A device and method for shortening the chordae tendineae by heating them. Shortening the chordae tendineae stabilizes the atrioventricular valve to which they are attached, thus reducing or halting valve prolapse. A catheter having a heating tip system is inserted in the heart of a patient and maneuvered by an operating surgeon so that the heating tip can induce or transfer heat to the chordae tendineae. The heating tip system heats the chordae tendineae by direct transmission of heat or by inducing heat in the chordae tendineae with electromagnetic radiation. The operating surgeon monitors the heating of the chordae tendineae and when the desired degree of shorting has been achieved, the catheter is removed from the patient.

The human heart is a pump comprising four chambers. The two smaller chambers, the left and right atria, are in the upper part of the heart and the two larger chambers, the left and right ventricles, are in the lower part. The heart pumps by the contractions of the strong muscles which make up the outer walls of the four chambers. During normal operation blood returning from the body is collected in the right atrium. When the right atrium becomes substantially full of blood, it contracts forcing blood through the tricuspid valve into the right ventricle. Contractions of the right ventricle send blood in pulses through the large pulmonary artery and into each lung where the blood becomes oxygenated before it returns through the pulmonary veins to fill the left atrium of the heart. Upon filling with blood, the left atrium contracts, squeezing blood through the mitral valve to the left ventricle. In the final step of the operational cycle of the heart, the powerful muscles of the left ventricle contract forcing oxygen-rich blood through the aorta, then into the smaller arteries to eventually deliver blood to tissue throughout the body. The left and right atria and left and right ventricles contract together, so that the heart functions essentially like two ganged two stage pumps.
Improper operation of the valves or failure of the valves to seal properly can significantly affect the efficiency of most pumps and the heart is no exception. Failure of the valves of the heart to function properly can lead to serious health problems or death. Fortunately, medical science has developed techniques to correct certain heart valve problems in some patients, but finding appropriate remedies for heart valve imperfections and failures while minimizing trauma to the patient is still a major challenge for cardiologists and cardiac surgeons.
Each atrioventricular valve is comprised of a number of cusps, many chordae tendineae which act as guy wires or like shrouds of a parachute to stabilize and keep the cusps in postion when closed, and papillary muscles to which the chordae are anchored. If the chordae tendineae are weak or stretched, the cusps which they stabilize may not seal when closed or may even pop backward into the atrium when the ventricle contracts, a condition known as prolapse.
The chordae tendineae may be divided into three groups or "orders". The first group extending from near the apices of the papillary muscles to near the edges of the valve which they stabilize. The tendinous cords subdivide into thinner strands as they near the valve edge. The chordae of the first order insert into the extreme edge of the valve by a large number of very fine strands. Their function seems to be merely to prevent eversion of the opposing borders of the cusps.
The chordae of the second order insert on the ventricular surface of the cusps approximately at the level of the noduli Albini or even higher. These are stronger and less numerous that those of the first order. These function as the mainstays of the valve and are comparable to the stays of an umbrella.
The chordae of the third order originate from the ventricular wall much nearer the origin of the cusps and often form bands or foldlike structures which may contain muscle. Occasionally, particularly on the left side, the chordae of the first two orders, even in normal hearts, may be wholly muscular, so that the papillary muscle seems to insert directly into the cusp. This is not surprising because the papillary muscle, the chordae tendineae, and major parts of the cusps are derived from the embryonic ventricular myocardium and therefore, were all muscular at one time (see Van Mierop, L. H. S. "Anatomy of the Heart," page 67, Clinical Symposia, Ciba-Geigy, Summit, N.J. (1965)).
Collagen-containing connective tissue is ubiquitous in the human body and demonstrates several unique characteristics not found in other tissue. It provides the cohesiveness of the musculoskeletal system, the structural integrity of the viscera as well as the elasticity of integument. Intermolecular cross links provide collagen connective tissue with unique physical properties of high tensile strength and substantial elasticity. Collagen fibers shrink and tighten when elevated in temperature. This unique molecular response to temperature elevation is the result of rupture of the collagen stabilizing cross links and immediate contraction of the collagen fibers to about one-third of their original linear dimension. Additionally, the caliber of the individual fibers increases greatly, over four fold, without changing the structural integrity of the connective tissue.
One technique for altering collagen connective tissue involves heating the tissue by means of infrared laser energy. Another technique involves inducing heat in the tissue by exposure to radio frequency (rf) or microwave radiation. It is also possible to heat the collagen tissue directly by contact with a warm probe or catheter heated with electricity or by circulation of a heated fluid. The use of infrared laser energy as a corneal collagen shrinking tool of the eye has been described and relates to laser keratoplasty (see U.S. Pat. No. 4,976,709). The importance of controlling the delivery of the energy, that is controlling the localization, time, and intensity, of the energy is critical to provide the soft tissue shrinkage effects without excess collateral damage. Another technique for altering collagen is taught in U.S. Pat. No. 5,458,596 to treat joints.
There is a need to remodel, e.g., strengthen, tighten, and shorten, tissue of the heart to correct faults and increase cardiac efficiency in certain patients. In particular, the chordae tendineae can be tightened to prevent valve prolapse. The art teaches that such remodeling in some cases can be achieved by surgical manipulation, usually requiring open heart surgery, a major, complicated operation. A less invasive remodeling procedure is needed which would lower the risk to the patient.
Copending U.S. patent application Ser. No. 08/739,820, filed Oct. 30, 1996, now U.S. Pat. No. 5,827,268 teaches heating heart tissue with a heat producing catheter as a treatment for Patent Ductus Arteriosus. Further, copending U.S. patent application Ser. No. 08/768,607, filed Dec. 18, 1996 discloses a device and treatment by heating collagen tissue of the heart to repair damage from a myocardial infarction.
It is an object of the present invention to provide a technique for improving atrioventricular valve performance and preventing prolapse by remodeling the corresponding chordae tendineae with heat. It is also an object to provide devices for this remodeling technique as well as methods of teaching the technique.

Method and device for soft tissue modification


A microcoil structure for use in an integrated circuit to produce magnetic flux for specific applications or to serve as an inductor is provided. Further, a method for forming the foregoing microcoil structure in a semiconductor wafer is provided. The microcoil structure includes a plurality of diffusion regions having two ends formed with ohmic contact regions. A field-oxide region and an overlaying insulating layer are formed over the diffusion regions. The field oxide region and the insulating layer together serve as a core member for the microcoil structure Further, a plurality of conductive layers are electrically connected between the diffusion regions in such a manner as to form a coil-like structure which winds around the core member to form the desired microcoil structure. When current is input to this microcoil structure, a magnetic flux is produced. The magnitude and polarity of the magnetic flux can be controlled by varying the magnitude and direction of the input current.

1. Field of the Invention
This invention relates to integrated circuits and semiconductor fabrication processes, and more particularly, to a microcoil structure for use in an integrated circuit and a method for forming the same in a semiconductor wafer.
2. Description of Related Art
A coil is an important circuit component which can be used to produce magnetic fluxes for specific purposes or to serve as an inductor in a circuit. The magnetic flux produced by a coil increases with an increase of the input current.
Microcoil is a coil of very small size that can be incorporated in circuits or instruments of very small scale, such as in integrated circuits. It can be used to separate magnetic substances from a mixed solution, to serve as a magnetic flux producing means in tiny medical instruments, or to serve as an inductor in an integrated circuit.

Method of forming microcoil structure for integrated circuits


A semiconductor device includes a plurality of field plate trenches formed in a semiconductor substrate, a plurality of gate trenches formed in the semiconductor substrate and spaced apart from the field plate trenches, and a plurality of device cells having a cell pitch defined by a distance from one side of a field plate trench to the same side of an adjacent field plate trench. Each device cell includes a first doped region of a first conductivity type and a second doped region of a second conductivity type adjacent the first doped region in a part of the semiconductor substrate disposed between the adjacent field plate trenches that define the cell pitch. At least some of the device cells have more than one gate trench per cell pitch.

Power MOSFETs (metal oxide semiconductor field effect transistors) have high switching speeds and require very little gate drive power because of the insulated gate. One drawback of power MOSFETs is on-resistance (Rdson) and its strong positive temperature coefficient. To achieve a high efficiency power MOSFET, Rdson*A must be reduced where the parameter 'A' represents device area. The main components of Rdson include the channel, accumulation layer, drift region, and parasitics (e.g. metallization, bond wires, package, etc.). Reduced Rdson*A is particularly important at low switching frequencies, where static losses dominate over dynamic losses.
In some applications such as automotive applications, power MOSFETs can switch too fast which causes problems. To mitigate these problems, Qgsth must be increased where Qgsth is the gate-source charge measured from 0V to the threshold voltage. In conventional solutions, increasing Qgd also increases the ratio Qgd/Qgsth where Qgsth is the gate charge in weak inversion. Increasing the ratio Qgd/Qgsth can cause capacitive turn-on and highly increased losses. Furthermore, due to overly high Qrr (reverse recovery charge) stored e.g. in the border of the die (chip), power MOSFETs are not sufficiently commutation-robust in some applications. Also, conventional power MOSFETs cause high losses because of high forward voltage in forward conduction mode. Accordingly, there is a need to reduce the channel resistance (Rchannel) in order to increase Qgd without significantly changing the ratio Qgd/Qgsth or increasing the risk of capacitive turn-on.

Multiple semiconductor device trenches per cell pitch


A private key generation apparatus for generating a private key corresponding to a user ID using the user ID as a public key is disclosed. When a user ID is input, an ID conversion unit outputs a specific element value of a discrete logarithm cyclic group having a one-to-one correspondence relationship with an input user ID. A private key calculation unit calculates a discrete logarithm result value based on the output specific element value of the discrete logarithm cyclic group and calculates a private key having a one-to-one correspondence relationship with the user ID. According to the present invention, it is possible to concretely propose a method of calculating a secret key, capable of guaranteeing a one-to-one correspondence relationship between the ID and the private key by applying a method of calculating a discrete logarithm using the pre-computation table in an ID-based encryption system.

1. Field of the Invention
The present invention relates to an apparatus and method for generating a private key for an ID-based encryption system and a recording medium having a program recorded thereon for causing a computer to execute the method, and more specifically, to an apparatus and method for generating a private key for an ID-based encryption system using a method of calculating a discrete logarithm using a pre-computation table and a recording medium having a program recorded thereon for causing a computer to execute the method.
2. Discussion of Related Art
With recent development of computer technology and rapid expansion of communication networks, a security of computer-related resources and transmitted data is a big issue. An encryption based system is used as one alternative capable of resolving such a problem. An encryption system making a greatest contribution so far is a public key encryption system. In this traditional public key system, it is necessary to authenticate a user's public key in advance, and a problematic certificate should be discarded even though a validity period remains. Accordingly, there is a difficulty associated with management of a certificate of the public key. Therefore, an identity (ID)-based public key system based on personal identity (ID) has been proposed.
Technical approaches for implementing ID-based encryption include a scheme using pairing of an elliptic curve, a quadratic residue use scheme, a lattice use scheme, a trapdoor discrete logarithm (TDL) group use scheme, etc. The scheme using pairing of an elliptic curve is a scheme that is currently feasible, most universal, and actively studied. However, since this technology includes a pairing operation, an implementation method is very complex and much computing power is necessary.
On the other hand, the method of calculating a discrete logarithm using a TDL group may be implemented using an exponentiation operation relatively simpler than the pairing. In particular, an ID-based encryption system having a feasible level computing power and speed may be built using a pre-computation table. However, implementation of an ID-based encryption using TDL technology is considered to be theoretically possible and an actual implementation method is not disclosed. This means that the TDL technology has only been applied to solve a general discrete logarithm problem. Accordingly, to apply the TDL technology to an ID-based encryption system, a specific method should be proposed.
Related arts will be described in detail. US Patent Publication No. 2005-0097316 (Title of Invention: Digital signature method based on identification information of group members, and method of acquiring identification information of signed-group member, and digital signature system for performing digital signature based on identification information of group members) is directed to data encryption using a public key encryption scheme based on identification information of one or more members belonging to the same group. The present invention is directed to an algorithm for deriving a real private key in applying a method of calculating a discrete logarithm using a pre-computation table, and differs from the related art in a configuration.
Further, a digital signature apparatus having different personal private keys in a plurality of financial institutions while storing one personal private key in a remote financial service authentication system is disclosed in Korean Patent Laid-Open Publication No. 2001-0003569 (Title of Invention: Apparatus for generating digital signature based on private-key/public-key). The present invention is directed to an algorithm for deriving a real private key in applying a method of calculating a discrete logarithm using a pre-computation table, and differs from the related art in a configuration.

Apparatus and method for generating secret key for ID-based encryption system and recording medium having program recorded thereon for causing computer to execute the method


Information handling system peripheral cables are managed with a cable clip guide that couples to and extends from the peripheral stand. The cable clip guide includes a support arm that extends substantially perpendicular from the support stand to maintain a ring in a substantially parallel alignment with the support stand. The ring inner circumference guides the cables from the support in a desired direction and is sized to allow movement of the cable through the ring in response to movement of the peripheral, such as the raising, lowering or rotating of a flat panel display. An opening formed from the inner to the outer circumference is sized to retain cables within the ring, however, flexing of the ring increases the opening to allow passage of cables into and out of the ring.

1. Field of the Invention
The present invention relates in general to the field of information handling system cable management, and more particularly to a system and method for managing information handling system adjustable cables.
2. Description of the Related Art
As the value and use of information continues to increase, individuals and businesses seek additional ways to process and store information. One option available to users is information handling systems. An information handling system generally processes, compiles, stores, and/or communicates information or data for business, personal, or other purposes thereby allowing users to take advantage of the value of the information. Because technology and information handling needs and requirements vary between different users or applications, information handling systems may also vary regarding what information is handled, how the information is handled, how much information is processed, stored, or communicated, and how quickly and efficiently the information may be processed, stored, or communicated. The variations in information handling systems allow for information handling systems to be general or configured for a specific user or specific use such as financial transaction processing, airline reservations, enterprise data storage, or global communications. In addition, information handling systems may include a variety of hardware and software components that may be configured to process, store, and communicate information and may include one or more computer systems, data storage systems, and networking systems.
Information handling systems often interact with each other and one or more external peripherals to display, print, communicate or otherwise process information. For instance, displays, speakers, printers, scanner and network cables typically extend from an information handling system to each respective peripheral to communicate information and in some cases power from the information handling system to each peripheral. Often, peripherals and their cables are positioned during set up of the information handling systems to remain in relatively permanent relation to each other, generally by fixing the cables in position with clamps. In large part, the relatively fixed position of peripherals and cables has resulted from the bulkiness and weight of information handling systems and peripherals, which has restricted the practicality of rearrangement of a set-up system. As an example, conventional cathode ray tube (CRT) displays generally have such large footprints and weights that movement of a CRT display in a desktop environment is not practical. This has become increasingly true as consumers have moved towards larger CRT displays having greater display space.
Recently, as the power of information handling system components has increased and their sized decreased, information handling system manufacturers have strived to reduce the size of installed systems and thus improve consumer flexibility in arranging a system. One innovation that has helped to reduce the footprint of installed information handling systems is the use of flat panel displays instead of CRT displays. Flat panel displays use liquid crystal display (LCD) or plasma pixels to display information instead of phosphors pixels used by CRT displays and thus do not require an electron gun to illuminate the pixels. As a result, flat panel displays have considerably reduced depth and weight when compared with CRT displays. The reduced bulkiness and weight of flat panel displays allows information handling system users to more easily adjust the position of a display on a desktop. In addition, flat panel displays are often mounted on a stand that allows rotation of the display, both vertically and horizontally, and adjustment of the height of the display area relative to the desktop. Thus, for instance, a user may rotate a panel by ninety degrees to present pages with enhanced size using the lengthiest portion of the display. However, one difficulty that often arises with movement of a flat panel display is that the cables running from the information handling system to the flat panel display tend to bind or bunch to either restrict movement of the display or require a user to move the display with two hands.

System and method for managing information handling system adjustable cables


A method for additive fabrication includes determining numerical calibration transforms for calibrating an imaging sensor and a printhead assembly to a common coordinate system, where at least some of the numerical calibration transforms include nonlinear transforms.

This invention relates to calibration of an additive manufacturing system.
Additive manufacturing is a set of methods that allows objects to be fabricated via selective addition of material. A typical additive manufacturing process works by slicing a digital model (for example, represented using an STL file) into a series of layers. Then the layers are sent to a fabrication apparatus that deposits the layers one by one from the bottom to the top. Additive manufacturing is rapidly gaining popularity in a variety of markets including automotive, aerospace, medical devices, pharmaceuticals, and industrial tooling.
Inkjet 3D printing is a method of additive manufacturing where printheads deposit droplets of printable material, also called ink. Printheads are typically mounted on a gantry system to allow deposition of printable liquid matrix material in different locations of the build volume. A build platform may also move with respect to the printheads, which may be stationary. The printable liquid matrix material is solidified using UV or visible-light radiation.
Multiple printheads may be used in one system in order to build objects with multiple base materials. For example, materials that have different optical, mechanical, thermal, electromagnetic properties can be used. These materials can be combined to achieve composite materials with a wide range of material properties.

Calibration for additive manufacturing by compensating for geometric misalignments and distortions between components of a 3D printer


The invention provides a method to screen for a modulator of enzymatic activity comprising the step of monitoring the association or dissociation of a pair of polypeptides which can associate as a dimer. The polypeptide pair comprises a first polypeptide comprising detection means and a site of post-translational modification and a second polypeptide comprising detection means.

The post-translational modification of proteins have been known for over 40 years and since then has become a ubiquitous feature of protein structure. The addition of biochemical groups to translated polypeptides has wide-ranging effects on protein stability, protein secondary/tertiary structure, enzyme activity and in more general terms on the regulated homeostasis of cells. Such additions include, but are not limited to, phosphorylation, glycosylation, ADP-ribosylation and ubiquitination.
Phosphorylation is a well-studied example of a post-translational modification of protein. There are many cases in which polypeptides form higher order tertiary structures with like polypeptides (homo-oligomers) or with unalike polypeptides (hetero-oligomers). In the simplest scenario, two identical polypeptides associate to form an active homodimer. An example of this type of association is the natural association of myosin II molecules in the assembly of myosin into filaments.
The dimerization of myosin II monomers is the initial step in seeding myosin filaments. The initial dimerization is regulated by phosphorylation the effect of which is to induce a conformational change in myosin II secondary structure resulting in the folded 10S monomer subunit extending to a 6S molecule. This active molecule is able to dimerize and subsequently to form filaments. The involvement of phosphorylation of myosin II in this priming event is somewhat controversial. Although in higher eukaryotes the conformational change is dependant on phosphorylation, in Ancanthoamoeba, a lower eukaryote, the post-translational addition of phosphate is not required to effect the initial dimerization step. It is of note that the dimerization domains in myosin II of higher eukaryotes contain the sites for phosphorylation and it is probable that phosphorylation in this region is responsible for enabling myosin II to dimerize and subsequently form filaments. In Dictyostelium this situation is reversed in that the phosphorylation sites are outside the dimerization domain and phosphorylation at these sites is required to effect the disassembly of myosin filaments. In contrast to both these examples, Acanthoamoeba myosin II is phosphorylated in the dimerization domain but this modification is not necessary to enable myosin II monomers to dimerize in this species.
By far the most frequent example of post-translational modification is the addition of phosphate to polypeptides by specific enzymes known as protein kinases. These enzymes have been identified as important regulators of the state of phosphorylation of target proteins and have been implicated as major players in regulating cellular physiology. For example, the cell-division-cycle of the eukaryotic cell is primarily regulated by the state of phosphorylation of specific proteins the functional state of which is determined by whether or not the protein is phosphorylated. This is determined by the relative activity of protein kinases which add phosphate and protein phosphatases which remove the phosphate moiety from these proteins. Clearly dysfunction of either the kinases or phosphatases may lead to a diseased state. This is best exemplified by the uncontrolled cellular division shown by tumor cells. The regulatory pathway is composed of a large number of genes that interact in vivo to regulate the phosphorylation cascade that ultimately determines if a cell is to divide or arrest cell division.
Currently there are several approaches to analysing the state of modification of target proteins in vivo:    1. In vivo incorporation of labeled (for example, radiolabeled) moieties (e.g., phosphate, ubiquitin or ADP-ribosyls, which are added to target proteins.    2. Back-labeling. The incorporation of a labeled moiety into a protein in vitro to estimate the state of modification in vivo.    3. The use of cell-membrane-permeable protein-modifying enzyme inhibitors (e.g., Wortmannin, staurosporine) to block modification of target proteins and comparable inhibitors of the enzymes involved in other forms of protein modification (above).    4. Western blotting, of either 1- or 2-dimensional gels bearing test protein samples, in which modification is detected using antibodies specific for modified forms of target proteins.    5. The exploitation of eukaryotic microbial systems to identify mutations in protein-modifying enzymes.
These strategies have certain limitations. Monitoring states of modification by pulse or steady-state labelling is merely a descriptive strategy to show which proteins are modified when samples are separated by gel electrophoresis and visualized by autoradiography. This is unsatisfactory, due to the inability to identify many of the proteins that are modified. A degree of specificity is afforded to this technique if it is combined with immunoprecipitation; however, this is of course limited by the availability of antibodies to target proteins. Moreover, only highly-expressed proteins are readily detectable using this technique, which may fail to identify many low-abundance proteins, which are potentially important regulators of cellular functions.
The use of enzyme inhibitors to block activity is also problematic. For example, very few kinase inhibitors have adequate specificity to allow for the unequivocal correlation of a given kinase with a specific kinase reaction. Indeed, many inhibitors have a broad inhibitory range. For example, staurosporine is a potent inhibitor of phospholipid/Ca+2 dependant kinases. Wortmannin is some what more specific, being limited to the phosphatidylinositol-3 kinase family. This is clearly unsatisfactory because more than one biochemical pathway may be affected during treatment making the assignment of the effects almost impossible.
Finally, yeast (Saccharomyces cervisiae and Schizosaccharomyces pombe) has been exploited as a model organism for the identification of gene function using recessive mutations. It is through research on the effects of these mutations that the functional specificities of many protein-modifying enzymes have been elucidated. However, these molecular genetic techniques are not easily transferable to higher eukaryotes, which are diploid and therefore not as genetically tractable as these lower eukaryotes.
An example of heterodimer association is described in patent application number WO92/00388. It describes an adenosine 3:5 cyclic monophosphate (cAMP) dependent protein kinase which is a four-subunit enzyme being composed of two catalytic polypeptides (C) and two regulatory polypeptides (R). In nature the polypeptides associate in a stoichiometry of R2C2. In the absence of cAMP the R and C subunits associate and the enzyme complex is inactive. In the presence of cAMP the R subunit functions as a ligand for cAMP resulting in dissociation of the complex and the release of active protein kinase. The invention described in WO92/00388 exploits this association by adding fluorochromes to the R and C subunits.
The polypeptides are labeled (or 'tagged') with fluorophores having different excitation/emission wavelengths. The excitation and emission of one such fluorophore effects a second excitation/emission event in the second fluorophore. By monitoring the fluorescence emission of each fluorophore, which reflects the presence or absence of fluorescence energy transfer between the two, it is possible to derive the level of association between the R and C subunits as a function of cAMP concentration. Therefore, the natural affinity of the C subunit for the R subunit has been exploited to monitor the concentration of a specific metabolite, namely cAMP.
The prior art teaches that intact, fluorophore-labeled proteins can function as reporter molecules for monitoring the formation of multi-subunit complexes from protein monomers; however, in each case, the technique relies on the natural ability of the protein monomers to associate.
Tsien et al. (WO97/28261) teach that fluorescent proteins having the proper emission and excitation spectra that are brought into physically close proximity with one another can exhibit fluorescence resonance energy transfer ("FRET"). The invention of WO97/28261 takes advantage of that discovery to provide tandem fluorescent protein constructs in which two fluorescent protein moieties capable of exhibiting FRET are coupled through a linker to form a tandem construct. In the assays of the Tsien application, protease activity is monitored using FRET to determine the distance between fluorophores controlled by a peptide linker and subsequent hydrolysis thereof. Other applications rely on a change in the intrinsic fluorescence of the protein as in the kinase assays of WO98/06737.
The present invention instead encompasses the use of FRET to monitor the association of polypeptides, as described herein, which are labeled with fluorescent moieties (protein and chemical); in the invention, FRET indicates the proximity of two labeled polypeptide binding partners, which labeled partners associate either in the presence or absence of a given post-translational modification to an engineered site which has been introduced into at least one of the partners, but not into the fluorophore, reflecting the modification state of one or both of the binding partners and, consequently, the level of activity of a protein-modifying enzyme.
There is a need in the art for efficient means of monitoring and/or modulating post-translational protein modification. Further, there is a need to develop a technique whereby the addition/removal of a modifying group can be monitored continuously during real time to provide a dynamic assay system that also has the ability to resolve spatial information.

This patent is subject to a terminal disclaimer.

Compositions and methods for screening for modulators of enzymatic activity


A container which has a watch in a top compartment and cosmetics in a compartment below the watch. The compartment for cosmetics has a removable tray which contains the cosmetics and an applicator, and has a release aperture which makes it convenient to remove the compartment containing the cosmetics in order to change the cosmetics.

This invention relates, in general, to containers for cosmetics, and, in particular, to a container in which the cosmetics are contained in a watch case.
Description of the Prior Art
In the prior art various types of cosmetics containers have been proposed. For example, U.S. Pat. No. 1,472,350 to Albanese discloses a watch case in one end of a container and a powder puff in another end.
U.S. Pat. No. 1,861,644 to Roberts discloses a watch case which has a vanity case beneath the watch.
U.S. Pat. No. 1,823,814 to Aiello discloses a combination watch and compact that is attached to a user's wrist by a strap.
U.S. Pat. No. 2,473,226 to Sheldon discloses a container that is worn on a person's wrist by a wrist band.
U.S. Pat. No. 5,611,362 to Duncan et al discloses a cosmetic container with a latch for holding the cover closed.


Provided are a single-card multi-mode multi-operator authentication method and device. An MAML in an AP receives an authentication request from a user, and authenticates hybrid networks found by a UE. Under the condition of determining that the authentication on each network in the hybrid networks is passed, the MAML receives terminal position information sent by the UE, and determines whether the terminal position information is the same as operator area information registered by the UE in advance, if so, the MAML acquires signal strength of each network and determines whether the signal strength of each network falls within a signal strength range covered by a base station under the network, if so, determines that all the networks in the hybrid networks are authenticated successfully, otherwise, determines that the authentication fails. The security in using an SIM card by a valid user and secure network service are ensured.

As the issuing of 3G (third generation mobile communications) license, the domestic 3G network is being constructed gradually, and three major mobile communication operators all come into the 3G era officially. Meanwhile, it has been presented to the schedule for constructing a long term evolution (referred to as LTE) network under the framework of 4G (fourth generation mobile communications), and some cities have started to construct a test network. The 3G network technology adopts the TD-SCDMA standard, which is a developing technical standard. WIFI network is also afoot in the industry while TD network construction is enhanced so as to promote the LTE process. Furthermore, in the above-mentioned process of implementing 3G and promoting 4G, an enhanced data rate for GSM evolution (referred to as EDGE) network, a TD-SCDMA, a WIFI network and an LTE network all play a key role.
In this case, the problem of how to deal with the relationships among four networks, i.e., EDGE, TD, WIFI, and LTE will arise subsequently. In order to enable the four networks, i.e., EDGE, TD, WIFI, and LTE to form an integrated network with progressive rates, progressive coverage distances and mutual complementation, the plan of integrating the four networks is proposed.
At present, the growing popularity of intelligent terminals and tablet personal computers officially proclaims the arrival of mobile Internet era. The mobile Internet is the development intersection of telecommunications industry and IT industry, and as the cover range of wireless local area network (referred to as WLAN) expands gradually, WIFI cover hotspots are established widely, and the LTE network construction is continuously completed, intelligent mobile phone terminals such as Android system have been accepted by more and more users, leading the trend of 3G terminal era. Under the situation that the four networks, i.e., EDGE, TD, WIFI, and LTE are integrated, the usage security of users starts to appear on the agenda.
Because of the coexistence of multiple mobile networks, the complexity of the network scenario, and the cover range difference of different mobile networks, it is easy to come across the situation that multiple networks coexist or there is only a certain single mobile network in the process of using a 3G terminal. However, so far, there is no secure authentication method capable of achieving an authentication mechanism under the conditions of the coexistence of multiple networks and the coexistence of multiple operators simultaneously regarding this case so as to ensure the security when the user uses a subscriber identity module (referred to as SIM) card, and the acquisition of services by a valid user and the normal operation of the network cannot be guaranteed.
In view of the problem that there is no secure authentication method capable of achieving an authentication mechanism under the conditions of the coexistence of multiple networks and the coexistence of multiple operators simultaneously in the relevant art, no effective solution has been proposed so far.

Single card multi-mode multi-operator authentication method and device


A container is disclosed including a plurality of seal blocks adapted to militate against the entry of a fluid into coolant channel headers formed in bipolar plates during a dip coating process, wherein the seal blocks are interconnected and include a fastening portion and a sealing portion, the sealing portion capable of being interchanged.

Fuel cell systems are increasingly being used as a power source in a wide variety of applications. Fuel cell systems have been proposed for use in power consumers, such as vehicles, as a replacement for internal combustion engines, for example. Fuel cells are electrochemical devices which combine a fuel such as hydrogen and an oxidant such as oxygen to produce electricity. The oxygen is typically supplied by an air stream. The hydrogen and oxygen combine to result in the formation of water. Other fuels can be used such as natural gas, methanol, gasoline, and coal-derived synthetic fuels, for example.
The basic process employed by the fuel cell system is efficient, substantially pollution-free, quiet, free from moving parts (other than an air compressor, cooling fans, pumps and actuators), and may be constructed to leave only heat and water as by-products. The term "fuel cell" is typically used to refer to either a single cell or a plurality of cells depending upon the context in which it is used. The plurality of cells is typically bundled together and arranged to form a stack with the plurality of cells commonly arranged in electrical series. Since single fuel cells can be assembled into stacks of varying sizes, systems can be designed to produce a desired energy output level providing flexibility of design for different applications.
Different fuel cell types can be provided such as phosphoric acid, alkaline, molten carbonate, solid oxide, and proton exchange membrane (PEM), for example. The basic components of a PEM-type fuel cell are two electrodes separated by a polymer membrane electrolyte. Each electrode is coated on one side with a thin catalyst layer. The electrodes, catalyst, and membrane together form a membrane electrode assembly (MEA).
In a typical PEM-type fuel cell, the MEA is sandwiched between "anode" and "cathode" diffusion mediums (hereinafter "DM's") or diffusion layers that are formed from a resilient, conductive, and gas permeable material such as carbon fabric or paper. The DM's serve as the primary current collectors for the anode and cathode, as well as provide mechanical support for the MEA. The DM's and MEA's are pressed between a pair of electronically conductive plates which serve as secondary current collectors for collecting the current from the primary current collectors. The plates conduct current between adjacent cells internally of the stack in the case of bipolar plates and conduct current externally of the stack (in the case of monopolar plates at the end of the stack).
The bipolar plates typically include two thin, facing metal sheets. One of the sheets defines a flow path on one outer surface thereof for delivery of the fuel to the anode of the MEA. An outer surface of the other sheet defines a flow path for the oxidant for delivery to the cathode side of the MEA. When the metal sheets are joined, the joined surfaces define a flow path for a cooling fluid. The plates are typically produced from a formable metal that provides suitable strength, electrical conductivity, and corrosion resistance, such as 316L alloy stainless steel, for example.
The bipolar plates may include at least one coating applied to the exterior. Typically, the at least one coating is applied using a dipping process. During the dipping process, the bipolar plates are submerged in at least one tank of fluid. Any amount of fluid that enters the coolant channels significantly affects the contact resistance and the uniformity of the current distribution throughout the active area of the bipolar plate, rendering the bipolar plate substantially unsuitable for stack builds. Therefore, the coolant channels of the bipolar plates are sealed during the dipping process. Generally, the fluid is prevented from entering the coolant channels by manually securing metal seal blocks over the coolant channel headers using a plurality of fasteners.
Such seal blocks require the use of additional equipment and tools to seal the coolant channels from the fluid. The additional equipment is excessively heavy and easily susceptible to improper installation.
It would be desirable to produce a container having integrated coolant seals for militating against entry of a fluid into internal coolant channels of bipolar plates during a dip coating process, wherein the container is economical to produce and the complexity of production and use thereof is minimized.


A method and apparatus is disclosed for managing storage used by a processor when processing instructions in which an estimate of register usage for program procedures or functions is generated and used to control the storage of the register bank in memory.

Computer processors use a primary storage facility to store the results of processed instructions and to pass data from one instruction to another. The primary storage reduces execution time of a program by providing immediate storage for the data being processed thus avoiding any delay in transferring the data to and from memory. In the Itanium™ architecture, for example, the primary storage is referred to as a Register Stack and in the Sun SPARC™ architecture the primary storage is referred to as a Register Window.
Generally, register stacks are finite in size and as a result, as call depth increases during the execution of a set of instructions the processor needs to save the data in some of the registers in a secondary storage facility or memory. Saving register contents to memory frees up registers for the processing of subsequent sets of instructions or procedure/function calls. The SPARC™ architecture generates a trigger in the software called a trap to initiate the saving of registers to create free registers. This method works well when the call depths are shallow or when the program depth does not change significantly to cause register window overflow. Traps are however computationally expensive as outlined in 64-bit CPUs: Alpha, SPARC, MIPS and Power, by Jim Turley, Feb. 21, 2002. The Itanium™ architecture avoids the use of traps by employing a Register Save Engine (RSE) which automatically saves registers to memory. The RSE can operate in two alternative modes. In the first mode is called the lazy mode and in this mode only a new stack frame allocation triggers the RSE to save registers to memory. The second mode is called the eager and in this mode registers are saved as and when the memory bus is idle. The register saves/restores in eager mode can occur any time, not necessarily during the procedure entry and exit.
The modes of operation of the Itanium™ RSE each have drawbacks in different scenarios. Given a register stack with more free registers than are required to complete the execution of the procedure sequence, the processor would not be required to perform any register stack saves (or restores). However, if the RSE mode of operation is set to eager, the processor will aggressively pursue saving and loading back registers across the procedure call/returns, resulting unnecessary memory traffic. If the RSE operation was set to lazy mode, and program execution requires more registers then are free, the operation of the RSE may stall the execution of the program whenever a procedure's register requirement exceeds the available clean registers in the register stack.
It is an object of the present invention to provide a method and apparatus for managing storage used by a processor when processing instructions, which avoids some of the above disadvantages or at least provides a useful alternative.

Method and apparatus for managing storage used by a processor when processing instructions

Hewlett-Packard Development Company, L.P.


A connecting rod (10) has a big end bearing eye (11) for connection to a crankshaft, a small end bearing eye (12) for connection to a piston of a cylinder, and an eccentric adjustment device (13) for adjusting an effective connecting rod length. The eccentric adjustment device (13) has an eccentric (36) that interacts with an eccentric lever (14), and eccentric rods (15, 16) that the eccentric lever (14). The eccentric lever (14) has segments (38, 39) positioned axially in front of and behind the small end bearing eye (12). Each eccentric lever segment (38, 39) is connected to the eccentric (36) for conjoint rotation. Each eccentric lever segment (38, 39), end faces (43, 44) of the small end bearing eye (10) adjacent the respective eccentric lever segment (38, 39), and adjacent end faces (45, 46) of the eccentric (36) have a beveled contours.

1. Field of the Invention
The invention relates to a connecting rod for an internal combustion engine and to an internal combustion engine.
2. Description of the Related Art
DE 10 2010 016 037 A1 and FIG. 1 herein show a known connecting rod of an internal combustion engine having an adjustable compression ratio. With reference to FIG. 1, the connecting rod 10 has a big end bearing eye 11 and a small end bearing eye 12. The big end bearing eye 11 is used to connect the connecting rod 10 to a crankshaft (not shown in FIG. 1) and the small end bearing eye 12 is used to connect the connecting rod 10 to a cylinder piston (not shown in FIG. 1) of the internal combustion engine. The connecting rod 10 is assigned an eccentric adjustment device 13, that has an eccentric (not shown in FIG. 1), an eccentric lever 14 and eccentric rods 15, 16. The eccentric lever 14 has a hole arranged eccentrically with respect to a central point 17 of the small end bearing eye 12 and has a central point 18. The hole in the eccentric lever 14 accommodates the eccentric and a hole in the eccentric accommodates a piston pin. The eccentric adjustment device 13 is used to adjust an effective connecting rod length leff, where the connecting rod length is to be taken to mean the distance between the central point 18 of the hole in the eccentric lever 14 and a central point 19 of the big end bearing eye 11. The eccentric rods 15, 16 can be moved to turn the eccentric body 14 and hence change the effective connecting rod length leff. Each eccentric rod 15, 16 is assigned a piston 20, 21 that is guided movably in a hydraulic chamber 22, 23. A hydraulic pressure prevails in the hydraulic chambers 22, 23 and acts on the pistons 20, 21 assigned to the eccentric rods 15, 16. Thus, repositioning of the eccentric rods 15, 16 is possible or not possible, depending on the quantity of oil in the hydraulic chambers.
Adjustment of the eccentric adjustment device 13 is initiated by action of inertia forces and load forces of the internal combustion engine acting on the eccentric adjustment device 13 during a power stroke of the internal combustion engine. The directions of action of the forces acting on the eccentric adjustment device 13 are constant during a power stroke. Hydraulic oil acts on the pistons 20, 21 and the pistons act on the eccentric rods 15, 16 to assist the adjusting movement. The pistons 20, 21 prevent resetting of the eccentric adjustment device 13 due to variations in the directions of action of the forces acting on the eccentric adjustment device 13. The eccentric rods 15, 16 interact with the pistons 20, 21 and are connected to each end of the eccentric body 14. The hydraulic chambers 22 and 23 in which the pistons 20, 21 are guided can be supplied with hydraulic oil via hydraulic oil lines 24 and 25, starting from the big end bearing eye 11. Check valves 26 and 27 prevent the hydraulic oil from flowing back out of the hydraulic chambers 23 and 24 into the hydraulic lines 24 and 25. A changeover valve 29 is accommodated in a hole 28 in the connecting rod 10. The operating position of the changeover valve 29 determines which of the hydraulic chambers 22 and 23 is filled with hydraulic oil and which of the hydraulic chambers 22 and 23 is emptied, with the direction of adjustment or direction of rotation of the eccentric adjustment device 13 depending thereon. In this arrangement, the hydraulic chambers 22 and 23 are in communication via fluid lines 30 and 31, respectively, with the hole 28 accommodating the changeover valve 29. An actuating means 32, a spring device 33 and a control piston 34 of the changeover valve 29 are shown schematically in FIG. 1, and the operation of these component elements of the changeover valve 29 is known from DE 10 2010 016 037 A1.
As explained above, the hydraulic oil that acts on the pistons 20, 21 guided in the hydraulic chambers 22, 23 is fed to the hydraulic chambers 22, 23, starting from the big end bearing eye 11, via hydraulic lines 24 and 25. The connecting rod 10 engages by means of the big end bearing eye 11 on the crankshaft (not shown in FIG. 1) in such a way that a connecting rod bearing shell 35 is arranged between the crankshaft, namely a crankshaft bearing journal thereof, and the big end bearing eye.
DE 10 2005 055 199 A1 discloses further details of an internal combustion engine with an adjustable compression ratio. More particularly, this prior art discloses inserting the eccentric lever on which the eccentric rods engage into a slot-type aperture in the small end bearing eye. The eccentric lever has a hole with internal toothing and is inserted into the slot-type aperture in the small end bearing eye. The eccentric has external toothing and is inserted into the hole in the eccentric lever. The eccentric and the eccentric lever are connected to one another by the intermeshing teeth. Connecting the eccentric to the eccentric lever by the intermeshing teeth requires a high outlay on production and assembly and is susceptible to wear.
It is an object of the invention to provide an internal combustion engine and a connecting rod with low production and assembly costs, improved wear, and a low weight.

Connecting rod and internal combustion engine

DR. ING. H.C.F. PORSCHE AKTIENGESELLSCHAFT


Methods and apparatus according to various aspects of the present invention comprise an on-off controller configured to control the flow of pressurized pneumatic fluid. In one embodiment, the on-off controller comprises a body having an inlet, at least one outlet, a vent, a vent passage, a rod positioned axially in the body, a position mechanism configured to move the rod axially in the body, a seal configured to sealably contact the rod and seal the vent passage, wherein the position of the rod may define operating states comprising: an on-state, an off-state, and a vent-state.

1. Field of the Invention
This invention pertains generally to methods and apparatus relating to pneumatic valves.
2. Description of Related Art
Pneumatic valves find uses in a variety of situations, such as, natural gas distribution systems, pneumatic tools, and controlling the flow of pressurized air to a paintball marker. Valves may benefit from a system that reduces the force that may be required to actuate the valve and a vent that may discharge the pneumatic fluid from the system when the fluid source is shut off.

Methods and apparatus for an on-off controller


A pretensioner clutch housing for a vehicle seat belt arrangement has a housing made of a first housing part arranged to mount a clutch, and a second housing part arranged to mount a pre-tensioning device. The first and second housing parts have cooperating members for connecting the parts together. The cooperating members preferably are a flange on one part and a slot on the other. The clutch housing part is thus universal and only the pretensioner housing part needs to be changed to alter the angle of extension of a tube containing a pretensioner's piston to facilitate different models of cars.

A pretensioner commonly has a piston mounted in a tube which is mechanically or electrically activated to pretension the seat belt by winding in the belt spool a predetermined amount upon the detection of a predetermined deceleration rate of the vehicle, which indicates a crash situation. The piston may be connected to one end of an actuator cable, the other end of which is connected to the belt spool via a clutch mechanism. Thus when the piston is activated, the cable is retracted, and the clutch engaged so as to wind in the belt spool. This may be achieved for example by the cable being wound around an annular member mounted on the axle of the spool so as to surround the axle, which annular member decreases in diameter when the cable is tightened so as to grip the spool axle, such that the axle then rotates with the annular member to wind in the belt.
The tube carrying the piston normally has an outer cover and is mounted on an upper part of the clutch housing. Some applications require that the tube extend from the clutch housing at a different angle, for example in order to fit different vehicles. It is therefore necessary to provide clutch housings with different configurations. This necessitates the provision of a new clutch housing tool for manufacturing the housing. Usually, clutch housings are required in left hand and right hand versions, for use on opposite sides of a vehicle, adding further to the cost and inconvenience of providing different housings.

Clutch housing for a seat belt pretensioner


An apparatus for a monolithic integrated circuit die is disclosed. In this apparatus, the monolithic integrated circuit die has a plurality of modular die regions. The modular die regions respectively have a plurality of power distribution networks for independently powering each of the modular die regions. Each adjacent pair of the modular die regions is stitched together with a respective plurality of metal lines.

Integrated circuits have become more "dense" over time, i.e., more logic features have been implemented in an IC of a given size. Therefore, power consumption has become an increasingly more significant issue, including power consumption during wafer-level testing. Hence, it is desirable to provide an IC having power consumption more suitable for wafer-level testing.

Monolithic integrated circuit die having modular die regions stitched together


A kit is provided for preferentially amplifying target RNA in a sample of tester RNA relative to non-target RNA in the sample, the kit driver sequences complementary to the non-target tester RNA under conditions where the driver sequences hybridize to the non-target RNA, a nucleic acid primer capable of hybridizing to the target RNA under conditions suitable for extension of the nucleic acid primer; and a promoter template capable of hybridizing to a DNA that is complementary to the target RNA under conditions suitable for extension of the complementary DNA such that a functional double promoter is formed.

This invention relates to a method for selectively amplifying target RNA relative to non-target RNA in a sample of tester RNA.
Disease is a deviation from the normal functioning of the body""s organs or systems. This deviation can arise in a number of ways: by either an abnormal gene being switched on or by a normal gene being switched off; by chromosomal mutations or rearrangements which frequently result in abnormal or missing gene products in congenital conditions; or by the presence of an infectious agent in genetically normal individuals. In some cases, the total complement of mRNA, the products of gene expression, in an abnormal cell Will be different from that in a normal cell. In other cases, there may be no apparent difference in the level of gene expression, but the genetic lesion may be a subtle point mutation giving rise to a defective gene product.
Identification of differences in genetic expression or sequence between normal and abnormal cells is a powerful diagnostic and/or prognostic tool. It can also be the first step in understanding a disease by revealing its underlying mechanism. Thus, identification of genetic differences between normal and abnormal cells can provide a clear path to the design of new diagnostic tests, new drugs or gene therapy.
Methods for identifying and isolating sequences present or actively expressed in one cell but diminished or absent in another cell, referred to as xe2x80x9cdifferential screeningxe2x80x9d and xe2x80x9cdifference cloningxe2x80x9d, have been applied to both genomic DNA and mRNA. Difference cloning is based upon subtractive hybridization, a method for isolating xe2x80x9ctargetxe2x80x9d sequences from one DNA population, referred to in this application as xe2x80x9ctesterxe2x80x9d, by using an excess of sequences from another DNA population, referred to in this application as xe2x80x9cdriverxe2x80x9d. One method of subtractive hybridization mixes a restriction endonuclease-digested tester DNA with an excess of randomly sheared driver DNA Lamar, et al., xe2x80x9cY-Encoded, Species-Specific DNA in Mice: Evidence That the Y Chromosome Exists in Two Polymorphic Forms in Inbred Strainsxe2x80x9d, Cell, Vol. 37, pp. 171-177, (1984). The DNA mixture is denatured, hybridized and ligated into a compatible restriction site in a cloning vector. Only a tester DNA fragment reannealled to its complement would have both of the correct ends required for cloning. Conversely, any tester DNA fragments that anneal to complementary driver DNA fragments would not have both of the required ends. The low yield of cloned target sequences in this method is due primarily to the slow reannealling of dilute tester sequences to their complements. In addition, the enrichment of unique target sequences from a background of sequences common to the driver is limited by the initial excess of driver to tester.
Other methods of subtractive hybridization are directed toward the preparation of subtracted probes for differential screening of cDNA libraries by in situ colony blot hybridization. In differential screening, differentially expressed nucleic acids are not cloned, but are used as hybridization probes to identify and characterize unenriched cDNA clones. One method described by Kuze, et al., xe2x80x9cA new vector and RNase H method for the subtractive hybridizationxe2x80x9d, Nucleic Acids Research, Vol. 17, No. 2, pp. 807, (1989). prepares subtracted RNA probes using hybridization to DNA, followed by digestion with RNase H to separate non-hybridized RNA from the hybrid. After the remaining RNA is purified, the subtractive hybridization process is repeated. Hybridization of immobilized DNA using the purified subtracted RNA probe indicates that the subtracted probes can be enriched at least 100 fold. Kuze et al. does not describe a way in which the sequences of the subtracted RNA itself may be cloned or amplified, or suggest a use for the subtracted RNA other than as a hybridization probe.
Some improvements to the subtractive hybridization methods, as applied to difference cloning, involve the use of nucleic acid amplification processes selectively to increase the copy number of a DNA segment having the target sequence. An improvement to subtractive hybridization described by Wieland, et al., xe2x80x9cA method for difference cloning: Gene amplification following subtractive hybridizationxe2x80x9d, Proc. Natl., Acad. Sci. USA, Vol. 87, pp. 2720-2724, (1990). Uses the xe2x80x9cpolymerase chain reactionxe2x80x9d (PCR) to increase the concentration of target sequences, and multiple steps of annealing tester DNA to excess driver DNA to further enrich the unique target sequences from a background of sequences common to the driver. in this procedure tester DNA fragments are first prepared for amplification by ligating to a xe2x80x9ctemplatexe2x80x9d oligonucleotide. A mixture of prepared tester DNA and a 200-fold excess of randomly sheared driver DNA is denatured and reannealled to 90% completion, after which the remaining single-stranded DNA containing target sequences is purified from the double-stranded DNA containing the driver. After three rounds of denaturation, annealing and purification, the remaining tester DNA is then amplified in PCR using primers that anneal to the template sequences. The double-stranded PCR products are then cloned and sequenced. The method gave a 100- to 700-fold enrichment of target sequences.
Another improvement to subtractive hybridization described by Lisitsyn, et al., xe2x80x9cCloning the Differences Between Two Complex Gnomesxe2x80x9d, Science, pp. 946-951, (1993). is a technique called xe2x80x9crepresentational difference analysisxe2x80x9d (RDA). RDA lowers the complexity of both tester and driver genomic DNA by using various restriction endonucleases, eg. BamHI, Bg/II and HindIII, to generate fragments of a particular length that can be efficiently amplified in PCR as xe2x80x9crepresentationsxe2x80x9d of the genome. The tester and driver fragments are ligated to dephosphorylated oligonucleotide adaptors such that an adaptor sequence is ligated to the 5xe2x80x2-end of each strand. The adapted fragments are then amplified in separate PCR reactions using the adaptor as a primer to achieve kinetic enrichment of a population of xe2x80x9campliconsxe2x80x9d that are below 1 kb in size. Finally, the tester and driver amplicons is digested with the same restriction endonuclease to remove the original adaptors.
The xe2x80x9cdifference analysisxe2x80x9d step of RDA is based upon the kinetic and subtractive enrichment of the tester amplicons in a second PCR. It begins with ligating different dephosphorylated oligonucleotide adaptors, this time only to the tester amplicons. An excess of driver amplicons are then mixed with the adapted tester fragments, denatured, and allowed to anneal. A portion of the annealed fragments is then treated with a DNA polymerase to allow extension of driver and adapted tester strands using the complementary driver or adapted tester strands as template. The annealed and extended amplicons are then amplified in PCR using the adaptor as a primer. Driver strands annealed to complementary driver strands will not be extended or amplified. Driver strands annealed to complementary adapted tester strands will be extended, but will lack a 5xe2x80x2-terminal adaptor sequence that is necessary to form a template for exponential amplification in PCR. Only the adapted tester strands that anneal to their complementary adapted tester strands and are extended prior to PCR will contain sequences on both 5xe2x80x2 and 3xe2x80x2 ends to enable exponential amplification.
Following 10 cycles of PCR, a portion of the amplified products is treated with a nuclease to specifically degrade single-stranded nucleic acids. After inactivating the nuclease, a portion of the remaining nucleic acids is further amplified in PCR using the same primer. After 15 to 20 more cycles of PCR, the double-stranded DNA products are digested with the restriction endonuclease, and the process of difference analysis is repeated. After sufficient rounds (typically 3 to 4, for example) of RDA are performed, the double-stranded DNA products are finally cloned and analyzed.
Although RDA was originally developed for genomic DNA, a variation of RDA has been more recently applied to cDNA Hubank, et al., xe2x80x9cIdentifying differences in mRNA expression by representational difference analysis of cDNAxe2x80x9d, Nucleic Acids Research, Vol. 22, No. 25, pp. 5640-5648, (1994). Since the cDNA population derived from a typical cell represents only 1-2% of the total genome, kinetic enrichment to reduce complexity should not be necessary for RDA when applied to cDNA. Dephosphorylated oligonucleotide adaptors are ligated to tester and driver cDNA fragments that are generated by digestion of the double-stranded cDNA with a frequently cutting restriction endonuclease, eg. DpnII. The adapted cDNA fragments are amplified separately by PCR and the resulting amplicons are digested with the same restriction endonuclease to remove the adaptor sequences. The tester cDNA amplicons are then subjected to multiple rounds of RDA as described for genomic DNA. Despite many successful applications of subtractive hybridization, the various methodologies have been described as xe2x80x9ctechnically difficult, time consuming and often either impractical or unreliablexe2x80x9d (Hubank and Schatz, 1994). One difficulty encountered with subtractive hybridization is the requirement for physical removal of hybridized tester sequences prior to amplification and/or cloning. Another limitation of subtractive hybridization is related to self-reassociation kinetics of complex genomes. Similarly, RDA shares some of the problems common to other subtractive hybridization methods. One is the requirement for self-reannealling of complementary tester sequences to enable amplification. For rare cDNA sequences (less than 1%, for example) this imposes kinetic limitations and requires lengthy hybridizations (of 20 hours, for example). Another limitation of RDA is the amplification of a double-stranded tester DNA that requires the use of an excess (typically 100-fold) of double-stranded driver DNA to compete with the re-annealing of complementary tester DNA strands. A single-stranded tester nucleic acid could more effectively hybridize with a complementary single-stranded driver DNA. To avoid physical removal of hybridized sequences, RDA requires the covalent activation and inactivation of tester sequences prior to amplification. [For subsequent rounds of RDA, these modifications must be removed with a restriction enzyme, new primers must be ligated and the newly modified tester must be subjected to hybridization with driver and activation/inactivation steps before the next application of PCR.] This multi-step process becomes a serious practical problem with RDA, in that the PCR amplification step is vulnerable to contamination by less enriched amplified products. Anti-contamination procedures cannot be implemented since the amplicons must remain intact and active as a template throughout the various steps of each round of RDA.
Thus, there is a need for a process for the enrichment of nucleic acid sequences which: 1) avoids hybrid removal prior to amplification; 2) does not require concentration-limiting self-annealing of tester nucleic acids; 3) utilizes single-stranded tester and driver nucleic acids; 4) avoids modification of the tester nucleic acids between rounds of subtractive hybridization and amplification; and 5) integrates hybridization, inactivation, and exponential amplification into a process with fewer steps, thus avoiding contamination.
The method of this invention, called subtractive amplification, represents a novel combination of subtractive hybridization and nucleic acid amplification. Subtractive amplification has the following advantages: 1) it avoids the physical removal of hybridized sequences before amplification; 2) it avoids a concentration-limiting self-annealing of tester nucleic acids in order to activate them for amplification; 3) it improves hybridization efficiency by using single-stranded tester and driver nucleic acids; 4) it avoids modification of the tester nucleic acids after the hybridization step to activate them for amplification; 5) it avoids modification of the tester nucleic acids between rounds of subtractive hybridization and amplification; 6) it allows a simple dilution procedure to prepare the products of one round of subtractive amplification reaction for the next round of subtractive amplification; and 7) it minimizes contamination by integrating multiple rounds of subtractive hybridization and exponential amplification into a process with fewer steps.
According to the method of the present invention, target RNA is preferentially amplified relative to non-target RNA in a sample of tester RNA. As used herein, tester RNA refers to RNA sequences which are to be subjected to subtractive amplification according to the present invention. Tester RNA includes both target and non-target RNA sequences, each containing terminal priming sequences to enable amplification. In the method, target RNA is preferentially amplified relative to any amplification of non-target RNA that may occur.
According to the method, a sample of tester RNA is contacted with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA. A nucleic acid primer is then extended using the target RNA as a template, forming a DNA template complementary to some or all of the target RNA. The DNA template is then rendered single-stranded to enable the hybridization to it of a promoter template, and extended using the promoter template to form a DNA template comprising a functional double-stranded promoter. Multiple copies of the target RNA sequence can now be transcribed from the DNA template through recognition of the contained functional double-stranded promoter by RNA polymerase.
According to the method, the driver sequences, nucleic acid primer, and promoter template may be DNA or RNA, and are preferably DNA.
According to the method, the hybridization of driver sequences to the non-target RNA interferes with the ability of the non-target RNA to serve as a template. As a result, the nucleic acid primer is preferentially extended using the target RNA as a template for forming the DNA template, as opposed to using the non-target RNA as template. In one embodiment of the method, non-target RNA is inactivated as a template for DNA synthesis prior to the formation of the DNA template. Inactivation of the non-target RNA may be performed by a variety of methods including, for example, digesting any non-target RNA which is hybridized to a driver sequence. Digestion of non-target RNA may be performed, for example, by any enzyme having ribonucleolytic activity analogous to that of ribonuclease H enzyme.
After its formation, the DNA template is preferably rendered single-stranded. This may be accomplished by digesting any target RNA hybridized to the DNA template, for example, using any enzyme having ribonucleolytic activity analogous to that of ribonuclease H enzyme. The DNA template may also be rendered single-stranded through strand separation, for example, by heat denaturation.
The promoter template includes a sequence capable of forming a functional promoter. In one embodiment, the promoter template comprises sequences capable of forming a functional promoter and a transcription initiation site from a bacteriophage. In a preferred embodiment, the bacteriophage is T7.
According to the present invention, the DNA template and/or the extended DNA template can be amplified prior to target RNA synthesis. This amplification can be performed by a variety of nucleic acid amplification methods known in the art and is preferably performed either by the PCR (polymerase chain reaction) or SDA (strand displacement amplification) or NASBA (nucleic acid sequence-based amplification) methodologies.
In one embodiment of the present invention, the synthetic target RNA is synthesized from the extended DNA complement, the single-stranded DNA template containing a functional double-stranded promoter, by transcription utilizing RNA polymerase. In a further embodiment, the synthetic target RNA so transcribed may be further amplified by a RNA amplification reaction, preferably by NASBA.
Subtractive amplification can be performed in multiple cycles where the sample of tester RNA being used in the subtractive amplification process is synthetic target RNA obtained from a prior subtractive amplification. Driver sequences for a subsequent cycle of subtractive amplification can also be obtained from a prior subtractive amplification.
In one particular embodiment, subtractive amplification is performed by:
a) contacting the sample of tester RNA with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA;
b) contacting the target RNA with nucleic acid primers under conditions suitable for hybridization;
c) extending those nucleic acid primers which hybridize to the target RNA to form a DNA template complementary to some or all of the target RNA;
d) making the DNA template single-stranded;
e) contacting, under hybridizing conditions, the single-stranded DNA template with a nucleic acid sequence capable of acting as a promoter template;
f) extending those DNA templates which hybridize to the promoter template to form extended DNA templates comprising both a functional double-stranded promoter portion and a single-stranded portion capable of acting as a template; and
g) transcribing the extended DNA template to form multiple copies of synthetic target RNA.
According to a preferred embodiment of the method, target RNA present in a sample of tester RNA is preferentially amplified relative to non-target RNA present in the tester, both target and non-target RNA containing terminal priming sequences to enable amplification, by performing the following reactions: (1) a hybridization reaction, wherein the sample of tester RNA-contacts driver sequences under conditions such that the driver sequences which are complementary to the non-target RNA hybridize, and prevent the hybridized non-target RNA from functioning as templates for nucleic acid synthesis; (2) a reverse transcription reaction, wherein nucleic acid primers hybridize to the target RNA from the hybridization reaction and are extended using a reverse transcriptase, thereby forming a DNA template; (3) a DNA conversion reaction, wherein DNA templates from the reverse transcription reaction are made single-stranded, hybridized to a promoter template, and extended using the DNA polymerase, thereby forming DNA templates with a functional double stranded promoter; and (4) a transcription reaction, wherein DNA templates with functional double stranded promoters from the DNA conversion reaction are transcribed using an RNA polymerase, thereby forming from each DNA template one or more copies of the target RNA.
According to one aspect of the preferred embodiment, in each step of the process all or a portion of one reaction may be added to a subsequent reaction. In another aspect of the preferred embodiment, the hybridization reaction is performed by adding the tester RNA to a medium comprising driver sequences under conditions such that the driver sequences hybridize with non-target RNA. In another aspect of the preferred embodiment, the reverse transcription reaction is performed by adding a portion of the hybridization reaction to a medium comprising a first primer and a reverse transcriptase under conditions such that DNA templates are formed. In another aspect of the preferred embodiment, the DNA conversion reaction is performed by adding a portion of the reverse transcription reaction to a medium comprising a DNA polymerase and a promoter template under conditions such that DNA templates with functional double-stranded promoters are produced. In another aspect of the preferred embodiment, the transcription reaction is performed by adding a portion of the DNA conversion reaction to a medium comprising an RNA polymerase under conditions such that target RNA is produced.
According to one embodiment of the process, the driver nucleic acid sequences are composed of DNA. In one aspect of this embodiment, a portion of the hybridization reaction is added to a degradation reaction medium comprising a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences that anneal to the DNA driver sequences (non-target RNA) are degraded, and a portion of the degradation reaction is added to the reverse transcriptase reaction, hence providing target RNA sequences. In another aspect of this embodiment, the hybridization reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences that anneal to the DNA driver sequences (non-target RNA) are degraded. Another aspect of this embodiment relates to the particular ribonucleases that may be used.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a degradation reaction medium comprising a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming a hybrid with the DNA templates are degraded, and a portion of the degradation reaction is added to the DNA conversion reaction, hence providing DNA templates.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a denaturation reaction under conditions such that the DNA templates are separated from the RNA tester sequences of the RNA:DNA hybrid, and a portion of the denaturation reaction is added to the DNA conversion reaction, hence providing DNA templates.
According to another embodiment of the process, the reverse transcription reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming a hybrid with the DNA template are degraded.
According to another embodiment of the process, a portion of the reverse transcription reaction is added to a DNA amplification reaction medium comprising the first primer, a second primer and a DNA polymerase under conditions such that the DNA template hybridizes to the second primer and is extended using the DNA polymerase to form a double-stranded DNA and the strands of the double-stranded DNA are separated, upon which a cycle ensues wherein: i) the first primer and the second primer each hybridize to their complementary DNA strands; ii) each primer is then extended using the DNA polymerase to form a double-stranded DNA; and iii) the complementary DNA strands of the double-stranded DNA are separated, and thereafter a portion of the DNA amplification reaction is added to the DNA conversion reaction, hence providing DNA templates.
In one aspect of this embodiment, the DNA amplification reaction is the polymerase chain reaction, wherein the complementary DNA strands of the double-stranded DNA are separated by adjusting the reaction conditions to cause denaturation. In another aspect of this embodiment, the DNA amplification reaction is by strand displacement amplification, wherein the reaction medium further comprises a restriction endonuclease under conditions that the restriction endonuclease nicks the primers of the double-stranded DNA and the complementary DNA strands of the double-stranded DNA are separated by the DNA polymerase extending the nicked primers to displace the complementary DNA strands.
According to another embodiment of the process, the DNA conversion reaction medium further comprises a first primer, and the promoter template therein further comprises a second primer under conditions such that the DNA template hybridizes to the second primer and is extended using the DNA polymerase to form a double-stranded DNA, upon which a cycle ensues wherein: i) the complementary DNA strands of the double-stranded DNA are separated; ii) the first primer and the second primer each hybridize to their complementary DNA strands; and iii) each primer is then extended using the DNA polymerase to form a double-stranded DNA, thereby forming DNA templates with functional double-stranded promoters.
In one aspect of this embodiment, the complementary DNA strands of the double-stranded DNA are separated by adjusting the reaction conditions to cause denaturation. In another aspect of this embodiment, the reaction medium further comprises a restriction endonuclease under conditions that the restriction endonuclease nicks the primers of the double-stranded DNA and the complementary DNA strands of the double-stranded DNA are separated by the DNA polymerase extending the nicked primers to displace the complementary DNA strands.
According to another embodiment of the process, the reverse transcription, DNA conversion and transcription reactions together comprise an RNA amplification reaction, wherein tester RNA sequences are provided in a medium comprising a first primer, a promoter template, a reverse transcriptase, a DNA polymerase and an RNA polymerase under conditions such that the tester RNA sequences hybridize to the first primer and provide templates for synthesis of DNA templates by extension of the annealed first primer using the reverse transcriptase, thereby forming RNA:DNA hybrids, the RNA strands of which are degraded; the DNA templates hybridize to the promoter template and are extended using the DNA polymerase, thereby forming DNA templates each with a functional double-stranded promoter; and the RNA polymerase recognizes each double-stranded promoter and synthesizes from the DNA templates copies of the target RNA sequences.
In one aspect of this embodiment, the DNA polymerase is reverse transcriptase. In another aspect of this embodiment, the RNA amplification reaction medium further comprises a ribonuclease that hydrolyzes the RNA of an RNA:DNA hybrid under conditions such that the RNA tester sequences forming hybrids with the DNA templates are degraded. In another aspect of this embodiment, a portion of the hybridization reaction comprising the tester RNA sequences that do not anneal to the driver sequences is added to the RNA amplification reaction, hence providing tester RNA sequences. In another aspect of this embodiment, target RNA sequences from one round of the RNA amplification reaction provide tester RNA sequences in a subsequent round of the RNA amplification reaction. In another aspect of this embodiment, a portion of the RNA amplification reaction comprising target RNA sequences from one round of the process is added to the hybridization reaction of a subsequent round of the process, hence providing tester RNA sequences.
According to another embodiment of the process, target RNA sequences from one round of the process provide tester RNA sequences in a subsequent round of the process. In one aspect of this embodiment, a portion of the transcription reaction comprising target RNA sequences from one round of the process is added to the hybridization reaction of a subsequent round of the process, hence providing tester RNA sequences.
According to another embodiment of the process, a mixture of RNA comprising target RNA sequences and each comprising terminal priming sequences are added to the hybridization reaction, hence providing tester RNA sequences.
According to another embodiment of the process, the hybridization reaction further comprises an RNA polymerase, and to which is added a mixture of DNA templates each with a functional double-stranded promoter, under conditions such that the RNA polymerase recognizes each double-stranded promoter and synthesizes from the DNA templates copies of a mixture of RNA comprising target RNA sequences and each comprising terminal priming sequences, hence providing tester RNA sequences. In one aspect of this embodiment, terminal sequences and a double-stranded promoter are appended to DNA templates from which tester RNA sequences are synthesized using an RNA polymerase.
According to another embodiment, a final round of the subtractive amplification process comprises: 1) a hybridization reaction; 2) a reverse transcription reaction; 3) a DNA duplexing reaction, wherein DNA templates from the reverse transcription reaction are provided in a medium comprising a DNA polymerase and a second primer under conditions such that the DNA templates hybridize to the second primer and provide templates for synthesis of double-stranded DNA templates by extension of the annealed second primer using the DNA polymerase.
According to one aspect of this embodiment, the DNA duplexing reaction is performed within a DNA amplification reaction wherein the DNA duplexing reaction medium further comprises a first primer under conditions such that the second primer hybridizes to the DNA templates and is extended using the DNA polymerase to form double-stranded DNA templates, upon which a cycle ensues wherein: i) the complementary DNA strands of each double-stranded DNA template is separated; ii) the first primer and the second primer each hybridizes to their complementary DNA template strands; and iii) each primer is then extended using the DNA polymerase to form double-stranded DNA templates.
According to another aspect of this embodiment, the double-stranded DNA templates of the duplexing reaction are characterized by cloning and sequencing. In one aspect of this embodiment, the characterized sequences are used to make hybridization probes or amplification primers. In another aspect of this embodiment, the characterized sequences are used to identify or characterize a useful nucleic acid sequence. In another aspect of this embodiment, the cloned DNA templates are used for the preparation of tester RNA sequences or driver nucleic acid sequences.
The present invention also relates to kits for performing subtractive amplification according to the present invention. In general, these kits may include any compilation of RNA and DNA sequences and enzymes used to perform subtractive amplification according to the method of the present invention. In one embodiment, the kit includes a nucleic acid primer capable of hybridizing to a target RNA under conditions suitable for extension of the nucleic acid primer, and a promoter template capable of hybridizing to a DNA complement of the target RNA under conditions suitable for extension of the DNA complement such that a functional promoter is formed which is capable of transcribing the DNA complement such that multiple copies of target RNA are synthesized. In this kit, the nucleic acid primer and promoter template are preferably DNA. The promoter template may include sequences capable of forming a promoter and transcription initiation site from a bacteriophage. One type of bacteriophage may be T7. The kit may also include one or more enzymes which are used to perform one or more of the steps of subtractive amplification according to the present invention. Examples of these enzymes include: reverse transcriptase; a ribonuclease capable of digesting non-target RNA hybridized to a driver sequence, such as an enzyme having the ribonucleolytic activity of ribonuclease H; a RNA polymerase; and a DNA polymerase.

Subtractive amplification kit useful in the diagnosis of genetic disease mutation or variation


A device for metered removal of flowable solid material comprises at least one delivery opening for the flowable solid material and an essentially horizontal intermediate storage floor for receiving the flowable solid material, arranged below said delivery opening. A collecting space for the flowable solid material is defined between the intermediate storage floor and the delivery opening. A lateral opening is positioned in the intermediate storage floor adjacent to the delivery opening. At least one removal rake with at least one blade is inserted into the collecting space and movable relative to the intermediate storage floor transverse to a delivery direction of the delivery opening for pushing the flowable solid material collected under the delivery opening into the lateral opening. A pivoting parallelogram-shaped rod arrangement with upper pivoting axes that are arranged in a common horizontal plane is provided for pivotably supporting the removal rake.

The invention relates to a device for the metered removal of flowable solid material (fluid bulk material), that is poured onto an essentially horizontal intermediate floor below at least one delivery opening, whereby a removal rake with at least one blade engages the space below the delivery opening and is arranged so as to be movable relative to the intermediate storage floor transverse to the pouring direction such that below the delivery opening the collected fluid bulk material is pushable into a lateral opening.
Adsorption medium reactors with a continuously or quasi-continuously moving bed of a pourable or flowable adsorption medium are increasingly used for flue gas scrubbing. The efficiency of the flue gas scrubbing depends primarily on the uniform exchange of the leaden adsorption medium particles for fresh and reactive particles. Fresh solid material particles are introduced into the reactor from the top to such an extent as the laden fluid bulk material particles, after passing the reactor and reaction with the fluid to be scrubbed, are removed from the delivery openings. The removal via rotating metering devices has been unsuccessful in practice because of the aggressiveness of the removed material as well as the non-uniform particle size and shape of the adsorption medium. With respect to especially large-scale technical adsorption apparatus, simple charging devices, such as flaps, slides, or rakes, of the aforementioned kind are used which with each cyclical movement release a predetermined amount of the fluid bulk material from each delivery opening.
From German patent application 3 406 413 a device of the aforementioned kind is known in which the removal rake is horizontally guided on the rectangular intermediate storage floor sections so that the intermediate storage floor as well as the lower edges of the removal rake are subjected to great wear. Furthermore, the possibility exists that the flowable bulk material to be removed blocks the rake movement. Operational disturbances as well as inaccurate metering due to limited strokes of the rake may occur and a substantial continuous service expenditure is required.
From French patent application 20 86 104 a device for the metered removal through a delivery opening is known in which below the delivery opening a curved intermediate storage plate is arranged. Two circular sector plates are pivotably mounted on the tube that defines the delivery opening and a single blade connected to the plates is pivotable at a distance over the curved intermediate storage plate in order to push the flowable bulk material collected on the intermediate storage plate over its edges. Because of the pivoting mounting with only one pivoting axis it is necessary to provide the intermediate storage plate with a great curvature. For a metered delivery from a plurality of delivery openings arranged in a row one after the other the known arrangement is not suitable.
It is an object of the invention to improve the device of the aforementioned kind such that, while maintaining an essentially horizontal guiding, a reliable metering function over an extended period of operation in a service-free manner is ensured under the aggravated conditions of removal of aggressive, sharp-edged, and granular fluid bulk material.

Device for metered removal of flowable solid material