Disclosed are systems, methods, and non-transitory computer-readable storage media for exchanging and processing tag information between users with different types of communications devices. In operation, tag information is received processed to provide an alternative tag communication to selected participants in the media event. That is, at least a portion of the tags of a media event are used to create an alternate tag communication that can be delivered to participants, together with or separate from the conventional or default tag communication associated with the media event. The alternate tag communications can be used to allow participation of users associated with communications devices incompatible with the media format of default tag communication. Further, the alternate tag information can also be used as a secondary means of communications during a media event.

1. Technical Field
The present disclosure relates to tagging of media event and more specifically to systems and methods for communicating tags for media events.
2. Introduction
A conventional tag cloud or word cloud (or weighted list in visual design) generally provides a visual depiction of user-generated tags, metadata, or content of a web site or other on-line content. Tags in a tag cloud are usually single words and are normally listed alphabetically, and the importance of a tag is shown with font size or color. Thus, it is possible to find a tag alphabetically and by popularity. The tags can also provide hyperlinks that lead to content, at the web site or other web sites, which is associated with the tags.
Tag clouds can also be used to provide a summary of tags for other types of media events, such as teleconferences, video conferences, and television broadcasts, in real time or thereafter. For example, during a concert, users viewing the event can tag portions of the concert with tags such as "nice guitar solo", "Hotel California", or "MP3 available at iTunes". Thereafter, the tags can be summarized as a tag cloud. Accordingly, a user can thereafter view the tag cloud and obtain a summary of the media event based on other users' tagging and the frequency and/or importance of the tags to these other users. Unfortunately, as tag clouds for media events are an offshoot of tag clouds for web sites and other on-line content, such tag clouds are typically limited to displaying an overall summary of tags associated with a media event.

System and method for communicating tags for a media event using multiple media types


The present disclosure generally relates to a data device which is mountable on the steering wheel of a vehicle. The device for mounting the data device on the steering wheel of the vehicle comprises a T-shaped arrangement including a member or members extending perpendicularly from the periphery of a frame of the data device, and connected to a further member disposed horizontally and in parallel with the periphery of the frame, that is, parallel to the width of the data device. As a result, the occupant of a vehicle is provided with the capability of quickly mounting the data device on, and quickly dismounting the data device from, the steering wheel of the vehicle. The data device is mounted on the steering wheel in such a manner as to provide for stability of the data device during its use. Preferably, the members extending perpendicularly from the periphery of the frame are connected by hinges to the periphery of the frame so that the device for mounting the data device on the steering wheel of the vehicle can be folded back against the rear or underside of the data device when the data device is not in use.

The use of a computer system, including data devices such as a keyboard and a computer/display unit, in a vehicle is becoming more prevalent. For example, such computer systems are employed in police vehicles or emergency vehicles. An example of such a vehicle mounted computer system is disclosed in U.S. Pat. No. 6,386,413 of Twyford, which is assigned to the assignee of the present invention. In such arrangements, the driver of the vehicle will sometimes find it more convenient to have the data device positioned directly in front of him/her.
Various arrangements have been developed for the purpose of mounting a tray or other support on the steering wheel of a vehicle. Examples of such arrangements are disclosed in the following U.S. Pat. Nos. 4,749, 161; 4,805,867; 4,915,035; 4,974,805; 5,060,581; 5,177,665; 5,413,035; 5,487,521; 5,749,305; Des. 257,589; Des. 292,445; and Des. 358,612.
However, such arrangements have disadvantages when used for the mounting of a data device on the steering wheel of a vehicle. Specifically, such devices are typically overly complicated to install in that they require multiple steps in order to secure the tray or support to the steering wheel. In addition, once the tray or support is mounted on the steering wheel, the data device is then simply placed on the tray. As a result, the data device can easily slide around on the tray during use, resulting in an increased number of typing mistakes which occur during data entry. Of course, one could provide some means for fixing the data device to the tray, but that would overly complicate the installation process, and make dismounting of the data device and/or tray too time-consuming. Many users do not have the time for lengthy installation of the data device on, or lengthy dismounting of the data device from, the steering wheel of the vehicle.
Therefore, there has been a need in the prior art for development of a data device which is quickly and easily mountable on, and quickly and easily dismountable from, the steering wheel of a vehicle, and which, once mounted on the steering wheel, will provide the user with a stable device which can be used with minimal occurrence of mistakes during use.

Data device mountable on the steering wheel of a vehicle


A system for automatically synchronizing, isolated clocks is disclosed. In the preferred embodiment, the internal clock of an automated external defibrillator (AED) or an external defibrillator (ED) is integrated with a radio broadcast to receive standard time. The system may also include a mechanism for synchronizing the clock or a computer tracking the time of emergency calls such as 911. Further, the system enables synchronization between the AED or ED internal clock and the 911 clock such that response times can be accurately determined.

AEDs are used by police officers, paramedics and other first-response emergency medical personnel to resuscitate cardiac arrest patients. As such, AFDs are typically used in responding to a 911 call (or a call to another local emergency number). Upon the receipt of such an emergency call, an emergency medical system (EMS) is usually activated, which then dispatches EMS personnel to the scene. EMS personnel are then responsible for reaching the patient and using the AED to defibrillate the patient. After cardiac stabilization the patient is usually transported to the hospital.
In order to adequately assess the response times and efficiencies of an emergency medical system, it is necessary to track the time between the receipt of the 911 call and the time at which care is first administered to the patient. Typically, the response time is calculated by comparing the time of the 911 call (as recorded on the 911 emergency computer) to the time of the first shock delivery (as recorded on the internal real time clock on the ALD). Unfortunately, it is often difficult to accurately measure this time due to inherent problems of comparing times on two independent clocks. This is especially true as the internal clock in an AED may differ with time relative to the 911 clock. As a result, the response times measured through the AED real time clock often included errors of many minutes. The magnitude of this error is extremely significant in light of studies which indicate that the chances for successful resuscitation diminish approximately ten percent per elapsed response minute. Accordingly, it is evident that there is a need for increased accuracy in measuring response times using AEDs.

Synchronization method and apparatus for isolated clock system


A method and system for homogenizing a fibrous, viscous food mass 1, in particular pasta filata, such as mozzarella, for example, wherein a feed quantity of the food mass 1 is initially fed continuously to a homogenizing device comprising a container, wherein in a subsequent step the food mass 1 exiting an outlet gap of the container is fed to a shaping and/or cooling device disposed underneath the container, wherein the shaping and/or cooling device forms a filling gap between two rollers, each of which, in particular, is equipped with a belt, and shapes the food mass 1 into a food strip, wherein the outlet gap of the container is oriented at least substantially parallel to the filling gap, wherein the opening width of the outlet gap 5 and/or the feed quantity are set such that the food mass located in the container can settle for the purpose of homogenization, wherein the fibers of the food mass entering the filling gap are aligned substantially in the processing direction.

The invention relates to a method for homogenizing a fibrous, viscous food mass, in particular "pasta filata", such as mozzarella, for example, wherein a feed quantity of the food mass is initially fed continuously to a homogenizing device comprising a container, wherein in a subsequent step the food mass exiting an outlet gap of the container is fed to a shaping and/or cooling device disposed underneath the container, wherein the shaping and/or cooling device forms a filling gap between two rollers, each of which, in particular, is equipped with a belt, and shapes the food mass into a food strip, wherein the outlet gap of the container is oriented at least substantially parallel to the filling gap.
In general, methods for forming a continuous strip of a food mass are known from the production of processed cheese. As shown in DE 103 28 905 A1, the hot cheese mass is fed via a conduit to a rolling and cooling device, in which the mass is shaped into a strip that is as uniform as possible. The thickness of the strip can be set via the filling gap. A device of the type in question is also shown in DE 22 19 629 C2, where the flowable processed cheese mass is introduced into the filling gap of the roller pair by way of a feed hopper. The feed hopper itself is filled by a melting device. Next, the continuous strip is cut into individual strips.
Uniform shaping and homogeneity within the strip and, therefore, the finished product, are considered to be an important quality criterium within production. For example, irregularities at the very beginning unfavorably affect the subsequent surface and, therefore, the appearance of the quality of the product. The known methods are used without exception for the processing of processed cheese that is fed in the liquid state and is cooled within the scope of processing. As known, such processed cheese has great homogeneity and can be processed relatively easily in the viscosity thereof, which can be influenced via temperature. The consistency of processed cheese, which is relatively fatty, is comparable to that of batter.
It has been shown, however, that the known methods are suitable only under certain conditions for shaping pasta filata cheese, such as mozzarella, for example, into a homogeneous strip. A pasta filata cheese is fibrous, aqueous, and has a rubbery consistency. As known, a pasta filata mass in the heated state behaves not like batter, but more like a yeast dough, which has a fibrous structure and limited flowability. In the heated state as well, these properties cause problems with homogeneity and therefore result in irregularities in the finished product.
The problem addressed by the present invention is therefore that of providing a method for producing a strip of a food mass having limited flowability in the molten state, such as pasta filata, in particular mozzarella, in which the food mass can be shaped into a homogeneous strip in a simple and low-cost manner. A further problem addressed by the invention is that of providing a device that is suitable therefor.
These problems are solved by the claimed methods and devices. Advantageous embodiments are set forth in the particular dependent claims.

Method and device for homogenising a fibrous, viscous food mass


To provide an inkjet printing apparatus that improves the smoothness of the ink layer surface and improves the image quality. Provided is an inkjet printing apparatus that prints with a UV curable ink, the inkjet printing apparatus including: an ejection unit configured to eject the UV curable ink onto the surface of a print medium; and a control unit configured to control the ejection of the UV curable ink from the ejection unit, the UV curable ink containing a thixotropy-imparting agent, and the control unit setting the viscosity of the UV curable ink to 1 mPa·s to 20 mPa·s at the time of ejection and to at least 80 mPa·s within five seconds of landing on the print medium.

An inkjet printing apparatus that prints a UV curable ink by ejection onto a print medium (medium) and emits ultraviolet light for curing is capable of printing onto a variety of media with a high-quality and stable image and is reliable. Such an inkjet printing apparatus also has the advantages of high-speed production because time is not needed for drying ink due to photocuring immediately after printing, and of a small installation space. However, inkjet printing has the disadvantage of poor smoothness due to the relatively large unevenness of the ejected UV curable ink on the printing face, and the printing face is generally matte (lusterless or having a matte base).
FIG. 8A is an explanatory view of an inkjet printing apparatus 201. The inkjet printing apparatus 201 is provided with a head (ejection unit) 210 for vertically ejecting UV curable ink toward a print medium 250, and an ultraviolet light source 220 for emitting ultraviolet light. The print medium 250 is mounted on a printing table 240 disposed at a position opposing the head 210, and the ejection unit 210 ejects UV curable ink droplets 260 to print on the print medium 250. When ultraviolet light 270 from an ultraviolet light source is emitted as in FIG. 8B immediately after UV curable ink 265 has landed on the print medium 250, the UV curable ink 265 cures while maintaining an ink layer surface S with large unevenness. More specifically, the ink layer surface S becomes matte.
In order to obtain a high-gloss printing face with favorable smoothness, a method can be considered in which the ink retains fluidity after landing for a longer period of time. However, if there is too much ink fluidity, the image quality deteriorates due to interference in the ink landing, i.e., mixing of the ink colors. With the current technology, there is a trade-off between improving the smoothness and improving the image quality.
When vinyl chloride is used as the medium, a solvent ink with a pigment dispersed in an organic solvent may be used instead of a UV curable ink in order to improve the weatherability, the abrasion resistance, and the adhesion. However, a strong irritant smell is produced that adversely affects the environment when the organic solvent vaporizes, time is needed for drying after printing, and the solvent resistance is low.
Patent Literature 1: Japanese Patent No. 5832946
Patent Literature 2: Japanese Patent No. 5614529
Patent Literature 3: Japanese Patent No. 6038831

Inkjet printing apparatus and inkjet printing method


A solid surface clamp is provided. The solid surface clamp has a top jaw and a bottom jaw. A connecting rod is attached to one of the jaws, and slideably extends through the other jaw. Alignment surfaces may be incorporated into the jaws to assist in accurately positioning the material to be laminated. A compression device is connected to the portion of the connecting rod that extends through the jaw. A shroud shields the compression device, thereby protecting the compression device from contamination by the laminating adhesive. An elongated stiffening member may be used to apply the clamping force over a longer surface, allowing for greater spacing between clamps and extended regions of even clamping pressure.

The demand for solid surface countertops such as granite, marble, engineered stone, and Corian® have steadily risen over the past decade. As the demand for solid surface countertops, vanities, tub-decks, fire place mantles and hearths continue to grow, the more imperative it is for solid surface fabrication companies to do the work faster, without sacrificing quality, in order to meet demand. In working with solid surfaces, it is often desirable to laminate two or more pieces of material together, especially at the visible edges of countertops. By laminating pieces together, it is possible to make the finished countertop appear thicker, and to provide a more substantial edge for the application of more elegant edge treatments. For example, much of the granite on the west coast comes in slabs that are only about 20 mm thick. In order to make the countertop appear thicker, a narrow strip of the slab is laminated to all visible edges, making the slab appear to be twice as thick. Also, the edge is now more substantial, allowing more complex and interesting edge treatments to be ground or cut into the edge. For example, when a 20 mm strip is laminated to a 20 mm thick countertop, an elegant full bullnose may be ground onto the edge. The process of laminating material is a regular and necessary task for most solid surface fabrication companies. Unfortunately, because this industry is still relatively young, the technology and tools used to laminate these materials are quite crude.
Solid surface material is often sold in slabs, which, in the case of granite, may be up to 9 feet by 6 feet in size, and in some cases, even larger. Solid surfaces may also be sold in tile form. These tiles are often available in standard sizes, such as 12″×12″, 16″×16″, or 18″×18″. It will be appreciated that solid material may be available in a variety of sizes, and may represent either a natural or man-made material.
Standard c-clamps are the most common tool employed by solid surface fabricators for joining two materials. There are many problems associated with using c-clamps, including the time it takes to use them, recurring replacement costs, poor lamination quality, and increased risk of repetitive motion injuries. When laminating using conventional tools, an adhering agent is applied between two pieces of material, the pieces are manually aligned, and then c-clamps are used to press the pieces together while the adhering agent cures. To achieve an even clamping pressure, the c-clamps must be spaced evenly and close together (as little as 3″), depending on the size of the c-clamp. Each c-clamp must be tightened to approximately the same torque as all the others. Even small differences in compression may result in a poor adhesion, or in one or both material pieces breaking. Additional fabricating operations are performed on the solid surfaces after they are joined and the adhering agent has cured such as machining, cutting, grinding, sanding, and polishing. Noticeable gaps between the two materials will appear after these other fabricating operations if even clamping pressure was not achieved during the joining process. Noticeable gaps are unacceptable and the completed work may be rejected, resulting in expensive material and labor loss due to rework and replacement efforts.
The use of conventional c-clamps for laminating has several undesirable effects. For example, it takes a long time to apply all the c-clamps and often requires more than one employee to tighten all the c-clamps before the adhering agent begins to cure. Also, as the c-clamps are tightened, the glue, epoxy, or other adhering agent may be squeezed from between the pieces. This adhering agent is, by nature, sticky and difficult to work with, and permanently hardens during the curing process. In this way, the screw threads on the c-clamps get contaminated with the adhering agent, rendering them inoperable and thus requiring recurring replacement costs. In addition, due to the highly concentrated pressure point of c-clamps, one or both pieces of material are often broken. Rejections due to uneven clamping pressure are also common and are often caused by user fatigue (c-clamps not tight enough or not evenly tightened) or the c-clamps being spaced too far apart. Finally, there is also an increased risk of repetitive motion injuries due to the high number of c-clamps and the force required to tighten each c-clamp manually by hand.

Solid surface clamp having multiple clamping jaws on a single elongated member


A chip carrier comprising a laminated layer and an oxidation protection layer is provided. The oxidation protection layer is a non-electrolytic metallic coating or an organic oxidation protection film on the surface of bonding finger pads or other contacts formed by deploying a simple, fast film-coating technique. Therefore, there is no need to plate a Ni/Au layer on the bonding pads or contacts using expensive electroplating equipment for preventing oxidation and there is no need to fabricate plating lines on the chip carrier or reserve space for laying out the plating lines. Thus, the cost for fabricating the chip carrier is reduced, the effective area of the chip carrier is increased and the electrical performance of the chip carrier is improved.

1. Field of the Invention
The present invention relates to a circuit substrate. More particularly, the present invention relates to a chip carrier and a chip package structure thereof.
2. Description of the Related Art
With the rapid progress in electronic technologies, high-tech products having more functions and humanized setup are introduced everyday. Furthermore, a light and compact body is the design trend in most electronic products. In the fabrication of semiconductors, chip carrier is one of the most common structural components. A chip carrier can be an organic dielectric substrate or an inorganic dielectric substrate comprising a plurality of alternately laid patterned conductive layers and dielectric layers. The dielectric layer is disposed between two adjacent patterned conductive layers and the patterned conductive layers can be electrically connected through a plating through hole (PTH) or a via passing through the dielectric layers. Since a chip carrier can provide a dense wiring, compact assembly and good electrical performance, it has become one of the preferred elements for fabricating chip packages.
According to the process of attaching and electrically connecting a chip to a chip carrier, chip packages are classified into wire-bonding package and flip-chip bonding packages. FIG. 1 is a schematic cross-sectional view showing part of a conventional wire-bonding chip package structure. The chip package structure 100 in FIG. 1 essentially comprises a chip 110, a plurality of bonding wires 116 and a chip carrier 120. Bonding pads 114 on the chip 110 are electrically connected to the bonding finger pads 124 on the surface 122 of the chip carrier 120 through bonding wires 116. Since the bonding wires 116 are broadly fabricated by gold, the cost of fabricating wire-bonding packages is high. Besides, in order to prevent the oxidation, a nickel/gold (Ni/Au) layer 128 serving as an anti-oxidation layer is formed over the surface of the bonding finger pads 124 outside the solder mask layer 140 or the surface of the lower contacts 126 for connecting with external devices. The nickel/gold layer 128 also serves as an adhesion layer for enhancing the bonding strength between a solder ball 160 and corresponding lower contact 126.
It should be noted that the conventional electroplating method of forming a nickel/gold layer has the following disadvantages:
1. The time and cost involved in performing an electroplating operation is too high. Therefore, plating a nickel/gold film over the bonding finger pad to serve as an oxidation protection layer increases the production cost of the package.
2. At least one plating line connecting all the bonding finger pads has to be fabricated on the chip carrier before the electroplating process. However, the plating line will occupy some layout area on the chip carrier and reduce the effective area for forming other circuits or devices.
3. The plating line may affect the electrical performance of the chip package.
4. Nickel has a relatively high resistance and may contain some impurities. Ultimately, the signal transmission quality may be affected.
In view of the foregoing, if the process of electroplating a nickel/gold film can somehow be unnecessary, the time and cost to fabricate a chip carrier can be reduced and the effective area of the chip carrier can be increased. Moreover, the electrical performance of the chip package is improved. That is why the present invention concerned to overcome.

Chip carrier with oxidation protection layer


In accordance with one embodiment, a printing apparatus comprises a light receiving section, a determination section and a printing section. The light receiving section receives light emitted from a light emitting section which emits light, and detects a voltage level corresponding to the quantity of the received light. The determination section determines the residual quantity of sheets according to the detected voltage level. The printing section carries out printing on a sheet.

Conventionally, in a printing apparatus such as a barcode printer and the like, printing is carried out on a sheet-like image receiving medium (hereinafter referred to as a "sheet") such as paper, and then the printed sheet is issued. The sheet to be subjected to printing processing by the printing apparatus is stored in a paper tray in the printing apparatus. Conventionally, a PE sensor (transmission sensor) and a plastic plate are arranged, and the existence of detection of the light emitted from the PE sensor is used to determine the existence of the sheet stored on the paper tray.
However, in a conventional printing apparatus, the degree of the residual quantity of the sheets cannot be detected. Thus, sheets are not replenished until the sheets stored on the paper tray are used up; as a result, downtime occurs frequently. Such a problem occurs not only in the printing apparatus but also in other apparatuses in which sheets are stored in a paper tray.

Printing apparatus, sheet residual quantity determination method and non-temporary recording medium


A support structure for a pet, wild bird or small animal treat which includes a stem having a first end and a second end. At the first end there is a disk and hanger that fit into a mold. At the second end is a branch member perpendicular to the stem. Food is packed into the mold so that is surrounds the stem and branch. The resulting treat is removed from the mold for packaging.

Bird treats such as bird bells which are formed from a combination of seeds (i.e. white millet, sunflower, red millet, milo), grains, bits of fruit and/and or nuts, and which are held together by a dried sticky material such as honey or dextrose, are commonly used to feed and attract wild bird species. Bird bells present a particularly difficult problem to support properly, in that they are loosely "glued" together since they are designed to feed birds. In the past, form-fitting net-type sacks have been utilized for hanging the treats.
In accordance with U.S. Pat. No. 5,033,708 to Brue et al., another type of bird treat support has been used which includes a disk for supporting the bottom of a bird bell, and a separate hanging tab that is connected to the disk and inserted into an axial slot within the bird bell. A top portion of the tab extends out above the bird bell for hanging. The hanging tab includes an aperture by which the bell support and bird bell may be hung. Centrally located in the disk is a slot into which a widened lower end of the hanging tab may be inserted and twisted to thereby connect to the disk. It has been found, however, that this two-piece construction can be comparatively expensive, and it is possible for the hanging tab and disk to become separated if they rotate with respect to each other, thereby allowing the bird bell to fall. Additionally, the bird bell cannot be molded around the support as it is formed, thus requiring a two-step manufacturing process of filling the mold and attaching the support to the molded treat.
Another type of bird treat support is disclosed in U.S. Pat. No. 5,323,995 to Grittman et al. This support is a unitary piece, having a movable suspension member at one end, connected to a stem with a hole at an opposite end. While this support is less expensive to manufacture and assemble than the two-piece design, it still requires a two-step manufacturing process.
Yet another type of bird treat support is disclosed in U.S. Pat. No. 6,085,691 to Loehndorf. A support structure to which a conglomeration of small animal food can be formed includes a stem having a top and bottom plate at opposite ends thereof. The top plate includes a hanging clip capable of securing the support to a small animal cage or other support structure. The bottom plate may have a securing clip capable of better securing the support to the cage. In order to improve the ability of the support structure to retain the conglomeration thereon when an animal picks at it, a plurality of protuberances are disposed along the length of the stem. Small animal food is disposed about the stem in the molding process, so that it is between the top and bottom plates. While this patent discloses a support that is used in one step molding process, it can only be used for shapes that allow the mold to be filled from the side of an elongated shape, not from an end. Therefore, for shapes such as a bell, ball, column or any other shape that does not have a flat side, this particular support cannot be used because such shapes are necessarily filled from one end.
Accordingly, a need exists for a bird treat support that is simple, relatively inexpensive and is inserted during the molding process.

Support for formed bird treat and method therefor


A method for controlling a manual transmission includes using a controller to determine a desired torque transmitted through an input clutch and a desired clutch slip for the desired gear after a shift lever is moved to a desired gear position and while a clutch pedal is being released for engaging the clutch; measuring torque in the vehicle drive assembly; using measured torque to determine actual clutch torque transmitted through the clutch; and reducing torque error using the controller to adjust a clutch actuator such that a difference between the desired torque and the actual torque is reduced. Slip error may also be reduced by adjusting the clutch actuator to reduce the difference between the desired slip and measured slip.

This invention relates generally to a method for controlling an input clutch of a manual transmission on the basis of direct, real-time torque measurements inside the transmission or in a vehicle driveline.
The market share of vehicles equipped with manual transmissions remains low in this country partly due to operating complexity involved in the gear shifting process. A typical manual transmission system requires a coordinated use of feet to operate the transmission input clutch and throttle pedals while moving a shift fork through a hand-operated shift lever. It also requires a foot-based control to slip the clutch for driving a vehicle at a low speed.
Missed clutch release timing, poor clutch slip control or missed engagement timing results in a stalled engine or undesirable noise, vibration and harshness (NVH) such as clutch shudder.
A robotized manual transmission system with electronically-controlled input clutch has been in use for limited production applications. Its clutch control primarily relies on clutch actuator position and slip speed measurements during clutch release, slip and engagement processes. However, these measurements cannot be directly utilized to accurately compute torque transmitted through the slipping input clutch. More specifically, there is no reliable method to accurately estimate clutch torque based on clutch actuator position and slip speed under all drive conditions because of limited understanding of dry clutch friction mechanisms. Accordingly, a conventional controller of the robotized manual transmission system is not designed to directly observe and use clutch torque in order to detect undesirable NVH behaviors and take corrective closed-loop actions in real-time during clutch release, slip and engagement processes.

Manual transmission clutch control using measured torque


An electromagnetic counter includes an electromagnetic counting mechanism in a frame, a plurality of number wheels at least one of which is configured to rotate through a predetermined angle in response to rotation of an electromagnetically motivated anchor, wherein each of the number wheels is disposed proximate a window provided in the front of a frame enclosing cover to form a readable display section. A mounting board supports a light emitting diode for illuminating the display section, and a wiring pattern that supplies electrical power to the light emitting diode. A reflective surface is provided on at least one of an internal surface of the cover member to reflect light from the light emitting diode toward the numbered wheels, the periphery of one or more of the numbered wheels, and indicia formed in the numbered wheels, to render the number wheels readily readable when illuminated by the light emitting diode.

The present invention relates to an electromagnetic counter such as that which is installed in various types of precision equipment such as gaming machines, copiers and printers for example, and more specifically to an improvement in an illumination arrangement for such an electromagnetic counter.
As described in Japanese Unexamined Patent Publication No. 57-93487, a known example of an electromagnetic counting mechanism is provided with an electromagnet, an anchor rotated by magnetization and demagnetization of the electromagnet, a lowest digit number wheel which is formed on its side with a ratchet gear that engages with a tab formed on the end of the anchor, and a predetermined number of additional digit number wheels having transmission gears formed on their sides, and pinions which are operatively interposed between these number wheels and which operatively interconnect the transmission gears.
In addition, this electromagnetic counting mechanism normally exhibits dust resistance via its disposition within a case so as to prevent obstruction of the rotation of the anchor due to adherence of iron filings or the like which are produced by the iron core magnetized by the electromagnet. The case may have, for example, a frame member, in which the electromagnet, anchor and number wheels are disposed at predetermined locations, and can be covered with a box-shaped cover member.
In this manner, an electromagnetic counter housing an electromagnetic counting mechanism facilitates handling and installation as a unit. However, it is often incorporated in various types of precision equipment such as gaming machines, copying machines or printers, which count, for example, the number of coins or number of pieces of paper fed, and thus frequently installed inside the aforementioned precision equipment, a problem arises that as the interior of the precision equipment has a complex structure and is dark, and the counter is not provided with a light emitting function, there are cases wherein it is difficult to read the values displayed on the counter. In such cases, there is no choice but to perform the bothersome task of reading the values by illuminating the inside of the precision equipment with a flashlight or the like.
In order to resolve such shortcomings, although the installation of a separate illumination lamp that illuminates the number wheels inside the precision equipment or the incorporation of a miniature light bulb inside the counter has been considered, due to the recent prominent trend of reducing the size of the precision equipment in which electromagnetic counters are installed, electromagnetic counters are also becoming increasingly small, thereby making it extremely difficult to install an illumination lamp either inside the precision equipment or inside the case of an electromagnetic counter. In addition, even if a confined space was secured and a miniature light bulb was installed therein, since the illumination service life of a miniature light bulb is short due to the use of a filament for the light source, it becomes necessary to periodically replace the light bulb, thereby resulting in the problems of bothersome maintenance work and its accompanying cost due to the confined installation space.
In addition, in the case of an electromagnetic counter of the type in which an electromagnetic, anchor and number wheels are housed within a case in which a cover member is fixed to a frame member, since replacement of the light bulb is essentially impossible, the problem arises in which it becomes necessary to replace the entire electromagnetic counter.
In order to solve these problems, the entity to which the instant application is assigned, previously filed patent for a novel electromagnetic counter having a built-in illumination means that can be housed in an extremely confined space within a compact electromagnetic counter, that requires hardly any maintenance and that is inexpensive (see Japanese Patent Application No. 2004-26803).
Irrespective of the improvements which are achieved by the arrangement disclosed in the above mentioned Japanese Patent Application No. 2004-26803, an examination of the technical contents of the electromagnetic counter with a built-in illumination means previously proposed by the present applicant revealed that there was still room for improvement with respect to the following points.
Namely, although the previously proposed electromagnetic counter is inserted into an enclosure such as a box-shaped cover member, and a frame member provided with an electromagnetic counting mechanism composed of an electromagnet, anchor, number wheels etc., and disposes a flexible board, on which is mounted a light emitting diode for illuminating the number wheels, in a confined space formed between the frame member and cover member, there is the risk that the mounted light emitting diode will provide insufficient illumination effects if only one or two used, while the mounting of a large number of light emitting diodes leads to increased costs. In addition, there are numerous cases in which it is difficult to mount a large number of light emitting diodes in light of the amount of space which is available.
In consideration of the circumstances of the prior art as described above, the object of the present invention is to further improve upon the previously proposed electromagnetic counter having a built-in illumination means that can be housed in an extremely confined space within a compact electromagnetic counter, requires hardly any maintenance and is inexpensive, by providing a novel electromagnetic counter with a built-in illumination means capable of obtaining efficient illumination effects using one or a few light emitting diodes.

Electromagnetic counter with built-in illumination means


A system analyzer may generate an estimated frequency response of a device, system, communication medium, or combination thereof by utilizing a stimulus signal that is robust against IQ modulator impairments. A stimulus generator may be used to generate a plurality of discrete tones according to a frequency spacing and a frequency offset. The frequency spacing and the frequency offset cause spectrally inverted spurs (generated by impairments of the IQ modulator) to occur at frequencies other than frequencies of said modulated signal that are associated with said plurality of discrete tones. Additionally, by implementing a Discrete Fourier Transform (DFT) to possess a frequency resolution equal to the frequency offset, there is no leakage of power associated with the spectrally inverted spurs into frequency bins of the DFT associated with the desired frequency components. Likewise, leakage between the desired frequency components and leakage associated with the local oscillator may be avoided.

When a radio frequency (RF)/intermediate frequency (IF) device or system is calibrated or analyzed, a signal of a known spectral content is provided to the device or system and the resulting signal is then analyzed to estimate the response of the device or system. Signals with precisely known amplitude and phase relationships may be readily created in the digital domain and converted into the analog domain through digital-to-analog converters (DACs). Such signals may be created utilizing a variety of methods and devices including arbitrary waveform generators that access waveform samples from memory in a circular manner.
Frequently, a signal consisting of a plurality of discrete sinusoids of known amplitude and phase are utilized. These signals typically are periodic with a period T. Fourier analysis indicates that the spectrum of such periodic signals will be composed of discrete tones spaced in the frequency domain at integer multiples of 1/T. Power spectrum 100 of such a signal is shown in FIG. 1 where ωp=2π/T. Spectrums such as power spectrum 100 are commonly referred to as "comb" spectrums, because the tones resemble the teeth of a comb.
As shown in FIG. 2, these signals may be used as stimuli in system 200 to perform RF/IF testing. System 200 includes stimulus generator 201 that generates the desired signal in the digital domain. The digital I and Q symbols are provided to DACs 202 to convert the signal into the analog domain. The analog channels of the periodic signal may be provided to IQ modulator 203 (and possibly up/down converters which are not shown) to be translated to a desired RF/IF frequency. IQ modulator 203 mixes the received analog channels with an oscillator signal of frequency ωLO from local oscillator (LO) 204. The modulated signal is provided to RF/IF channel 205. RF/IF channel 205 is the communication medium, system, or device being tested or calibrated. RF/IF channel 205 may also include any suitable up-converters and/or down-converters. As discussed herein, a "channel" shall refer to any system, device, communication medium, or combination thereof that may transmit and/or process a suitable signal in a manner appropriate for measurement, analysis, or calibration.
The signal that results after communication through and/or processing by RF/IF channel 205 may then be converted to the digital domain by analog-to-digital converter (ADC) 206. Alternatively, the resulting signal may be processed by an IQ demodulator (not shown) before conversion into the digital domain. The digital version of the resulting signal may then be analyzed by signal analysis system 207. In general, the frequency content of the resulting signal is determined by employing a Discrete Fourier Transform (e.g., the Fast Fourier Transform (FFT)). From the frequency representation of the resulting signal, the frequency response of RF/IF channel 205 at the corresponding frequencies (which may be translated) may be determined.
It shall be appreciated that if IQ modulator 203 does not maintain the desired amplitude and phase relationships of the stimulus signal, errors in the measured frequency response will occur. In practice, IQ modulators commonly suffer from several dominant impairments such as LO leakage and amplitude and phase imbalance. LO leakage manifests itself as an undesired spectral spur at the local oscillator frequency (ωLO). LO leakage may be modeled as undesired DC offsets in the I and Q inputs. Amplitude imbalance occurs when the gain of the I channel and the gain of the Q channel are not equal. Phase imbalance occurs when the quadrature split of the LO signal for multiplication by the I and Q signals is not precisely at 90 degrees. As known in the art, amplitude and phase imbalance are manifested through the generation of a spectrally inverted image of the desired signal.
An example of artifacts generated by IQ modulator impairments is shown in FIG. 3. Impaired spectrum 300 comprises three spectral components 301, 302, and 303 that result from the application of the complex excitation associated with an Upper Sideband (USB) tone at frequency ωS to an impaired IQ modulator that utilizes a local oscillator of frequency ωLO. Spectral component 302 (at ωS+ωLO) is the desired spectral component to be produced by the IQ modulation. However, impaired spectrum 300 further comprises spectral component 301 (located at ωLO) due to the LO leakage of the IQ modulator. Spectral component 303 is observed at the image frequency (ωS−ωLO) as the result of the amplitude and/or phase imbalance of the IQ modulator.
The performance of IQ modulators is commonly characterized by the level of suppression of the undesired image as a measure of the amplitude and phase balance and the amount of the LO feedthrough. Greater suppression of these artifacts is associated with better modulators. Common values for the suppression of these terms is 20-30 dB suppression of the LO signal and 20-40 dB suppression for the inverted image. However, for some measurements or calibration functions, these levels need to be reduced to 40-50 dB (or greater) to achieve the desired accuracy. The cost (if even possible) of implementing modulators satisfying these levels of suppression may be prohibitive.

Systems and methods of using IQ modulation for calibration or measurement


For the purpose of coding or decoding motion information in a picture with a small overhead, a coding method of performing compression coding of a moving picture by motion compensated prediction is configured to have a motion compensated prediction step (processing by motion detector 2, motion compensator 5, spatial predictor 9, etc.) of performing the motion compensated prediction while assigning one or more motion vectors (e.g., up to two motion vectors) in units of sub-blocks obtained by sub-dividing each of macroblocks resulting from division of each frame of the moving picture; and an assignment information coding step (processing by variable length encoder 23 and others) of outputting information about an assignment situation of the one or more motion vectors to the sub-blocks in a multiplexed form on a bitstream.

The image coding and decoding method based on the H.26L coding method is known as a case example of the conventional video coding methods, which is described in International Standardization Working Document ITU-T SG16 VCEG-M81, "H.26L Test Model Long Term Number 7 (TML-7)" draft0 (http://standard.pictel.com/ftp/video-site/0104—Aus/VCEG-M81d0.doc).
In this H.26L video coding, many motion division patterns shown in FIG. 4A to FIG. 4G are prepared as inter prediction modes so as to enable elaborate motion compensation. This ingenuity presents such effect that it is feasible to efficiently capture motions in a case where fine motions occur inside macroblocks, and to increase prediction efficiency.
However, the method of preparing the many motion division patterns as inter prediction modes has the problem that more motion vector information needs to be transmitted with increase in the number of motion divisions, so as to result in increase of the overhead. FIG. 5 shows the volume of transmitted information necessary for each of the modes of FIG. 4A to FIG. 4G. In this FIG. 5 "mode bit" represents a code length of a variable length code for expressing each prediction mode, "minimum motion vector bit" represents the minimum information amount that can be taken as the amount of information about motion vectors to be transmitted in each macroblock unit, and "minimum requisite bit count" represents the sum of the mode bit and the minimum motion vector bit. The motion vector bit is the total bit count of two values in the horizontal and vertical directions, and the case yielding the minimum is one wherein the motion vector agrees with a motion vector predicted value in both the horizontal and vertical directions (a predicted value of a motion vector is calculated from near motion vectors according to a predetermined rule and the difference between the motion vector value and the motion vector predicted value is coded). However, that is normally a rare case, and it is necessary to transmit more information than the bit count. It is seen from this FIG. 5 that the overhead becomes very large in Mode 4 or 5 or higher.
On the other hand, in the case of ordinary images, it is believed that there exist cases wherein sufficient motion compensation can be achieved by a comprehensive way of capturing the motions by division into two segments or so, without elaborate motion detection based on segmentalization of each macroblock region into four or more segments. However, the fixed motion assignment in the 16×8 and 8×16 pixel units prepared in the H.26L coding method had the problem that it was not possible to accurately express motion divisions inside each macroblock, which was the reason why the seven inter prediction modes of FIG. 4A to FIG. 4G were prepared.
The present invention has been accomplished in order to solve the above problem and an object of the present invention is to provide a coding method, a decoding method, a coding apparatus, a decoding apparatus, an image processing system, a coding program, and a decoding program enabling coding or decoding of motion information in a picture with the overhead being small.

Coding method, decoding method, coding apparatus, decoding apparatus, image processing system, coding program, and decoding program


A method of configuring a printer is disclosed, wherein a set of printing parameters is stored in a control unit of the printer. The method includes the steps of storing the set of printing parameters, which is adapted to a specific functional component of the printer, in a storage device addressable over the Internet at a predetermined URI (Universal Resource Identifier); and, when the printer is to be configured, getting access to the URI and downloading the set of printing parameters directly into the control unit of the printer.

1. Field of the Invention
The invention relates to a method of configuring a printer, wherein a set of printing parameters is stored in a control unit of the printer. The invention further relates to a printer suitable for executing this method and to an ink cartridge for use with an ink jet printer of this type.
2. Discussion of Background Art
A printer typically has at least one functional component, e.g. a nozzle head or an ink cartridge in case of an ink jet printer, an LED array, a photoconductor or a toner cartridge in case of a laser printer, which influences the settings of printing parameters. Thus, when such a functional component is replaced, it may be necessary to reconfigure the printer, i.e. to change the parameter settings, in order to assure a proper operation of the printer and/or to achieve the best possible image quality. One example of printing parameters which need to be reconfigured upon insertion of a new print head into the printer are the voltages with which the individual nozzles of an ink jet nozzle head or the individual LEDs of a laser printer are to be energized. Another example is the compensation of positional errors of the individual nozzles or LEDs. In a scanning-type ink jet printer or laser printer, the nozzles and LEDs, respectively, are typically arranged in a linear array which extends orthogonal to the main scanning direction. Due to manufacturing tolerances, the positions of the nozzles or LEDs in the main scanning direction may slightly deviate from one another. Such position errors can be compensated by providing suitable time offsets in the timings at which the nozzles or LEDs are fired during the scan motion of the print head. The nozzles of print heads manufactured in one same production series will normally have the same position errors, so that the corresponding time offsets will be the same for all print heads belonging to this series. However, when a print head from another production series is used, it may be necessary to reconfigure the printer in order to provide that corrected time offsets. Other parameters may, for example, be related to the transportation speed of the recording paper, compensation of changes in the slip and/or thickness of the recording paper, colour correction routines and the like.
Some printing parameters such as voltages, operating temperatures and the like may also depend on the age of the ink or toner being used.
For more convenient configuring or re-configuring of a printer having an interchangeable print head, it has been known for long that the printing parameters pertinent for a specific print head may be encoded or stored in a memory element that is provided on the print head. When the print head is mounted in a printer, the information stored in the memory element is automatically read by the control unit of the printer, and thus, the new printing parameters are stored. Methods of this type are disclosed for example in "Xerox Disclosure Journal" Vol. 8, No. 6, 1983, page 503; U.S. Pat. No. 4,872,027 and U.S. Pat. No. 5,049,898. These known methods all have the drawback that, once the printing parameters have been stored in the memory element, it is no longer possible or very cumbersome to adapt the configuration of the printer, when the operating conditions have changed.

Method of configuring a printer and ink cartridge


This invention relates to antiviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) and further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy, particularly nucleoside reverse transcriptase inhibitor therapy using phenotypic susceptibility assays or genotypic assays.

Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
This invention relates to antiretroviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). The invention further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy using phenotypic or genotypic susceptibility assays. The Invention also relates to novel vectors, host cells and compositions for carrying out phenotypic susceptibility tests. The invention further relates to the use of various genotypic methodologies to identify patients whose Infection has become resistant to a particular antiretroviral drug regimen. This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhabit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of nucleoside reverse transcriptase inhibitor resistance using phenotypic susceptibility tests and/or genotypic tests.
HIV infection is characterized by high rates of viral turnover throughout the disease process, eventually leading to CD4 depletion and disease progression. Wei X, Ghosh S K, Taylor M E, et al. (1995) Nature 343, 117-122 and Ho D D, Naumann A U, Perelson A S, et al. (1995) Nature 373, 123-126. The aim of antiretroviral therapy is to achieve substantial and prolonged suppression of viral replication. Achieving sustained viral control is likely to involve the use of sequential therapies, generally each therapy comprising combinations of three or more antiretroviral drugs. Choice of initial and subsequent therapy should, therefore, be made on a rational basis, with knowledge of resistance and cross-resistance patterns being vital to guiding those decisions. The primary rationale of combination therapy relates to synergistic or additive activity to achieve greater inhibition of viral replication. The tolerability of drug regimens will remain critical, however, as therapy will need to be maintained over many years.
In an untreated patient, some 100 new viral particles are produced per day. Coupled with the failure of HIV reverse transcriptase (RT) to correct transcription errors by exonucleolytic proofreading, this high level of viral turnover results in 104 to 103 mutations per day at each position in the HIV genome. The result is the rapid establishment of extensive genotypic variation. While some template positions or base pair substitutions may be more error prone (Mansky L M, Temin H M (1995) J Virol 69, 5087-5094) (Schinazi R F, Lloyd R M, Ramanathan C S, et al. (1994) Antimicrob Agents Chemoter 38, 268-274), mathematical modeling suggests that, at every possible single point, mutation may occur up to 10,000 times per day in infected individuals.
For antiretroviral drug resistance to occur, the target enzyme must be modified while preserving its function in the presence of the inhibitor. Point mutations leading to an amino acid substitution may result in change in shape, size or charge of the active site, substrate binding site or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh M K, Ching W T W, et al. (1993) Proc Natl Acad Sci USA 90, 25-29) (Nxc3xa1jera I, Richman D D, Olivares I, et al. (1994) AIDS Res Hum Retroviruses 10, 479-1488) (Nxc3xa1jera I, Holguin A, Quixc3x1ones-Mateu E, et al. (1995) J Virol 69, 23-31). However, these mutant strains represent only a small proportion of the total viral load and may have a replication or competitive disadvantage compared with wild-type virus. (Coffin J M (1995) Science 267, 483-489). The selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost S D W, McLean A R (1994) AIDS 8, 323-332) (Kellam P, Boucher C A B, Tijnagal J M G H (1994) J Gen Virol 75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
Nucleoside Reverse Transcriptase Inhibitors
Seven nucleoside analogue HIV reverse transcriptase Inhibitors (zidovudine (ZVD: Retrovir, Glaxo Wellcome, Uxbridge, UK), zalcitabine (ddC: HIVID, Hoffman-LaRoche, Basle, Switzerland), didanosine (ddI: Videx, Bristol-Myers Squibb, Syracuse, N.Y., USA), stavudine (d4T: Zerit, Bristol-Myers Squibb, Syracuse, N.Y., USA), and lamivudine (3TC, Epivir), abacavir (ABC, Ziagen, Glaxo Wellcome), and adefovir (ADV, Preveon, Gilead Sciences) are currently licensed in Europe and the USA. Additionally, three NNRTIs, nevirapine (Viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany) and delavirdine (Rescriptor, Pharmacia and Upjohn, Kalamazoo, Mich., USA) and efavirenz (EPV,) are licensed in the USA. All these agents have demonstrated at least short-term antiviral activity and, therefore, it is not surprising that, as they exert a selective pressure on HIV, drug-resistant mutants arise during therapy. Whilst these drugs are normally used in combination regimens, many of the available resistance data arise from phase I/II monotherapy studies. Mutations observed during monotherapy may not accurately reflect mutations responsible for resistance that develops in the presence of pressure from several agents acting at the same site and, hence, on the same gene.
Novel Mutations
Whilst patterns of genotypic mutations associated with changes in phenotypic resistance to the leading reverse transcriptase inhibitors (RTIs) are established from both in-vitro and in-vivo work, other, rarely reported, resistance mutations may arise occasionally during clinical studies. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 115 have been identified in patients receiving prolonged combination therapy with ZDV plus ddI or ddC: these isolates are resistant to both drugs and there is cross-resistance to stavudine and partial cross-resistance to 3TC. No consistent genotypic change has been associated with phenotypic d4T resistance or, indeed, loss of virological effect of this compound.
Mutations to Nucleoside Analogue RT Inhibitors Zidovudine
HIV variants with decreased susceptibility to ZDV were first reported in 1989; in some isolates, greater than 100-fold increases in the concentration of ZDV were required to inhibit viral replication by 50% (Larder B A, Darby G, Richman D D (1989) Science 243, 1731-1734). The ZDV-resistant phenotype appears to be reasonably stable in vivo, with resistant virus sometimes being detected up to 1 year after cessation of therapy, (Boucher C A, O""Sullivan E, Mulder J W et al. (1992) J Infect Disease 165, 105-110) and despite treatment with didanosine (Smith M S, Koerber K L, Pagano J S, (1994) J Infect Disease 169, 184-188).
Nucleotide sequencing of HIV RT has revealed a number of mutations which can influence viral sensitivity to ZDV and which may be used as genotypic markers for the presence of ZDV resistance (Kellam P, Boucher C A B, Tijnagal J M G H et al. (1994) J Gen Virol 75, 341-351) (Boucher C A B, Tersmette M, Lange J M A, et al. (1990) Lancet 336, 585-590) (Lopez-Galindez C, Rojas J M, Najera R, et al. (1991) PNAS 88, 4280-4284). A range of mutants with increasing levels of resistance appear in an ordered manner, with the sequential appearance of these mutations being associated with incremental reductions in viral sensitivity to ZDV (id) (Larder B A, Kellam P, Kemp S D, (1991) AIDS 5, 137-144). A substitution at codon 70 (Arg70xe2x86x92Lys) may be transiently dominant and appears critical to virological failure during ZDV monotherapy (DeJong M D, Veenstra J, Stilianakis N I, et al. (1996) PNAS 93, 9501-9506). Continued ZDV therapy selects for a further mutation at codon 215, which appears to be a more stable variant, though both Thr215xe2x86x92Tyr and Thr215xe2x86x92Phe substitutions have been described and may coexist (Mayers D L, McCutchan F E, Sanders-Buell E E, et al. (1992) J Acq Imm Def Synd 5, 749-759). Virus with additional mutations may then appear, most commonly a substitution at codon 41 (Met41xe2x86x92Leu), followed by further additional mutations at codons 67, (Asp67xe2x86x92Asn) and 219 (Lys219xe2x86x92Gln) or the reappearance of the codon 70 mutation
Site-directed mutagenesis techniques have been used to assess the interactions resulting from the different mutations (id). These demonstrated that high-level resistance to ZDV (IC50 greater than 1 xcexcM) is typically associated with the presence of multiple mutations. Although frequently synergic, mutations may also be antagonistic. For example, a mutation at codon 74 (Leu74xe2x86x92Val) observed during therapy with ddI or ddC has been noted to be antagonistic to the ZDV 215 mutation in vitro, reducing the degree of resistance to ZDV (St Clair M H, Martin J L, Tudor-Williams G, et al. (1991) Science 253, 1557-1559). Antagonism of the 215 mutation in vitro has also been reported by the codon 181 mutation selected for by most NNRTIs and the mutation at codon 184 seen with lamivudine and, less frequently, ddC and ddI (Larder B A, (1992) Antimicrob Agents Chemother 36, 2064-2669) (Boucher C A B, Cammack N, Schipper P, et al. (1993) Antimicrob Agents Chemother 37, 2231-2234) (Tisdale M, Kemp S D, Parry N R, et al. (1993) PNAS 90, 5653-5656) (Larder B A, Kemp S D, Harrigan P R (1995) Science 269, 696-699) (Zhang D, Caliendo A M, Eron J J, et al. (1994) Antimicrob Agents Chemother 38, 282-287). However, novel mutation patterns or additional xe2x80x98compensatoryxe2x80x99 mutations may be observed in vivo during combination therapy facilitating dual or multi-drug resistance (see below).
Viral strains resistant to ZDV exhibit cross-resistance to other nucleoside analogues containing the 3xe2x80x2-azido group such as 3xe2x80x2-azido-2xe2x80x2,3xe2x80x2-dideoxyuridine (AZU) (Rooke R, Parniak M A, Tremblay M, et al. (1991) Antimicrob Agents Chermother 35, 988-991). Cross-resistance to stavudine, a thymidine-based analogue which lacks a 3xe2x80x2-azido moiety, has also been reported by one group in both a laboratory strain of HIV and one of 11 clinical isolates (ibid). Most investigators have found no evidence that mutations selected for during ZDV monotherapy influence sensitivity to ddI, ddC or 3TC (Rooke R, Tremblay M, Soudeyns H, et al. (1989) AIDS 3, 411-415) (id) (Larder B A, Chesebro B, Richman D D (1990) Antimicrob Agents Chemother 34, 436-441) (id) (Dimitrov D H, Hollinger F B, Baker C J, et al. (1993) J Infect Disease 167, 818-823). However, resistance to ddI has been rarely reported after prolonged ZDV therapy (id) (Japour A J, Chatis P A, Eigenrauch H A, et al. (1991) PNAS 88, 3092-3096), and one report has suggested that, for each ten-fold decrease in ZDV sensitivity in clinical isolates, there is a corresponding 2.2-fold reduction in susceptibility to ddI and two-fold decrease in sensitivity to ddC (Mayers D L, Japour A J, Arduino J M, et al. (1994) Antimicrob Agents Chemother 38, 307-314). Furthermore, patients with ZDV resistance at baseline are significantly less likely to achieve an RNA response after the addition of ddC or ddI than those with wild-type virus at baseline (Holodniy M, Katzenstein D, Mole L, et al. (1996) J Infect Disease 174, 854-857).
Zalcitabine And Didanosine
Resistance to ddI is mediated through a Leu74xe2x86x92Val mutation which produces a six-fold to 26-fold reduction in sensitivity, but may partially restore susceptibility to ZDV in vitro by antagonism of the codon 215 mutation. This mutation also reduces sensitivity to ddC by around ten-fold (id). The frequency of the codon 74 mutation has been reported to have increased from zero at the start of therapy to 56% at week 24 in a group of 64 persons with a mean baseline CD4 cell count of 129/mm who switched to ddI having previously received ZDV (Kozal M J, Kroodsma K, Winters M A, et al. (1994) Annals Intern Med 121, 263-268). Similarly, in a mixed population of both treatment-naive and ZDV-experienced patients with CD4 cell counts of 200-500/mm who received ddI monotherapy in the ACTG 143 study, 17 of 26 isolates had mutations at codon 74 at 1 year. Mutant codon 74 arose in only two of the 55 patients in this study who received ZDV/ddI combination therapy (Shafer R W, Iversen A K N, Winters M A, et al. (1995) J Infect Disease 172, 70-78).
Virus with a mutation at codon 65 (Lys65xe2x86x92Arg) has been isolated from several patients receiving long-term treatment with ddI or ddC. This is associated with a three-fold to five-fold increase in the IC50 of ddI with a five-fold to ten-fold reduction in ddC sensitivity and a 20-fold reduction in susceptibility to 3TC (id) (Gu Z, Gao Q, Fang H, et al. (1994) Antimicrob Agents Chemother 38, 275-281. A mutation at codon 69 (Thr69xe2x86x92Asp), which leads to a five-fold reduction in sensitivity to ddC but does not appear to result In cross-resistance to other nucleoside analogues, is the most frequent mutation selected for by ddC in vivo (id) (Fitzgibbon J E, Howell R M, Haberzettl C A, et al. (1992) Antimicrob Agents Chemother 36, 153-157). The development of resistance to ddC has recently been reviewed elsewhere (Craig C, Moyle G (1997) AIDS 11, 271-279).
Combination Therapyxe2x88x92Zidovudine+Zalcitabine or Didanosine
Combination therapy with ZDV/ddC or ZDV/ddI may influence the rate of emergence of resistance and may suppress some of the mutations observed during monotherapy but may result in the appearance of novel (and hence possibly more compromised) mutational patterns.
Novel mutation patterns may emerge during combination therapy. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 115 have occasionally been identified in patients receiving prolonged combination therapy with ZDV plus ddI or alternating ZDV/ddC: these are resistant to both drugs (id) (Shafer R W, Kozal M J, Winters M A, et al. (1994) J Infect Disease 169, 722-729) (Shirasaka T, Kavlick M F, Ueno T, et al. (1995) PNAS 92, 2398-2402) and confer cross-resistance to stavudine and partial cross-resistance to 3TC. The frequency in persons treated for  greater than 1 year with ZDV. ddU ranges from 0 to  greater than 10% (ibid). Mutations selected by 3TC (184Val) and nevirapine (181Cys) may readily be added to this background in vitro (Shafer R W, Winters M A, Iversen A K N, et al. (1996) Antimicrob Agents Chemother 40, 2887-2890) and 184Val and 103Asp (for loviride resistance) being reported in vivo (Schmit J C, Cogniaux J, Hermans P, et al. (1996) J Infect Disease 174, 962-968). While these virus mutations appear to be replication competent in the presence of drug, the likely reason these novel mutations are not seen during monotherapy probably relates to their failure to compete with those mutants that become dominant.
Lamivudine
Resistance to 3TC occurs rapidly in vivo with a substitution at codon 184 (most commonly Met184xe2x86x92Val) (id) (Kuritzkes D R, Quinn J B, Benoit S L, et al. (1996) AIDS 10, 975-981) (Bartlett J A, Benoit S L, Johnson V A, et al. (1996) Annal Intern Med 125, 161-172) (Eron J J, Benoit S L, Jemsek J, et al. (1995) NEJM 333, 1662-1669) (Katlama C, Ingrand D, Loveday C, et al. (1996) JAMA 276, 118-125) (Staszewski S, Loveday C, Picazo J J, et al. (1996) JAMA 276, 111-117) being observed during both monotherapy and combination therapy and its appearance being temporally associated with at least partial virological failure (id) (Moyle G J (1996) Drugs 52, 168-185) (Goulden M G, Cammack N, Hopewell P L, et al. (1996) AIDS 10, 101-102). This mutation leads to high-level resistance to 3TC (500-fold to 1000-fold increase in IC50), as well as some cross-resistance to both ddI and ddC (four-fold to eight-fold reductions in susceptibility) (id) (Gu Z, Gao Q, Li X, et al. (1992) J Virol 66, 7128-7135) In vitro this mutation may antagonize ZDV (id) (id) (id), although dual ZDV/3TC resistance has been reported both in vitro and in clinical isolates (id). Other, possibly compensatory, mutations such as at codon 135 or 333 may be required for dual ZDV/3TC resistance, an issue that is currently under investigation (id). When 3TC was added to patients pre-treated with ZDV in study NUCA3002, phenotypic 3TC resistance developed in 82% of 33 patients by week 12. Of the ten patients with ZDV resistance at baseline (as defined by an IC50 greater than 0.2 mM) who developed 3TC resistance, four had isolates that were more sensitive to ZDV whilst six patients had dual ZDV/3TC resistance, suggesting that viral resensitization to ZDV is not universal in vivo.
Stavudine
In vitro selection of HIV resistant to d4T, confirmed by site-directed mutagenesis, has identified a mutation at codon 75 (Val75xe2x86x92Thr) which confers a seven-fold increase in IC50, as well as reduced susceptibility to both ddI and ddC (Lacey S F, Larder B A (1994) Antimicrob Agent Chemother 38, 1428-1432). A mutation at codon 50 leading to a 30-fold reduction in d4T sensitivity, but which does not appear to confer cross-resistance to other nucleoside analogues has also been observed in vitro (Gu Z, Gao Q, Fank H, et al. (1994) Leukemia 8, Suppl. 1, 166-169). In vivo, however, a range of amino acid changes, including the codon 75 mutation but not the codon 50 substitution, have been reported. The maximum decrease in sensitivity to d4T seen in 13 ZDV-naive patients followed for 18 to 22 months was 12-fold. However, five patients developed nine-fold to 176-fold reductions in ZDV sensitivity and three subjects developed seven-fold to 29-fold decreases in susceptibility to ddI (Lin P F, Samanta H, Rose R E, et al. (1994) J Infect Disease 170, 1157-1164), suggesting use of d4T may limit subsequent therapeutic options in some patients. No consistent mutation pattern for resistance to d4T has, therefore, been established.
Abacavir
In vitro selection of HIV strains resistant to abacavir, confirmed by site-directed mutagenesis, has shown that individual mutations cause only low level resistance to abacavir. Multiple mutations (at least three) are required to produce 10-fold resistance. M184V is the most common resistance mutation selected in vitro in the presence of abacavir and results in a 2-5 fold decrease in susceptibility. Mutations at L74V and F115Y were also shown to contribute to loss of susceptibility to abacavir (Tisdale M, Alnadaf T, Cousens D (1997) Antimicrob Agent Chemother 41, 1094-1098). Cross resistance to ddC and ddI were observed but not to d4T or ZDV. Resistance in HIV derived from patient virus populations has been ascribed to mutations previously associated with NRTI-resistance. A combination of ZDV-resistance mutations (M41L, L210W, T215Y) plus a 3TC-resistance mutation (M184V) showed an eight fold reduction in susceptibility to abacavir. The multi-nucleoside resistance complex (A62V, V75I, F77L, Y116F and Q115M) was associated with a 17 fold reduction in susceptibility (Lanier R, Danehower S, Daluge S, et. al. (1998) 2nd International Workshop on HIV Drug Resistance and Treatment Strategies).
Adefovir
In vitro selection in the presence of adefovir resulted in either a K65R or a K70E mutation appearing which confers 16- or 9-fold reduction in susceptibility to adefovir. Studies in patients have reported the appearance of the K70E mutation but not the K65R mutation. Many AZT-resistant, 3TC-resistant and multi-drug resistant viruses remain sensitive to adefovir (Mulato A S, Lamy P D, Miller M D et. al. (1998) Antimicrob Agent Chemother 42, 1620-1628).
Clinical Significance of Resistance
Choice of initial and subsequent therapy for HIV infection should be uncompromising in terms of activity but also planned and based rationally on knowledge of resistance and cross-resistance patterns to maintain a wide base of future therapy options.
It is an object of this invention to provide a drug susceptibility and resistance test capable of showing whether a viral population in a patient is resistant to a given prescribed drug. Another object of this invention is to provide a test that will enable the physician to substitute one or more drugs in a therapeutic regimen for a patient that has become resistant to a given drug or drugs after a course of therapy. Yet another object of this invention is to provide a test that will enable selection of an effective drug regimen for the treatment of HIV infections and/or AIDS. Yet another object of this invention is to provide the means for identifying the drugs to which a patient has become resistant, in particular identifying resistance to nucleoside reverse transcriptase inhibitors. Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds that act on specific viruses, viral genes and/or viral proteins particularly with respect to viral drug resistance associated with nucleoside reverse transcriptase inhibitors. It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility. This and other objects of this invention will be apparent from the specification as a whole.
The present invention relates to methods of monitoring, using phenotypic and genotypic methods, the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy. The invention is also based, in part, on the discovery that genetic changes in HIV reverse transcriptase (RT) which confer resistance to antiretroviral therapy may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods. The methods utilize nucleic acid amplification based assays, such as polymerase chain reaction. Herein after such nucleic acid amplification based assays will be referred to as PCR based assays. Alternatively, methods evaluating viral nucleic acid or viral protein in the absence of an amplification step could utilize the teaching of this invention to monitor and/or modify antiretroviral therapy. This invention is based in part on the discovery of a mutation/insertion at codon 69 either alone or in combination with a mutation at codon 41 and 215 of HIV reverse transcriptase in nucleoside reverse transcriptase inhibitor (NRTI) treated patient(s) in which the presence of the mutations correlate with a decrease in d4T susceptibility, and a decrease in susceptibility to AZT, ddC, ddI, 3TC, and abacavir. The mutations were found in plasma HIV RNA after a period of time following initiation of therapy. The development of the mutation/insertion at codon 69 in addition to the mutation at codon 41 and 215 in HIV RT was found to be an indicator of the development of resistance and ultimately of immunological decline. More specifically the mutation/insertion at codon 69 in RT (T69SSA, T69SSG, T69SSS) may also be associated with mutations associated with resistance to AZT (e.g. M41L, L210W, T215Y) and 3TC (M184V) or ddI/ddC (L74V) which correlate with a decrease in NRTI-susceptibility, including a decrease in d4T susceptibility and a decrease in susceptibility to AZT, ddC, ddI, 3TC and abacavir. It was also found the mutation/insertion at codon 69 in RT (T69SSA, T69SSG, T69SSS) may be associated with mutations associated with resistance to multi-NRTIs at codon 62 (e.g. A62V) and/or a novel mutation at codon 75 (e.g. V75M). It was observed for the first time that the mutation/insertion at codon 69 (T69SSG) and the mutation at codon 75 (V75M) was associated with decreased susceptibility to d4T (three fold) and substantial decreases in AZT susceptibility (thirty fold). This invention is based in part on the discovery of mutations associated with multi-NRTI resistance at codons 62, 75, 77, 116, and 115 of RT discovered to occur in nucleoside reverse transcriptase inhibitor (NRTI) treated patients in which the presence of the mutation correlates with decreased susceptibility to d4T, ddC, ddI and AZT. It has also been discovered that mutations specifically associated with resistance to: AZT at codons 41, 67, 210, 215 and 219 (e.g. M41L, D67N, L210W, T215Y, K219Q); 3TC at codon 184 (M184V); ddC at codon 69 (T69D); or a novel mutatIon at codon 215 (T215V) may accompany the mutations associated with multi-NRTI resistance at codons 62, 75, 77, 116, or 115 which correlate with decreased susceptibility to d4T, ddC, ddI and AZT. This invention is based in part on the discovery of four or more mutations associated with AZT resistance selected from the group consisting of codons 41, 67, 70, 210, 215 and/or 219 (e.g. M41L, D67N, K70R, L210W, T215Y/F, K219Q) either alone or in combination with a mutation at codon 74 (associated with ddI resistancexe2x80x94V74I), 69 (associated with ddC resistancexe2x80x94T69D), 75 (V75M, V75S) and/or 219 (K219N) of HIV reverse transcriptase in nucleoside reverse transcriptase inhibitor treated patient(s) in which the presence of the mutations correlate with a decrease in d4T susceptibility. The mutations were found in plasma HIV RNA after a period of time following initiation of NRTI therapy. It was observed through the construction, by site directed mutagenesis, of resistance test vectors containing the single site mutation at codon 75 (V75I) did not alter d4T susceptibility but increased AZT susceptibility. It was also observed using site directed mutagenesis that the single site mutation at codon 115 (Q151M) reduced d4T and AZT susceptibility. Yet an additional observation of the present invention was that the double site mutation at codons 75 and 115 (V75I+Q151M) reduced d4T and AZT susceptibility. It was also discovered using site directed mutagenesis that resistance test vectors containing five (M41L, D67N, K70R, T215Y, K219Q) or six (M41L, D67N, K70R, L210W, T215Y, K219Q) AZT resistance associated mutations showed reduced susceptibility to d4T and AZT. It was also observed that resistance test vectors containing single site mutations at codons 62, 69 and 75 (A62V, T69SSA, V75I, V75V) did not reduce d4T susceptibility. However, it was observed that the single site mutation at codon 75 (V75I) or (V75V) increased AZT susceptibility slightly. The T69SSA single site mutation reduced AZT susceptibility slightly while the A62V mutation had no effect on AZT susceptibility. In yet further studies using site directed mutagenesis, it was observed that resistance test vectors containing double site mutations at codons 62 and 69 (e.g. A62V+T69SSA) did not reduce d4T susceptibility more than the T69SSA mutation alone, but further reduce AZT susceptibility due to the T69SSA mutation alone. In still further studies using site directed mutagenesis, it was observed that resistance test vectors containing double site mutations at codons 62 and 75 (e.g. A62V+V75I) had no effect on d4T susceptibilIty. It was also found that the A62V mutation did not alter the reduced AZT susceptibility caused by the V75I mutation. In yet further studies using site directed mutagenesis, it was observed that a combination of three mutations at codons 62, 69 and 75 (e.g. A62V+T69SSA+V75I) did not reduce d4T susceptibility more than the T69SSA mutation alone. It was also observed in the case of the combination of three mutations at codons 62, 69 and 75 (e.g. A62V+T69SSA+V75I) that the V75I mutation completely suppressed the reduced AZT susceptibility caused by the combination of A62V and T69SSA mutations. In still further studies using site directed mutagenesis, it was observed that resistance test vectors containing three mutations at codons 41, 69 and 215 (e.g. M41L+T69SSA+T215Y) showed a significant decrease in both d4T and AZT susceptibility. In yet further studies using site directed mutagenesis, it was observed that a combination of four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) did reduce d4T susceptibility more than the T69SSA mutation alone or the T69SSA+A62V double mutant. It was also observed in the case of the combination of four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) that the combination of all four mutations reduced AZT susceptibility more than the combination of M41L and T215Y mutations alone. In yet further studies using site directed mutagenesis, it was observed that a combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g. M41L+A62V+T69SSA+M184V+T215Y) did reduce d4T susceptibility more than the T69SSA mutation alone or the A62V+T69SSA double mutant. It was also observed in the case of the combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g. T41L+A62V+T69SSA+M184V+T215Y) that the M184V mutation suppressed the reduced AZT susceptibility caused by the combination of M41L, A62V, T69SSA and T215Y mutations. In yet another study using site directed mutagenesis the T69SSA mutation in a clone of a patient""s virus was reverted (T69SSAxe2x86x92SSA69T). Reversion of the T69SSA mutation reduced d4T resistance (i.e. increased susceptibility) and also reduced AZT resistance (i.e. increased susceptibility).
In yet further studies using site directed mutagenesis, it was observed that the introduction of the L210W mutation with mutations at 41, 69, and 215 (e.g. M41L+T69SSA+T215Y) resulted in a substantial decrease in susceptibility to AZT compared to the 140-fold decrease in susceptibility observed for AZT without the 210 mutation. In still further studies using site directed mutagenesis, it was observed that four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) showed a substantial decrease in AZT susceptibility (greater than 1000-fold) and only slight decreases in susceptibility to the other NRTIs. In yet further studies using site directed mutagenesis, it was observed that the introduction of the L210W mutation with four mutations at codons 41, 62, 69 and 215 (e.g. M41L+A62V+T69SSA+T215Y) had little effect on drug susceptibility and showed a resistance profile similar to the profile obtained for when only the four mutations were present. In yet further studies using site directed mutagenesis, it was observed that the introduction of the T215Y mutation with mutations at 62 and 69 (e.g. A62V+T69SSA) resulted in a substantial decrease in susceptibility to AZT (greater than 1000-fold) compared to the 7-fold decrease in susceptibility observed for AZT without the 215 mutation. It was also observed that the introduction of the L74V mutation with mutations at 62 and 69 (e.g. A62V+T69SSA) resulted in a shift back to wild-type susceptibility for AZT. In yet further studies using site directed mutagenesis, it was observed that the introduction of the V75M mutation with four mutations at codons 41, 69, 210 and 215 (e.g. M41L+T69SSA+L210W+T215Y) had little effect on drug susceptibility and showed a resistance profile similar to the profile obtained when only the four mutations were present.
In a further embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. More specifically in yet another embodiment of the invention PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 (T69SSA, T69SSG, T69SSS) in combination with mutations at other codons including 41 (M41L), 210 (L210W), 215 (T215Y), 184 (M184V) and/or 74 (L74V) of HIV RT which correlate, as described herein, with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. In yet another embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 62, 75, 77, 116 or 115 either alone or in combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or 215 of HIV HT which correlate, as described herein, with resistance to antiretroviral therapies and immunologic decline. Examples of the mutations at the aforementioned codons include, but are not limited to (A62V, V75I, F77L, F116Y, Q151M) and (M41L, D67N, L210W, T215Y, K219Q, M184V/I, T69D, T215Y). In yet another embodiment of the invention, PCR based assays, including phenotypic and genotypic assays, may be used to detect four or more mutations in RT at codons in the group consisting of 41, 67, 70, 210, 215 and/or 219 (e.g. M41L, D67N, K70R, L210W, T215Y/F, K219Q) either alone or in combination with mutation at codon 74 (V74I), 69 (T69D), 75 (V75M, V75S) and/or 219 (K219N) of HIV RT which correlates, as described herein, with resistance to antiretroviral therapy and immunologic decline. Once mutations at codon 69 either alone or in combination with mutation at codon 41 and 215 of HIV RT in a patient undergoing NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once mutations at codon 69 and/or 41, 210, 215, 184 and/or 74 have been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once mutations at codon 62, 75, 77, 116 and/or 115 either alone or in combination with mutations associated with resistance to AZT, 3TC, ddC or a mutation T215V has been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. Similarly, once four or more mutations associated with AZT resistance selected from the group consisting of 41, 67, 70, 210, 215 and/or 219 either alone or in combination with a mutation at codon 74 (V741), 69 (T69D), 75 (V75M, V75S) and/or 219(K219N) has been detected in a patient undergoing certain NRTI antiretroviral therapy, an alteration in the therapeutic regimen must be considered. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. Similarly, PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41, 219, 215, 184 and/or 74 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 62 and/or 75 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. PCR based assays, including phenotypic and genotypic assays, may be used to detect mutations at codon 62, 75, 77, 116 and 115 either alone or in combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or T215V of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline. The timing at which a modification of the therapeutic regimen should be made, following the assessment of the antiretroviral therapy using PCR based assays, may depend on several factors including the patient""s viral load, CD4 count, and prior treatment history.
In another aspect of the invention there is provided a method for assessing the effectiveness of a nucleoside reverse transcriptase antiretroviral drug comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a); (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a)-(c) are carried out in the absence of the NRTI anti-HIV drug, wherein a test concentration of the NRTI, anti-HIV drug is presented at steps (a)-(c); at steps (b)-(c); or at step (c).
This invention also provides a method for assessing the effectiveness of non-nucleoside reverse transcriptase antiretroviral therapy in a patient comprising: (a) developing a standard curve of drug susceptibility for an NRTI anti-HIV drug; (b) determining NRTI anti-HIV drug susceptibility in the patient using the susceptibility test described above; and (c) comparing the NRTI anti-HIV drug susceptibility in step (b) with the standard curve determined in step (a), wherein a decrease in NRTI anti-HIV susceptibility indicates development of anti-HIV drug resistance in the patient.
This invention also provides a method for evaluating the biological effectiveness of a candidate HIV antiretroviral drug compound comprising: (a) introducing a resistance test vector comprising a patient-derived segment and an indicator gene into a host cell; (b) culturing the host cell from step (a); (c) measuring expression of the indicator gene in a target host cell wherein expression of the indicator gene is dependent upon the patient derived segment; and (d) comparing the expression of the indicator gene from step (c) with the expression of the indicator gene measured when steps (a)-(c) are carried out in the absence of the candidate anti-viral drug compound, wherein a test concentration of the candidate anti-viral drug compound is present at steps (a)-(c); at steps (b)-(c); or at step (c)
The expression of the indicator gene in the resistance test vector in the target cell is ultimately dependent upon the action of the patient-derived segment sequences. The indicator gene may be functional or non-functional.
In another aspect this invention is directed to antiretroviral drug susceptibility and resistance tests for HIV/AIDS. Particular resistance test vectors of the invention for use in the HIV/AIDS antiretroviral drug susceptibility and resistance test are identified.
In yet another aspect this invention provides for the identification and assessment of the biological effectiveness of potential therapeutic antiretrovlral compounds for the treatment of HIV and/or AIDS. In another aspect, the invention is directed to a novel resistance test vector comprising a patient-derived segment further comprising one or more mutations on the RT gene and an indicator gene.

Means and methods for monitoring nucleoside reverse transcriptase inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS


D(+)-threo-2-hydroxy-3-(2-aminophenylthio)-3-(4-methoxyphenyl)propionic acid, an important intermediate in the synthesis of diltiazem hydrochloride, is obtained in better yields and high optical purity by a novel resolution process comprising the reaction of 2 moles of the (.+-.) racemic acid with 1 mole of quinine and 1 mole of a base in a suitable polar solvent system to form a salt of the D(+) acid and quinine which precipitates; separating said salt and decomposing it with a strong base.

Diltiazem, a well known drug with calcium antagonist activity has the formula (I) ##STR2##
As seen in formula (I), Dilitiazem has two asymmetric carbon atoms (marked with (*)), the configuration of Dilitiazem at its two asymmetric carbons being (2S, 3S).
Numerous methods are known for the synthesis of Dilitiazem from the intermediate threo-propionic acid, and particularly from the optical isomer D(+)-threo-propionic acid.
Several methods have been proposed for the optical resolution of (.+-.) threo-propionic acid (II). Thus, U.S. Pat. No. 4,416,819, JP 103,356 and EP 381,570 describe the use of D(+)-phenylethylamine as the resolving agent for (.+-.)-threopropionic acid.
Other optically active amines such as tolylethylamine (JP 097,606), R(+)-naphtylethylamine (JP 61,207,371) or threo-1-(4-X-phenyl)-2-amino-1,3-propanediol (EP 89,113,135) were also used for the same purpose, while in accordance with JP 8,822,556, IT 023,769 and WO 9100270 L-lysine or phenyl-glycine amide were used instead of the optically active amines as resolving agents.
JP 8,910,617 describes the resolution (.+-.) of erythro-propionic acid by selective hydrolysis of its methyl ester. In this procedure, purification of the separated isomer is necessary, resulting in a reduced yield.
None of the above methods have been found satisfactory, because of low yields, difficult reaction conditions and/or poor recovery of the expensive catalyst or resolving agent.

Optical resolution of threo-2-hydroxy-3-(2-aminophenylthio)-3-(4-methoxyphenyl)-propionic acid