A method and system of emulating serial com port communication. A computer processing system has computer-executable operating system instructions including first instructions that interact with a first serial device according to a predefined input/output (I/O) hardware interface. A first serial device has a receive port and a transmit port and has the predefined (I/O) hardware interface. A second serial device has a receive port and a transmit port. The transmit port of the first serial device is in serial communication with the receive port of the second serial device, and the receive port of the first serial device is in serial communication with the transmit port of the second serial device. Computer-executable instructions emulate serial communication port device communication and include instructions that transmit information over another medium in response to receive requests from the second serial device. The transmitted information includes information representative of the receive request from the second device. Transmissions to the first serial device by the operating system instructions are transmitted over the other medium and serial communication port device communication is thereby emulated over the other medium. The emulation instructions further include instructions, responsive to a communication received from the other medium, that transmit information representative of the received communication to the transmit port of the second serial device. The emulation system communicates with at least one console device. The other medium comprises a packet-based network. The other medium comprises a cell-based network. The operating system communicates directly and solely with the predefined I/O hardware interface.

1. Field of the Invention
The present invention relates to serial port communication and, more specifically, to efficiently emulating serial port communication.
2. Description of the Related Art
Conventional computing systems use a serial port to communicate with basic peripherals like the console display and keyboard. The console is used, for example, for system administration messages, kernel error messages, security events, debugging, and/or other purposes.
FIG. 1 depicts the architecture and components of an exemplary arrangement of serial communication. Communication may originate in the upper layers of operating system software 102 (or perhaps even in applications, not shown). The upper layers 102 transmit such requests to corresponding device driver software 108 via a predefined driver model interface 104. The device driver 108, in turn, communicates send (S) and receive (R) messages to the serial com port device 112 via a predefined hardware interface 113. The device 112 transmits and receives such messages via path 116 to connected peripheral 118. The connected peripheral is typically a computer with a serial port and attached keyboard and video devices running terminal emulation software, such as HyperTerm to provide a console and keyboard interface. Analogously, communication may also be conducted via BIOS 110, through the predefined BIOS interface 106. The BIOS logic for serial communication uses the same hardware interface 113 for the serial port device.
In some computing platforms, the processor environment does not include the basic peripherals such as a console display or keyboard. Instead these devices are typically located remotely. For example, FIG. 2 is a high-level architectural diagram of the BladeFrame™ computing platform, as described in U.S. patent application Ser. No. 10/038,354, filed Jan. 4, 2002, entitled Address Resolution Protocol System and Method in a Virtual Network, published on Oct. 24, 2002, which is hereby incorporated by reference in its entirety. Processor nodes 205 have processors 207 which inter-communicate with each other via a communication fabric (e.g., Giganet fabric) 210 and 215. Control nodes may also communicate on the communication fabric and also communicate with management logic 235 which (for present purposes) may be considered to have a keyboard and display to act as a console for a processing network.
For environments, such as FIG. 2, serial communication normally associated with the console may be achieved by a system as depicted in FIG. 3. The upper layers of the OS 102′ transmit communication messages to driver 302 via the conventional driver model interface 104 for the serial port driver. The driver 302 has a device driver front end 308 that "looks like" and "acts like" the conventional serial com port driver (e.g., driver 108 of FIG. 1), at least from the perspective of the OS 102. The driver 302, however, includes emulation logic 310 that communicates over an alternative medium 312 (i.e., not a conventional serial link) to serial devices 118. In the example of FIG. 2 the other medium 312 includes the communication fabric 210. For example, the emulation logic 310 in driver 302 may "wrap" the serial port communication in special packets to be delivered to the control blade where they can be "unwrapped" and the data therein may be used to communicate with the appropriate peripheral. It should be appreciated that this other medium may be quite complicated. For example, in FIG. 2, the control nodes 220 may include special forwarding logic for detecting serial communication requests and re-formatting and forwarding such requests yet again on a different physical medium or using a different protocol. Notice in the environment of FIG. 3 there is no serial com port device 112.
For certain processing contexts, the above approach of emulation via a device driver is incomplete. FIG. 4, for example, illustrates the situation presented in some environments. Some operating systems 102′, and other low-level software, expect to communicate directly 402 with the hardware interface 113 of the serial port device, bypassing any device drivers and Basic Input/Output System (BIOS). Consequently, emulating serial communication with an approach like FIG. 3 will not work for such direct communication.

System and method for emulating serial port communication


An improved structure of energy-efficient lamp base includes a fixing base, conductors, two elastic elements, and a bottom cover. The conductors are disposed and fixed in the fixing base by the elastic elements. The conductors go into the wire holes of the fixing base using back hooks, thereby fixing the wires. The bottom cover is inside the fixing base. A blocking part is used to block the wire inside the wire hole and the connecting pin of the energy-efficient lamp. The blocking part serves the purposes of double-blocking and preventing inappropriate contacts between the wires and the energy-efficient lamp. This improves the safety of the invention.

1. Field of Invention
The invention relates to an improved structure of the energy-efficient lamp base and, in particular, to an energy-efficient lamp base that can be easily connected to a power outlet.
2. Related Art
A usual lamp base, as shown in FIG. 3, has a base 9, two springs 8, two conductive plates 7, and a cover 6. The inner side of the base 9 has two prong holes 91 for accommodating the connecting prongs of the energy-efficient lamp. The two conductive plates 7 are disposed inside the prong holes 91 of the base. They are fixed inside the base 9 under the push of the springs 8. Each of the two conductive plates 7 has a prong 71 connecting with a wire (not shown). The wire is clutched by two pinch plates 711 of the prong 71 of each conductive plate 7. The wire is then connected to the prong 71. Finally, a cover 6 covers the entire apparatus.
To obtain the above-mentioned conventional lamp base structure, each prong 71 holds one end of the wire before being disposed inside the base 9. In this case, the wire punching process of the prong 71 has to be finished first. Therefore, an appropriate length of the wire has to be determined before hand. Such a structure requires a complicated assembly procedure and cannot effectively control the wire length. It only increases the assembly cost and the stocking risk of the products.


A method, apparatus and system for detecting structural variations is provided. A management apparatus divides a test sequence according to loci of chromosomes to obtain at least two portions of detection tasks, sends respective detection tasks to respective detection apparatuses and activates the respective detection tasks; detects detection task completion situations of detection apparatuses and determines whether the number of uncompleted tasks is reduced to a preset proportion threshold of a total number of tasks; when the number is reduced to a preset proportion threshold of the total number of tasks, the management apparatus sends, to detection apparatuses that have not yet completed detection tasks, an instruction message to kill uncompleted detection tasks; the management apparatus further divides the uncompleted detection tasks into at least two portions, sends respective detection tasks to respective detection apparatuses, and activates said respective detection apparatuses to continue to perform detection of structural variations.

In current human being whole-genome re-sequencing, structural variation detection needs to be performed for massive raw data to perform data analysis.
As the amount of current data increases, a scheme that may be supported comprises a non-concurrent scheme in the current structural variation detection. This scheme is adapted to perform detection for all test sequences in a single-unit single-process manner. In this scheme, computer resources are not used sufficiently, and the testing duration is very long. To reduce the testing duration, the prior art makes improvements to the non-concurrent scheme and proposes a concurrent scheme for detecting structural variations. In this scheme, the test sequence is segmented according to chromosomes. Upon processing, each apparatus may process chromosomes in a designated chromosome segment. As such, processing may be executed concurrently between multiple segments of chromosomes, and one chromosome is processed with one process. To further use computer resources, in the prior art there is further proposed a simple concurrent calculation scheme in the chromosomes. According to the scheme, the test sequence is divided according to chromosomes, then each chromosome is sliced into N portions according to loci, and each portion activates a process for concurrent execution.
However, a processing duration varies with different loci of chromosomes, and a processing duration between segments also varies. This causes allocation of concurrent tasks uneven: some concurrent tasks are completed very quickly, and some require a very long period of time. As a result, there are idle computer resources that are not used sufficiently, and the testing duration of the whole test sequence is relatively long.

Method, apparatus and system for detecting structural variations

BEIJING BAIDU NETCOM SCIENCE AND TECHNOLOGY CO., LTD.


Various exemplary illustrations of a camshaft assembly for actuating valves of an engine are disclosed. The camshaft assembly may include a camshaft having a plurality of lobes, including at least one phase adjustable lobe configured to be selectively rotated with respect to the camshaft. The assembly may further include a hydraulic valve actuator in communication with a first lobe of the camshaft. The hydraulic valve actuator may be configured to selectively actuate at least one valve in communication with the hydraulic valve actuator in response to the at least one cam lobe.

Camshaft phasing mechanisms allow selective adjustment of valve timing for internal combustion engines by selectively advancing or retarding the positions at least some of the lobes on a camshaft, thereby allowing associated valve movements to occur either earlier or later in the gas exchange cycle. For example, engines may operate more efficiently or effectively during one set of operating conditions when the valve timing is advanced, i.e., such that a valve(s) movement occurs earlier during the combustion cycle. Additionally, it may be desirable during a second set of operating conditions to retard the valve timing, i.e., such that a valve(s) movement occurs later during the gas exchange cycle. Adjusting the relative positions of at least some of the lobes on a camshaft allows internal combustion engines to operate with improved fuel economy, torque, and emissions.
Lobes of a camshaft may be used to open and close valves or to actuate pushrods which in turn open and close valves of an engine. While cam phasing mechanisms are useful, they may still suffer from inherent limitations of mechanical valve actuation systems. For example, lift and duration of a valve may be generally incapable of being adjusted during engine operation. As a result, valve opening and/or closing parameters of an engine may not be ideal across all engine operating conditions.
Accordingly, there is a need for a camshaft assembly that addresses the above problems.

Variable valve phasing lift and duration


A binocular telescope with a photographing function comprises a pair of telescopic optical systems, a photographing optical system, and a pair of reticle elements, on which reticles are formed. The reticle elements are provided in the telescopic optical systems for focusing the telescopic optical systems. The distance between the optical axes of the telescopic optical systems can be adjusted, so that the reticle images are fused. The shapes of the fused reticle images are geometrically non-coordinate with each other.

1. Field of the Invention
The present invention relates to a binocular telescope with a photographing function, which has a pair of observation optical systems and a photographing optical system, and is constructed in such a manner that a focusing mechanism for the pair of observation optical systems and a focusing mechanism for the photographing optical system are operated in association with each other so that the pair of observation optical systems is utilized as a focusing device for the photographing optical system.
2. Description of the Related Art
As is well known, binocular telescopes are used for watching sports, wild birds, and so on. When using a binocular telescope, it is often the case that the user sees something that he or she would like to photograph. Typically, he or she will fail to photograph the desired scene because he or she must change a camera for the binocular telescope and during this time the chance is lost. For this reason, a binocular telescope containing a camera is proposed, whereby a photograph can be taken immediately by using the camera contained in the binocular telescope while continuing the observation through the binocular telescope.
For example, Japanese Unexamined Utility Model Publication (JUUMP) (KOKAI) No. 6-2330 discloses a binocular telescope with a photographing function, i.e., a combination of a binocular telescope and a camera, in which the camera is simply mounted in the binocular telescope. The binocular telescope is provided with a pair of telescopic optical systems for observing an observed object in an enlarged state, and a photographing optical system for photographing the observed image. Namely, in the binocular telescope with a photographing function, the pair of telescopic optical systems functions not only as a viewfinder optical system for the photographing optical system, but also as a telescopic binocular system.
Generally, in an observation optical system such as a binocular telescope, when the rear focal point of the objective lens system and the front focal point of the ocular lens system roughly coincide with each other, an observed object at infinity (i.e., distant view) can be observed in an in-focus state through the observation optical system. Accordingly, for observing an observed object at a shorter distance than infinity (i.e., close-range view) in an in-focus state, a focusing operation is needed for focusing on the close-range view. In such a focusing operation, the objective lens system and the ocular lens system are separated from the in-focus state of the distant view. Therefore, in the observation optical system, a focusing mechanism is mounted, which moves the objective lens system and the ocular lens system to adjust the distance therebetween. Concretely, the focusing mechanism comprises a rotary wheel, disposed adjacent to the observation optical system, and a movement conversion mechanism for converting a rotational movement of the rotary wheel into a relative back-and-forth movement of the objective lens system and the ocular lens system.
In the binocular telescope with a photographing function disclosed in the above-described JUUMP '330, however, there is no description of the focusing operation of the pair of observation optical systems. Further, as described above, the pair of observation optical systems functions as a viewfinder optical system for indicating an observed range, and '330 does not indicate how the photographing optical system focuses on an object to be photographed.
U.S. Pat. No. 4,067,027 discloses another type of binocular telescope with a photographing function, which is provided with a pair of observation optical systems and a photographing optical system. In this binocular telescope with a photographing function, a focusing mechanism for the pair of observation optical systems is provided with a mechanism for performing a focusing operation of the photographing optical system. Namely, by rotating the rotary wheel of the focusing mechanism manually, the objective lens system and the ocular lens system are moved relative to each other in each of the observation optical systems, which causes the photographing optical system to move relative to a surface of a silver halide film, and thus, the focusing operations are performed for the pair of observation optical systems and the photographing optical system. Thus, when an observed object is observed in an in-focus state through the pair of observation optical systems, the object is also in an in-focus state in the photographing optical system. Therefore, if a photographing operation is carried out when the observed object is observed in an in-focus state through the pair of observation optical systems, the object image is focused on a surface of the silver halide film.
When different users observe an observed object in an in-focus state through an observation optical device such as a binocular telescope, the observation optical system is not necessarily observed with the same dioptric power for each user. This is because, generally, human eyes have the ability to adjust, so that an object in a range from 15 cm to infinity ahead of the eyes can be focused. This ability to adjust depends upon the age of the observer, so that the range in which the eyes can focus on an object is different depending upon the observer. Thus, even if the dioptric power of the observation optical system is offset from a usual value, a human can still observe the observed object image through the observation optical system as a focused image. Therefore, in the binocular telescope with the photographing function described in U.S. Pat. No. '027, even if the observed object image is observed through the pair of observation optical systems in an in-focus state after manual operation of the rotary wheel, the observed object image is not necessarily focused by the photographing optical system. Thus, it may happen that, although the observed object image is observed through the pair of observation optical systems in an in-focus state, the photographed image is not in an in-focus state.
To solve the problem described above, it is proposed in Japanese Examined Patent Publication (KOKOKU) No. 36-12387 that a reticle element (or focusing index element) be movably provided at an in-focus position of the ocular optical system of the observation optical system so that the observation optical system is always focused with a constant dioptric power. The reticle element is a transparent glass plate, for example, on which an index having a proper shape (e.g., a cross) is formed. If the index element is positioned at an in-focus position of the ocular optical system of the observation optical system, the user observes the observed object in an in-focus state at the position of the index. Namely, the observed object is always observed with a constant dioptric power. Therefore, when the observation optical system reaches an in-focus state, the photographing optical system is adjusted in an in-focus state in association with the observation optical system. Thus, in the binocular telescope with a photographing function, the observation optical system can be utilized as a focusing device for the photographing optical system.
Generally, the reticle element is not assembled in each optical system of a pair of observation optical systems, but provided only in one of the observation optical systems. This is because, when the interpupillary distance of the pair of observation optical systems is adjusted so as to fuse the right and left observed images, it is difficult to completely superpose the right and left reticle images on each other. Namely, although a binocular telescope is designed in such a manner that, when the interpupillary distance of the pair of observation optical systems is perfectly adjusted, the optical axes of the pair of observation optical systems are completely coincident with each other, it is very difficult to make the optical axes completely coincident. Therefore, when the reticle element is assembled in each of the observation optical systems, the pair of reticles are observed slightly offset from each other, when fusing the right and left observed images, and the offset reticle images are distracting to the user. Accordingly, the reticle element is assembled in one of the observation optical systems In practice, the reticle element is often provided in the right observation optical system, since, in most people (around 80%), the right eye is more skillful than the left eye.
In the binocular telescope with a photographing function, in which a reticle element is provided, it is necessary to perform a dioptric power adjustment depending upon the eyesight of the user. Therefore, the ocular optical systems of a pair of observation optical systems are constructed in such a manner that their positions relative to the in-focus positions of the objective optical systems can be adjusted. Namely, first, the user adjusts the positions of the ocular lens systems so as to be able to observe the reticle image in an in-focus state. In this state, when performing a focusing operation of the pair of observation optical systems to focus on the observed image, the observed image is formed in an in-focus state in the photographing optical system as a photographed image, and thus, a focused photographed image can be obtained.
In a binocular telescope in which a reticle element is assembled in the right observation optical system, although the position of the ocular lens system can be adjusted relative to the in-focus position of the objective optical system to adjust the dioptric power in the left observation optical system, a reticle element is not assembled in the left observation optical system, and therefore, it is difficult to perform a dioptric power adjustment properly in the left observation optical system. Thus, in the left observation optical system, the observed image may be observed in a state which is offset from a proper dioptric power with which the left eye, in a relaxed state, of the user observes the observed image. If an observation using the binocular telescope is carried out for a long time in the offset state, the eyes will become tired since the adjusting performance of the eyes is continually carried out.
On the other hand, for a user whose left eye is more skillful than the right eye, since the focusing operation for the observed image is forcedly performed using the right observation optical system, the eyes will become more strained. Further, when a user whose left eye is more skillful than the right eye is not consciously focusing an observed image using the right observation optical system, the focusing operation for the observed image tends to be performed using the left observation optical system. In this focusing operation, since there is no reticle element in the left observation optical system, the adjusting operation of the left eye occurs, so that the dioptric powers of the right and left observation optical systems are offset. Thus, in the photographing optical system, the observed image cannot be formed in an in-focus state.

Binocular telescope with photographing function


A valve member including a pair of substantially hemispherical shell components fixed together to form a substantially spherical, hollow shell. The shell has an exterior surface, an interior surface, and two opposing annular openings. The valve member further includes a sleeve having a first end and a second end. The sleeve is positioned between the annular openings of the shell to define a central bore between the annular passages. The sleeve is in contact with and fixed to the shell solely at the first end and the second end thereof in a way that a continuous void is defined between the sleeve and the interior surface of the shell from the first end of sleeve to the second end of the sleeve.

Valves that utilize a valve member in the form of a ball with a passage extending from one side to another have long been known for controlling the flow of fluid. Further, it has long been desired to reduce the cost of manufacturing valve members of this type, particularly valve members of larger sizes.
It has been proposed to form a hollow sphere with a cylindrical sleeve extending through the sphere. By fabricating the valve member as a hollow sphere, the amount of steel used to make the valve member is reduced, thereby reducing manufacturing costs. However, it has been found that the hollow sphere is susceptible to being crushed when exposed to high working pressures. To overcome such susceptibility, reinforcement structures have been added between the cylindrical sleeve and the interior surface of the sphere. The inclusion of the reinforcement structures increases the cost of the valve.
To this end, a need exists for a floating ball valve member that is capable of being used in a medium to high pressure environment, yet is cost effective to manufacture and repair. It is such a valve member that the inventive concepts disclosed herein are directed.


Methods, systems, and apparatus, including computer programs encoded on a computer storage medium, for extending application access across devices. In some implementations, an electronic device receives a request to provide access to the electronic device to a particular user that is not registered as a user of the electronic device. The electronic device receives authentication credentials for the particular user. The electronic device provides the authentication credentials to a server system and receives data from the server system that (i) indicates that the providing access to the electronic device in a guest mode is authorized, and (ii) indicates a state of an instance of an application installed on a second device. The electronic device provides access to the electronic device in the guest mode that provides an interface that at least partially recreates the state of the instance of the application installed on the second device.

As phones and other mobile devices have become increasingly capable, people rely on them more and more. Many people use their phones as their primary means of communication and organization, using their phones for email, messaging, scheduling and calendar management, note taking, navigation, and more. As a result, many users would be severely inconvenienced if they lost access to their phones, for example, if the battery runs out of power or if the user does not have the phone present. In particular, without his phone, a user would generally lose access to the user's applications installed on the phone and the communication ability that the applications provide.

Extending application access across devices


The present disclosure relates to a cell potential detection device, a method of manufacturing the cell potential detection device, and an information processing system that enable prevention of culture solution for a cell from leaking. The cell potential detection device includes: a cell potential detection chip including an electrode unit that detects potential of a cell; a substrate on which the cell potential detection chip is implemented; a first member sealing a connection electrically connecting the cell potential detection chip and the substrate; and a second member layered on the first member, the second member forming a liquid-storage portion that stores culture solution for the cell, together with the first member. The present technology can be applied to, for example, a semiconductor module in which packaged is a chip that detects the potential at an action-potential source point due to a chemical change of a cell.

Until now, it has been proposed that, with an integrated cell setting device including an integrated composite electrode including a plurality of microelectrodes and the lead patterns thereof provided on a glass plate, a cylindrical polystyrene frame having been centered on the center portion of the plurality of microelectrodes is secured to the glass plate and then the polystyrene frame is filled with culture solution (refer to Patent Document 1).

Cell potential detection device, method of manufacturing cell potential detection device, and information processing system

SONY SEMICONDUCTOR SOLUTIONS CORPORATION


A slide type cellular phone having two separate bodies automatically or manually sliding with respect to each other includes a first main body having a motor with a shaft, an elastic member elastically supporting the motor, and a switch activating the motor, a power transmitting unit coupled to the shaft of the motor to receive a rotation power from the motor, a pinion coupled to the power transmitting unit to be rotated by the rotation power, a second main body slidably coupled to the first main body, having a rack coupled to the pinion, and sliding with respect to the first main body according to movements of the pinion and the rack, and a sliding detecting unit detecting a termination of a sliding operation of the second main body to control the motor. The pinion, the rack, and the power transmitting member to operates by a rotation of the motor to automatically or manually slide the second main body with respect to the first main body, thereby providing a more convenient cellular phone.

1. Field of the Invention
The present invention relates to a cellular phone, and more particularly, to a slide type cellular phone having two separate bodies which manually or automatically slide with respect to each other, and a method thereof.
2. Description of the Related Art
Generally, a cellular phone (wireless or mobile phone) is a terminal which provided with a mobile service which is useful in a predetermined distance of a service area of the mobile service by assigning a portion of telephone lines of subscribers located in the service area to mobile phone lines. While a conventional telecommunication service is only useful in a limited service area using phone line numbers of telephone lines, the cellular phone is used for phone calls without limiting the limited service area using logical numbers regardless of the phone line numbers of the telephone lines.
The cellular phone is very compact to carry with and very convenient to use with, and demands increase tremendously. Moreover, the cellular phone has been developed to be minimized in size and to be multi-functional in operation to satisfy users having various demands.
Generally, the cellular phone is classified into one of a bar type having a key pad exposed to an outside of the cellular phone, a flip type covering the key pad using a cover, and a folder type having a folder folded onto a main body. Currently, the folder type cellular phone having the folder unfolded from and folded onto the main body becomes popular.
FIG. 1 is a perspective view of a conventional folder type cellular phone. As shown in FIG. 1, the cellular phone includes a main body 10 and a folder 20. The main body includes a plurality of main components for receiving and transmitting operations, a microphone 12, a key pad 14m and a battery pack B to supply electrical power to the cellular phone.
The folder 20 includes a speaker 22, a display window 24 displaying various communication information and functional information, and other components.
In the folder type cellular phone, the folder 20 is rotatably connected to the main body 10 through a rotation unit 30 having a circular shape to hinge with respect to the main body 10. The main body 10 is uncovered and covered by the folder 20 according to a rotation of the rotation unit 30 in opposite (forward and reverse) directions.
The folder 20 is folded to cover the main body 10 in a standby mode and is unfolded to uncover the main body 10 when the cellular phone is in a calling mode and a receiving mode.
However, the folder type cellular phone is disadvantageous to unfold the folder 20 from the main body 10 by inserting a user finger between the folder 20 and the main body 10 to unfold the folder 20 from the main body 10.
In order to solve the above problem, another cellular phone having a folder automatically folded onto and unfolded from the main body has been developed. However, a new demand on a new type of the cellular phone increases since the user is fed up with the conventional folder type cellular phone as the conventional folder type cellular type becomes popular.

Slide type cellular phone and sliding method thereof


The present invention relates to methods and apparatus for the encapsulation of biologically-active substances in various cell populations. More particularly, the present invention relates to a method and apparatus for the encapsulation of biologically-active substances in various cell populations in blood by electroporation to achieve therapeutically desirable changes in the physical characteristics of the various cell populations in blood.

Red Blood Cells
In the vascular system of an adult human being, blood has a volume of about 5 to 6 liters. Approximately one half of this volume is occupied by cells, including red blood cells (erythrocytes), white blood cells (leukocytes), and blood platelets. Red blood cells comprise the majority of the cellular components of blood. Plasma, the liquid portion of blood, is approximately 90 percent water and 10 percent various solutes. These solutes include plasma proteins, organic metabolites and waste products, and inorganic compounds.
The major function of red blood cells is to transport oxygen from the lungs to the tissues of the body, and transport carbon dioxide from the tissues to the lungs for removal. Very little oxygen is transported by the blood plasma because oxygen is only sparingly soluble in aqueous solutions. Most of the oxygen carried by the blood is transported by the hemoglobin of the erythrocytes. Erythrocytes in mammals do not contain nuclei, mitochondria or any other intracellular organelles, and they do not use oxygen in their own metabolism. Red blood cells contain about 35 percent by weight hemoglobin, which is responsible for binding and transporting oxygen.
Hemoglobin is a protein having a molecular weight of approximately 64,500 Daltons. It contains four polypeptide chains and four heme prosthetic groups in which iron atoms are bound in the ferrous state. Normal globin, the protein portion of the hemoglobin molecule, consists of two α chains and two β chains. Each of the four chains has a characteristic tertiary structure in which the chain is folded. The four-polypeptide chains fit together in an approximately tetrahedral arrangement, to constitute the characteristic quaternary structure of hemoglobin. There is one heme group bound to each polypeptide chain, which can reversibly bind one molecule of molecular oxygen. When hemoglobin combines with oxygen, oxyhemoglobin is formed. When oxygen is released, the oxyhemoglobin is reduced to deoxyhemoglobin.
Although the secondary and tertiary structure of various hemoglobin subunits are similar, reflecting extensive homology in amino acid composition, the variations in amino acid composition that do exist impart marked differences in hemoglobin's oxygen carrying properties. In addition, the quaternary structure of hemoglobin leads to physiologically important allosteric interactions between the subunits, a property lacking in monomeric myoglobin, which is otherwise very similar to the -subunit of hemoglobin.
Comparison of the oxygen-binding properties of myoglobin and hemoglobin illustrate the allosteric properties of hemoglobin that results from its quaternary structure and differentiate hemoglobin's oxygen-binding properties from that of myoglobin. The curve of oxygen-binding to hemoglobin is sigmoidal typical of allosteric proteins in which the substrate, in this case oxygen, is a positive homotropic effector. When oxygen binds to the first subunit, of deoxyhemoglobin, it increases the affinity of the remaining subunits for oxygen. As additional oxygen is bound to the second and third subunits, oxygen-binding is further, incrementally, strengthened, so that at the oxygen tension in lung alveoli, hemoglobin is fully saturated with oxygen. As oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally unloaded and the affinity of hemoglobin for oxygen is reduced. Thus at the lowest oxygen tensions found in very active tissues the binding affinity of hemoglobin for oxygen is very low allowing maximal delivery of oxygen to the tissue. In contrast the oxygen-binding curve for myoglobin is hyperbolic in character indicating the absence of allosteric interactions in this process. When the affinity for oxygen is decreased, the sigmoidal curve is shifted to the right. This shift of the curve is commonly known as a "right shift".
The allosteric oxygen-binding properties of hemoglobin arise directly from the interaction of oxygen with the iron atom of the heme prosthetic groups and the resultant effects of these interactions on the quaternary structure of the protein. When oxygen binds to an iron atom of deoxyhemoglobin it pulls the iron atom into the plane of the heme. Since the iron is also bound to histidine F8, this residue is also pulled toward the plane of the heme ring. The conformational change at histidine F8 is transmitted throughout the peptide backbone resulting in a significant change in tertiary structure of the entire subunit. Conformational changes at the subunit surface lead to a new set of binding interactions between adjacent subunits. The latter changes include disruption of salt bridges and formation of new hydrogen bonds and new hydrophobic interactions, all of which contribute to the new quaternary structure.
The latter changes in subunit interaction are transmitted, from the surface, to the heme binding pocket of a second deoxy subunit and result in easier access of oxygen to the iron atom of the second heme and thus a greater affinity of the hemoglobin molecule for a second oxygen molecule. The tertiary configuration of low affinity, deoxygenated hemoglobin (Hb) is known as the taut (T) state. Conversely, the quaternary structure of the fully oxygenated high affinity form of hemoglobin Hb(O2)4 is known as the relaxed (R) state.
Delivery of oxygen to tissues depends upon a number of factors including, but not limited to, the volume of blood flow, the number of red blood cells, the concentration of hemoglobin in the red blood cells, the oxygen affinity of the hemoglobin and, in certain species, on the molar ratio of intra-erythrocytic hemoglobins with high and low oxygen affinity. The oxygen affinity of hemoglobin depends on four factors as well, namely: (1) the partial pressure of oxygen, (2) the pH, (3) the concentration of the allosteric effector 2,3-diphosphoglycerate (DPG) in the hemoglobin, and (4) the concentration of carbon dioxide. In the lungs, at an oxygen partial pressure of 100 mm Hg, approximately 98% of circulating hemoglobin is saturated with oxygen. This represents the total oxygen transport capacity of the blood. When fully oxygenated, 100 ml of whole mammalian blood can carry about 21 ml of gaseous oxygen.
The effect of the partial pressure of oxygen and the pH on the ability of hemoglobin to bind oxygen is best illustrated by examination of the oxygen saturation curve of hemoglobin. An oxygen saturation curve plots the percentage of total oxygen-binding sites present in a unit of blood, or a sample that are occupied by oxygen molecules when solutions of the hemoglobin are in equilibrium with varying partial pressures of oxygen.
As stated above, the oxygen saturation curve for hemoglobin is sigmoid. Thus, binding the first molecule of oxygen increases the affinity of the remaining hemoglobin for binding additional oxygen molecules. As the partial pressure of oxygen is increased, a plateau is approached at which each of the hemoglobin molecules is saturated and contains the upper limit of four molecules of oxygen.
The reversible binding of oxygen by hemoglobin is accompanied by the release of protons, according to the equation:HHb++O2HbO2+kH+Thus, an increase in the pH will pull the equilibrium to the right and cause hemoglobin to bind more oxygen at a given partial pressure. A decrease in the pH will decrease the amount of oxygen bound.
In the lungs, the partial pressure of oxygen in the air spaces is approximately 90 to 100 mm Hg and the pH is also high relative to normal blood pH (up to 7.6). Therefore, hemoglobin will tend to become almost maximally saturated with oxygen in the lungs. At that pressure and pH, hemoglobin is approximately 98 percent saturated with oxygen. On the other hand, in the capillaries in the interior of the peripheral tissues, the partial pressure of oxygen is only about 25 to 40 mm Hg and the pH is also relatively low (about 7.2 to 7.3). Because muscle cells use oxygen at a high rate thereby lowering the local concentration of oxygen, the release of some of the bound oxygen to the tissue is favored. As the blood passes through the capillaries in the muscles, oxygen will be released from the nearly saturated hemoglobin in the red blood cells into the tissue. Hemoglobin will release about a third of its bound oxygen as it passes through the muscle capillaries, so that when it leaves the muscle, it will be only about 64 percent saturated. In general, the hemoglobin in the venous blood leaving the tissue cycles between about 65 and 97 percent saturation with oxygen in its repeated circuits between the lungs and the peripheral tissues. Thus, oxygen partial pressure and pH function together to affect the release of oxygen by hemoglobin
A third important factor in regulating the degree of oxygenation of hemoglobin is the allosteric effector 2,3-diphosphoglycerate (DPG). DPG is the normal physiological effector of hemoglobin in mammalian erythrocytes. DPG regulates the oxygen-binding affinity of hemoglobin in the red blood cells in relationship to the oxygen partial pressure in the lungs. In general, the higher the concentration of DPG in the cell, the lower the affinity of hemoglobin for oxygen.
When the delivery of oxygen to the tissues is chronically reduced, the concentration of DPG in the erythrocytes is increased in normal individuals. For example, at high altitudes the partial pressure of oxygen is significantly less. Correspondingly, the partial pressure of oxygen in the tissues is less. Within a few hours after a normal human subject moves to a higher altitude, the DPG level in the red blood cells increases, causing more DPG to be bound and the oxygen affinity of the hemoglobin to decrease. Increases in the DPG level of red blood cells also occur in patients suffering from hypoxia. This adjustment allows the hemoglobin to release its bound oxygen more readily to the tissues to compensate for the decreased oxygenation of hemoglobin in the lungs.
As normally isolated from blood, hemoglobin contains a considerable amount of DPG. When hemoglobin is "stripped" of its DPG, it shows a much higher affinity for oxygen. When DPG is increased, the oxygen-binding affinity of hemoglobin decreases. A physiologic allosteric effector such as DPG is therefore essential for the normal release of oxygen from hemoglobin in the tissues.
While DPG is the normal physiologic effector of hemoglobin in mammalian red blood cells, phosphorylated inositols are found to play a similar role in the erythrocytes of some birds and reptiles. Although IHP is unable to pass through the mammalian erythrocyte membrane, it is capable of combining with hemoglobin of mammalian red blood cells at the binding site of DPG to modify the allosteric conformation of hemoglobin, the effect of which is to reduce the affinity of hemoglobin for oxygen. For example, DPG can be replaced by inositol hexaphosphate (IHP), which is even more potent than DPG in reducing the oxygen affinity of hemoglobin. IHP has a 1000-fold higher affinity to hemoglobin than DPG (R. E. Benesch et al., Biochemistry, Vol. 16, pages 2594–2597 (1977)) and increases the P50 of hemoglobin up to values of 96.4 mm Hg at pH 7.4, and 37° C. (J. Biol. Chem., Vol. 250, pages 7093–7098 (1975)).
The oxygen release capacity of mammalian red blood cells can be enhanced by introducing certain allosteric effectors of hemoglobin into erythrocytes, thereby decreasing the affinity of hemoglobin for oxygen and improving the oxygen economy of the blood. This phenomenon suggests various medical applications for treating individuals who are experiencing lowered oxygenation of their tissues due to the inadequate function of their lungs or circulatory system.
Because of the potential medical benefits to be achieved from the use of these modified erythrocytes, various techniques have been developed in the prior art to enable the encapsulation of allosteric effectors of hemoglobin in erythrocytes. Accordingly, numerous devices have been designed to assist or simplify the encapsulation procedure. The encapsulation methods known in the art include osmotic pulse (swelling) and reconstitution of cells, controlled lysis and resealing, incorporation of liposomes, and electroporation. Current methods of electroporation make the procedure commercially impractical on a scale suitable for commercial use.
The following references describe the incorporation of polyphosphates into red blood cells by the interaction of liposomes loaded with IHP: Gersonde, et al., "Modification of the Oxygen Affinity of Intracellular Haemoglobin by Incorporation of Polyphosphates into Intact Red Blood Cells and Enhanced O2 Release in the Capillary System", Biblthca. Haemat., No. 46, pp. 81–92 (1980); Gersonde, et al., "Enhancement of the O2 Release Capacity and of the Bohr-Effect of Human Red Blood Cells after Incorporation of Inositol Hexaphosphate by Fusion with Effector-Containing Lipid Vesicles", Origins of Cooperative Binding of Hemoglobin, (1982); and Weiner, "Right Shifting of Hb-O2 Dissociation in Viable Red blood cells by Liposomal Technique," Biology of the Cell, Vol. 47, (1983).
Additionally, U.S. Pat. Nos. 4,192,869, 4,321,259, and 4,473,563 to Nicolau et al. describe a method whereby fluid-charged lipid vesicles are fused with erythrocyte membranes, depositing their contents into the red blood cells. In this manner, it is possible to transport allosteric effectors such as inositol hexaphosphate into erythrocytes, where, due to its much higher binding constant, IHP replaces DPG at its binding site in hemoglobin.
In accordance with the liposome technique, IHP is dissolved in a phosphate buffer until the solution is saturated and a mixture of lipid vesicles is suspended in the solution. The suspension is then subjected to ultrasonic treatment or an injection process, and then centrifuged. The upper suspension contains small lipid vesicles containing IHP, which are then collected. Erythrocytes are added to the collected suspension and incubated, during which time the lipid vesicles containing IHP fuse with the cell membranes of the erythrocytes, thereby depositing their contents into the interior of the erythrocyte. The modified erythrocytes are then washed and added to plasma to complete the product.
The drawbacks associated with the liposomal technique include poor reproducibility of the IHP concentrations incorporated in the red blood cells and significant hemolysis of the red blood cells following treatment. Additionally, commercialization is not practical because the procedure is tedious and complicated.
In an attempt to solve the drawbacks associated with the liposomal technique, a method of lysing and the resealing red blood cells was developed. This method is described in the following publication: Nicolau, et al., "Incorporation of Allosteric Effectors of Hemoglobin in Red Blood Cells. Physiologic Effects," Biblthca. Haemat., No. 51, pp. 92–107, (1985). Related U.S. Pat. Nos. 4,752,586 and 4,652,449 to Ropars et al. also describe a procedure of encapsulating substances having biological activity in human or animal erythrocytes by controlled lysis and resealing of the erythrocytes, which avoids the RBC-liposome interactions.
The technique is best characterized as a continuous flow dialysis system which functions in a manner similar to the osmotic pulse technique. Specifically, the primary compartment of at least one dialysis element is continuously supplied with an aqueous suspension of erythrocytes while the secondary compartment of the dialysis element contains an aqueous solution, which is hypotonic with respect to the erythrocyte suspension. The hypotonic solution causes the erythrocytes to lyse. The erythrocyte lysate is then contacted with the biologically active substance to be incorporated into the erythrocyte. To reseal the membranes of the erythrocytes, the osmotic and/or oncotic pressure of the erythrocyte lysate is increased and the suspension of resealed erythrocytes is recovered.
In related U.S. Pat. Nos. 4,874,690 and 5,043,261 to Goodrich et al., a related technique involving lyophilization and reconstitution of red blood cells is disclosed. As part of the process of reconstituting the red blood cells, the addition of various polyanions, including inositol hexaphosphate, is described. Treatment of the red blood cells according to the process disclosed results in a cell with unaffected activity. Presumably, the IHP is incorporated into the cell during the reconstitution process, thereby maintaining the activity of the hemoglobin.
In U.S. Pat. Nos. 4,478,824 and 4,931,276 to Franco et al., a second related method and apparatus is described for introducing effective agents, including inositol hexaphosphate, into mammalian red blood cells by effectively lysing and resealing the cells. The procedure is described as the "osmotic pulse technique." In practicing the osmotic pulse technique, a supply of packed red blood cells is suspended and incubated in a solution containing a compound, which readily diffuses into and out of the cells, the concentration of the compound being sufficient to cause diffusion thereof into the cells so that the contents of the cells become hypertonic. Next, a trans-membrane ionic gradient is created by diluting the solution containing the hypertonic cells with an essentially isotonic aqueous medium in the presence of at least one desired agent to be introduced, thereby causing diffusion of water into the cells with a consequent swelling and an increase in permeability of the outer membranes of the cells. The increase in permeability of the membrane is maintained for a period of time sufficient only to permit transport of at least one agent into the cells and diffusion of the compound out of the cells. These fluxes must be coupled because polyanionic compounds do not simply diffuse across membranes but exchange for ions of equal charge.
Polyanions which may be used in practicing the osmotic pulse technique include pyrophosphate, tripolyphosphate, phosphorylated inositols, 2,3-diphosphoglycerate (DPG), adenosine triphosphate, heparin, and polycarboxylic acids which are water-soluble, and non-disruptive to the lipid outer bilayer membranes of red blood cells.
The osmotic pulse technique has several shortcomings including the fact that the technique is tedious, complicated and unsuited to automation. For these reasons, the osmotic pulse technique has had little commercial success.
Another method for encapsulating various biologically-active substances in erythrocytes is electroporation. Electroporation has been used for encapsulation of foreign molecules in different cell types including IHP red blood cells as described in Mouneimne, et al., "Stable rightward shifts of the oxyhemoglobin dissociation curve induced by encapsulation of inositol hexaphosphate in red blood cells using electroporation," FEBS, Vol. 275, No. 1, 2, pp. 117–120 (1990).
The process of electroporation involves the formation of pores in the cell membranes, or in any vesicles, by the application of electric field pulses across a liquid cell suspension containing the cells or vesicles. During the poration process, cells are suspended in a liquid media and then subjected to an electric field pulse. The medium may be electrolyte, non-electrolyte, or a mixture of electrolytes and non-electrolytes. The strength of the electric field applied to the suspension and the length of the pulse (the time that the electric field is applied to a cell suspension) varies according to the cell type. To create a pore in a cell's outer membrane, the electric field must be applied for such a length of time and at such a voltage as to create a set potential across the cell membrane for a period of time long enough to create a pore.
Electroporation has been used effectively to incorporate allosteric effectors of hemoglobin in erythrocytes. In the article by Mouneime, et al., supra, it was reported that right shifts of the hemoglobin-oxygen dissociation curve in treated erythrocytes having incorporated IHP can be achieved. Measurements at 24 and 48 hours after loading with IHP showed a stable P50 value indicating that resealing of the erythrocytes was permanent. Furthermore, it was shown that red blood cells loaded with inositol hexaphosphate have a normal half life of eleven days. However, the results obtained by Mouneimne and his colleagues indicate that approximately 20% of the re-transfused cells were lost within the first 24 hours of transfusion. U.S. Pat. Nos. 5,720,921, 6,090,617, and 6,074,605 are incorporated herein by reference in their entirety and all disclose an apparatus and method for flow electroporation.
The electroporation methods disclosed in the prior art are not suitable for processing large volumes of sample, nor use of a high or repetitive electric charge. In addition, the stability of the P50 right shift as well as the stability of the red blood cells has not proved adequate for clinical use. Furthermore, the methods are not suitable for use in a continuous or "flow" electroporation chamber. Available electroporation chambers are designed for static use only; namely, processing of samples by batch. Continuous use of a "static" chamber results in over heating of the chamber and increased cell lysis. Furthermore, the existing technology is unable to incorporate a sufficient quantity of IHP in a sufficient percentage of the cells being processed to dramatically change the oxygen carrying capacity of the blood. In addition, the prior art methods require elaborate equipment and are not suited for loading red blood cells of a patient at the point of care. Thus, the procedure is time consuming and not suitable for use on a commercial scale.
What is needed is a simple, efficient and rapid method for encapsulating biologically-active substances in erythrocytes in sufficient volume while preserving the integrity and biologic function of the cells. The potential therapeutic applications of biologically altered blood cells suggests the need for simpler, and more effective and complete methods of encapsulation of biologically-active substances, including allosteric effectors of hemoglobin in intact erythrocytes.
There are numerous clinical conditions that would benefit from treatments that would increase of oxygen bound to hemoglobin. For example to tissue, the leading cause of death in the United States today is cardiovascular disease. The acute symptoms and pathology of many cardiovascular diseases, including congestive heart failure, ischemia, myocardial infarction, stroke, intermittent claudication, and sickle cell anemia, result from an insufficient supply of oxygen in fluids that bathe the tissues. Likewise, the acute loss of blood following hemorrhage, traumatic injury, or surgery results in decreased oxygen supply to vital organs. Without oxygen, tissues at sites distal to the heart, and even the heart itself, cannot produce enough energy to sustain their normal functions. The result of oxygen deprivation is tissue death and organ failure. Another area that would benefit from treatments that would increase delivery of oxygen bound to hemoglobin to tissue is racing animals, athletes, etc.
Another area is in treating diseases such as adult respiratory distress syndrome because administration of blood that is capable of increased delivery of oxygen to the peripheral tissues will ease the pressure of loading hemoglobin in the lungs.
Although the attention of the American public has long been focused on the preventive measures required to alleviate heart disease, such as exercise, appropriate dietary habits, and moderation in alcohol consumption, deaths continue to occur at an alarming rate. One approach to alleviate the life-threatening consequences of cardiovascular disease is to increase oxygenation of tissues during acute stress. The same approach is also appropriate for persons suffering from blood loss or chronic hypoxic disorders, such as congestive heart failure.
Another condition that could benefit from an increase in the delivery of oxygen to the tissues is anemia. A significant portion of hospital patients experience anemia or a low "crit" caused by an insufficient quantity of red blood cells or hemoglobin in their blood. This leads to inadequate oxygenation of their tissues and subsequent complications. Typically, a physician believes that he or she can temporarily correct this condition by transfusing the patient with units of packed red blood cells.
Enhanced tissue oxygenation may also reduce the number of heterologous transfusions and allow use of autologous transfusions in more cases. The current method for treatment of anemia or replacement of blood loss is transfusion of whole human blood. It is estimated that three to four million patients receive transfusions in the U.S. each year for surgical or medical needs. In situations where there is more time or where the religious beliefs of the patient forbid the use of heterologous blood for transfusions, it is advantageous to completely avoid the use of donor or heterologous blood and instead use autologous blood.
Often the amount of blood that can be drawn and stored prior to surgery limits the use of autologous blood. Typically, a surgical patient does not have enough time to donate a sufficient quantity of blood prior to surgery. A surgeon would like to have several units of blood available. As each unit requires a period of several weeks between donations and can not be done less than two weeks prior to surgery, it is often impossible to sequester an adequate supply of blood. By processing autologous blood with IHP, less blood is required and it becomes possible to completely avoid the transfusion of heterologous blood.
As IHP-treated red blood cells transport 2–3 times as much oxygen as untreated red blood cells, in many cases, a physician will need to transfuse fewer units of IHP-treaded red blood cells. This exposes the patient to less heterologous blood, decreases the extent of exposure to viral diseases from blood donors and minimizes immune function disturbances secondary to transfusions. The ability to infuse more efficient red blood cells is also advantageous when the patient's blood volume is excessive. In other more severe cases, where oxygen transport is failing, the ability to rapidly improve a patient's tissue oxygenation is life saving.
Although it is evident that methods of enhancing oxygen delivery to tissues have potential medical applications, currently there are no methods clinically available for increasing tissue delivery of oxygen bound to hemoglobin. Transient elevations of oxygen deposition (6 to 12 hours) have been described in experimental animals using either DPG or molecules that are precursors of DPG. The natural regulation of DPG synthesis in vivo and its relatively short biological half-life, in addition to its lower efficiency in modulating Hb properties, however, limit the DPG concentration and the duration of increased tissue PO2, and thus limit its therapeutic usefulness.
What is needed is a simple, efficient and rapid method for encapsulating biologically-active substances, such as IHP, in erythrocytes without damaging the erythrocytes beyond their ability to produce a clinical effect. An important requirement for any system of introducing IHP into red blood cells is that the right shift of the sigmoidal oxygen-binding curve be substantially stable and the red blood cell must be substantially similar to untreated red blood cells.
Gene Transfection
Efforts to develop human gene therapies have their roots in the 1950s, when early successes with kidney transplantation led to speculation that it might be possible to transplant cells from a normal individual into a patient suffering from a genetic disease. Soon after the discovery of the enzymatic defects in Gaucher's and Niemann-Pick disease, scientists considered organ and bone marrow transplantation and enzyme supplementation to treat rare genetic disorders (Brady, R., NEJM 275:312 (1966)). By the late 1960s and early 1970s, several investigators speculated that it also might be possible to introduce genes into a patient's own cells, and the cloning of the first human genes only a few years later intensified work in the field.
Until recently, almost all of the theoretical and experimental work on human gene therapy was centered on extremely rare genetic diseases, and gene therapy has come to mean, to many in the field, the modification of a patient's genes to treat a genetic disease. However, gene therapy has far wider applications than simply treatment of a genetic disease. Gene therapy is perhaps more appropriately described as medical intervention in which cells, either from the individual to be treated or another appropriate source, are modified genetically to treat or cure any condition, regardless of etiology, that will be ameliorated by the long-term delivery of a therapeutic protein. Gene therapy can therefore be thought of as an in vivo protein production and delivery system, and almost all diseases that are currently treated by the administration of proteins are candidates for treatment using gene therapy.
Gene therapy can be divided into two areas: germ cell and somatic cell gene therapy. Germ cell gene therapy refers to the modification of sperm cells, egg cells, zygotes, or early stage embryos. On the basis of both ethical and practical criteria, germ cell gene therapy is inappropriate for human use. In contrast to germ cell gene therapy, somatic cell gene therapy would affect only the person under treatment (somatic cells are cells that are not capable of developing into whole individuals and include all of the body's cells with the exception of the germ cells). As such, somatic cell gene therapy is a reasonable approach to the treatment and cure of certain disorders in human beings.
In conventional somatic cell gene therapy system, somatic cells (e.g., fibroblasts, hepatocytes, or endothelial cells) are removed from the patient, cultured in vitro, transfected with the gene(s) of therapeutic interest, characterized, and reintroduced into the patient. The means by which these five steps are carried out are the distinguishing features of a given gene therapy system.
Presently-available approaches to gene therapy include the use of infectious vectors, such as retroviral vectors, which include the genetic material to be expressed. Such approaches have limitations, such as the potential of generating replication-competent viruses during vector production; recombination between the therapeutic virus and endogenous retroviral genomes, potentially generating infectious agents with novel cell specificities, host ranges, or increased virulence and cytotoxicity; independent integration into large numbers of cells, increasing the risk of a tumorigenic insertional event; limited cloning capacity in the retrovirus (which restricts therapeutic applicability) and short-lived in vivo expression of the product of interest. A better approach to providing gene products, particularly one that avoids the risks associated with presently available methods and provides long-term production, would be valuable.
A method for encapsulating genetic material in various cell populations is electroporation. Electroporation has been used for encapsulation of foreign molecules in different cell types including IHP red blood cells as described in Mouneimne, et al supra. The electroporation methods disclosed in the prior art are not suitable for processing large volumes of sample, nor use of a high or repetitive electric charge. One of the problems with electroporation of cells has always been the excess heat that occurs during the electroporation process. This heat generated by the electroporation process causes extensive damage to living cells. In addition, the stability of the transformed cells has not proved adequate for clinical use. Furthermore, the methods are not suitable for use in a continuous or "flow" electroporation chamber. Most available electroporation chambers are designed for static use only. Namely, processing of samples by batch. Continuous use of a "static" chamber results in over heating of the chamber and increased cell lysis. Furthermore, the existing technology is unable to incorporate a sufficient quantity of genetic material in a sufficient percentage of the cells being processed. In addition, the prior art methods require elaborate equipment and are not suited for transforming cells of a patient at the point of care. Thus, the procedure is time consuming and not suitable for use on a commercial scale.
Prior Art Flow Cells
U.S. Pat. No. 5,676,646 discloses a flow electroporation apparatus with a flow cell comprising two electrodes separated by a non-conductive spacer, the spacer defining a flow path. The major problem with this flow cell as well as other prior art flow cells is that the surface area of the electrode is not sufficient to dissipate heat as the cells are being electroporated. Thus, the heat buildup in the prior art flow cells is very large as the cells are being electroporated. This heat build up can cause damage to cells and cell components and decrease the efficiency of the electroporation process.
What is needed is a simple, efficient and rapid method for encapsulating genetic material in cells in sufficient volume in real-time, while preserving the integrity and biologic function of the cells. The potential therapeutic applications of transformed cells suggests the need for simpler, and more effective and complete methods of encapsulation of genetic material in intact cells. In addition, a flow cell is needed that is capable of removing heat so that damage to living cells that are being electroporated is kept to a minimum.

Apparatus and method for electroporation of biological samples


There is provided a resin-coated carrier with a coating layer attached to the surface of a core material in a stable manner, the electrophotographic carrier which can provide an image with excellent quality. The resin-coated carrier of the present invention comprises a resin-coating layer comprising, on a core material, a polymer comprising a unit represented by the formula (1):

1. Field of the Invention
The present invention relates to a resin-coated carrier for an electrophotographic developer which constitutes a two-component developer used for development of an electrostatic latent image such as electrophotography, electrostatic recording or electrostatic printing, and to a two-component developer and a developer for replenishment using the same.
2. Related Background Art
Polymers having a hydrophilic group such as a sulfonic acid group are expected to be used for various applications.
The polymers containing a sulfonic acid group are generally synthesized only by using a specific vinyl monomer containing a sulfonic acid functional group. Specific examples of the monomer include sulfonated styrene and AMPS (2-acrylamido-2-methylpropanesulfonic acid). Such monomers are described in Japanese Patent Application Laid-Open No. 2002-351147.
Electrostatic printing comprising forming an electric latent image on the surface of a photoconductive material by electrostatic means and developing the latent image to form an image has been conventionally known, and various methods thereof are known. Specifically, electrostatic printing generally involves forming an electric latent image on a photosensitive member by various means using a photoconductive substance; then attaching a highly pulverized electroscopic material called toner, which is carried and transported by a developer carrying member, to the latent image to form a toner image corresponding to an electrostatic latent image; transferring the toner image to the surface of an image supporting member such as paper as required; and subsequently fixing the toner image with heat, pressure or solvent vapor to obtain a copy.
As a development method for visualizing an electric latent image with a toner, a powder cloud method, a cascade development method, a magnetic brush method, a method employing a conductive magnetic toner, etc. are known. In addition to the above methods, a so-called J/B development method comprising applying a bias electric field composed of an alternating current component and a direct current component to a space between a developer carrying member (development sleeve) and a photoconductive layer to carry out development is known. A typical example of the development method is a magnetic brush method. The magnetic brush method involves use of a two-component developer composed of a toner and a magnetic carrier. By using magnetic particles of steel, ferrite, etc. as a carrier, a developer comprising the magnetic carrier is retained on a developer carrying member having a magnet incorporated therein, and the developer is arranged in the shape of a brush on the developer carrying member by the action of the magnetic field of the magnet. When the magnetic brush is brought into contact with the electrostatic latent image surface on a photoconductive layer, only the toner in the developer is attracted from the brush to the electrostatic latent image, so that the electrostatic latent image is developed.
Carriers which constitute a two-component developer applied to the above development method are roughly classified into conductive carriers and insulating carriers. As a conductive carrier, an oxidized or non-oxidized iron powder is usually used. However, a developer having the iron powder carrier as a constituent triboelectrically charges a toner in an unstable manner. On the other hand, a typical insulating carrier is a resin-coated carrier comprising a carrier core material composed of a ferromagnetic material such as iron, nickel or ferrite having a surface uniformly coated with an insulating resin, generally. A developer with such an insulating resin-coated carrier very rarely causes adhesion of toner particles to the carrier surface as compared with the case of the conductive carrier as described above, exhibits easily controlled triboelectric charging between a toner and a carrier, and has excellent durability and a long useful life, advantageously, and is thus particularly suitable for a high-speed electronic copier.
Various properties are required for an insulating carrier. Particularly important properties include appropriate chargeability, impact resistance, abrasion resistance, good adhesion of a coating material to a core, and a uniform charge distribution. There has been proposed use of a resin with a small surface energy as a material for a coating layer to prevent spending of a carrier such as toner deposition, thereby improving durability of a developer. Specifically, it is said that a carrier coated with a silicone resin, a fluorine resin or the like is spent only with difficulty, and provides a developer with a long life. Japanese Patent Application Laid-Open No. S62-121462 discloses that a resin layer is improved by adding various silane coupling agents to a condensation reaction-type silicone resin.
Other problems in an electrophotographic carrier relate to endurance stability and environmental stability. Generally, problems with endurance stability include slow initial rising of the charge quantity, production of fog in the initial image, and a tendency toward an increased image density. Problems with endurance stability in the long-term copying include a reduced charge quantity, production of fog in the image and a tendency toward an increased image density. Generally, problems with environmental stability in a high humidity environment include a tendency toward a reduced charge quantity, production of fog in the image, an increase in the image density, and a tendency toward scattering of a toner. Problems with environmental stability in a low humidity environment include a tendency toward an increased charge quantity and a tendency toward a reduced image density.

Carrier for electrophotographic developer


A system and method is provided for revising the time stamp information in an MPEG-2 transport stream after content in the transport stream is modified. According to one method, successive PCR time stamps previously added to the transport stream are used to determine PCRSYNC time stamps for synchronization points in the transport stream by interpolation. The PCRSYNC time stamps are substituted into transport packets within the transport stream. The PCRSYNC is also used to determine modified time of arrival (TOA) stamps to be added to transport packets. According to a second method, PCR interpolation is used to determine the values of PCRSYNC. TOA interpolation of TOA stamps previously added to the transport stream are used to determine modified TOA stamps to be substituted into the transport stream for the previously added TOA stamps.

This invention is described in the context of audio-video programs, which include at least one audio signal or one video signal. However, those of ordinary skill in the art will appreciate the applicability of this invention to other types of program signals.
The MPEG-2 Systems specification, ISO/IEC 13818-1, describes a standardized method and data format of packetizing and multiplexing compressed digital audio-visual information for serial transmission applications. This format is called the transport stream format and can be used to multiplex compressed data from one or more audio-visual programs into a single stream. It exhibits a hierarchical structure in which the compressed audio-visual data is present at the lower, compression level, and the packetization and multiplexing of this information is carried out at the higher, systems level. The raw compressed representation of one audio or video signal is referred to as an elementary stream (ES). Compression formats for elementary streams include—but are not restricted to—MPEG-1 Video (ISO/IEC 11172-2 and 11172:3), MPEG-2 Video (ISO/IEC 13818-2), MPEG-4 Video (Part 2 or 10), H.263++, H.26L and the draft H.264/MPEG-4 Part 10 for encoding video data, and MPEG-1 Audio, MPEG-2 Audio (ISO/IEC 13818-3), and MPEG-4 Audio and Dolby-AC-3 for encoding audio data.
The MPEG-2 PES and transport streams encapsulating MPEG-2 video will be used herein as a model for illustrating the invention. The MPEG-2 PES streams and transport streams will be used as a specific example of the systems layer. Those skilled in the art will appreciate that other types of elementary streams, such as encoded audio, MPEG-4 video, etc. may be encapsulated in the PES and transport streams rather than MPEG-2 video.
Audio-visual programs are obtained by using an appropriate combination of one or more elementary streams for storage or transmission of data. For example, one audio elementary stream and one video elementary stream may be combined, or one video elementary stream and multiple audio elementary streams may be combined. The transport stream format enables both single program transport streams (SPTS) in which the elementary streams of a single audio-visual program are multiplexed together into a serial stream, and multiple program transport streams (MPTS), in which the component elementary streams of multiple audio-visual programs are all multiplexed together into a single serial stream.
Referring to FIG. 1, to form a transport stream, each of N elementary streams 100 (including ES1, ES2, through ESN) is first packetized into N packetized elementary streams of (PES) packets 110, independent of its underlying compression format. Each PES packet is comprised of a PES packet header and a segment of a single elementary stream as a payload, which contains data for only a single elementary stream. However, a PES packet may contain data for more than one decoding unit (e.g., data for more than one compressed picture or for more than one compressed audio frame). A variety of packetization strategies for forming PES packets from an elementary stream are permitted.
PES packets from each elementary stream are further packetized into fixed size (188 byte) transport stream (TS) packets 120. Each TS packet 120, as shown in FIG. 2, consists of a fixed 4 byte packet header 121, an optional adaptation field 122 of variable length, and the remaining bytes containing the PES packet data as payload 123. The fixed packet header 121 contains a field called Packet IDentifier (PID), which is a unique numeric identifier or tag for each elementary stream 100 carried in a transport stream 120. For example, one PID is assigned to a video ES of a particular program, a second, different PID is assigned to the audio ES of a particular program, etc.
TS packets 120 from multiple underlying elementary streams 100 are then multiplexed together according to the rules for transport streams set forth in the MPEG-2 Systems specification. This includes insertion of special TS packets 130 containing System Information (SI) which include tables specifying the different programs within the transport stream as well the PIDs which belong to each program. Thus, the transport stream format consists of a lower compression layer, comprising the component elementary streams, and a higher system layer, comprising the PES and TS packets.
The system layer contains important timing information which enables the receiver to play back the audio-visual information in a time-synchronized manner. This includes a Presentation Time Stamp (PTS) in the PES packet header which indicates the time instants at which the associated audio or video presentation unit (an audio or video frame) of a given audio-visual program should be decoded and presented to the user. This PTS is relative to the System Time Clock used by the transmitting encoder. The TS packets also carry samples of this encoder clock called Program Clock References (PCR) in a quasi-periodic manner to enable the receiver to synchronize its system time clock to that of the encoder. This enables the receiver to decompress and present the audio and video data at the correct times, thereby recreating the original presentation.
A requirement for MPEG-2 transport streams is that the PCR for each program must be sent at least once every 100 ms. In the case of the DVB extension (Specification of Service Information (SI) in DVB Systems, ETSI Standard EN 300 468, May 2000) to MPEG-2, these PCR packets are to be sent at least once every 40 ms. PCR information, along with other optional information, is carried in the TS packet inside the adaptation field 122. The PCRs for a given program can be carried in the TS packets carrying any one of the component elementary streams 100 of that program (as identified by its PID), or they can be carried in separate TS packets with a unique PCR PID. Typically, PCRs are carried in the video PID of a program.
In the MPEG-2 context, there are many applications that require one or more audio-visual programs carried inside a MPEG-2 transport stream to be modified at the elementary stream level, using stream processing devices. The prior art teaches a number of "stream processors" or devices, such as transcoders, editors and splicers, that process previously generated transport streams. A transcoder receives an already encoded elementary stream and re-encodes it, e.g., at a different bit rate, according to a different encoding standard, at a different resolution, using different encoding options, changing the audio sampling rate or video frame rate, etc. while maintaining the underlying content with as much fidelity as possible. A splicer is a device that appends one signal to another, inserts that signal in the middle of the first, or replaces part of the signal at a given instant. For example, a splicer may append one encoded elementary stream at the end of another elementary stream in a program so that they will be presented seamlessly and in sequence. Alternatively, the splicer could insert one program in the middle of another, e.g., in the case of inserting a commercial in the middle of a television show. An editor is a device that edits (modifies) an elementary stream and produces an edited encoded elementary stream. Examples of these devices are described in U.S. Pat. Nos. 6,141,447, 6,038,256, 6,094,457, 6,192,083, 6,005,621, 6,229,850, 6,310,915, and 5,859,660.
In such stream processing, the underlying bit positions of various parts of the elementary stream have been changed. For instance, video or audio transcoding tends to change the amount of information (number of bits) needed to represent each presentable portion of the video or audio. This is especially true for a transcoder that changes the bit rate of the output signal but is also true of a transcoder which, for example, re-encodes the elementary stream according to a different standard than it was originally prepared. Likewise, a splice or edit tends to change the relative location of two points (namely, the end point of the original encoded video signal portion that precedes the inserted elementary stream information and the beginning point of the original encoded video signal portion that follows the inserted elementary stream information) in the originally encoded video signal. Therefore, the modified elementary streams must be re-packetized and re-multiplexed into a syntax-compliant transport stream for serial transmission.
One of the critical requirements in transport stream output packetization and delivery is that the inherent information content in the outgoing elementary streams retain the same timing relationship as that of the input. This is required to enable the receiver to play back the underlying audio-visual presentation in a time-synchronized manner. Since the relationship between input and output elementary stream bits is invalidated by the process of stream processing, the output packetization process must somehow re-create the original timing relationship.
Existing approaches to this problem address this by using a full-fledged multiplexer at the output. This involves first recovering the original encoder clock for each modified program using clock recovery techniques like phase locked loops. Thereafter, the presentation times and decoding times of each outgoing audio or video frame are determined and re-stamped and inserted into the PES packets, and each outgoing TS packet is emitted in a manner that complies with the T-STD buffer model. Finally, PCR values are inserted into the emitted TS packets at the required frequency by looking up the recovered encoder clock at the instant of departure of the PCR-bearing TS packets. Since the timing information is completely regenerated and inserted, non-modified elementary streams in any processed program need to be de-packetized to their elementary stream levels, re-packetized, and re-transmitted. All these tasks, especially the need to obey T-STD buffer model requirements, impose a large implementation overhead, thereby increasing the complexity and cost of the stream processing system.

This patent is subject to a terminal disclaimer.

Method and system for re-multiplexing of content-modified MPEG-2 transport streams using PCR interpolation


A projector mount is provided, which includes a foundation, a base, a Y axis translation unit, a rod, an X axis translation unit, a telescopic element, a Z axis translation unit and an angle adjusting unit. The base is connected to the foundation. The base is moved in the Y axis relative to the foundation via the Y axis translation unit. The rod is connected to the base. The rod is moved in the X axis relative to the base via the X axis translation unit. The telescopic element is connected to the rod. The telescopic element is moved in the Z axis relative to the rod via the Z axis translation unit. The angle adjusting unit is connected to the telescopic element.

1. Field of the Invention
The present invention relates to a mount, and in particular to a projector mount.
2. Description of the Related Art
Conventional projector mounts only provide five degrees of freedom, which cannot position the projector properly. The relative angle and position between two parts of the conventional projector mount are changed manually first, and then screwing the two parts together tightly. However, the conventional modification process cannot provide fine-tuned functionality. Additionally, when the two parts are being fixed by screws, the projector may sway and/or swerve, and the shape and dimension of the projection image may be changed.


An insulated electrical connector is used with an associated switch. The switch includes three protruding co-planar switch terminals parallel and spaced from one another. The connector includes first, second, and third contacts positioned within an insulating housing that has at least three cavities parallel to one another. Each contact is positioned within the housing so that the connector is configured to receive external conductors and the associated switch terminals.

The present invention relates generally to an insulated connector, and more particularly to insulated connectors for use with switches. The connector may be used with sub-miniature switches, and more particularly, with push button sub-miniature switches.
Switches are well known and can be used in various electro-mechanial devices, such as video games, vending machines, and control apparatuses. Push button switches are one type of switch that can incorporate normally open, normally closed and common terminals. Push button switches are often used in vending machines, video games, slot machines and the like. These switches can be used to allow a user to operate a computer animated figure in a video game, to order a refreshment beverage in a vending machine, or to play a xe2x80x98handxe2x80x99 in a slot machine.
Many of these devices have a limited amount of space for switch placement, and the switches are generally part of an electrical circuit that requires external conductors to remain in electric contact with the switch terminals. Typically, the conductors are soldered onto the terminals of the switches, and the ends of the conductors are terminated at the terminals. It is imperative that the conductors are soldered onto the appropriate switch terminals to allow for proper switch functionality. Because space within the devices is limited, this process is very tedious and can result in manual error during installation.
To protect the switch or adjacent components from early burn out, a circuit element, such as a diode, is often placed across two of the switch terminals. Typically, circuit element leads are soldered onto the switch terminals. It is crucial that the circuit element leads are connected onto the appropriate switch terminals for proper switch functionality. Again, because space within the devices is limited, this process is tedious and can result in manual error.
In addition, the switches often times work in conjunction with another electrical device, such as a lamp as part of an electrical assembly. Often, the lamp is positioned vertically above or below the switch and has two terminals protruding therefrom. Like the switch, the lamp requires external conductors to remain in electric contact with the lamp terminals. Some circuits require the lamp to be connected to the circuit via an electrical circuit element and an external conductor. The conductors and/or circuit element leads are typically soldered onto the lamp terminals and then terminated. Similar to the switch, the conductors and/or circuit element leads must be connected to the appropriate lamp terminals for proper functionality of the lamp in the electrical circuit. This process is also very tedious and can result in manual error during installation.
In the past, external conductors and/or circuit element leads were manually soldered onto the appropriate switch and electrical device terminals, increasing manual error and labor manufacturing costs.
Accordingly, there continues to be a need for an insulated electrical connector that eases the process of connecting a switch to a circuit within an apparatus. Preferably, such a connector eliminates most, if not all of the circuit element soldering otherwise required for these devices. Further, such a connector also avoids the need for multiple, individual connectors for each switch or lamp terminal.
The present invention relates generally to insulated connectors. A first embodiment of a connector of the invention is configured for use with an associated switch. The switch includes three protruding co-planar switch terminals parallel and spaced from one another. The connector comprises first, second, and third contacts. Each contact includes a terminal receiving portion configured to cooperate with one of the associated three switch terminals and an external conductor receiving portion configured to cooperate with an external conductor.
The first embodiment of the connector also includes an insulating housing having at least three cavities parallel to one another and defining a cavity plane. Each cavity includes a terminal receiving portion and a first conductor opening formed therein.
Each contact is positioned within the housing so that the terminal receiving portions of each contact and cavity are substantially aligned. The conductor receiving portion and the conductor openings of the contacts and cavities are substantially aligned and configured to receive an external conductor transverse to the cavity plane.
A second embodiment of the invention is an insulated connector for use with an associated electrical assembly that has upper and lower terminals vertically spaced from one another. The insulated connector comprises at least one upper contact, at least one lower terminal contact, and at least one lower circuit element contact. The second embodiment also includes an insulated housing that has at least one upper cavity, at least one lower terminal cavity, and at least one lower circuit element cavity. The second embodiment also includes at least one circuit element that has first and second leads.
The upper contacts, lower terminal contacts, and lower circuit element contacts are positioned within the upper cavities, lower terminal cavities, and lower circuit element cavities of the housing so that the connector is configured to receive upper and lower terminals of the associated electrical assembly, and external conductors. The first and second leads of the circuit element are interposed within the contacts so that the upper contacts and lower circuit element contacts are in electrical contact with one another.
Other features and advantages of the present invention will be apparent from the following detailed description, the accompanying drawings, and the appended claims.


A system and method for communication information between origination equipment and destination equipment, in which switching signal information is received from a calling party telephone network. Additionally, information is also received from origination equipment for use by destination equipment for displaying calling party numbers. A display for use with the destination equipment receives the information from the calling party telephone network. From the plurality of information received, a first code from the calling party telephone network identifies calling party number information, and a second code from the calling party telephone network identifies partitioning information of the first code relating to a convention of parsing of telephone numbers associated with the calling party telephone network. The parsing of the calling party numbers into fields introduces field separator characters between the fields including delimiters such as dashes, spaces, parenthetical, graphical characters and the like to separate destination codes from subscriber numbers according to international, country, national or regional fields, such as the North American Numbering Plan.

1. Field of the Invention
The present invention relates to calling party number display systems and, more particularly, to a calling party number display system in which information is communicated between the origination equipment and destination equipment for parsing of telephone numbers to be displayed to increase the readability of international numbers and calls from different area codes.
2. Description of the Related Art
Presently, calling party number delivery systems, also known as caller-ID systems, typically only provide an unparsed telephone number of the calling party sent via the originating switching equipment and transported through the telecommunications network to the terminating switching equipment for display at the telephone receiver's device. Partitioning information is not sent through the network. Some terminating switching equipment attempts to provide partitioning of the calling party number using a best guess algorithm for partitioning the received calling party number digits. However, these algorithms often fail to provide the correct partitioning for all calls.
Known algorithms that can provide correct partitioning on a high percentage of calls are generally complicated and require the maintenance of an ever-changing database of numbering plan information, especially for international numbers. Thus, it would be desirable to take advantage of the switching equipment providing the calling party number which is best suited for determining the proper partitioning since it "knows" the numbering plan of the originating user.
Partitioning of displayed information for calling party numbers is a feature that may be used in the telecommunications field. To this end, it would be further desirable to apply such partitioning to a public (National or International) or private network telecommunication systems to improve readability of the calling party number displayed at the telephony users device by partitioning the number into logical fields separated by the field separators commonly used at the location of the display. Such partitioning may be especially helpful for use with international numbers which are of a more flexible format and often more difficult to read without the partitioning. Therefore, it would be advantageous to provide calling party number delivery systems in which information between origination equipment and destination equipment is communicated so as to identify a convention of parsing of telephone numbers associated with the calling party telephone network.

Partitioning of display for calling party numbers

Siemens Information and Communication Networks, Inc.