μs pulses of 100 microseconds showed only marginal conductivity changes of the tissue. Accordingly, the second set of experiments explored the effect of pulse length on conductivity change. Pulse length was varied from 100 microseconds to 10 milliseconds using eight 400V/cm pulses at I Hz while maintaining a constant voltage-to-distance ratio of 400V/cm. This limit was chosen because, under observation with the oscilloscope, the pulse shape began to loose integrity at lengths larger than 10 ms.
Several experiments (2–5 per sample) were conducted using eight pulses at a frequency of IHz with a pulse length of 10 milliseconds. Dramatic changes were observed, which appeared to diminish over time. Since the time between the last pulse and the measurement was only accurate within a half second precision, the results are presented as a five second average, 10–15 seconds after the last pulse.
Experiments were conducted at 200(2), 250(2), 300(5), 350(2), 400(4), 450(3) and 500(3) V/cm, where the number in parenthesis is the number of trials at that gradient for a total of 32 experiments. The results showed an average drop in resistivity of 3.2�0.8 for 200 V/cm; 12.1�0.3 for 250 V/cm; 14.212.5 for 300 V/cm; 19.211.2 for 35 V/cm; 22.711.4 for 400 V/cm; 31.4+4.7 for 450 V/cm; and 34.8�6.5 for 500 V/cm. Standard deviation in the results was relatively small.
With this set of experiments, resistance was recorded through the electroporation electrodes themselves. Measurements were taken by applying a IV pulse through these electrodes one second before the electroporation pulses and one second after. As mentioned earlier, this is generally not desirable in bioimpedance studies because the electrodes, which inject current, should not also be the measuring electrodes because of the induced uncertainty from the contact resistance.
This experiment made it possible to (1) estimate the absolute resistivity value of the tissue from the resistance measurement (because of the essentially rectangular geometry of the tissue samples); and (2) because no switching of the electrodes was required, measurements could be recorded within 1 s after the delivery of the final pulse.
Measurements were taken one second after the last electroporation pulse using the electroporation electrodes themselves, followed 15–20 s afterwards by the bioimpedance electrodes to explore transient effects. The results are summarized in
Reversible electroporation is a transient phenomenon as shown above. The initial drop in resistance should eventually recover if the pulses are reversible.
A simplified two-dimensional isotropic cross-section of a liver 7 cm in diameter is used for the simulation. The surface of the analyzed tissue is assumed to be electrically isolated with a zero flux boundary condition to simulate a situation in which the liver is temporarily placed in an insulating, semi-rigid electrode array fixture during a surgical procedure. To collect data for the reconstruction algorithm, 32 electrodes are equally spaced around the periphery of the virtual phantom, i.e. electroporation conductivity map.
For computational reasons and for model demonstration, we used the data from the 10 ms pulses in this study. Specifically, with these experiments, eight 10 ms pulses at a frequency of 1 Hz were delivered for electric fields of 0, 200, 300, 400 and 500V cm″1. The threshold gradient required to induce electroporation was taken from the experimental data and was shown to be 200V cm″1. The impedance change was measured with a 2.5 kHz, 0.3 mA rms sinusoidal current for 5 seconds, 10 seconds after the electroporation pulse. It was shown in earlier studies that permeability in tissues persists for periods longer than 15 seconds after delivery of the pulses. Three trials were performed for each gradient for a total of 15 experiments. These experimental results enable us to generate conductivity maps with five conductivity levels, including four degrees of electroporation in the tissue. A summary of the experimental results used as input into the electroporation models is included in
The electroporation simulation evaluates the potential and gradient distribution in the tissue due to an electrical pulse using the finite element method. To account for the change in electrical properties of the tissue during electroporation, the algorithm iteratively solves the Poisson equation with a zero source term:
where σ is the electrical conductivity and φ is the electrical potential. The conductivity of the tissue is assumed to be initially at 0.13 S m−1, which agrees with results for dog liver in situ at IkHz and measured from one sample. At locations where the voltage gradient surpasses a threshold gradient, the conductivity increases as prescribed by experimental results in
In addition to the baseline example, three electroporation electrode geometrical configurations were investigated, which are modeled after in vivo electroporation experiments conducted on rabbit liver and murine skeletal muscle.
The first configuration which is show in
The reconstruction algorithm consisted of a 116-element mesh with 96 peripheral elements and 20 elements defining-the electroporated area. The outer region maintained a conductivity of 0.13 S/m while the inner region used a weighted conductivity value of 0.2 S/m. The algorithm initially assumes the electroporated region to be a circle with a 6 mm radius centered between the electroporation electrodes. The boundary conditions were zero flux for periphery segments except for the electrode source and sink. The BIT electrode element widths were 1 mm, and the source/sink current was +/−1 mA.
The variables used by the reconstruction algorithm, namely 1) the number of elements defining the electroporated area, 2) the conductivity of the inner region, 3) the initial radius of the inner region, 4) the domain size, and 5) the number of HIT electrodes, were studied parametrically. The parameters from the first three examples were used as control using the electroporation electrode configuration from the baseline model. The control model used in the reconstruction algorithm consisted of a 180-element mesh with 160 peripheral elements and 20 elements defining the electroporated area. The inner region had a conductivity value of 0.2 S/m and was initially assumed to be a circle with a 6 mm radius centered between the electroporation electrodes. There were four electroporation electrodes located in the center of the tissue placed in a square configuration, 10 mm from one another, with the top electrodes set to 1300V and the bottom electrodes set to ground.
There are two primary applications for in vivo electroporation: antitumor electrochemotherapy (ECT) and electrotransfer as a novel approach of non-viral gene therapy. Lower field strengths with long pulses are generally used, in addition to permeabilizing the membrane, to electrophoretically push the macromolecules into the cell. This is not required for small drugs such as bleomycin, which enter a permeabilized cell via diffusion. For example, the protocols for porcine skeletal muscle are 450V/cm with 100 us pulses for ECT but only 90V/cm with 20 ms pulses for gene therapy.
Initial trials with 100μs pulses suggested conductivity changes of less than 3%. With 10 ms pulses, at 10 ms, significantly larger changes in conductivity were observed.
As the voltage gradient increased, the ability of the tissue to recover to its original conductivity decreased, resulting in an increasing residual conductivity change. This suggests the presence of both reversible and irreversible electroporation. Furthermore, these results also show that EIT monitoring to be more applicable for applications requiring longer pulses such as gene therapy, which inherently needs more precision than ECT. Because the conductivity change is essentially permanent and more significant for irreversible electroporation, these results also show that EIT is suitable to image irreversible electroporation.
The results in
The second example is difficult to image as the reconstruction algorithm currently is written. This is because of its convex shape and two local maxima, i.e. two separate regions with 500V/cm thresholds centered at each electrode. This problem should be resolvable with future iterations of the algorithm. The other examples had concentric conductivity regions, which is much easier to image with one average conductivity value.
To image example 2 appropriately, the algorithm can be written to incorporate multiple embedded conductivity regions.
Examples may derive the electroporated tissue conductivity from in vitro cell experimental results taken while the pulse was applied. If the transient ex vivo experimental results presented above and in
The results in
In summary, with electroporation, as in all other molecular medicine techniques, there is no method to actively monitor or control the process in real-time, i.e., to determine during the procedure that the cell membranes have become temporarily permeabilized in the desired area of the tissue. However, the above Examples demonstrate that monitoring of molecular medicine in vivo can be achieved through the synthesis of two fairly well understood techniques, electroporation and BIT.
The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.
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Crysis patch delayed until jan!!!!
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Join Date: Aug 2005
Location: Europe
Posts: 3,866
Re: Crysis patch delayed until jan!!!!
Originally Posted by
Also + huge wads of cash from Intel and nVidia, companies that both advertise within the game and use Crysis to sell their hardware in online adverts. Are they going to be happy with Crytek boosting performance on their old lines of hardware, when that same boost could be applied to the new hardware to make it seem that bit faster than before? Take note especially of the fact that Crysis is seen by The Industry as being the current Game Of The Future, meaning that any hardware capable of running Crysis smoothly is, effectively, future-proof.
In general I don't believe into conspiracy theories but your point isn't a bad one because the (computer) industry always took bigger jumps forward once a game or engine has been released that was "ahead of its time". The results were always faster hardware and a cheaper price and we already saw that with the release of cards like the 8800GT, 3850, 3870 etc., all excellent cards with impressive price/performance ratios.
At the same time you have to remember what Crytek did with FarCry for instance with the HDR engine upgrade (the first "high precision" HDR game IIRC) and they have promised to give Crysis a similar visual upgrade with more advanced shaders and higher settings ("Ultra High" settings).
These "Ultra High" settings could very well be reserved for the "next generation" of video cards (Nvidia D9E etc.) and if they do it this way there actually speaks nothing against boosting performance on current hardware ("Very High" or lower settings) because the new cards would get their "exclusive" visual upgrade just like current cards would get their performance enhancements at lower settings.
The real question to me is actually "how" they want to increase performance because in general you need to decrease image quality IF there isn't enough "untouched" performance left in the engine. I do think there is room for improvement left with multi-card solutions (10-20% gain w/o image quality decrease should be possible) but when it comes to single cards I'm not that optimistic other than lowering quality of certain effects or move some to other settings. Although a visual decrease would speak against what Crytek is about (pushing visuals to the limit even if it hurts) so I would be disappointed by that move.
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"We're off to a very strong start," Creatura said. "The dominant reason is the resolution of the fiscal cliff. But January is usually a strong month, as investors all shift money into the market at the same time. When the calendar flips, it's as if you're allowed to begin the race anew."
Economists had warned that the full force of the fiscal cliff could drag the country into a recession. The law passed late Tuesday night averted that outcome for now, but other fiscal squabbles are likely in the months ahead. Congress must raise the government's borrowing limit soon or be forced to choose between slashing spending and paying its debts.
In other Thursday trading, prices of U.S. government bonds fell, sending their yields higher. The yield on the benchmark 10-year Treasury note rose to 1.90 percent from 1.84 percent late Wednesday, a sign that some bond traders believe the Fed minutes hinted at an early end to its bond buying.
Family Dollar Stores sank 13 percent after reporting earnings that fell short of analysts' projections. The company also forecast a weaker outlook for the current period and full year. Family Dollar's stock lost $8.30 to $55.74.
Nordstom Inc. surged 3 percent after the department-store chain reported strong holiday sales, especially in the South and Midwest. Nordstrom's stock was up $1.64 to $55.27.
Among other stocks making big moves:
— Transocean jumped $2.96 to $49.20. The owner of the oil rig that sank in the Gulf of Mexico in 2010 after an explosion killed 11 workers reached a $1.4 billion settlement with the Justice Department.
— Hormel Foods, known for making Spam and other meat products, said that it's buying Skippy, the country's No. 2 peanut butter brand, from Unilever for about $700 million. Hormel's stock jumped $1.19 to $33.20.


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Best Burgers 2015: Sideswiped
From housemade pickles to truffle popcorn, there is life beyond fries.
Best Burgers 2015: Let it Slide
These five sliders—with bold, global flavors—are good eats in small packages.
Get Your Goetta Game On
In honor of the legions of All-Star Game visitors who will descend upon the city this month, a handful of restaurants will feature goetta specials from July 11 to 14.
2015 Best Burgers: Honorable Mentions
Did we leave your favorite burger joint off our list? Stop sharpening your pitchforks. Here are 20 extra spots we just can't quit.
The 45 Best Burgers in the Queen City
After 123 burgers, epic levels of face-stuffing, and a few bouts of the meat sweats, our intrepid eaters narrowed the field to the top 45. Only one (very big) question remains: Which one will you try first?
New Kid On The Block: Rooted Juicery + Kitchen
Oakley Square's newest juice bar ups the ante on Cincinnati's health food scene.
Power Steer-ing at Le Bar a Boeuf
Top-of-the-line ingredients abound at Le Bar A Boeuf, but service occasionally sputters.
Spotted: The Peach Truck
As far as I'm concerned, the only peaches worth eating are those that require leaning over the kitchen sink. For those peaches, you're gonna want to start cyber-stalking The Peach Truck.
Try This: Hong Kong Fried Chicken at Yat Ka Mein
Put down the bucket: This definitely isn't Kentucky fried chicken.
- See More
High Spirits
Better Know An Industry Pro
Best Restaurants 2015
There are five bridges that cross the river into Northern Kentucky. Pick one and make your way to Bouquet posthaste.
Pontiac Is An Instant Classic
(And the BBQ shack of your dreams.)
Cheap Eats
Chicken on the Run
Bar or restaurant? When you're unsure, it's time to consult the golden ratio: kids at tables to grizzled old guys hunched over the bar. On a recent Saturday mid-afternoon visit to Chicken on the Run, the ratio was 6:5.
Best of the City
Pierogi, Korean BBQ, and "old fashion tub butter"—get the best local food finds.


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Few American institutions have been subjected to such a consistent stream of vitriol and assault as the minimum wage, which celebrates its 74th birthday this week. The first federal minimum wage was established when FDR signed the Fair Labor Standards Act (FLSA) on June 25, 1938. The FLSA also established the eight-hour day, paid overtime and child labor protections into federal law. Since then, it has been amended nine times to expand coverage and to raise the wage to keep it in line with the nation's economic growth.
Business leaders, industry associations, politicians and more recently think tanks have opposed the FLSA and every legislative amendment since. They said it would destroy American civilization, kill jobs and hurt black people. Business owners predicted they would be forced into bankruptcy.
One business opponent of the 1938 legislation even warned that the minimum wage would lead to the decline of the American empire. In 1937, Guy Harrington of the National Publishers Association testified before a congressional committee that "Rome, 2,000 years ago, fell because the government began fixing the prices of services and commodities. We, however, know what has always happened when governments have tried to superintend the industry of private persons. The final result has always been distress, misery and despair."
That same year the National Association of Manufacturers asserted that the FLSA "constitutes a step in the direction of communism, bolshevism, fascism, and Nazism."
Fast forward to 1960 and, despite those warnings, American democracy was alive and well – with the minimum wage at $1 an hour. The American middle class was the largest in world history. That didn't stop the doom-saying.
Business lobbies claimed that a 1961 proposal to increase the wage would shutter thousands of businesses across the American heartland. Ernest Kuhn, the manager of the Hanford Hotel in Mason City, Iowa, said that the hotel would be forced to close if the wage were increased to $1.25 per hour. "There is not enough mechanization or automation yet developed to save my business from the minimum wage horror. If minimum wage legislation is passed by Congress, you will be able to buy hotels cheap."
Perhaps he was right. The Hanford Inn, operated by the Kuhn Hotel Corp., finally closed in 2009. If it is a deadly institution, the minimum wage is clearly a slow-motion killer. It has been increased six times since 1961.
Similarly, Joseph E. Chastain, owner of Lintz's 10 department stores inTexas and Oklahoma, said that a proposed 1966 increase from $1.25 to $1.75 per hour would devastate his business. "No company our size can live under such circumstances. Undoubtedly we would have to liquidate, which is a distressing situation to confront a solvent company that has operated profitably for over 60 years."
In yet more evidence of the slow-motion minimum wage disaster, Lintz's Texas store closed in 2000 and the entire chain closed all their stores in 2007.The assault has continued unabated ever since.
Economist Milton Friedman, interviewed in Playboy in 1973 said, "I've often said the minimum-wage rate is the most anti-Negro law on the books." It's hard to believe that Friedman hadn't heard of the Jim Crow laws throughout the South.
In 1980, presidential candidate Ronald Reagan claimed that "The minimum wage has caused more misery and unemployment than anything since the Great Depression."
In 1993, the owner of Mega Management Company that owns 95 Burger King franchises in the U.S., Canada and the Netherlands said that "creeping socialism begins at the $5.05 level." But as New York Times columnist Joe Nocera reported, the Burger King Corporation was purchased, leveraged and resold by Goldman Sachs and two private equity firms (including, of course, Bain Capital). The private equity firms pulled $1 billion out of the fast food chain, funds that according to one Wall Street expert could have been used to help the struggling company – and certainly to pay its employees a higher minimum wage.
And in 1995, Jack Farris, president of the National Federation of Independent Business, claimed that President Clinton's proposed 90-cent per hour minimum wage hike from $4.25 to $5.15 was "a regressive and job-killing scheme which will put a big dent in small-business hiring." According to County Business Patterns data, employment in businesses with fewer than 20 employees grew by almost two million workers between 1995 and 2000. Oops!
Despite these (and more) constant predictions of doom, the minimum wage remains wildly popular in the eyes of the American people. They understand basic economics – when wages go up, people spend more. Without minimum wage laws, employers pay less. They understand what I'll call "Chris Rock-onomics," the economic theory the comedian and social commentator described recently this way: "I used to work at McDonald's making minimum wage. You know what that means when someone pays you minimum wage? You know what your boss was trying to say? It's like, 'Hey if I could pay you less, I would, but it's against the law.'"
Happy Birthday, Minimum Wage!


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text:
Image: http://www.sayradio.net
We're going to come right out and say it. Actually, we'll let Magic say it... the only way the Lakers have a kismet, is if the entire team steps up to the ball, and plays hoops. It can't be the Kobe Bryant show. Not this time. Thus far in the playoffs, Kobe has averaged 26.9 points per game. With Kobe's hurt ankle, finger, and knee, it's absolutely imparative his teammates pick up the slack. Otherwise, it's going to be a long road for the Glam-Star Lakers to the finals.
Conference: Pacific Division, Western Conference
Next Game: Mon, May 17 vs. Phoenix, 9:00 pm (est)
Founded: 1946
Next Game: Mon, May 17th @ LA Lakers


text:
Patricia Velasco
4th & 5th Grade Teacher
Hello! I grew up in Reno, Nevada, after my family immigrated to the U.S. from the Philippines. My parents raised the five of us siblings to value education. Having that upbringing really instilled in me a discipline and appreciation for education, and its power to change people's lives for the better.
For college, I stayed in Nevada and went south to Las Vegas, where I majored in Print Journalism and minored in Anthropology. After graduation and some various news related positions, I worked at the Girl Scouts.
I moved to Portland to explore more opportunities working with youth. Through volunteering at the Immigrant Refugee and Community Organization (IRCO), I met a family of Burmese refugees who I tutored in English. That meaningful experience is what helped cement my decision to pursue teaching as a career. I obtained my Masters in Teaching at Lewis and Clark College and student taught in 3rd and 5th grade with Beaverton School District.
My most recent position was coordinating an after-school enrichment program that included family engagement nights, parent education, a weekly food distribution and other wrap around social services with Schools Uniting Neighborhoods (SUN) at an elementary school in Southeast Portland.
I was inspired by the progressive teaching philosophies we learned at Lewis and Clark and am excited about the opportunity to apply what I learned in my class at Trillium! I believe that each child is unique and has an innate desire to ask questions and find their own solutions. My hope for my students is that they leave my classroom with the ability to think critically about the world and a commitment to creating positive change in their communities and globally.
When I'm not teaching, I enjoy eating, cooking, reading, camping, seeing live music, running, playing tennis, dancing to Zumba (though I can't salsa or anything) and kickboxing. I volunteer at the Oregon Food Bank assisting in cooking classes. I'm hoping to do some service learning with my students this year!
Bijal Makadia
4th & 5th Grade Teacher
Bio Coming Soon!
Michael Koopman
4th & 5th Grade Teacher
My name is Michael Koopman and I have been working in education for over six years. I grew up in a small town in Eastern Oregon called La Grande. It's a quiet beautiful farm town nestled in the Blue Mountains surrounded by river, lakes and forests. I was born in this town and stayed through High School. In High School I was involved in our leadership program and took the position of class representative for three years. I played trumpet in our marching band and sang in our acapella choir. I started performing on stage in our musicals and began to love acting and theatre.
I went to college at the University of Oregon where I began to gravitate towards Theatre Arts as my major. Through the theatre program I was able to travel abroad in London for four months where I took acting classes and watched many plays.
My experience in London motivated a move to NYC where I resided for two years. In New York I waited tables at a high end restaurant while auditioning and booking for acting and modeling jobs. After my NYC stint I decided the east coast was amazing but not meant for me as a home. I returned to my home and felt that Portland fit me better than the big apple.
In Portland I pursued my acting career further and appeared in many television commercials, print ads and local film projects. I longed for something else in my life so I decided to take a part time job working with children at Kids Community Learning Center in northwest Portland. At that time everything changed. I found a profession I cared about more than acting and devoted my life to education with children.
I worked for KCLC for a total of 6 years, taking one year off to go to Lewis and Clark and receive my teaching credentials.
My current position at Trillium is a new adventure that I am thrilled about. My goals are to be able to apply my life experience as well as my years of work with youth to my classroom position. My ultimate goal was to be a full time classroom teacher and I am honored to begin that journey here at Trillium Charter School!


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31 Halloween Craft Projects
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Ghost Window Card
By: Debi Adams for Sizzix
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A ghost peaks out from behind the window of a haunted house in this unique Halloween card from Sizzix. This impressive Halloween card is simple to create with die-cut shapes.It's quick and easy so have at it!
Brand Items
- 654407 - Sizzix Clearlits Die - Circle 1 1/2"
- 654780 - Sizzix Movers & Shapers XL Die Set - Card, Horizontal A2 & Four Window Panes (Kit #1)
- 655553 - Sizzix Sizzlits Die Set 3PK - Spider Set
- 655564 - Sizzix Bigz Die - Ghosts
Other Supplies
- Plain Paper
- Sewing Machine
- Ribbon
Die-cut Card out of colored paper. Line inside of Card with contrasting paper. Die-cut shapes and adhere to the inside of the Card, showing through the window. Machine stitch across the front of the card. Print greeting on a piece of white paper and punch in the shape of a circle to create a tag. Embellish with ribbon and adhere to the Card.
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8 MB/s, 16 MB/s (Compressed), 100 GB Native Capacity, 200 GB Compressed Capacity, 3.5 lbs
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Cisco MDS 9124 for IBM System Storage - Switch - 8 x 4Gb Fibre Channel - rack-mountable - Express
IBM 3628 - Tape drive - LTO Ultrium ( 800 GB / 1.6 TB ) - Ultrium 4 - SAS-2 - external - for BladeCenter HS21
The QLogic iSCSI Single Port PCIe HBA for IBM System x delivers the first PCI Express single port iSCSI Initiator adapter for iSCSI Storage solutions for System x.
IBM System Storage N6060 Model A12 - NAS server - rack-mountable - RAID 6, DP - Gigabit Ethernet / 4Gb Fibre Channel - iSCSI - 6U
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MacMall is the #1 Apple Direct Reseller. We sell more Apple-branded products than any other direct Apple reseller. Since 1987, MacMall has been supplying Mac computers, Mac software, iPods, iPad accessories, and more Apple brand products and consumer electronics to consumers, businesses, and creative professionals. At MacMall, we understand the increasing demand for the best quality consumer products at huge discounted prices. That's why we are proud to offer the lowest prices on our extensive selection of best-selling products, and hard-to-find Mac and iPod products you crave. Get yourself the new iPad or the new Apple TV for your family. Looking to upgrade your MacBook? Or just want to find out the best suited iPod/iPhone accessories for your hot gadget? Keep your iPad up-to-date with function and technology with our line of top-brand iPad accessories. Compare brands. Equip your Apple products. More than the innovative products, MacMall goes beyond commerce and into affordable, high-quality business solutions. From customized business sites to financing & leasing to software licensing, technology certifications, configuration services, packaged service programs, and more. Enjoy easy management, easy installation, and easy implementation. Discover a whole new way to create and re-create your business. Plus, with our 24/7 customer service hotline, we're always just a phone call away no matter when you want to reach us. Get the MacMall Advantage. Don't miss out on the best deals, special prices, rebate savings, exclusive bundles, and dedicated service you won't find anywhere else!
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For Financial Aid and Scholarships information, please refer to the
Financial Aid and Scholarships page under the Student Services
section. Questions may be emailed to us at
The college reserves the right to change tuition rates without prior notice.
The in-county rate applies to residents of Worcester, Wicomico and Somerset counties. The out-of-county
rate applies to Maryland residents who live outside of Worcester, Wicomico and Somerset
counties. The out-of-state rate applies to residents of any state other than Maryland.
Non-U.S. citizens who meet the requirements of the Maryland Dream Act can pay in-state
tuition rates. Go to www.worwic.edu/MarylandDreamAct
for eligibility information.
At the time of admission or whenever a student reports a change in student information, he or she must
confirm his or her domicile for tuition classification purposes. As used here, "domicile" is defined
as "a student's permanent place of abode, where physical presence and possessions are maintained with
the intention of remaining indefinitely" or "the permanent place of abode of any person or persons contributing
more than one-half of the student's financial support during the most recently-completed year." To establish
domicile, an individual must permanently reside in the state or in the county for three months prior
to the beginning of the term. However, in some cases, this does not automatically guarantee domicile
status and supporting evidence of residency may be required in the form of income tax papers, driver's license,
vehicle registration, insurance policies, voter's registration, rent receipts and/or other requested information.
Tuition (not including fees) is waived for Maryland residents who are 60 years old or older or who are
retired as a result of a disability as defined by the Social Security or Railroad Retirement Act, who
enroll in any credit class that has at least 10, or is being continued with less than 10, regularly-enrolled
students. Members of the Maryland National Guard who have two or more years of service remaining are
eligible for a 50 percent tuition waiver. Appropriate documentation for a Maryland National Guard waiver must
be presented each term. In order to receive a tuition waiver based on retirement due
to a disability, the student must provide appropriate documentation from the Social Security Administration.
In order to receive a tuition waiver based on disability, students must submit documentation of the disability
and apply for federal financial aid annually. The disability waiver is applied only when the financial aid awarded to
the student does not fully cover tuition charges. The senior citizen tuition waivers are provided on a space-available basis. In order
to qualify for a senior citizen tuition waiver, the student must provide proof that his or her 60th
birthday has occurred prior to the first class session. Tuition waiver forms are available in the counseling
The College and Career Readiness and College Completion Act of 2013 offers qualified
dual enrollment students discounts for tuition. Public high school students who
qualify for free and reduced meals (FARM) can enroll in Wor- Wic classes tuition-free,
only having to pay for books and fees. Public high school students who do not qualify
for FARM pay 90 percent of the 75 percent tuition rate.
Students attending any Worcester, Wicomico or Somerset public high school, the Salisbury
School, Salisbury Christian School or Holly Grove Christian School, as well as Delmar
High School students who live in Maryland, can provide certification by a high school
official that they meet the school's dual enrollment eligibility requirements to
receive a discount on the regular tuition rate.
The college reserves the right to change fees without prior notice.
Wor-Wic accepts VISA, MasterCard and Discover. Payment can be made online or in
person at the cashier's office on campus. Payment can also be mailed to: Cashier's
Office, Wor-Wic Community College, 32000 Campus Dr., Financial Information/33 Salisbury,
MD 21804. Students can view an itemized list of charges and financial aid awards
that have been applied to their accounts by accessing the myWor-Wic portal. Students
who have not made full payment or alternative payment arrangements (installment
plan enrollment, pending financial aid or third-party payment confirmation) by the
stated deadlines will have their registrations dropped. Students who register after
the stated deadlines must make full payment or alternative payment arrangements
at the time of registration.
Students whose tuition is being paid by a third party, such as an employer, must
provide a letter from the third party guaranteeing payment to the cashier by the
stated deadlines.
An installment plan is available to all students who register for fall or spring classes and whose bill
for tuition is at least equal to the in-county cost of six credit hours. Students who register online
and set up an installment plan can pay the balance online with a credit card or to the cashier on campus.
Students must pay at least half of the tuition bill before the specified payment deadline,
with the balance due by the end of the sixth week of classes. Students who do not make full payment
by the end of the sixth week of classes are charged a late payment fee. These students cannot obtain grades
or transcripts, register for classes or participate in commencement until full payment is made.
Dropping or withdrawing from a course does not eliminate the student's financial obligations to
the college.
Students who have delinquent accounts with the college, the bookstore or the libraries at Salisbury
University or the University of Maryland Eastern Shore cannot obtain grades or transcripts, register
for classes or participate in commencement.
Refunds are provided only to students who complete the drop process. Students who are eligible for online registration can complete the drop process online, while those who are not eligible for online registration must meet with their advisor. Refunds are calculated based on the days the college is open for business (Monday through Saturday, excluding holidays). All tuition and fees are refunded to students who complete the drop process within the following time frames:
In addition to the standard student services offered by the college, veterans can receive added assistance
from the regional office of the Veterans Administration (VA). The tuition and fees of veterans eligible
for the Post 9-11 GI-Bill and vocational rehabilitation are paid directly to the college by the VA. Since educational benefits for
other veterans are paid directly to the veterans, they are directly responsible to the college for the
payment of their tuition and fees, regardless of the timeliness in which their VA checks are received.
Wor-Wic recognizes and adheres to VA standards and strives to assist veterans as much as possible in
resolving discrepancies that relate to educational programming.
American Opportunity Credit/Hope and lifetime learning tax credits are available to eligible students
under federal legislation. The American Opportunity Credit/Hope enables students to receive a tax credit
of up to $2,500 per year for four years of postsecondary education. The lifetime learning tax credit
is available for an unlimited number of years for up to $2,000 each year. The business office sends
1098-T tuition statements to students by Jan. 31 each year.
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First Symetra National Life Insurance Company of New York is committed to helping self-funded employers reduce their medical claim costs and has the expertise needed to deliver results.
Medical stop loss helps employers who self fund their medical plan cap their risk while providing the flexibility needed to meet today's health care challenges.
A best-in-class policyThe First Symetra policy mirrors the employer's plan document without internal limits or definitions that may conflict with the plan. We do not have provisions in our policy like late enrollee wording, medical necessity or experimental/investigational definitions.Outstanding claims supportWe work with out TPA and cost-containment partners to help claimants receive the best possible treatment with cost savings. And our team of registered nurses assist with high-risk events such as treatment for kidney disease or hemophilia, transplants and premature births.Fast, accurate claims handlingMost specific stop loss claims are reimbursed within five working days of receipt. Aggregate claims payments also average five working days.Technical referral assistance programAccess to this unique program helps the policyholder research questionable or complex treatment plans at no cost.National and regional health care programsFirst Symetra provides voluntary access to many of the nation's transplant centers of excellence through our partner vendors.Fairness and partnershipOur policy is to act fairly and responsibly toward policyholders. We won't terminate or non-renew a stop loss policy because of poor claims experience.
COVERAGE TYPESSpecific Stop Loss CoverageHelps protect against catastrophic claims above the specific deductible.Aggregate Stop Loss CoverageHelps protect a self-funded plan in the event that actual claims for a group exceed planned, budgeted claims.
First Symetra Stop Loss (filed as the Excess Loss policy) is insured by First Symetra National Life Insurance Company of New York, New York, NY.


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Originally written on Lady At The Bat | Last updated 10/14/12
Daily News PhotoIn little more than the space of a calendar day, the Yankees went from being triumphant series winners to being down 0-1 in the ALCS. They also went from following Mr Postseason to being a bunch of leaderless parts that don't seem to fit together.The parts don't seem to fit together because there is not enough glue. That glue is offense. If the Yankees can find that glue and spread it in the right places, they can work just fine, in spite of Derek Jeter being lost for the remainder of the postseason. After all, they got this far despite all of the following happening:* Mariano Rivera was lost for the season with a knee injury.*Brett Gardner was out for most of the regular season with an elbow injury.* CC Sabathia had two stints on the disabled list, for groin and elbow problems respectively.* Andy Pettitte was out almost two months with a broken bone in his leg.* Alex Rodriguez was out for more than a month with a broken hand.* Mark Teixeira was out for about a mon...
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text: Business iWatch on the way? Apple buys wireless chip maker Passif Semiconductor Image Credit: afagen August 1, 2013 2:03 PM Tom Cheredar 0 Apple has acquired wireless chip manufacturing startup Passif Semiconductor for an undisclosed amount of money, the company confirmed to AllThingsD earlier today. Passif specializes in producing low-power chips for smaller devices, such as iPhones, iPods, health care devices, and maybe even wearable gadgets like the rumored iWatch that Apple may be producing. The company's technology uses a radio that works with a low-energy version of Bluetooth called Bluetooth LE, according to former Wall Street Journal reporter Jessica Lessin, who first reported the news of Passif being acquired. While Apple may not be making as many moves as Yahoo, its purchase of Passif is the latest in a string of acquisitions over the last few months. Last week, the company acquired local data management startup Locationary and transit data service HopStop.


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Sebi also turned down requests from brokers to allow their clients to transfer funds into the brokers' accounts ahead of opening of public offerings.
If a hacker can wipe out your demat account details, it would be equivalent to your money getting vanished.
Sebi passed the order against NDTV yesterday and it can be challenged by the company before the Securities Appellate Tribunal (SAT).
Sebi has taken action against various entities engaged in luring investors through SMSes and phone calls to their fraudulent schemes with a promise of high returns.
Leading brokerage Emkay Global today settled a Sebi probe into alleged violations of broker regulations by paying about Rs 11 lakh towards settlement charges.
Sebi has made it easier for banks and public financial institutions to act as trustees, while a new code of conduct has been put in place to safeguard investors' interest.
Capital markets regulator Sebi today said it expects the proposed merger of commodities regulator FMC with it to be completed by September-end.
Partly heeding to govt's request on OFS route for PSU disinvestment, Sebi allowed companies to disclose such plans two banking days prior to the share sale.


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The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 β-1,6-N-acetylglusaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas
1 Department of Veterinary Clinical Sciences, University of Minnesota, Room C339, Veterinary Medical Center, 1352 Boyd Avenue, St. Paul, MN 55108, USA
2 Department of Laboratory Medicine and Pathology, University of Minnesota, 420 Delaware St SE, Minneapolis, MN 55455, USA
3 Biostatistics and Informatics Shared Resource, University of Minnesota, 420 Delaware, Minneapolis, MN 55455, USA
4 Department of Veterinary and Biomedical Sciences, University of Minnesota, 1988 Fitch Avenue, St Paul, MN 55108, USA
BMC Cancer 2009, 9:79 doi:10.1186/1471-2407-9-79Published: 6 March 2009
The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).
We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.
Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.
C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.


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Events - Mountain Parks Foundation
Saturday, May 16, 2015
Mountain Parks Foundation hosts "The Tramp and the Roughrider", a theatrical presentiation in Big Basin Redwoods State Park starring Lee Stetson as John Muir and Alan Sutterfield as President Teddy Roosevelt. Ticket availability and more information coming soon about this not-to-be-missed event!
Saturday, September 12, 2015
Come to the 30th Annual Ohlone Day at Henry Cowell Redwoods State Park! Celebrate the past, present & future of Ohlone culture. Storytelling, native games, traditional dance and song, food and much more! Activities are FREE. Parking $10.
Saturday, October 10, 2015
Pre-sale tickets are on sale now! Don't wait, this wildly popular event sells out every year! Check it out here.